Activity-regulatable il-12 fusion protein and use thereof

Abstract

Provided are an activity-regulatable IL-12 fusion protein and the use thereof. The fusion protein comprises a first structural unit, a second structural unit, and a third structural unit. The first structural unit is selected from a first Fc fragment, or a first antibody or an antigen-binding fragment thereof, wherein the first antibody or the antigen-binding fragment thereof has the binding activity to a tumor antigen or an immune checkpoint; the second structural unit is selected from a cleavable linker or a non-cleavable linker; and the third structural unit comprises an IL-12 cytokine or a functional fragment thereof. The second structural unit mediates the steric hindrance of the first structural unit to mask the activity of the IL-12 cytokine in the third structural unit, thereby reducing the toxic side effects of in-vivo use of the IL-12 cytokine. Compared with a wild-type IL-12, the fusion protein has the advantage of high safety, and can be further fused with an antibody Fab, scFv or VHH, an antigen-binding peptide or a recombinant protein, a polypeptide, etc. to obtain a multifunctional fusion protein that can be conditionally released and activated.

Description

Activity-regulatable IL-12 fusion protein and its use Technical Field

[0001] The present invention belongs to the field of medical technology and specifically relates to an activity-regulatable IL-12 fusion protein and its uses. More specifically, it relates to a fusion protein, nucleic acid molecule, expression vector, recombinant cell, fusion protein complex, immunotherapy cell and pharmaceutical composition containing the IL-12 cytokine, and their uses in the preparation of drugs for treating or preventing cancer, infectious diseases or autoimmune diseases. Background Art

[0002] Interleukin-12 (IL-12) is a multipotent cytokine encoded by two independent genes, IL-12A (p35) and IL-12B (p40). It forms an active p70 heterodimer linked by two disulfide bonds. Activated dendritic cells (DCs), macrophages, and neutrophils produce IL-12 locally during T cell priming. IL-12 acts directly on cytotoxic immune effector cells, including natural killer (NK) cells, natural killer T (NKT) cells, and CD8+ T cells, stimulating their proliferation and increasing their cytotoxic function. IL-12 also induces nascent helper T cells to differentiate toward a Th1 phenotype and produce cytokines (most notably IFNγ). IFNγ itself has cytostatic / cytotoxic and anti-angiogenic properties, and can upregulate the expression of MHC I and II on tumor cells to enhance recognition and lysis. Consequently, even relatively low concentrations of IL-12 in clinical use can have potent stimulatory activity on immune cells in normal tissues, leading to severe systemic side effects and a narrow therapeutic window. Furthermore, IL-12 has a rapid clearance serum half-life, necessitating frequent dosing to maintain therapeutically relevant exposure.

[0003] Antibody-cytokine fusion proteins, also known as immune cytokines, can effectively achieve the enrichment of cytokines at the tumor site by utilizing the targeting properties of antibodies, thereby reducing the dosage and reducing the toxic side effects of drugs. Currently, there are studies fusing IL-12 with monoclonal antibodies targeting tumor antigens or tumor necrosis to construct antibody-cytokine bifunctional fusion proteins. IL-12 can stimulate the proliferation of CD8+ T cells and NK cells to exert anti-tumor effects, and antibodies can serve as "missiles" to carry IL-12, achieving the enrichment of IL-12 at the tumor site. However, due to the significant biological activity of IL-12, even small off-target effects can lead to strong toxic side effects. The currently reported immune cytokines still cannot completely solve the problem of IL-12 systemic toxicity. Therefore, there is an urgent need to develop a drug product that can deliver IL-12 to the target tissue in the body and release it. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a fusion protein containing IL-12 cytokine.

[0005] In the first aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein includes a first structural unit, a second structural unit and a third structural unit. The first structural unit is selected from a first Fc fragment, or a first antibody or an antigen-binding fragment thereof, and the first antibody or antigen-binding fragment thereof has tumor antigen or immune checkpoint binding activity; the second structural unit is selected from a cleavable linker or a non-cleavable linker, and the cleavable linker can be cleaved by a protease expressed in tumor tissue; the third structural unit comprises an IL-12 cytokine or a functional fragment thereof; wherein the first structural unit is connected to one or more of the third structural units, and the third structural unit is connected to the C-terminus of the first structural unit through the second structural unit. The third structural unit of the fusion protein of the present invention contains an IL-12 cytokine or a functional fragment thereof, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies. Therefore, the fusion protein does not need to contain any IL-12 cytokine antibodies or receptors, and can reduce the toxic side effects caused by the IL-12 cytokine. The fusion protein has the advantages of strong safety and simple structure.

[0006] In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The nucleic acid molecule of the present invention can encode the fusion protein described in the first aspect.

[0007] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect. Thus, the expression vector of the present invention can effectively express the fusion protein described in the first aspect, thereby enabling large-scale production of the fusion protein in vitro.

[0008] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the nucleic acid described in the second aspect or the expression vector described in the third aspect; or expressing the fusion protein described in the first aspect. Under suitable conditions, the recombinant cell can effectively express the fusion protein described in the first aspect within the cell.

[0009] In the fifth aspect of the present invention, the present invention proposes a fusion protein complex. According to an embodiment of the present invention, the fusion protein complex includes the fusion protein described in the first aspect. As mentioned above, the third structural unit of the fusion protein described in the first aspect contains IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve the shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies. Therefore, the fusion protein does not need to contain any antibodies or receptors for IL-12 cytokines, and can reduce the toxic and side effects caused by IL-12 cytokines. The fusion protein has advantages such as strong safety. Therefore, the fusion protein complex of the present invention has advantages such as strong safety and can be used to treat cancer, infectious diseases or autoimmune diseases.

[0010] In the sixth aspect of the present invention, the present invention proposes an immunotherapy cell. According to an embodiment of the present invention, the immunotherapy cell expresses the fusion protein described in the first aspect. As mentioned above, the third structural unit of the fusion protein described in the first aspect contains IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve the shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies. Therefore, the fusion protein does not need to contain any antibodies or receptors for IL-12 cytokines, and can reduce the toxic and side effects caused by IL-12 cytokines. The fusion protein has advantages such as strong safety. Therefore, the immunotherapy cell containing the above-mentioned fusion protein has advantages such as strong safety and can be used to treat cancer, infectious diseases or autoimmune diseases.

[0011] In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the fusion protein complex described in the fifth aspect, or the immunotherapy cell described in the sixth aspect. As can be seen from the above, the third structural unit of the fusion protein described in the first aspect contains IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies. Therefore, the fusion protein does not need to contain any antibodies or receptors for IL-12 cytokines, and can reduce the toxic and side effects caused by IL-12 cytokines. The fusion protein has the advantages of strong safety. Therefore, the pharmaceutical composition containing the above-mentioned fusion protein has the advantages of strong safety and can be used to treat cancer, infectious diseases or autoimmune diseases.

[0012] In the eighth aspect of the present invention, the present invention proposes a use of the fusion protein described in the first aspect, the fusion protein complex described in the fifth aspect, the immunotherapy cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect in the preparation of a drug for treating cancer, infectious diseases or autoimmune diseases.

[0013] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0015] Figure 1 is a schematic diagram of the structure of the fusion protein of the present invention, wherein LIC401 is a C-indestructible L-Fc with only Fc as the first structural unit; LIC402 is an Fc-indestructible LC with only Fc as the first structural unit; LIC403 is an Fc-bi-cleavage LC with only Fc as the first structural unit; LIC404 and LIC405 are Fc-single-cleavage LC with only Fc as the first structural unit; LIC406 is an IgG-indestructible LC with a monoclonal antibody as the first structural unit; LIC407 is an IgG-bi-cleavage LC with a monoclonal antibody as the first structural unit; LIC408 and LIC409 are IgG-single-cleavage LC with a monoclonal antibody as the first structural unit; LIC410 is two identical ScFv -Fc as the first structural unit of C-uncleavable L-Fc-ScFv; LIC411 is two identical ScFv-Fc as the first structural unit of ScFv-Fc-uncleavable LC; LIC412 is two identical ScFv-Fc as the first structural unit of ScFv-Fc-double cleavage LC; LIC413, LIC414 are two identical ScFv-Fc as the first structural unit of ScFv-Fc-single cleavage LC; LIC415 is two different ScFv-Fc as the first structural unit of C-uncleavable L-Fc-ScFv; LIC416 is two different ScFv-Fc as the first structural unit of ScFv-Fc-uncleavable LC; LIC417 is a ScFv-Fc-double-cleavage LC with two different ScFv-Fc as the first structural unit; LIC418 and LIC419 are ScFv-Fc-single-cleavage LC with two different ScFv-Fc as the first structural unit; LIC420 is a (Fab)2-uncleavable LCL-Fc with two Fab as the first structural unit and Fc as the fourth structural unit; LIC421 is a (Fab)2-double-cleavage LCL-Fc with two Fab as the first structural unit and Fc as the fourth structural unit; LIC422 and LIC423 are (Fab)2-single-cleavage LCL-Fc with two Fab as the first structural unit and Fc as the fourth structural unit; C424 is a Fab-uncleavable LCL-Fc with a single Fab as the first structural unit and an Fc as the fourth structural unit; LIC425 is a Fab-double-cleavage LCL-Fc with a single Fab as the first structural unit and an Fc as the fourth structural unit; LIC426 and LIC427 are Fab-single-cleavage LCL-Fc with a single Fab as the first structural unit and an Fc as the fourth structural unit; LIC428 is an IgG-uncleavable L-C2 with a monoclonal antibody as the first structural unit and containing two molecules of cytokines; LIC429 is a (Fab)2-uncleavable L-C2-L-Fc with two Fabs as the first structural unit and an Fc as the fourth structural unit and containing two molecules of cytokines;LIC430 is a (Fab)2-bilobate L-C2-L-Fc with two Fabs as the first structural unit and Fc as the fourth structural unit, and contains two molecules of cytokine; LIC431 is a (Fab)2-singlelobate L-C2-L-Fc with two Fabs as the first structural unit and Fc as the fourth structural unit, and contains two molecules of cytokine; LIC432 is a single Fab as the first structural unit and Fc as the fourth structural unit, and contains two molecules of cytokine Fab-uncleavable L-C2-L-Fc; LIC433 is a single Fab as the first structural unit and Fc as the fourth structural unit, and contains two molecules of cytokine LIC436 is a monoclonal antibody as the first structural unit and an IgG-uncleavable LC with cytokine subunits in series; LIC437 is a (Fab)2-uncleavable LCL-Fc with two Fabs as the first structural unit, Fc as the fourth structural unit and cytokine subunits in series; LIC438 is a two-unit (Fab)2-uncleavable LCL-Fc with two Fabs as the first structural unit, Fc as the fourth structural unit and cytokine subunits in series; The same ScFv as the first structural unit, Fc as the fourth structural unit and cytokine subunits are connected in series. ScFv-uncleavable LCL-Fc; LIC439 is two different ScFv as the first structural unit, Fc as the fourth structural unit and cytokine subunits are connected in series. ScFv-uncleavable LCL-Fc; LIC440 is two identical ScFv as the first structural unit, Fc as the fourth structural unit and cytokine subunits are connected in series. Fc-uncleavable LCL-ScFv; LIC441 is two different ScFv as the first structural unit, Fc as the fourth structural unit and cytokine subunits are connected in series. LIC442 is a ScFv-Fc-non-cleavable LC with two identical ScFv-Fc subunits in series; LIC443 is a ScFv-Fc-non-cleavable LC with two different ScFv-Fc subunits in series as the first structural unit; LIC444 is an IgG-non-cleavable LC with a monoclonal antibody as the first structural unit and cytokine subunits in series, but in a different order than LIC436; LIC445 is an IgG-double-cleaved LC with a monoclonal antibody as the first structural unit and containing two cytokine molecules. Among them, single-cleavage L represents a fusion protein with only one chain containing a cleavable linker; double-cleavage L represents a fusion protein with two chains containing a cleavable linker; C represents a fusion protein containing one cytokine molecule, and C2 represents a fusion protein containing two cytokine molecules; L represents the non-cleavable linker at the C-terminus of the IL12 subunit.

[0016] FIG2 is a diagram of protein A affinity chromatography purification of LIC401 fusion protein.

[0017] Figure 3 is an SDS-PAGE electrophoresis of the purified LIC401 fusion protein. NR represents the non-reduced sample, and R represents the reduced sample. The mother liquor represents the collected cell supernatant, NaOH represents the impurities eluted with NaOH solution after chromatography, and the flowthrough represents the flowthrough from the protein A affinity chromatography process. The eluted fraction represents the LIC401 fusion protein.

[0018] Figure 4 shows Western blot analysis of the LIC401 fusion protein. The plasmids were transfected at a 1:1 ratio, and R represents the reduced sample.

[0019] FIG5 is a diagram of protein A affinity chromatography purification of LIC402 fusion protein.

[0020] Figure 6 shows Western blot analysis of the LIC402 fusion protein. The plasmids were transfected at a 1:1 ratio, and R represents the reduced sample.

[0021] FIG7 is a photograph showing the stimulation of mouse spleen by LIC401 and LIC402 fusion proteins.

[0022] FIG8 is a statistical graph of spleen weights in mice treated with PBS, LIC401, and LIC402 fusion proteins.

[0023] FIG9 is a diagram of protein A affinity chromatography purification of LIC403 fusion protein.

[0024] Figure 10 is an SDS-PAGE electrophoresis of the purified LIC403 fusion protein. NR is the non-reduced sample, and R is the reduced sample. Flowthrough is the flowthrough from the protein A affinity chromatography process, and the eluted product is the LIC403 fusion protein.

[0025] Figure 11 is a graph showing the in vitro activity assay of the purified LIC403 fusion protein, a graph showing the in vivo activity assay of the LIC401, LIC402, LIC402-1, and LIC403 fusion proteins, and a graph showing changes in mouse body weight after administration of the LIC401, LIC402, LIC402-1, and LIC403 fusion proteins.

[0026] FIG12 is a diagram showing the protein A affinity chromatography purification of the LIC404 fusion protein.

[0027] Figure 13 is an SDS-PAGE electrophoresis of the purified LIC404 fusion protein. NR is the non-reduced sample, and R is the reduced sample. Flowthrough is the flowthrough from the protein A affinity chromatography process, and the eluted liquid is the LIC404 fusion protein.

[0028] FIG14 is a diagram showing the protein A affinity chromatography purification of the LIC405 fusion protein.

[0029] FIG15 is a graph showing the results of in vitro activity assays of LIC404, LIC405, and a control fusion protein.

[0030] FIG16 is a diagram showing the protein A affinity chromatography purification of LIC407 fusion protein.

[0031] FIG17 is an ELISA test chart showing the affinity of LIC407 fusion protein for human PDL1 antigen.

[0032] Figure 18 is a graph showing in vitro activity assays of LIC407, LIC408, and LIC409 fusion proteins, in vivo activity assays of LIC406 to LIC409 fusion proteins, and weight changes of mice after administration of LIC406 to LIC409 fusion proteins.

[0033] FIG19 is a diagram showing the protein A affinity chromatography purification of LIC412 fusion protein.

[0034] FIG20 is a graph showing in vitro activity assays of LIC412 and control fusion proteins.

[0035] FIG21 is a diagram showing the protein A affinity chromatography purification of LIC417 fusion protein.

[0036] FIG22 is a graph showing in vitro activity assays of LIC417 and control fusion proteins.

[0037] FIG23 is a diagram showing the protein A affinity chromatography purification of the LIC421 fusion protein.

[0038] FIG24 is a graph showing the in vitro activity assay of the purified LIC421 fusion protein.

[0039] FIG25 is a graph showing the in vitro activity assay of the single-cleavage form of LIC422 and LIC423 fusion proteins.

[0040] FIG26 is a diagram showing the protein A affinity chromatography purification of LIC425 fusion protein.

[0041] FIG27 is a schematic diagram of SDS-PAGE of LIC425 fusion protein after in vitro enzymatic cleavage.

[0042] FIG28 is a graph showing the in vitro activity assay of the LIC425 fusion protein.

[0043] FIG29 is an ELISA test chart showing the affinity of LIC425 fusion protein for human PDL1 antigen.

[0044] FIG30 is an ELISA assay showing the distribution of LIC425 fusion protein in various tissues of tumor-bearing mice.

[0045] FIG31 is a diagram showing the protein A affinity chromatography purification of the LIC425-1 fusion protein.

[0046] FIG32 is a graph showing changes in mouse body weight after administration of LIC425-1 and control fusion proteins.

[0047] FIG33 is a statistical graph showing the spleen weight of mice at the end point after administration of LIC425-1 and control fusion proteins.

[0048] FIG34 is a graph showing the counts of CD8+ T cells and NK cells in peripheral blood at the end point of administration of LIC425-1 and control fusion proteins.

[0049] FIG35 is a schematic diagram of the proportion of CD8+ T cells in the spleen at the end point of administration of LIC425-1 and control fusion proteins.

[0050] FIG36 is a graph showing the in vitro activity assay of the single-cleavage form of LIC426 and LIC427 fusion proteins.

[0051] FIG37 is a diagram showing protein A affinity chromatography purification of LIC430 fusion protein.

[0052] FIG38 is a graph showing the results of detecting the activity of LIC430 fusion protein in promoting IFN-γ secretion by mouse spleen cells before and after uPA enzyme cleavage.

[0053] FIG39 is a diagram showing protein A affinity chromatography purification of LIC433 fusion protein.

[0054] FIG40 is a graph showing in vitro activity assays of LIC433 and control fusion proteins.

[0055] FIG41 is a diagram showing protein A affinity chromatography purification of LIC436 fusion protein.

[0056] FIG42 is a diagram showing the protein A affinity chromatography purification of the LIC444 fusion protein.

[0057] FIG43 is a graph showing the results of detecting the activity of LIC436 and LIC444 fusion proteins in promoting the secretion of IFN-γ by mouse spleen cells.

[0058] FIG44 is a graph showing the results of LIC438 and LIC441 fusion proteins stimulating IFN-γ secretion in vitro.

[0059] Figure 45 is a diagram showing the protein A affinity chromatography purification of the humanized IL12 form LIC445 fusion protein.

[0060] FIG46 is a graph showing the results of LIC445 fusion protein stimulating human PBMC to secrete IFN-γ in vitro.

[0061] FIG47 is a graph showing a comparison of the shielding effects of fusion proteins with different structures in Example 37 of the present invention.

[0062] Figure 48 is a graph showing changes in mouse body weight after administration of fusion proteins of different structures in Example 38 of the present invention. DETAILED DESCRIPTION

[0063] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0064] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0065] Detailed description of the invention

[0066] Definitions and General Terms

[0067] It should be noted that the endpoints of the scope disclosed in this article and any value are not limited to this accurate scope or value, and these scopes or values ​​should be interpreted as comprising the value approaching these scopes or value.For numerical range, between the endpoint values ​​of each scope, between the endpoint values ​​of each scope and an independent point value, and between the independent point value, can be combined with each other and obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed in this article.Unrepresented specific technology or condition in the embodiment, according to the technology or condition described in the document in this area or according to product specification sheet, carry out.Agents therefor or instrument do not indicate manufacturer, are the conventional products that can be obtained by commercial purchase.

[0068] It should be noted that, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the corresponding fields.

[0069] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0070] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0071] As used herein, the term "fragment" refers to a target protein or polypeptide, as well as a target protein or polypeptide with N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or internal deletion.

[0072] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0073] As used herein, the term "fusion protein" refers to a novel protein (e.g., a biologically active polypeptide and an effector molecule) formed by the fusion of at least two proteins or polypeptides. The fusion operation can usually be achieved by techniques such as genetic engineering, chemical methods, or other appropriate methods, for example, the expression product of two genes recombined by DNA recombination technology. If necessary, the fusion molecule can be fused at one or several positions via an amino acid linker. The fusion protein can exist in the form of a monomer or a polymer (e.g., a dimer). In this article, it refers to a fusion state between the first structural unit, the second structural unit, and the third structural unit comprising the IL-12 cytokine or a functional fragment thereof.

[0074] The fusion proteins described herein are typically prepared by biosynthetic methods. Based on the nucleotide sequences described herein, those skilled in the art can readily produce the encoding nucleic acids of the present invention using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis. For specific methods, see J. Sambrook, Molecular Cloning: A Laboratory Manual. As one embodiment of the present invention, the encoding nucleic acid sequences of the present invention can be constructed by synthesizing nucleotide sequences in segments followed by overlap extension PCR.

[0075] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a light chain (L chain) and a heavy chain (H chain). In a general sense, a heavy chain can be understood as the polypeptide chain with the larger molecular weight in an antibody, while a light chain refers to the polypeptide chain with the smaller molecular weight in an antibody. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3), while each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs). The variable regions (VH and VL) of each heavy chain / light chain pair respectively form the antibody binding site. The term "antibody" is not limited to any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG or its mutants, IgA1, IgA2, IgD, IgE or IgM antibodies. In some embodiments of the present invention, the antibody that binds to a tumor antigen or immune checkpoint is a full-length immunoglobulin, including IgG1, IgG2, IgG3 and IgG4.

[0076] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment comprising a portion or all of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an antigen-binding portion. For example, the fragment may comprise a portion or all of an antibody CDR. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some embodiments of the present invention, the antigen-binding fragment of the antibody is in the form of Fab, Fab', (Fab')2, Fv, scFv, or VHH, or Fab, Fab', (Fab')2, Fv, scFv, VHH, and scFv. In the present invention, "Fab" is a fragment obtained by treating an antibody molecule with papain (cleaving the amino acid residue at position 224 of the H chain), wherein approximately half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds. In the present invention, "F(ab')2" is an antibody fragment with a molecular weight of approximately 100,000 Da, obtained by digesting the portion below the two disulfide bonds in the hinge region of an antibody molecule with pepsin, and comprising two Fab regions connected at the hinge position. In the present invention, "Fab'" is an antibody fragment obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds to an antigen with a reducing agent such as dithiothreitol. In the present invention, "Fv" is the smallest functional fragment of an antibody molecule that retains an antigen-binding site and is composed of a light chain variable region and a heavy chain variable region, which are bound together by non-covalent bonds. In the present invention, "scFv" refers to an antibody fragment comprising the heavy chain variable region (VH) and light chain variable region (VL) of an antibody. ScFv is generally 1 / 6 the size of an intact antibody and preferably consists of a single amino acid sequence encoded by a single nucleotide chain. In the present invention, "VHH" is the single variable region of a heavy chain antibody that has antigen-binding ability.

[0077] As used herein, the terms "full-length antibody," "full-length monoclonal antibody," or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0078] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the nucleic acid molecule contains a nucleotide sequence that can express a target protein, and the nucleic acid molecule can be transferred into a host cell and / or between host cells. The expression vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0079] As used herein, the term "recombinant cell" generally refers to a cell that has been modified or reorganized using genetic engineering techniques or cell fusion techniques to modify or reorganize the genetic material of a recipient cell (or host cell) to obtain a cell with a unique trait of stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., an expression vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0080] As used herein, the term "pharmaceutical composition" generally refers to a pharmaceutical in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active fusion protein with a liquid carrier, a finely divided solid carrier, or both.

[0081] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. Except to the extent that any conventional excipient is incompatible with the fusion protein of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0082] In this article, the term "administration" refers to introducing a predetermined amount of material into a patient by a certain suitable mode. Fusion protein or pharmaceutical composition of the present invention can be administered by any common approach, as long as it can reach the expected tissue. The various modes of administration are expected to include peritoneal, intravenous, intramuscular, subcutaneous injections, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, compositions of the present invention are administered by intravenous or subcutaneous injection.

[0083] As used herein, the term "treatment" refers to a method for obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a fusion protein or pharmaceutical composition to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing the fusion protein described herein to an individual in need.

[0084] Detailed description of the cytokine fusion protein of the present invention and its use

[0085] The present invention provides a fusion protein, nucleic acid molecule, expression vector, recombinant cell, fusion protein complex, immunotherapy cell, and pharmaceutical composition containing an IL-12 cytokine, as well as their use in preparing a medicament for treating or preventing cancer, infectious diseases, or autoimmune diseases, each of which is described in detail below.

[0086] Fusion protein

[0087] In the first aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein includes a first structural unit, a second structural unit and a third structural unit, the first structural unit is selected from a first Fc fragment, or a first antibody or an antigen-binding fragment thereof, the first antibody or its antigen-binding fragment having tumor antigen or immune checkpoint binding activity; the second structural unit is selected from a cleavable linker or a non-cleavable linker, the cleavable linker can be cleaved by a protease expressed in tumor tissue; the third structural unit comprises an IL-12 cytokine or a functional fragment thereof; wherein the first structural unit is connected to one or more of the third structural units, and the third structural unit is connected to the C-terminus of the first structural unit through the second structural unit. The third structural unit of the fusion protein of the present invention contains an IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies, so the fusion protein does not need to contain any IL-12 cytokine antibodies or receptors, and can reduce the toxic side effects caused by the IL-12 cytokine, and the fusion protein has the advantages of strong safety.

[0088] Furthermore, the inventors found that the N-terminus of the third structural unit is affected by the steric hindrance of the first structural unit, and its binding to the receptor is restricted in the fused state, resulting in lower biological activity. After the cleavable linker of the second structural unit is cut, the N-terminus or the N-terminus and C-terminus of the third structural unit are released, and the affinity for the receptor is restored, and the biological activity is restored to a higher level, thereby reducing its toxic side effects on non-target tissues while improving the therapeutic effect on the target.

[0089] Herein, the term “X1 is connected to the C-terminus of X3 via X2” means that the sequence from N-terminus to C-terminus is X3-X2-X1, for example, “the third structural unit is connected to the C-terminus of the first structural unit via the second structural unit” means that the sequence from N-terminus to C-terminus is first structural unit-second structural unit-third structural unit; the term “X1 is connected to the N-terminus of X3 via X2” means that the sequence from N-terminus to C-terminus is X1-X2-X3, for example, “the third structural unit is connected to the N-terminus of the first structural unit via the second structural unit” means that the sequence from N-terminus to C-terminus is third structural unit-second structural unit-first structural unit.

[0090] In some embodiments of the present invention, "IL-12" refers to interleukin 12, including any natural (wild type) IL-12, its functional fragment or mutant, preferably mammalian interleukin 12. In some embodiments, mammalian interleukin 12 includes but is not limited to interleukin 12 derived from humans, mice, pigs, rabbits, sheep, monkeys, and cats. In some embodiments, the IL-12 cytokine is selected from a murine IL-12 cytokine or a human IL-12 cytokine. All structures of the present invention are applicable to murine or human IL-12 cytokines, that is, the IL-12 cytokine is at least one of murine or human.

[0091] According to an embodiment of the present invention, when the first structural unit is connected to one of the third structural units, the second structural unit is selected from a non-cleavable linker.

[0092] In some embodiments of the present invention, in the fusion protein of the present invention, the first structural unit is connected to the C-terminus of the third structural unit via the non-cleavable linker (cytokine C-terminus) of the second structural unit. The fusion protein structure is shown in Figure 1 as LIC401, LIC410, and LIC415, and comprises, from N-terminus to C-terminus: Chain A: P35 subunit, non-cleavable linker (cytokine C-terminus), antibody Knob sequence; Chain B: P40 subunit, non-cleavable linker (cytokine C-terminus), antibody Hole sequence; wherein the two antibody chains can have the same or different targeting. In certain embodiments, different cytokine subunits fused to Fc-Knob or Fc-Hole can be interchanged without affecting their structure or function. For example, in LIC401, the Fc-Knob is linked to the IL-12P35 subunit via a flexible linker, and the Fc-Hole is linked to the IL-12P40 subunit via a flexible linker. Replacing these with the Fc-Knob linked to the IL-12P40 subunit via a flexible linker and the Fc-Hole linked to the IL-12P35 subunit via a flexible linker does not alter IL-12 activity. The present invention discovered that IL-12 is more active when located at the N-terminus of the fusion protein. Injection into mice significantly stimulates spleen enlargement, thus allowing LIC401 to serve as a positive control molecule.

[0093] In some embodiments of the present invention, the first structural unit of the fusion protein of the present invention is connected to the N-terminus of the third structural unit via the non-cleavable linker of the second structural unit (cytokine N-terminus). In certain embodiments, the fusion protein structure is as shown in Figure 1, LIC402, LIC406, LIC411, and LIC416. The fusion protein comprises, from N-terminus to C-terminus: Chain A: antibody Knob sequence, non-cleavable linker (cytokine N-terminus), P35 subunit; Chain B: antibody Hole sequence, non-cleavable linker (cytokine N-terminus), P40 subunit; wherein the targets of the two antibody chains may be the same or different. In other embodiments, the fusion protein structure is as shown in FIG1 LIC428, LIC436, LIC442, LIC443, LIC444, the fusion protein comprises, from N-terminus to C-terminus, an antibody, a non-cleavable linker (cytokine N-terminus), and the P35 subunit-P40 subunit of IL-12; wherein the fusion order of the P35 subunit-P40 subunit can be the P35 subunit at the N-terminus and the P40 subunit at the C-terminus, or the P40 subunit at the N-terminus and the P35 subunit at the C-terminus; in addition, the non-cleavable linker and the P35 subunit-P40 subunit fusion protein can be symmetrically linked to the C-terminus of the two Fc chains of the antibody, or to the C-terminus of only one Fc chain. The present application found that compared to the fusion protein with IL-12 located at the N-terminus, the activity of IL-12 located at the C-terminus of the fusion protein was significantly lower. Secondly, the activity of the heterologous IL-12 form at the C-terminus (such as LIC406) was weaker than that of the homologous IL-12 form (such as LIC428).

[0094] According to an embodiment of the present invention, when the first structural unit is connected to one of the third structural units, the second structural unit is a cleavable linker.

[0095] According to an embodiment of the present invention, when the first structural unit is connected to a plurality of the third structural units, the second structural unit connected to the plurality of the third structural units contains at least one cleavable linker, and preferably, the second structural units connected to the plurality of the third structural units are all cleavable linkers.

[0096] According to an embodiment of the present invention, when the first structural unit is connected to a plurality of the third structural units, the plurality of the second structural units are all cleavable linkers.

[0097] In some embodiments of the present invention, the first structural unit of the fusion protein of the present invention is linked to the N-terminus of the third structural unit via a cleavable linker of the second structural unit. The fusion protein structures are shown in Figure 1 for LIC403, LIC407, LIC412, and LIC417. The fusion protein comprises, from N-terminus to C-terminus, the following: Chain A: antibody Knob sequence, cleavable linker, P35 subunit; Chain B: antibody Hole sequence, cleavable linker, P40 subunit. The targets of the two antibody chains may be the same or different. In other embodiments, the fusion protein structure is shown in Figure 1 for LIC445. The fusion protein comprises, from N-terminus to C-terminus, an antibody, a cleavable linker, and an IL-12 P35 subunit-P40 subunit fusion protein. The order of the P35-P40 subunit fusion can be either P35 at the N-terminus and P40 at the C-terminus, or P40 at the N-terminus and P35 at the C-terminus. The targets of the two antibody chains may be the same or different. The present application found that the cleavable fusion protein of IL-12 at the C-terminus exhibits different activities before and after cleavage and has better safety.

[0098] According to an embodiment of the present invention, when the first structural unit is connected to a plurality of the third structural units, the plurality of the second structural units contain at least one cleavable linker.

[0099] In some embodiments of the present invention, the first structural unit of one chain of the fusion protein of the present invention is connected to the N-terminus of the third structural unit through the non-cleavable linker of the second structural unit (the N-terminus of the cytokine). The first structural unit of the other chain is connected to the N-terminus of the third structural unit through the cleavable linker of the second structural unit. The fusion protein structure is shown in Figure 1 as LIC404, LIC405, LIC408, LIC409, LIC413, LIC414, LIC418, and LIC419. The fusion protein comprises, from N-terminus to C-terminus: Chain A: antibody Knob sequence, non-cleavable linker, P35 subunit; Chain B: antibody Hole sequence, cleavable linker, P40 subunit; or Chain A: antibody Knob sequence, cleavable linker, P35 subunit; Chain B: antibody Hole sequence, non-cleavable linker, P40 subunit; wherein the targets of the two chains of the antibody may be the same or different. The present application found that compared with the non-cleavable fusion protein of IL-12 at the C-terminus, the single-chain cleavable fusion protein of IL-12 at the C-terminus has stronger stimulatory activity on lymphocytes after cleavage.

[0100] According to an embodiment of the present invention, the fusion protein does not include an antibody or an antigen-binding fragment thereof to the IL-12 cytokine.

[0101] According to an embodiment of the present invention, the first antibody or antigen-binding fragment thereof includes a first full-length antibody, a first Fab fragment, a first ScFv fragment or a first VHH fragment.

[0102] According to an embodiment of the present invention, the tumor antigen or immune checkpoint is selected from at least one of Her2, EGFR, VEGF, VEGFR, Claudin 18.2, B7-H3, Nectin-4, MSLN, Trop2, Siglec-9, Siglec-15, PD-L1, PD-1, TIGIT, LAG3, TIM-3, CTLA-4, BTLA, and VISTA.

[0103] "EGFR," as used herein, refers to the epidermal growth factor receptor, whose mutation or overexpression generally triggers tumors. EGFR dimerization activates downstream cellular signaling pathways, contributing to the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. Antibodies or antigen-binding fragments that bind to EGFR can inhibit EGFR activity, preventing its phosphorylation and, consequently, suppressing activation of downstream signaling pathways, thereby achieving anti-tumor effects.

[0104] In the present invention, "VEGF" refers to vascular endothelial growth factor, which acts on endothelial cells through a paracrine mechanism, playing an important role in promoting angiogenesis, inhibiting endothelial cell apoptosis, and increasing vascular permeability. VEGF is overexpressed in nearly all human tumors and tumor cell lines. Antibodies or antigen-binding fragments that bind to VEGF can inhibit the binding of VEGF to its receptors VEGFR-1 and VEGFR-2 on endothelial cells, thereby inactivating VEGF's biological activity and reducing tumor angiogenesis, thereby inhibiting tumor growth.

[0105] "Claudin 18.2," as used in this invention, refers to the 18.2 isoform of the tight junction protein claudin. It is a highly specific cell surface molecule that, in normal tissue, is expressed only on differentiated gastric epithelial cells. It is highly expressed in solid tumors such as gastric cancer, pancreatic cancer, ovarian cancer, biliary cancer, and lung adenocarcinoma. Antibodies that bind to claudin 18.2 can specifically recognize tumor cells, triggering antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, and inhibition of cell proliferation, demonstrating its powerful ability to eliminate cancer cells and control disease.

[0106] In this invention, "PD-L1" stands for programmed death receptor-ligand 1, an immune checkpoint protein. Tumor cells overexpress PD-L1, continuously activating the PD-1 / PD-L1 signaling pathway and causing various immune suppression effects. Antibodies or antigen-binding fragments that bind to PD-L1 act as immune checkpoint protein blockers, inhibiting the PD-1 / PD-L1 pathway and blocking the co-inhibitory function of CD80 and PD-L1, thereby promoting comprehensive T cell activation and cytokine production.

[0107] In the present invention, "PD-1" refers to programmed death protein 1, which is a co-inhibitory receptor that can be induced to express on the surface of T cells, B cells, monocytes, and natural killer cells.

[0108] In the present invention, "TIGIT" refers to a protein containing T cell immunoglobulin and ITIM domains. Its abnormal expression can be found in a variety of tumors, which can lead to immune cell dysfunction and is associated with tumor progression and poor prognosis. By blocking TIGIT, the functional failure of immune cells can be reversed and anti-tumor effects can be exerted. It is a new generation of immunotherapy target.

[0109] In the present invention, "LAG3" is lymphocyte activation gene-3, "TIM-3" is T cell immunoglobulin mucin molecule 3, and "CTLA-4" is cytotoxic T lymphocyte-associated antigen-4. All three act as immunosuppressive receptors and participate in T cell immune regulation.

[0110] According to an embodiment of the present invention, the IL-12 cytokine or a functional fragment thereof includes at least one of a P35 fragment of IL-12 or a functional fragment thereof, and a P40 fragment of IL-12 or a functional fragment thereof.

[0111] It should be noted that when the IL-12 cytokine or its functional fragment includes the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, it is the P70 fragment of IL-12 or its functional fragment.

[0112] According to an embodiment of the present invention, the IL-12 cytokine or a functional fragment thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 2 and 70 to 71.

[0113] According to an embodiment of the present invention, the IL-12 cytokine or its functional fragment includes two subunit fragments: a P35 fragment of IL-12 or its functional fragment, and a P40 fragment of IL-12 or its functional fragment. The third structural unit further includes a non-cleavable connecting peptide, and the two subunit fragments are connected by the connecting peptide.

[0114] According to an embodiment of the present invention, the IL-12 cytokine or its functional fragment includes two subunit fragments: a P35 fragment of IL-12 or its functional fragment, and a P40 fragment of IL-12 or its functional fragment. The C-terminus of one of the subunit fragments is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the other subunit fragment.

[0115] According to an embodiment of the present invention, the connecting peptide includes at least one of (GS)n, (GGS)n, (GGSG)n, (GSSG)n, (GGGS)n, (GGGGS)n and (GSGGS)n, where n is any integer between 1 and 10.

[0116] In an optional embodiment of the present invention, n is 2, 3, 4, 5, 6, 7 or 8.

[0117] According to an embodiment of the present invention, the amino acid length of the connecting peptide is 1-60, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or any two point values ​​therebetween as a range value between the endpoint values.

[0118] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in (GGGGS)n, where n is 3, 4, 5 or 6.

[0119] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in any one of SEQ ID NO: 75.

[0120] According to an embodiment of the present invention, the third structural unit has an amino acid sequence as shown in SEQ ID NO: 1, 2, 70, 71, 76 or 77.

[0121] In an optional embodiment of the present invention, the second structural unit is a cleavable linker.

[0122] In an optional embodiment of the present invention, the second structural unit is a cleavable linker.

[0123] In an alternative embodiment of the present invention, the fusion protein comprises two monomers, and the two second structural units are respectively a cleavable linker and a non-cleavable linker. In an alternative embodiment of the present invention, the fusion protein comprises two monomers, and the two second structural units are both cleavable linkers. In an alternative embodiment of the present invention, the fusion protein comprises two monomers, and the two second structural units are both non-cleavable linkers.

[0124] According to an embodiment of the present invention, the protease expressed in the tumor tissue includes matrix metalloproteinase, serine protease, or asparagine endopeptidase.

[0125] According to an embodiment of the present invention, the cleavable linker comprises a protease substrate sequence and optionally a flexible peptide segment.

[0126] According to an embodiment of the present invention, the flexible peptide segment includes at least one of (GS)n, (GGS)n, (GGSG)n, (GSSG)n, (GGGS)n, (GGGGS)n and (GSGGS)n, where n is any integer between 1 and 10.

[0127] According to an embodiment of the present invention, the amino acid length of the flexible peptide segment is 1-60, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or any two point values ​​therebetween as a range value between the endpoint values, for example, 5 to 30, 5 to 25 or 5 to 20.

[0128] In some embodiments of the present invention, the second structural unit of the present application comprises a peptide linker that can be cleaved by a matrix metalloproteinase. Peptide linkers known in the art that can be cleaved by matrix metalloproteinases, such as those disclosed in WO2019010219A, can be used in the present application, and the above-mentioned document is incorporated herein by reference. In some embodiments, the amino acid sequence of the second structural unit includes QLLGFLTA, a substrate of the matrix metalloproteinase MMP-2 / 9 / 14.

[0129] In some embodiments of the present invention, the second structural unit of the present application comprises a peptide linker that can be cleaved by a serine protease, such as urokinase or trypsin. Peptide linkers that can be cleaved by a serine protease known in the art can be used in the present application. In some embodiments, the amino acid sequence of the second structural unit includes the urokinase substrate LSGRSDNH.

[0130] In some embodiments of the present invention, the second structural unit of the present application comprises a peptide linker that can be cleaved by asparagine endopeptidase. Peptide linkers that can be cleaved by asparagine endopeptidase known in the art can be used in the present application. In some embodiments, the amino acid sequence of the second structural unit includes the asparagine endopeptidase substrate AANL.

[0131] According to an embodiment of the present invention, the protease substrate sequence is selected from QLLGFLTA, LSGRSDNH, GFFY, RQARAVGG or AANL.

[0132] According to an embodiment of the present invention, the cleavable linker is a flexible peptide segment-protease substrate sequence, or a flexible peptide segment-protease substrate sequence-flexible peptide segment.

[0133] According to an embodiment of the present invention, the amino acid length of the cleavable linker is 15 to 60, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or any two point values ​​therebetween as a range value between the endpoint values, for example, 15 to 50, 15 to 45 or 15 to 40.

[0134] In an optional embodiment of the present invention, the cleavable linker is a flexible peptide segment-protease substrate sequence-flexible peptide segment; wherein the amino acid length of each flexible peptide segment is independently 10 to 20 amino acids (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the protease substrate sequence is selected from QLLGFLTA, LSGRSDNH, GFFY, RQARAVGG, or AANL. In an optional embodiment of the present invention, the amino acid sequence of the flexible peptide segment is GSSGG, GSSGGSGGSGGSG, GSSGGSGGSGGS, SSGGSGGSGGSG, or GSSGGSGGSGGSGSSGGSGG.

[0135] According to an embodiment of the present invention, the cleavable linker has an amino acid sequence as shown in SEQ ID NO: 7, 8, 78, 79, 80, 83 or 84.

[0136] According to an embodiment of the present invention, the non-cleavable linker includes at least one of (GS)n, (GGS)n, (GGSG)n, (GSSG)n, (GGGS)n, (GGGGS)n and (GSGGS)n, where n is any integer between 1 and 10.

[0137] According to an embodiment of the present invention, the amino acid length of the non-cleavable linker is 1 to 60, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or any two point values ​​therebetween as a range value between the endpoint values, for example, 5 to 50, 5 to 40, 5 to 30 or 10 to 20.

[0138] According to an embodiment of the present invention, the non-cleavable linker has an amino acid sequence as shown in (GGGGS)n, where n is 3, 4, 5 or 6.

[0139] According to an embodiment of the present invention, the non-cleavable linker has an amino acid sequence as shown in SEQ ID NO: 5 or 6.

[0140] According to an embodiment of the present invention, the first structural unit is a first ScFv fragment, the C-terminus of the first structural unit is connected to the N-terminus of the second structural unit, the C-terminus of the second structural unit is connected to the N-terminus of the third structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit.

[0141] According to an embodiment of the present invention, the C-terminus of the first structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the third structural unit.

[0142] According to an embodiment of the present invention, the first structural unit is a first Fc fragment or a first VHH fragment, the first Fc fragment or the first VHH fragment is connected to one or two third structural units, and each of the third structural units is connected to the first Fc fragment or the first VHH fragment through the second structural unit.

[0143] According to an embodiment of the present invention, the C-end of the first structural unit is connected to the N-end of the second structural unit, and the C-end of the second structural unit is connected to the N-end of the third structural unit, or the C-end of the third structural unit is connected to the N-end of the second structural unit, and the C-end of the second structural unit is connected to the N-end of the first structural unit.

[0144] According to an embodiment of the present invention, the C-terminus of the first structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the third structural unit.

[0145] According to an embodiment of the present invention, when the first Fc fragment or the first VHH fragment is connected to one of the third structural units, the IL-12 cytokine or its functional fragment in the third structural unit includes two subunit fragments, namely, the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, preferably the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment.

[0146] According to an embodiment of the present invention, when the first Fc fragment or the first VHH fragment is connected to two of the third structural units, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker.

[0147] According to an embodiment of the present invention, both of the two second structural units are cleavable linkers, both are non-cleavable linkers, or one is a cleavable linker and the other is a non-cleavable linker.

[0148] According to an embodiment of the present invention, when the first Fc fragment or the first VHH fragment is connected to two third structural units, the IL-12 cytokines or functional fragments thereof in the two third structural units are the same or different.

[0149] According to an embodiment of the present invention, the IL-12 cytokines or functional fragments thereof in the two third structural units are respectively the P35 fragment of IL-12 or a functional fragment thereof, and the P40 fragment of IL-12 or a functional fragment thereof.

[0150] According to an embodiment of the present invention, when the fusion protein is composed of a first structural unit, a second structural unit and a third structural unit, and the first structural unit is a first Fc fragment, the IL-12 cytokine or its functional fragment of the third structural unit includes two subunit fragments, namely, a P35 fragment of IL-12 or its functional fragment, and a P40 fragment of IL-12 or its functional fragment; the two subunit fragments are respectively connected to the two fragments of the first Fc fragment.

[0151] According to an embodiment of the present invention, the first structural unit is a first full-length antibody, the first full-length antibody is connected to one or more third structural units, and each of the third structural units is connected to the first full-length antibody through the second structural unit.

[0152] According to an embodiment of the present invention, the first full-length antibody is connected to a third structural unit, and the third structural unit is connected to the C-terminus of the heavy chain of the first full-length antibody through the second structural unit;

[0153] According to an embodiment of the present invention, the first full-length antibody is connected to a plurality of the third structural units, and the plurality of the third structural units are respectively connected to the C-terminus of the heavy chain in the first full-length antibody through the second structural unit.

[0154] According to an embodiment of the present invention, the first full-length antibody is connected to two of the third structural units, and the two third structural units are respectively connected to the C-termini of the two heavy chains in the first full-length antibody through the second structural unit.

[0155] According to an embodiment of the present invention, when the first full-length antibody is connected to one of the third structural units, the IL-12 cytokine or its functional fragment in the third structural unit includes two subunit fragments, namely, the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, preferably the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment.

[0156] According to an embodiment of the present invention, when the first full-length antibody is connected to a plurality of the third structural units, the plurality of the second structural units are each independently selected from a cleavable linker or a non-cleavable linker, preferably all are cleavable linkers.

[0157] According to an embodiment of the present invention, when the first full-length antibody is connected to two third structural units, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker.

[0158] According to an embodiment of the present invention, both of the two second structural units are cleavable linkers, both are non-cleavable linkers, or one is a cleavable linker and the other is a non-cleavable linker.

[0159] According to an embodiment of the present invention, when the first full-length antibody is connected to two of the third structural units, and the two third structural units are respectively connected to the C-termini of the two heavy chains in the first full-length antibody through the second structural unit, the IL-12 cytokines or their functional fragments in the two third structural units are the same or different, preferably, the IL-12 cytokines or their functional fragments in the two third structural units are the P35 fragment or its functional fragment of IL-12, and the P40 fragment or its functional fragment of IL-12, respectively.

[0160] According to an embodiment of the present invention, the first structural unit is a first Fab fragment, the first Fab fragment is connected to one or two third structural units, and each of the third structural units is connected to the C-terminus of the first Fab fragment through the second structural unit.

[0161] According to an embodiment of the present invention, the first Fab fragment is connected to a third structural unit, and the third structural unit is connected to the C-terminus of the CH1 fragment or the C-terminus of the CL fragment in the first Fab fragment through the second structural unit.

[0162] According to an embodiment of the present invention, the first Fab fragment is connected to two of the third structural units, and the two third structural units are respectively connected to the C-terminus of the CH1 fragment and the C-terminus of the CL fragment in the first Fab fragment through the second structural unit.

[0163] According to an embodiment of the present invention, when the first Fab fragment is connected to one of the third structural units, the IL-12 cytokine or its functional fragment in the third structural unit includes two subunit fragments, namely, the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, preferably the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment.

[0164] According to an embodiment of the present invention, when the first Fab fragment is connected to two of the third structural units, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker.

[0165] According to an embodiment of the present invention, both of the two second structural units are cleavable linkers, both are non-cleavable linkers, or one is a cleavable linker and the other is a non-cleavable linker.

[0166] According to an embodiment of the present invention, when the full first Fab fragment is connected to two of the third structural units, the IL-12 cytokines or their functional fragments in the two third structural units are the same or different, and preferably, the IL-12 cytokines or their functional fragments in the two third structural units are respectively the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment.

[0167] According to an embodiment of the present invention, when the first Fab fragment is connected to one of the third structural units, and the third structural unit is connected to the C-terminus of the CH1 fragment in the first Fab fragment through the second structural unit, the fusion protein includes two monomers.

[0168] According to an embodiment of the present invention, in the two monomers, the IL-12 cytokine or its functional fragment of the third structural unit is respectively the P35 fragment or its functional fragment of IL-12, and the P40 fragment or its functional fragment of IL-12, and the P35 fragment or its functional fragment of IL-12 and the P40 fragment or its functional fragment of IL-12 are connected by a disulfide bond.

[0169] According to an embodiment of the present invention, the fusion protein further includes a fourth structural unit, and the fourth structural unit is selected from a second Fc fragment, or a second antibody or its antigen-binding fragment, and the second antibody or its antigen-binding fragment has tumor antigen or immune checkpoint binding activity.

[0170] As used herein, unless otherwise specified, an Fc fragment (e.g., the first Fc fragment and / or the second Fc fragment) includes a CH2, a CH3 region, and optionally a hinge region. In one embodiment of the present invention, the C-terminus of the CH2 region is connected to the N-terminus of the CH3 region. In another embodiment of the present invention, the C-terminus of the hinge region is connected to the N-terminus of the CH2 region, and the C-terminus of the CH2 region is connected to the N-terminus of the CH3 region. In some embodiments, the two heavy chains of the fusion protein of the present invention may be identical or different, for example, to avoid mispairing, an Fc fragment containing a "knob in hole" mutation may be used.

[0171] Herein, the "knob into hole structure" refers to a knob and hole mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate heavy chain engagement to form a heterodimer. For example, Fc-Knob is selected from S354C and T366W in the CH3 of human IgG1-Fc; Fc-Hole is selected from T366S, L368A, Y407V, and optionally Y349C in the CH3 of human IgG1-Fc. In certain embodiments, different subunits of the cytokine fused with Fc-Knob or Fc-Hole can be interchangeable without affecting their structure and function. For example, the Fc-Knob of LIC401 is connected to the IL-12P35 subunit via a flexible linker, and the Fc-Hole is connected to the IL-12P40 subunit via a flexible linker. Replacing them with the Fc-Knob connected to the IL-12P40 subunit via a flexible linker, and the Fc-Hole connected to the IL-12P35 subunit via a flexible linker, does not change the activity of IL-12.

[0172] In some embodiments, the Fc fragment (e.g., the first Fc fragment and / or the second Fc fragment) is derived from human IgG1 or IgG4 and includes one or more amino acid substitutions, additions, or deletions compared to the wild type, such that the fusion protein weakens or even eliminates ADCC and / or CDC function, thereby reducing nonspecific immune response.

[0173] In an optional embodiment of the present invention, the first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure.

[0174] In an optional embodiment of the present invention, the amino acid sequence of the Fc-Knob fragment is shown in SEQ ID NO: 3; the amino acid sequence of the Fc-Hole fragment is shown in SEQ ID NO: 4.

[0175] In some embodiments of the present invention, the third structural unit is located at the N-terminus of the fusion protein, as shown in LIC401, LIC410, and LIC415 in FIG1 .

[0176] In some embodiments of the present invention, the third structural unit is located at the C-terminus of the fusion protein, as shown in LIC402-409, LIC411-414, LIC416-419, LIC428, LIC436, and LIC442-445 in FIG1 .

[0177] In some embodiments of the present invention, the third structural unit is located between the first structural unit and the fourth structural unit of the fusion protein, as shown in LIC420-427, LIC429-435, and LIC437-441 in FIG1 .

[0178] According to an embodiment of the present invention, the second antibody or antigen-binding fragment thereof includes a second full-length antibody, a second Fab fragment, a second ScFv fragment or a second VHH fragment.

[0179] According to an embodiment of the present invention, the first structural unit is a first Fc fragment, the fourth structural unit is selected from a second Fab fragment, a second ScFv fragment, or a second VHH fragment, or the first structural unit is selected from a first Fab fragment, a first ScFv fragment, or a first VHH fragment, the fourth structural unit is a second Fc fragment, and the third structural unit is located between the first and fourth structural units. That is, the fusion protein includes the first structural unit, the third structural unit, and the fourth structural unit in order from the N-terminus to the C-terminus, thereby forming a "sandwich" structure.

[0180] According to an embodiment of the present invention, the C-terminus of the fourth structural unit is connected to the N-terminus of the first structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the fourth structural unit.

[0181] According to an embodiment of the present invention, the fusion protein further includes a fifth structural unit, and the fourth structural unit is connected to the first structural unit or the third structural unit through the fifth structural unit.

[0182] According to an embodiment of the present invention, the fifth structural unit is selected from a cleavable linker or a non-cleavable linker. The definition of the cleavable linker or non-cleavable linker of the fifth structural unit refers to the definition of the cleavable linker or non-cleavable linker in the second structural unit.

[0183] It should be noted that the second structural unit and the fifth structural unit in the present invention are each independently selected from a cleavable linker or a non-cleavable linker.

[0184] According to an embodiment of the present invention, when the third structural unit is located between the first and fourth structural units, the third structural unit is connected to the first structural unit via the second structural unit, and the third structural unit is connected to the fourth structural unit via the fifth structural unit. That is, the fusion protein includes the first structural unit, the second structural unit, the third structural unit, the fifth structural unit, and the fourth structural unit in sequence from the N-terminus to the C-terminus, thereby forming a "sandwich" structure.

[0185] In an optional embodiment of the present invention, in the above-mentioned "sandwich" structure, for the dimeric fusion protein, at least one second structural unit is a cleavable linker.

[0186] In some embodiments of the present invention, the first structural unit of the fusion protein of the present invention is connected to the N-terminus of the third structural unit via the non-cleavable linker (cytokine N-terminus) of the second structural unit, and the fourth structural unit is connected to the C-terminus of the third structural unit via the non-cleavable linker (cytokine C-terminus) of the fifth structural unit. The fusion protein structure is shown in Figure 1 as LIC420, LIC424, LIC429, and LIC432. The fusion protein comprises, from the N-terminus to the C-terminus, an antibody or antibody fragment such as Fab, a non-cleavable linker (cytokine N-terminus), a fusion protein of the P35 subunit or the P40 subunit or both, a non-cleavable linker (cytokine C-terminus), and an antibody or antibody fragment such as Fc; wherein the Fab segment can be two or one. The present invention has found that the masking ability of a single Fab at the N-terminus is stronger than the steric hindrance masking of two Fabs for IL-12 activity.

[0187] In some embodiments of the present invention, the first structural unit of the fusion protein of the present invention is connected to the N-terminus of the third structural unit through the cleavable linker of the second structural unit, and the fourth structural unit is connected to the C-terminus of the third structural unit through the non-cleavable linker (cytokine C-terminus) of the fifth structural unit. The fusion protein structure is shown in Figure 1 as LIC421, LIC425, LIC430, and LIC433. The fusion protein comprises, from N-terminus to C-terminus, an antibody or antibody fragment such as Fab, a cleavable linker, a fusion protein of the P35 subunit or the P40 subunit or both, a non-cleavable linker (cytokine C-terminus), an antibody or antibody fragment such as Fc; wherein the Fab segment can be two or one. The present invention found that after the cleavable linker of the second structural unit is cleaved by enzyme, the activity of IL12 is greatly enhanced.

[0188] In some embodiments of the present invention, the first structural unit of one chain of the fusion protein of the present invention is connected to the N-terminus of the third structural unit via the cleavable linker of the second structural unit, and the fourth structural unit is connected to the C-terminus of the third structural unit via the non-cleavable linker (cytokine C-terminus) of the fifth structural unit; the first structural unit of the other chain is connected to the N-terminus of the third structural unit via the non-cleavable linker (cytokine N-terminus) of the second structural unit, and the second fourth structural unit is connected to the C-terminus of the third structural unit via the non-cleavable linker (cytokine C-terminus) of the fifth structural unit. The fusion protein structure is shown as LIC422, LIC423, LIC426, LIC427, LIC431, LIC434, and LIC435 in Figure 1. The fusion protein comprises, from N-terminus to C-terminus, the following: an antibody or antibody fragment such as Fab, a (non-)cleavable linker, a P35 subunit or a P40 subunit or a fusion protein of the two, a non-cleavable linker (cytokine C-terminus), an antibody or antibody fragment such as Fc; wherein the Fab can be two or one. The present invention found that after the cleavable linker of the second structural unit was cleaved by enzyme, the activity of IL12 was enhanced.

[0189] In some embodiments of the present invention, the first structural unit of one chain of the fusion protein of the present invention is linked to the N-terminus of a fragment of the fourth structural unit via the non-cleavable linker of the second structural unit (cytokine subunits in series); the first structural unit of the other chain is linked to the N-terminus of the third structural unit via the non-cleavable linker of the second structural unit (cytokine N-terminus), and another fragment of the fourth structural unit is linked to the C-terminus of the third structural unit via the non-cleavable linker of the fifth structural unit (cytokine C-terminus). The fusion protein structures are shown in Figure 1 as LIC437, LIC438, LIC439, LIC440, and LIC441. From N-terminus to C-terminus, the fusion protein comprises the following: Chain A: antibody or antibody fragment, non-cleavable linker (cytokine subunits in series), antibody or antibody fragment; Chain B: antibody or antibody fragment, non-cleavable linker (cytokine subunit N-terminus), P35-P40 subunit fusion protein or P40-P35 subunit fusion protein, non-cleavable linker (cytokine C-terminus), antibody or antibody fragment. The targets of both antibody chains can be the same or different.

[0190] According to an embodiment of the present invention, when the first structural unit is a first Fc fragment or a first full-length antibody, the fourth structural unit is selected from a second Fab fragment, a second ScFv fragment or a second VHH fragment, and the C-terminus of the fourth structural unit is connected to the N-terminus of the first structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the fourth structural unit; preferably, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, and the C-terminus of the fifth structural unit is connected to the N-terminus of the first structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the fifth structural unit, and the C-terminus of the fifth structural unit is connected to the N-terminus of the fourth structural unit.

[0191] According to an embodiment of the present invention, when the first structural unit is a first Fab fragment, a first ScFv fragment or a first VHH fragment, the fourth structural unit is selected from a second Fc fragment, and the C-terminus of the third structural unit is connected to the N-terminus of the fourth structural unit; preferably, the C-terminus of the third structural unit is connected to the N-terminus of the fifth structural unit, and the C-terminus of the fifth structural unit is connected to the N-terminus of the fourth structural unit.

[0192] According to an embodiment of the present invention, when the first structural unit is a first Fab fragment, a first ScFv fragment or a first VHH fragment, the fourth structural unit is selected from a second Fc fragment, and the N-terminus of each fragment in the fourth structural unit is connected to the C-terminus of the third structural unit; preferably, the N-terminus of each fragment in the fourth structural unit is connected to the C-terminus of the third structural unit through a fifth structural unit.

[0193] According to an embodiment of the present invention, the fusion protein comprises:

[0194] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 24 (i.e., LIC402);

[0195] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 25, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 26 (i.e., LIC403);

[0196] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 26 (i.e., LIC404);

[0197] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 25, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 24 (i.e., LIC405);

[0198] an amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 27, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 28, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC406);

[0199] an amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 29, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 30, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC407);

[0200] an amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 27, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 30, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC408);

[0201] An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 29, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 28, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC409);

[0202] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 33, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 34 (i.e., LIC411);

[0203] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 36 (i.e., LIC412);

[0204] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 36 (i.e., LIC413);

[0205] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 34 (i.e., LIC414);

[0206] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 38 (i.e., LIC416);

[0207] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 39 (i.e., LIC417);

[0208] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 39 (i.e., LIC418);

[0209] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 38 (i.e., LIC419);

[0210] An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 40, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 41, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC420);

[0211] An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 42, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 43, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC421);

[0212] An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 40, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 43, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC422);

[0213] An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 41, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 42, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC423);

[0214] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:44, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:40 (i.e., LIC424);

[0215] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 45, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 43 (i.e., LIC425);

[0216] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:44, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:43 (i.e., LIC426);

[0217] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:45, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:40 (i.e., LIC427);

[0218] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:46, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:9 (i.e., LIC428);

[0219] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:47, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:9 (i.e., LIC429);

[0220] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 48, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 9 (i.e., LIC430);

[0221] An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 49, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 50, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC431);

[0222] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 51, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 52 (i.e., LIC432);

[0223] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 53, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 50 (i.e., LIC433);

[0224] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 53, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 52 (i.e., LIC434);

[0225] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 51, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 50 (i.e., LIC435);

[0226] an amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 54, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 12, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC436);

[0227] an amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 55, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 52, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC437);

[0228] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 56, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 57 (i.e., LIC438);

[0229] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 56, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 58 (i.e., LIC439);

[0230] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 59, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 60 (i.e., LIC440);

[0231] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 59, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 61 (i.e., LIC441);

[0232] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 62, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 63 (i.e., LIC442);

[0233] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 62, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 64 (i.e., LIC443);

[0234] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 66, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 65 (i.e., LIC403-1);

[0235] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 81, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 82 (i.e., LIC403-2);

[0236] The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 67, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 68 (i.e., LIC425-1);

[0237] an amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 69, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 12, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC444);

[0238] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 74, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 73 (ie, LIC445).

[0239] That is, the connection order of the domains of the fusion protein of the present invention from N-terminus to C-terminus is any one selected from the following:

[0240] The third structural unit comprises an IL-12 cytokine or a functional fragment thereof, the second structural unit comprises a non-cleavable linker (the C-terminus of the cytokine), and the first structural unit comprises an Fc-Knob (Hole) or an Fc-Knob (Hole) + ScFv. The two ScFv targets may be the same or different (as shown in FIG1 , LIC401, LIC410, and LIC415).

[0241] The first structural unit is Fc-Knob (Hole), full-length monoclonal antibody, or ScFv + Fc-Knob into Hole (the two ScFv targets can be the same or different), the second structural unit is a non-cleavable linker (cytokine N-terminus), and the third structural unit is IL-12 cytokine or a functional fragment thereof (as shown in Figure 1 LIC402, LIC406, LIC411, LIC416, LIC428, LIC436, LIC442, LIC443);

[0242] The first structural unit is Fc-Knob (Hole), full-length mAb, or ScFv + Fc-Knob into Hole (the two ScFv targets can be the same or different), the second structural unit is a cleavable linker, and the third structural unit is an IL-12 cytokine or a functional fragment thereof (as shown in FIG1 , LIC403, LIC407, LIC412, and LIC417);

[0243] One chain comprises a first structural unit Fc-Knob (Hole), a full-length monoclonal antibody, or ScFv + Fc-Knob into Hole (the two ScFv targets may be the same or different), a second structural unit a non-cleavable linker (the N-terminus of the cytokine), and a third structural unit IL-12 cytokine or a functional fragment thereof; the other chain comprises a first structural unit Fc-Knob (Hole), a full-length monoclonal antibody, or ScFv + Fc-Knob into Hole (the two ScFv targets may be the same or different), a second structural unit a cleavable linker, and a third structural unit IL-12 cytokine or a functional fragment thereof (such as LIC404, LIC405, LIC408, LIC409, LIC413, LIC414, LIC418, and LIC419 in FIG1 );

[0244] The first structural unit is a Fab segment (single or double), the second structural unit is a non-cleavable linker (cytokine N-terminus), the third structural unit is an IL-12 cytokine or a functional fragment thereof, the fifth structural unit is a non-cleavable linker (cytokine C-terminus), and the fourth structural unit is an Fc segment N-terminus (as shown in FIG1 LIC420, LIC424, LIC429, and LIC432);

[0245] The first structural unit comprises a Fab segment (single or double), a second structural unit comprises a cleavable linker, a third structural unit comprises an IL-12 cytokine or a functional fragment thereof, a fifth structural unit comprises a non-cleavable linker (the C-terminus of the cytokine), and a fourth structural unit comprises an N-terminus of the Fc segment (as shown in FIG. 1 , LIC421, LIC425, LIC430, and LIC433);

[0246] One chain Fab or VL and / or VH segment, a second structural unit cleavable linker, a third structural unit IL-12 cytokine or a functional fragment thereof, a second structural unit non-cleavable linker (cytokine C-terminus), and a first structural unit Fc segment; another chain Fab or VL and / or VH segment, a second structural unit non-cleavable linker (cytokine C-terminus), and a third structural unit IL-12 cytokine or a functional fragment thereof (as shown in LIC422, LIC423, LIC426, LIC427, LIC431, LIC434, and LIC435 in FIG1 );

[0247] One chain comprises a first structural unit Fab or ScFv, a second structural unit non-cleavable linker (cytokine subunits in series), and a fourth structural unit Fc fragment; the other chain comprises a first structural unit Fab or ScFv, a second structural unit non-cleavable linker (cytokine N-terminus), a third structural unit IL-12 cytokine or a functional fragment thereof, a fifth structural unit non-cleavable linker (cytokine N-terminus), and a fourth structural unit Fc another fragment (as shown in LIC437, LIC438, and LIC439 in FIG1 ); the two Fabs or ScFvs may be the same or different.

[0248] One chain comprises a fragment of the Fc segment of the first structural unit, a second structural unit non-cleavable linker (cytokine subunits in series), and a fourth structural unit Fab or ScFv; the other chain comprises another fragment of the Fc segment of the first structural unit, a second structural unit non-cleavable linker (cytokine N-terminus), a third structural unit IL-12 cytokine or a functional fragment thereof, a fifth structural unit non-cleavable linker (cytokine N-terminus), and a fourth structural unit Fab or ScFv (as shown in LIC440 and LIC441 in Figure 1); the two Fabs or ScFvs may be the same or different.

[0249] The fusion protein of the present invention can be selected from any of the following structures:

[0250] In an optional manner, the C-terminus of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit, and the second structural unit is a cleavable linker. Taking the first structural unit as an antibody Fc fragment, the second structural unit as a cleavable linker, and the third structural unit as IL-12P35 or IL-12P40 as an example, the fusion protein comprises an Fc fragment, a cleavable linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (as shown in LIC403 in Figure 1). The present invention found that Fc and the linker form a steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC403 being masked and relatively low before cleavage. After cleavage, IL-12 is released, the steric hindrance is lifted, and the activity is restored. This significantly improves the safety of LIC403 compared to the IL-12 monomer.

[0251] In one optional embodiment, the C-terminus of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from an Fc fragment, the C-terminus of one fragment of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit; the C-terminus of another fragment of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit; the two second structural units are respectively a cleavable linker and a non-cleavable linker. For example, taking the first structural unit as an antibody Fc fragment, the second structural unit as one cleavable linker and one non-cleavable linker (cytokine N-terminus), and the third structural unit as IL-12P35 or IL-12P40, the fusion protein comprises, from N-terminus to C-terminus, an Fc fragment, a cleavable (non-cleavable) linker, and IL-12P35 or IL-12P40 (as shown in LIC404 and LIC405 in Figure 1). The present invention discovered that the Fc and linker create steric hindrance to IL-12, thereby affecting IL-12 activity. This results in the masking of the activity of LIC404 and LIC405 before cleavage, resulting in relatively low activity. However, after cleavage, the individual IL-12 subunits are released, partially relieving the steric hindrance and partially restoring their activity. Compared to complete cleavage, single-chain release can have a longer half-life. This significantly improves the safety of LIC404 and LIC405 compared to IL-12 monomer.

[0252] In an optional manner, the C-terminus of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit, and the second structural unit is a cleavable linker. Taking the first structural unit as an antibody targeting a tumor antigen or an immune checkpoint, the second structural unit as a cleavable linker, and the third structural unit as IL-12P35 or IL-12P40 as an example, the fusion protein comprises a full-length antibody, a cleavable linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (as shown in LIC407 in Figure 1). The present invention found that the full-length antibody and the linker form a steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC407 being masked before cleavage and relatively low, and after cleavage, IL-12 is released, the steric hindrance is lifted, and the activity is restored. This significantly improves the safety of LIC407 compared to the IL-12 monomer.

[0253] In one optional manner, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from a full-length antibody, the C-terminus of one CH3 fragment of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; the C-terminus of another CH3 fragment of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; and the two second structural units are a cleavable linker and a non-cleavable linker, respectively. Taking the first structural unit as a full-length antibody targeting a tumor antigen or PD-L1 function, the second structural unit as a cleavable linker and an uncleaved linker (cytokine N-terminus), and the third structural unit as IL-12P35 or IL-12P40, the fusion protein comprises a full-length antibody, a cleavable (non-cleavable) linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (as shown in LIC408 and LIC409 in Figure 1). The present invention found that the full-length antibody and the linker form a steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC408 and LIC409 being masked before cleavage and relatively low. After cleavage, the single IL-12 subunit is released, the steric hindrance is partially relieved, and the active part is restored. Compared with complete cleavage, single-chain release can have more stable targeting and a longer half-life. This significantly improves the targeting and safety of LIC408 and LIC409 compared to IL-12 monomers.

[0254] In one optional embodiment, the C-terminus of the fourth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from an Fc fragment. In one chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; in the other chain, the C-terminus of the other CH3 fragment of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; both second structural units are cleavable linkers. Taking the fourth structural unit as scFv, the first structural unit as the antibody Fc fragment, the second structural unit as the cleavable linker, and the third structural unit as IL-12P35 or IL-12P40, respectively, as an example, the fusion protein comprises scFv, Fc fragment, cleavable linker, IL-12P35 or IL-12P40 from N-terminus to C-terminus (as shown in LIC412 of FIG1 ). The two scFvs may be different (as shown in LIC417 of FIG1 ). The present invention found that the antibody scFv+Fc and linker form a steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC412 being masked and relatively low before cleavage, and after cleavage, IL-12 is released, the steric hindrance is lifted, and the activity is restored. This significantly improves the safety of LIC412 and LIC417 compared to the IL-12 monomer.

[0255] In one optional embodiment, the C-terminus of the fourth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from an Fc fragment. In one chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; in the other chain, the C-terminus of the other CH3 fragment of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; the two second structural units are respectively cleavable linkers or non-cleavable linkers. The fourth structural unit is an scFv, and the first structural unit is an antibody Fc fragment, a cleavable (non-cleavable) linker, IL-12P35, or IL-12P40 (as shown in LIC413 and LIC414 in Figure 1). The two ScFvs may be different (as shown in LIC418 and LIC419 in Figure 1). The present invention found that the antibody ScFv+Fc and the linker form a steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC413, LIC414, LIC418, and LIC419 being masked before cleavage, which is relatively low. After cleavage, the single IL-12 subunit is released, the steric hindrance is partially relieved, and the active part is restored. Compared with complete cleavage, single-chain release can have more stable targeting and a longer half-life. This significantly improves the targeting and safety of LIC413, LIC414, LIC418, and LIC419 compared to IL-12 monomers.

[0256] In one optional manner, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from an Fc fragment, and in one chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; in the other chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; both second structural units are non-cleavable linkers, and both fifth structural units are cleavable linkers. Taking the first structural unit as an Fc fragment, the second structural unit as a non-cleavable linker, the third structural unit as IL-12P35 or IL-12P40, the fourth structural unit as Fab (one or two), and the fifth structural unit as a cleavable linker as an example, the fusion protein comprises Fab, a cleavable linker, IL-12P35 or IL-12P40, a non-cleavable linker (cytokine C-terminus), and Fc from N-terminus to C-terminus (two Fabs are shown in LIC421 of Figure 1, and a single Fab is shown in LIC425 of Figure 1). The present invention found that the two Fabs and the linker form a steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC421 being masked before cleavage and relatively low, and after cleavage, IL-12 is released, the steric hindrance is lifted, and the activity is restored. The disulfide bond masking ability in a single Fab is stronger than the steric hindrance of two Fabs, which significantly improves the safety of LIC421 and LIC425 compared to the IL-12 monomer.

[0257] In one optional manner, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from an Fc fragment. In one chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; in the other chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; both second structural units are non-cleavable linkers, and the two fifth structural units are a cleavable linker and a non-cleavable linker, respectively. Taking the first structural unit as an Fc fragment, the second structural unit as a non-cleavable linker, the third structural unit as IL-12P35 or IL-12P40, the fourth structural unit as Fab (one or two), and the fifth structural unit as a cleavable linker as an example, the fusion protein comprises Fab, a cleavable linker, IL-12P35 or IL-12P40, a non-cleavable linker (cytokine C-terminus), and Fc from N-terminus to C-terminus (two Fabs are shown as LIC422 and LIC423 in Figure 1, and a single Fab is shown as LIC426 and LIC427 in Figure 1). The present application found that the two Fabs and the linker form a steric hindrance to IL-12, thereby affecting the activity of IL-12. The disulfide bond masking ability in a single Fab is stronger than the steric hindrance of the two Fabs, resulting in the activity of LIC422, LIC423, LIC426, and LIC427 being masked before cleavage, which is relatively low. After cleavage, the single IL-12 subunit is released, the steric hindrance is partially lifted, and the active part is restored. Compared to complete cleavage, single-chain release allows for more stable targeting and a longer half-life, significantly improving the targeting and safety of LIC422, LIC423, LIC426, and LIC427 compared to IL-12 monomers.

[0258] In one optional manner, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from an Fc fragment, and in one chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; in the other chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; both second structural units are non-cleavable linkers, and both fifth structural units are cleavable linkers. Taking the example of a first structural unit being an Fc fragment, a second structural unit being a non-cleavable linker, a third structural unit being IL-12P70, a fourth structural unit being Fab (one or two), and a fifth structural unit being a cleavable linker, the fusion protein comprises Fab, a cleavable linker, IL-12P70, a non-cleavable linker (cytokine C-terminus), and Fc from N-terminus to C-terminus (two Fabs are shown as LIC430 in Figure 1, and a single Fab is shown as LIC433 in Figure 1). The present invention found that Fab and Fc form a steric hindrance to IL-12, thereby affecting the activity of IL12, resulting in the activity of LIC430 being masked before cleavage and being relatively low. After cleavage, IL12 is released, the steric hindrance is lifted, and the activity is restored. The disulfide bond masking ability in a single Fab is stronger than the steric hindrance of two Fabs, which significantly improves the safety of LIC430 and LIC433 compared to the IL12 monomer.

[0259] In one optional manner, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit. In an optional embodiment of the present invention, the third structural unit is selected from an Fc fragment. In one chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; in the other chain, the C-terminus of the fourth structural unit is linked to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is linked to the N-terminus of the third structural unit, the C-terminus of the third structural unit is linked to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is linked to the N-terminus of the first structural unit; both second structural units are non-cleavable linkers, and the two fifth structural units are a cleavable linker and a non-cleavable linker, respectively. Taking the example of a first structural unit being an Fc fragment, a second structural unit being a non-cleavable linker, a third structural unit being IL-12P70, a fourth structural unit being Fab (one or two), and a fifth structural unit being a cleavable linker, the fusion protein comprises Fab, a cleavable linker, IL-12P70, a non-cleavable linker (cytokine C-terminus), and Fc from N-terminus to C-terminus (two Fabs are shown as LIC431 in FIG1 , and a single Fab is shown as LIC434 and LIC435 in FIG1 ). The present invention found that Fab and Fc form steric hindrance to IL-12, thereby affecting the activity of IL-12. The disulfide bond masking ability in a single Fab is stronger than the steric hindrance of two Fabs, resulting in the activity of LIC431, LIC434, and LIC435 being masked before cleavage and relatively low. After cleavage, a single IL-12 subunit is released, the steric hindrance is partially relieved, and the active portion is restored. Compared with complete cleavage, single-chain release can have more stable targeting and a longer half-life. This significantly improves the targeting and safety of LIC431, LIC434, and LIC435 compared to IL-12 monomer.

[0260] In an optional manner, the third structural unit is connected to the first structural unit and the fourth structural unit through its N-terminus and C-terminus, respectively. Taking the first structural unit as Fab, ScFv or Fc, the second structural unit as a non-cleavable linker, the third structural unit as IL-12P70, the fourth structural unit as Fab, ScFv or Fc, and the fifth structural unit as a non-cleavable linker as an example, the fusion protein A chain from N-terminus to C-terminus sequentially comprises Fab or ScFv or Fc, a non-cleavable linker (in series between cell subunits), Fc or Fab or ScFv; the B chain sequentially comprises Fab or ScFv or Fc, a non-cleavable linker (cytokine N-terminus), IL-12P70, Fc or Fab or ScFv (as shown in LIC437-LIC441 in Figure 1). The present application found that Fab and Fc form steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the weakening of IL-12 activity in LIC437-LIC441, which significantly improves safety compared to IL-12 monomers.

[0261] Nucleic acid molecules, expression vectors and recombinant cells

[0262] In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The nucleic acid molecule of the present invention can encode the fusion protein described in the first aspect.

[0263] The nucleic acid of the present invention can be a DNA molecule or an RNA molecule, or a nucleic acid analog. The nucleic acid molecule in the present invention can contain naturally occurring nucleic acid residues or artificially generated nucleic acid residues. The nucleic acid molecule of the present invention can be single-stranded or double-stranded, linear or circular, natural or synthetic, and unless otherwise indicated, there is no size limitation. The nucleic acid molecule can also contain a promoter, which can be homologous or heterologous.

[0264] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include either or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is provided in most cases, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies disclosure of the other.

[0265] In its third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect. When linking the nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself or exogenous, that is, not derived from the expression vector itself. Of course, it is sufficient that the nucleic acid molecule and the control elements are operably linked.

[0266] "Operably connected" herein refers to connecting the foreign gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, etc., can bring into play the function of transcribing and translating the foreign gene of its expected regulation. Conventional expression vectors can for example be plasmids, cosmids, viruses, phages and other commonly used vectors in genetic engineering. In some embodiments, these vectors are applicable to transformed cells, eukaryotic cells such as fungal cells, microbial cells such as yeast or prokaryotic cells. In a preferred embodiment, these vectors are applicable to the stable transformation of bacterial cells, for example, to transcribe nucleic acid molecules of the present invention.

[0267] According to some specific embodiments of the present invention, after the expression vector is introduced into suitable recipient cells, it can effectively express the fusion protein described in the first aspect under the mediation of the regulatory system, thereby achieving large-scale in vitro production of the fusion protein.

[0268] In one embodiment, the expression vector may contain a marker gene and a replication origin, a promoter, and a transcription termination signal to ensure replication in the selected host. Between the promoter and the termination signal, preferably, there is at least one restriction site capable of inserting the desired expressed nucleic acid sequence / molecule.

[0269] According to an embodiment of the present invention, the expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0270] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector or a viral expression vector, such as a lentiviral expression vector. Preferably, the expression vector of the present application is selected from the group consisting of pET series expression vectors, pGEX series expression vectors, pcDNA series expression vectors, and pCMV series expression vectors. More preferably, the expression vector of the present application is a pMF09 expression vector.

[0271] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the nucleic acid described in the second aspect or the expression vector described in the third aspect; or expressing the fusion protein described in the first aspect. Under suitable conditions, the recombinant cell can effectively express the fusion protein described in the first aspect within the cell.

[0272] According to some specific embodiments of the present invention, the recombinant cells can efficiently and massively express the fusion protein under appropriate conditions. The fusion protein has stronger specificity, longer half-life and higher efficacy, lower toxic side effects and higher safety.

[0273] It should be noted that "suitable conditions" refer to conditions suitable for the expression of the fusion protein of the present invention. Those skilled in the art will readily appreciate that conditions suitable for the expression of the fusion protein include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, healthy host cells, suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the fusion protein described above based on the specific laboratory environment.

[0274] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the expression vector described in the third aspect into a host cell.

[0275] In one embodiment, the fusion protein is expressed in a recombinant cell, subsequently isolated, and typically purified to a pharmaceutically acceptable purity. For protein expression, a nucleic acid encoding the protein is inserted into an expression vector by standard methods. Expression is performed in a suitable stable host cell, and the protein is recovered from the cell (supernatant or lysed cells).

[0276] In another embodiment, nucleic acid molecules of the present invention and / or vectors containing nucleic acid molecules of the present application can be transduced, transformed or transfected or otherwise imported into a host cell. For example, the host cell is a eukaryotic or prokaryotic cell, preferably a eukaryotic cell. As a non-limiting example, the host cell is a mammalian cell. The host cell of the present application can be people, yeast or fungal cells, such as Chinese hamster ovary cells (CHO), kidney cells of baby hamsters (BHK, ATCC CCL 10), Sertoli cells of baby mice (Sertoli cells), kidney cells of monkeys (COS cells), kidney CVI cells of monkeys transformed by SV40 (COS-7, ATCC CRL 1651), embryonic kidney cells (HEK-293) of people, monkey kidney cells (CVI, ATCC CCL-70), kidney cells of African green monkeys (VERO-76, ATCC CRL-1587), cervical cancer cells of people (HELA, ATCC CCL-2) etc. Preferably, the host cell of the present invention is a human HEK293 cell.

[0277] The present invention also provides a method for preparing the fusion protein described above, comprising the steps of culturing the host cells of the present application under conditions suitable for expressing the fusion protein, and recovering the fusion protein from the cells or cell culture supernatant. In one embodiment, the method for preparing the fusion protein of the present invention comprises the steps of constructing an expression vector comprising a gene encoding the fusion protein, constructing a host cell comprising the expression vector by transiently transfecting the host cell, culturing the host cell and collecting the cell supernatant, and purifying the fusion protein by affinity chromatography using Protein A / G as a filler.

[0278] Suitable conditions for expressing the fusion protein should be known to those skilled in the art. Those skilled in the art can select applicable culture medium based on experience and culture under conditions suitable for host cell growth. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time. The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly.

[0279] Methods for isolating and purifying fusion proteins are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant, centrifugation, osmotic shock, ultrafiltration, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography, and various other liquid chromatography techniques, and combinations thereof.

[0280] Fusion protein complexes, immunotherapy cells and pharmaceutical compositions

[0281] In the fifth aspect of the present invention, the present invention proposes a fusion protein complex. According to an embodiment of the present invention, the fusion protein complex includes the fusion protein described in the first aspect. As mentioned above, the third structural unit of the fusion protein described in the first aspect contains IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve the shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies. Therefore, the fusion protein does not need to contain any antibodies or receptors for IL-12 cytokines, and can reduce the toxic and side effects caused by IL-12 cytokines. The fusion protein has advantages such as strong safety. Therefore, the fusion protein complex of the present invention has advantages such as strong safety and can be used to treat cancer, infectious diseases or autoimmune diseases.

[0282] One embodiment of the present application relates to a fusion protein complex that can be a dimer formed by the binding of two different or identical fusion proteins; that is, the fusion protein complex can be a homodimer or a heterodimer. The fusion proteins in the fusion protein complex can be linked by interchain bonds formed between Fc fragments, preferably covalently linked to form a dimer via disulfide bonds formed between the Fc fragments. In some embodiments, the fusion protein complex comprises two fusion proteins of the present invention.

[0283] In the sixth aspect of the present invention, the present invention proposes an immunotherapy cell. According to an embodiment of the present invention, the immunotherapy cell expresses the fusion protein described in the first aspect. As mentioned above, the third structural unit of the fusion protein described in the first aspect contains IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve the shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies. Therefore, the fusion protein does not need to contain any antibodies or receptors for IL-12 cytokines, and can reduce the toxic and side effects caused by IL-12 cytokines. The fusion protein has advantages such as strong safety. Therefore, the immunotherapy cell containing the above-mentioned fusion protein has advantages such as strong safety and can be used to treat cancer, infectious diseases or autoimmune diseases.

[0284] In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the fusion protein complex described in the fifth aspect, or the immunotherapy cell described in the sixth aspect. As can be seen from the above, the third structural unit of the fusion protein described in the first aspect contains IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity of the IL-12 cytokine in the third structural unit, especially without the need for traditional affinity shielding strategies. Therefore, the fusion protein does not need to contain any antibodies or receptors for IL-12 cytokines, and can reduce the toxic and side effects caused by IL-12 cytokines. The fusion protein has the advantages of strong safety. Therefore, the pharmaceutical composition containing the above-mentioned fusion protein has the advantages of strong safety and can be used to treat cancer, infectious diseases or autoimmune diseases.

[0285] In some embodiments, the pharmaceutical composition of the present invention further comprises monoclonal antibodies targeting tumor antigens or immune checkpoints, such as antibodies against PD-1, PD-L1, LAG3, TIM-3, CTLA-4, Her2, EGFR, Claudin 18.2, VEGF, CD20, CD33, etc., which, when used in combination with the fusion protein of the present invention, can exert a synergistic anti-tumor effect and improve the therapeutic effect of the disease.

[0286] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient. Examples of suitable pharmaceutical excipients are well known in the art. Pharmaceutical compositions comprising such carriers can be formulated by known conventional methods. In some embodiments, the pharmaceutical composition of the present application may also contain other active ingredients for treatment.

[0287] Use or treatment method

[0288] In the eighth aspect of the present invention, the present invention proposes a use of the fusion protein described in the first aspect, the fusion protein complex described in the fifth aspect, the immunotherapy cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect in the preparation of a drug for treating cancer, infectious diseases or autoimmune diseases.

[0289] The present invention proposes the use of the fusion protein described in the first aspect, the fusion protein complex described in the fifth aspect, the immunotherapy cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect in the treatment of cancer, infectious diseases or autoimmune diseases.

[0290] The present invention provides a fusion protein as described in the first aspect, a fusion protein complex as described in the fifth aspect, an immunotherapy cell as described in the sixth aspect, or a pharmaceutical composition as described in the seventh aspect, for use in treating cancer, infectious diseases or autoimmune diseases.

[0291] The above-mentioned fusion protein, fusion protein complex, immunotherapy cell, pharmaceutical composition of the present invention can be administered in different ways, for example, via injection (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal). Preferably, the pharmaceutical composition of the present invention is in the form of a lyophilized preparation or an aqueous solution. The clinical dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's physique, body surface area, age, drug to be administered, sex, administration time and route, general health, and other drugs administered simultaneously. The fusion protein, fusion protein complex, immunotherapy cell, pharmaceutical composition of the present invention can be administered locally or systemically. Preferably, it can be administered intravenously or subcutaneously. The fusion protein, fusion protein complex, immunotherapy cell, pharmaceutical composition of the present invention can also be directly administered to the target site, for example, by targeted administration to an internal or external target site.

[0292] Among them, cancer includes but is not limited to leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythrocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), macroglobulinemia and solid tumors, such as sarcoma and malignant tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Infectious diseases include, but are not limited to, smallpox virus infection, HIV infection, bacterial infection, fungal infection, and HBV infection. Autoimmune diseases include, but are not limited to, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, Crohn's disease, gastritis, and mucositis.

[0293] In the present invention, the drug includes therapeutic cells, such as CAR T, CAR NK, etc. The fusion protein of the present invention is expressed or modified on the surface of the cells, helping to improve the survival and activity of the cells in the tumor site, thereby improving the efficacy of cell therapy for the disease.

[0294] In an eighth aspect, the present invention provides a method for treating and / or preventing cancer, infectious diseases, or autoimmune diseases. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable dose of the fusion protein of the first aspect, the fusion protein complex of the fifth aspect, the immunotherapy cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0295] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0296] Example 1: Preparation of IL-12-uncleaved-Fc (LIC401) fusion protein

[0297] 1. Construction of expression vector encoding LIC401 fusion protein

[0298] A nucleic acid sequence encoding the LIC401 fusion protein (the corresponding amino acid sequences are shown in SEQ ID NO:21 and SEQ ID NO:22) was prepared by gene synthesis and PCR. As shown in Figure 1, the LIC401 fusion protein contains IL-12 at the N-terminus, linked to the Fc fragment via a non-cleavable linker. IL-12 comprises both the p35 and p40 subunits.

[0299] The obtained nucleic acid sequence was inserted into the pMF09 plasmid between the Hind III and BamH I restriction sites via homologous recombination. The homologous recombination product was then transformed into the DH5α strain to generate the expression vector pMF09 / LIC401. The correct construction of the vector was verified by sequencing. After culturing to the logarithmic growth phase, the strain was supplemented with glycerol to a final concentration of 20% and stored at -80°C.

[0300] 2. Plasmid amplification

[0301] The volume of the bacteria expressing the pMF09 / LIC401 expression vector was expanded by 1:100 times the inoculum volume. The plasmid was extracted by alkaline lysis using the EZNA Endo-free Plasmid Maxi Kit (Cat. No.: D6926-03) provided by OMEGA. The plasmid was filtered through a sterile filter membrane and stored at -20°C.

[0302] 3. Cell Culture and Transient Transfection to Express LIC401 Fusion Protein

[0303] 1) Cell preparation

[0304] Human HEK293E cells were cultured in suspension using a medium consisting of SFM4 HEK293 Medium (Hyclone, USA) and Freestyle 293 Expression Medium (Invitrogen, USA) mixed at a volume ratio of 1:1, followed by the addition of fetal bovine serum at a final concentration of 2%. 6The cells were passaged at a density of / mL into fresh culture medium and cultured in suspension at 125 rpm in a 37°C constant temperature shaker (5% CO2).

[0305] One day before transfection, cells were passaged into 500 mL conical culture flasks and the cell density was adjusted to 4 × 10 6 / mL, culture volume 200mL.

[0306] The cell density at the time of transfection was 6×10 6 / mL, transfection was performed when the cell viability was above 95%; 100mL of culture medium was taken, cells were collected by centrifugation, and washed once with Freestyle 293 medium. Cells were resuspended with 100mL of Freestyle 293 medium;

[0307] 2) Plasmid dilution

[0308] 0.5 μg plasmid / 10 6 Take the plasmid from the cells, dilute it to 40 ng / μL with Freestyle 293 medium, and mix well.

[0309] 3) PEI-encapsulated plasmid

[0310] If the mass ratio of plasmid to PEI is 1:5, take 1.5 mL of PEI (polyethyleneimine, transfection reagent, purchased from Polysciences) and add it to the diluted plasmid, mix well, and let it sit for 10-15 minutes. Control the time and the mixture will become turbid.

[0311] 4) PEI transfection

[0312] Add the PEI-wrapped plasmid to the cells and culture them on a shaker.

[0313] 5) Fluid replacement after 4 hours

[0314] After 4 h, an equal volume of 100 mL SFM4 HEK293 medium, 1 mL VPA (200X, 750 mmol / L, final concentration of 3.75 mmol / L), and 400 μL G418 (50 mg / mL) were added.

[0315] 6) Fluid replacement after 24 hours

[0316] 200 μL of anti-clumping (1000X) and 5.0 mL of 20% TN1 (final concentration 0.5%) were added.

[0317] 7) Monitor the changes in cell viability. After culturing for 6-7 days, when the cell viability drops below 50%, collect the cell culture supernatant for the next purification step.

[0318] 4. Purification of LIC401 fusion protein

[0319] 1) Sample preparation: Centrifuge the cell suspension at 7000 rpm for 30 minutes, discard the pellet, and filter the supernatant through a 0.45 μm filter for later use.

[0320] 2) Rinse the protein purification system with 20% ethanol, and rinse and equilibrate the protein A column with 10 column volumes of double-distilled water and loading buffer, respectively;

[0321] 3) Load the sample using the peristaltic pump of the AKTA start protein purifier. The flow rate is set according to the column volume, generally not exceeding 1 column volume / min, and the flow-through is collected.

[0322] 4) After loading, re-equilibrate the protein A column with 10 column volumes of loading buffer.

[0323] 5) Elution was performed using elution buffer (citric acid-sodium citrate buffer, pH = 2.8). The eluate was collected in separate tubes, with each 1 mL collected in one tube. The elution peak was observed based on UV absorbance at 280 nm. An appropriate amount of 1 M Tris-HCl (pH 9.0) was added to the collection tube containing the elution peak to adjust the pH of the target protein solution to approximately 7.0 to 8.0. The protein A affinity chromatography purification of the fusion protein LIC401 is shown in Figure 2. A UV absorbance peak exceeding 1000 mAU was observed, indicating that the target protein was eluted by the elution buffer.

[0324] 6) Rinse the Protein A column with 150 mM NaOH solution; then rinse the Protein A column with 10 column volumes of double-distilled water and 20% ethanol, ensuring that the column packing is completely submerged in ethanol.

[0325] 7) The purity of each elution tube was tested by SDS-PAGE. The elution tubes were combined and, after ultrafiltration and buffer exchange, the protein was dissolved in PBS for the next step. The SDS-PAGE electrophoresis of the fusion protein LIC401 after purification by affinity chromatography is shown in Figure 3. The target protein was obtained after affinity chromatography. The molecular weight of the non-reduced sample was between 100 kDa and 150 kDa, and the single-chain molecular weight of the reduced sample was between 45 kDa and 75 kDa. There were two bands, representing the polypeptide chains of the amino acid sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively.

[0326] 5. Western Blot Verification of LIC401 Fusion Protein Expression

[0327] 1) Take an appropriate amount of affinity chromatography-purified LIC401 fusion protein and run it on SDS-PAGE. Electrophoresis conditions: 90V, 20min; 100V, 70min;

[0328] 2) Transfer the target protein to a PVDF membrane using a wet transfer method. Transfer conditions: 200 mA, 90 min;

[0329] 3) After transfer, block the PVDF membrane with 5% skim milk powder at room temperature for 2 hours or at 4°C overnight.

[0330] 4) Use goat anti-human heavy and light chain HRP antibodies as secondary antibodies to verify the expression of antibody fragments in the LIC401 fusion protein. Incubation time: 1 hour at room temperature;

[0331] 5) After membrane washing and ECL development, the results shown in Figure 4 were obtained, demonstrating that the non-reduced LIC401 fusion protein band was located between 100 and 150 kDa, while the reduced fusion protein had molecular weights between 45 and 75 kDa, consistent with the SDS-PAGE results of the previous protein purification. Furthermore, Western blot analysis confirmed the expression of the LIC401 fusion protein fragment by binding of the secondary antibody to the antibody fragment.

[0332] Example 2: Preparation of Fc-uncleaved-IL-12 (LIC402, LIC402-1) fusion protein

[0333] LIC402 and LIC402-1 fusion proteins were prepared using the same method as in Example 1. DNA fragments encoding the amino acid sequences set forth in SEQ ID NOs: 23 and 24 (LIC402) and SEQ ID NOs: 86 and 87 (LIC402-1) were prepared by chemical gene synthesis and PCR techniques, and plasmids were constructed. As shown in Figure 1 , the Fc fragment in the LIC402 fusion protein is located at the N-terminus and is linked to IL-12 via a non-cleavable linker. IL-12 comprises p35 and p40 subunits. The Protein A affinity chromatography elution peak and Western blot of the prepared fusion protein are shown in Figures 5 and 6 , respectively. A UV absorption peak exceeding 1000 mAU is observed, indicating that the target protein was eluted by the elution buffer. The reduced fusion protein has a molecular weight between 45 kDa and 75 kDa, exhibiting two bands.

[0334] Example 3: Determination of spleen stimulating activity of LIC401 and LIC402 fusion proteins

[0335] The spleen stimulatory activity of LIC401 prepared in Example 1 and LIC402 prepared in Example 2 was evaluated in 6-8 week old Balb / c female mice. Mice were intravenously injected with PBS or LIC401 (0.1 mg / kg) or LIC402 (0.1 mg / kg) on ​​Days 1, 4, and 7. Mice were sacrificed on Day 9, and their spleens were isolated, observed, and weighed. As shown in Figures 7 and 8, LIC401 significantly stimulated splenomegaly at a dose of 0.1 mg / kg, while LIC402 fusion protein did not induce significant splenomegaly at a dose of 0.1 mg / kg. This result suggests that IL-12 at the N-terminus of the fusion protein has strong biological activity, while the activity of IL-12 at the C-terminus of the fusion protein is masked by steric hindrance from the Fc.

[0336] Example 4: Preparation of Fc-bilobate-IL-12 (LIC403) fusion protein

[0337] LIC403 fusion protein, LIC403-1 fusion protein, and LIC403-2 fusion protein were prepared using the same method as in Example 1 (the three proteins differ only in the second structural unit). DNA fragments were prepared and plasmids constructed using chemical gene synthesis and PCR techniques. After expression and purification steps, the amino acid sequences of the LIC403 fusion protein were shown in SEQ ID NOs:25 and 26, the amino acid sequences of the LIC403-1 fusion protein were shown in SEQ ID NOs:65 and 66, and the amino acid sequences of the LIC403-2 fusion protein were shown in SEQ ID NOs:81 and 82. As shown in Figure 1, the Fc fragment of the LIC403, LIC403-1, and LIC403-2 fusion proteins is located at the N-terminus and connected to IL-12 via a cleavable linker. IL-12 comprises a p35 subunit and a p40 subunit. All three fusion proteins were successfully prepared.

[0338] This example demonstrates the detection results of the LIC403 fusion protein. The Protein A affinity chromatography elution peak and SDS-PAGE analysis of the prepared LIC403 fusion protein are shown in Figures 9 and 10, respectively. A UV absorption peak exceeding 1000 mAU is visible, indicating that the target protein is eluted by the elution buffer. The reduced fusion protein has a molecular weight between 45 kDa and 75 kDa, showing two bands.

[0339] Example 5: Activity detection and in vivo safety evaluation of LIC401-LIC403

[0340] 1. In vitro activity assay of LIC403, LIC403-1, and LIC403-2 fusion proteins

[0341] The fusion proteins LIC403, LIC403-1 or LIC403-2 prepared in Example 4 were added to EP tubes with urokinase uPA at a mass ratio of 20:1, and lysed in a 37°C water bath for 12 h. The lysed LIC403 was then diluted 3-fold to obtain lysed LIC403.

[0342] Spleens from normal BALB / c mice were isolated and single-cell suspensions were prepared. Specifically, the mice were dislocated by cervical dislocation and immersed in an alcohol cup for 5 minutes. The spleens were removed using autoclaved scissors and forceps. 4 ml of sterile RPMI 1640 medium was placed in a 35 mm culture dish. The spleens were triturated using a sterile syringe plunger. The resulting cell suspension was centrifuged and resuspended in culture medium (RPMI 1640 + 10% FBS + 1% Pen / Strep + 4 mm Glutamax + 50 mm β-mercaptoethanol).

[0343] 4 × 10 cells were seeded per well in a 96-well plate. 5 Splenocytes were then added with gradient dilutions of varying concentrations of LIC403, cleaved LIC403, and control fusion proteins (LIC401 prepared using the method of Example 1 and LIC402 prepared using the method of Example 2) and cultured in a 37°C, 5% CO2 incubator for 60 hours. The 96-well plate was centrifuged at 300×g for 10 minutes, and the cell supernatant was collected. IFNγ production was measured using a mouse IFNγ AlphaLISA assay kit. The results showed that the fusion proteins LIC403, LIC403-1, or LIC403-2 all exhibited improved safety and biological activity, demonstrating significant anti-tumor effects while reducing systemic toxic side effects.

[0344] This example shows the detection results of LIC403 fusion protein, in which as shown in FIG11A , the positive control LIC401 has a stimulatory activity against splenocytes EC 50 The EC value of LIC403 was calculated after cleavage. 50 The Fc fragment at the N-terminus can shield the activity of the IL-12 cytokine when not cleaved, and the IL-12 released after linker cleavage has high biological activity. Therefore, the LIC403 fusion protein has better safety and biological activity, and can significantly reduce systemic toxic side effects while achieving significant anti-tumor effects.

[0345] 2. In vivo activity detection and in vivo safety evaluation of LIC401-LIC403

[0346] The fusion protein LIC401 prepared in Example 1, the fusion proteins LIC402 and LIC402-1 prepared in Example 2, and the fusion protein LIC403 prepared in Example 4 were subjected to the following test:

[0347] 2.1 Culture mouse colon cancer cells MC-38 and resuspend in PBS to 5×10 6 Cells / ml, 100ul / mouse were subcutaneously inoculated into 6-8 week old C57BL / 6 female mice. The average tumor volume of the mice was about 75mm on the 9th day after tumor bearing. 3 The mice were randomly divided into 5 groups: IgG, LIC401-13.3pmol, LIC402-13.3pmol, LIC401-66.7pmol, and LIC402-66.7pmol, with 10 mice in each group. The mice were administered with the tail vein on the 9th, 12th, and 19th days after tumor loading. The tumor growth and body weight changes of the mice were continuously observed and recorded. The tumor volume reached 2000mm. 3 As the end of survival.

[0348] See Figure 11B for test results. In terms of safety, LIC401 exhibited significant toxicity in the MC38 model. At a dose of 66.7 pmol, mice experienced a 22% decrease in body weight, with four mice dying after the second dose. At a dose of 13.3 pmol, mice experienced a 5% decrease in body weight. In contrast, mice in the LIC402 group showed no weight loss.

[0349] 2.2 Culture mouse colon cancer cells MC-38 and resuspend in PBS to 5×10 6 100 μL cells / ml were subcutaneously inoculated into 6-8 week old C57BL / 6 female mice. The average tumor volume of the mice was about 86 mm on the 9th day after tumor bearing. 3 The mice were randomly divided into 5 groups: IgG, LIC401-66.7pmol, LIC402-66.7pmol, LIC403-66.7pmol, and LIC402-1-66.7pmol, with 6 mice in each group. The mice were administered with the tail vein on the 9th, 12th, and 15th days after tumor loading. The tumor growth and body weight changes of the mice were continuously observed and recorded. The average tumor volume in the IgG group reached 1000mm 3 The tumor weight was measured during dissection.

[0350] See Figure 11C for test results. In the MC38 model, LIC402-1 at a dose of 66.7 pmol resulted in a body weight decrease of approximately 6%, while LIC403 at the same dose only decreased body weight by 4%. Furthermore, LIC402-1 achieved an 80% tumor inhibition rate, while LIC403 achieved a 99% inhibition rate. At a dose of 66.7 pmol, LIC402-1 exhibited a higher tumor inhibition rate than LIC402.

[0351] 2.3 Culture mouse prostate cancer cells RM-1, resuspend in PBS to 5×10 6 100 μl of cells / ml were subcutaneously inoculated into 6-8 week old C57BL / 6 male mice. The average tumor volume of the mice was about 100 mm on the 10th day after tumor bearing. 3 The mice were randomly divided into 4 groups: IgG, LIC401, LIC402, and LIC403, with 10 mice in each group. The first tail vein administration was performed once every three days for a total of 3 times. The dose of LIC401-LIC403 was 13.3 pmol. The tumor growth and weight changes of the mice were continuously observed and recorded. The tumor volume reached 2000 mm 3 As the end of survival.

[0352] The test results are shown in Figure 11D. In the RM-1 model, LIC401 and LIC403 demonstrated strong antitumor effects, with tumor inhibition rates of 92% and 93%, respectively. Regarding systemic toxicity, LIC401 caused a 5% weight loss in mice, while mice in the LIC402 and LIC403 groups experienced no weight loss.

[0353] Example 6: Preparation of Fc-single cleaved-IL-12 (LIC404) fusion protein

[0354] The LIC404 fusion protein was prepared using the same method as in Example 1. DNA fragments encoding the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:26 were prepared by chemical gene synthesis and PCR, and plasmids were constructed. As shown in Figure 1, the Fc fragment of the LIC404 fusion protein is located at the N-terminus, one chain is linked to IL-12P35 via a non-cleavable linker, and the other chain is linked to IL-12P40 via a cleavable linker. The Protein A affinity chromatography elution peak and SDS-PAGE of the prepared fusion protein are shown in Figures 12 and 13, respectively. A UV absorption peak exceeding 1000 mAU is observed, indicating that the target protein was eluted by the elution buffer. The reduced fusion protein has a molecular weight between 45 kDa and 75 kDa, showing two bands.

[0355] Example 7: Preparation of Fc-single cleaved-IL-12 (LIC405) fusion protein

[0356] The LIC405 fusion protein was prepared using the same method as in Example 1. DNA fragments encoding the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:24 were prepared by chemical gene synthesis and PCR, and plasmids were constructed. As shown in Figure 1, the Fc fragment of the LIC405 fusion protein is located at the N-terminus. One chain is linked to IL-12P35 via a cleavable linker, and the other chain is linked to IL-12P40 via a non-cleavable linker. The elution peak of the prepared fusion protein after Protein A affinity chromatography is shown in Figure 14. A UV absorbance peak exceeding 800 mAU is observed, indicating that the target protein is eluted by the elution buffer.

[0357] Example 8: In vitro activity assay of LIC404, LIC405 and control fusion proteins

[0358] The same method as in Example 5 was used to determine the in vitro activity of the LIC404 fusion protein prepared in Example 6 and the LIC405 fusion protein prepared in Example 7, and LIC401 was used as a positive control. The results are shown in FIG15 . The stimulatory activity of the positive control LIC401 on splenocytes EC 50 The stimulatory activity of LIC404 without urokinase cleavage was weak, and the EC 50 The activity of LIC404 can be partially restored after single-strand cleavage, and its EC is calculated. 50 The activity of LIC405 is similar to that of LIC404. After enzymatic cleavage of the single chain, one subunit is released, which increases the flexibility of the cytokine while retaining the fusion of the Fc segment. This results in a significantly longer half-life than the IL-12 monomer.

[0359] Example 9: Preparation of PDL1 mAb-bilobite-IL-12 (LIC407) fusion protein

[0360] 1. LIC403 can be fused with antibodies targeting different antigens to produce more bifunctional proteins, such as the PDL1 monoclonal antibody. The LIC407 fusion protein was prepared using the same method as in Example 1. DNA fragments encoding the amino acid sequences of SEQ ID NO:9, SEQ ID NO:29, and SEQ ID NO:30 were prepared by chemical gene synthesis and PCR, and plasmids were constructed. As shown in Figure 1, the PDL1 antibody in the LIC407 fusion protein is located at the N-terminus and connected to IL-12P35 and IL-12P40 via cleavable linkers. The elution peak of the prepared fusion protein by Protein A affinity chromatography is shown in Figure 16. A UV absorbance peak exceeding 1000 mAU is observed, indicating that the target protein is eluted by the elution buffer.

[0361] 2. ELISA determination of the affinity of PDL1 monoclonal antibody or LIC407 for PDL1 antigen

[0362] 1) Human PD-L1 antigen coating: Dilute PD-L1 to 1.0 μg / mL with ELISA coating buffer, add 100 μL to each well of a 96-well plate, and coat overnight at 4°C.

[0363] 2) Block, discard the coating solution, wash 4 times with PBST, add 250 μL / well of blocking solution (5% BSA, PBST), and incubate at room temperature for 2 hours.

[0364] 3) Incubate with primary antibody, discard blocking solution, wash 4 times with PBST, add serially diluted PDL1 monoclonal antibody or LIC407 fusion protein to the plate at 100 μL / well, and incubate at room temperature for 2 h.

[0365] 4) Secondary antibody incubation: Discard the primary antibody and wash four times with PBST. Dilute HRP-labeled secondary antibody (goat anti-human IgG (H+L)) in PBST at a ratio of 1:10,000, add 100 μL / well to the plate, and incubate at room temperature for 1 hour.

[0366] 5) For color development, add TMB substrate solution at a volume of 100 μL / well and incubate at 37°C in the dark for 5 min. Terminate the reaction by adding stop solution (2 M H₂SO₄) at a volume of 50 μL per well. Measure absorbance at 450 nm. Data were processed using GraphPad 8.0 and are shown in Figure 17. As can be seen, the PDL1 monoclonal antibody, after fusion with IL-12, still retains a high affinity for PD-L1 similar to that of the PDL1 monoclonal antibody.

[0367] Example 10: Preparation of PDL1 mAb-uncleaved-IL-12 (LIC406, LIC408, LIC409) fusion protein

[0368] LIC406, LIC408, and LIC409 fusion proteins were prepared using the same method as in Example 1. DNA fragments were prepared and plasmids constructed using chemical gene synthesis and PCR techniques. The amino acid sequences of LIC406, LIC408, and LIC409 were expressed and purified, as shown in SEQ ID NOs: 27, 28, and 9; the amino acid sequences of LIC408, LIC409, and LIC409 are shown in SEQ ID NOs: 27, 30, and 9; and the amino acid sequences of LIC409, LIC406, LIC408, and LIC409 are shown in SEQ ID NOs: 29, 28, and 9. As shown in Figure 1, the PDL1 monoclonal antibody fragment in the LIC406 fusion protein is located at the N-terminus and is linked to IL-12P35 and IL-12P40 via a non-cleavable linker. The PDL1 monoclonal antibody fragment in the LIC408 fusion protein is located at the N-terminus, with one chain linked to IL-12P35 via a non-cleavable linker and one chain linked to IL-12P40 via a cleavable linker. The PDL1 monoclonal antibody fragment in the LIC409 fusion protein is located at the N-terminus, one chain is connected to IL-12P35 through a cleavable linker, and the other chain is connected to IL-12P40 through a non-cleavable linker.

[0369] Example 11: Activity Assay and In Vivo Safety Evaluation of LIC406, LIC407, LIC408, and LIC409 Fusion Proteins

[0370] 1. In vitro activity assay of LIC406, LIC407, LIC408, and LIC409 fusion proteins

[0371] The in vitro activity of the LIC406, LIC407, LIC408, and LIC409 fusion proteins prepared in Example 9 or Example 10 was determined using the same method as in Example 5, with LIC401 used as a positive control. The results are shown in FIG18 . Compared with the positive control LIC401, the LIC407, LIC408, and LIC409 fusion proteins had weaker stimulatory activity on splenocytes. 50 Both were greater than 400 pM. This suggests that the PDL1 monoclonal antibody can mask the activity of IL-12 when located at the N-terminus of IL-12. This form of the molecule can also enhance the targeting of IL-12.

[0372] 2. In vivo activity detection and in vivo safety evaluation of LIC406, LIC407, LIC408, and LIC409

[0373] The fusion protein LIC401+αPDL1 prepared in Example 1 and LIC406, LIC407, LIC408 and LIC409 prepared in Example 9 or Example 10 were tested in the same manner as in Example 5.

[0374] Mouse prostate cancer cells RM-1 were cultured and resuspended in PBS to 5 × 10 6 100 μl of cells / ml were subcutaneously inoculated into 6-8 week old C57BL / 6 male mice. The average tumor volume of the mice was about 100 mm on the 10th day after tumor bearing. 3 The mice were randomly divided into 6 groups: IgG, LIC401+αPDL1, LIC406, LIC407, LIC408, and LIC409, with 8 mice in each group. The first dose was administered via the tail vein once every three days for a total of 3 times. The dose of LIC401 was 0.1 mg / kg, and the doses of αPDL1, LIC406, LIC407, LIC408, and LIC409 were all 3 mg / kg. The tumor growth and weight changes of the mice were continuously observed and recorded. The tumor volume reached 2000 mm. 3 As the end of survival.

[0375] The test results are shown in Figure 18. In terms of the safety of each molecule, as reflected by weight changes, LIC401 at a dose of 0.1 mg / kg caused a significant weight loss of approximately 20% in mice. LIC406 significantly improved the safety of IL-12, achieving a balance between safety and anti-tumor efficacy by introducing different linkers. LIC407 reduced the weight loss to 10%, while LIC408 and LIC409 caused almost no weight loss in mice.

[0376] Example 12: Preparation of PDL1-ScFv-Fc-double (single) cleft-IL-12 (LIC412, LIC413 and LIC414) fusion protein

[0377] LIC412, LIC413, and LIC414 fusion proteins were prepared using the same method as in Example 1. DNA fragments were prepared and plasmids constructed using chemical gene synthesis and PCR techniques. Following expression and purification, the amino acid sequences of the LIC412 fusion protein, the amino acid sequences of the LIC413 fusion protein, and the amino acid sequences of the LIC414 fusion protein were shown in SEQ ID NOs: 35 and 36, 33 and 36, and 35 and 34, respectively. As shown in Figure 1 , the PDL1-ScFv-Fc fragment in the LIC412, LIC413, and LIC414 fusion proteins is located at the N-terminus and linked to IL-12P35 and IL-12P40 via a cleavable linker (or non-cleavable linker). The protein A affinity chromatography elution peak of the prepared LIC412 fusion protein is shown in Figure 19 . A UV absorbance peak exceeding 1000 mAU is observed, indicating that the target protein was eluted by the elution buffer.

[0378] Example 13: Activity determination of LIC412 and its control fusion protein

[0379] The in vitro activity of the LIC412, single-cleaved LIC413, and LIC414 fusion proteins prepared in Example 12 was determined using the same method as in Example 5, with LIC410 used as a positive control and LIC411 as a negative control. As shown in Figure 20, compared to the positive control LIC410, the LIC412, LIC413, and LIC414 fusion proteins exhibited weaker stimulatory activity on splenocytes, with EC50 values ​​significantly lower than those of the positive control. LIC412 activity was restored after cleavage by urokinase, demonstrating that the ScFv+Fc combination at the N-terminus of IL-12 can mask the activity of IL-12.

[0380] Example 14: Preparation of PD1 / PDL1-ScFv-Fc-double (single) cleft-IL-12 (LIC417, LIC418 and LIC419) fusion protein

[0381] LIC417, LIC418, and LIC419 fusion proteins were prepared using the same method as in Example 1. DNA fragments were prepared and plasmids constructed using chemical gene synthesis and PCR techniques. The amino acid sequences of the LIC417 fusion protein, the LIC418 fusion protein, and the LIC419 fusion protein were expressed and purified, respectively. The amino acid sequences are shown in SEQ ID NOs: 35 and 39, 33 and 39, and 35 and 38, respectively. As shown in Figure 1 , the PD1 / PDL1-ScFv-Fc fragment in the LIC417, LIC418, and LIC419 fusion proteins is located at the N-terminus and linked to IL-12P35 and IL-12P40 via a cleavable linker (or non-cleavable linker). The protein A affinity chromatography elution peak of the prepared LIC417 fusion protein is shown in Figure 21 . A UV absorption peak exceeding 1000 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0382] Example 15: Activity determination of LIC417 and its control fusion protein

[0383] The in vitro activity of the LIC417, single-cleaved LIC418, and LIC419 fusion proteins prepared in Example 14 was determined using the same method as in Example 5, using LIC415 as a positive control and LIC416 as a negative control. The results, as shown in Figure 22, show that compared to the positive control LIC415, the LIC417, LIC418, and LIC419 fusion proteins exhibited weaker stimulatory activity against splenocytes. This suggests that ScFv+Fc with different targets can mask IL-12 activity when located at the N-terminus of IL-12. This molecule also enhances the targeting of IL-12.

[0384] Example 16: Preparation of PDL1-(Fab)2-double (single) cleft-IL-12-Fc (LIC421, LIC422 and LIC423) fusion protein

[0385] LIC421, LIC422, and LIC423 fusion proteins were prepared using the same method as in Example 1. DNA fragments were prepared and plasmids constructed using chemical gene synthesis and PCR techniques. The amino acid sequences of the LIC421 fusion protein, the LIC422 fusion protein, and the LIC423 fusion protein were expressed and purified. The amino acid sequences are shown in SEQ ID NOs: 9, 42, and 43, respectively. The amino acid sequences of the LIC422 fusion protein are shown in SEQ ID NOs: 9, 40, and 43, respectively. The amino acid sequences of the LIC423 fusion protein are shown in SEQ ID NOs: 9, 42, and 41. As shown in Figure 1 , the PDL1-Fab fragment in the LIC421, LIC422, and LIC423 fusion proteins is located at the N-terminus and the Fc fragment is located at the C-terminus. These proteins are connected to IL-12P35 and IL-12P40 via a cleavable linker (or non-cleavable linker). The protein A affinity chromatography elution peak of the prepared LIC421 fusion protein is shown in Figure 23 . A UV absorption peak exceeding 500 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0386] Example 17: Activity Assay of PDL1-(Fab)2-Bisected-IL-12-Fc (LIC421) Fusion Protein

[0387] The in vitro activity of the LIC421 fusion protein prepared in Example 16 was determined using the same method as in Example 5, and LIC401 was used as a positive control. The results are shown in Figure 24. The EC50 of the positive control LIC401 on spleen cells was 2.2 pM. The stimulatory activity of LIC421 without urokinase cleavage was weaker, with an EC50 of 2.2 pM. 50 The activity of LIC421 was almost completely restored after UPA cleavage, and its EC value was calculated. 50 The molecular form of IL-12 located in the middle of the fusion protein has high masking activity, and the activity is fully restored after cleavage. The Fc fusion is retained after cleavage, which can extend the half-life of the cytokine and exert a more lasting therapeutic effect.

[0388] Example 18: Activity Assay of PDL1-(Fab)2-Single-Cleavage-IL-12-Fc (LIC422, LIC423) Fusion Protein

[0389] The in vitro activity of the LIC422 and LIC423 fusion proteins prepared in Example 16 was determined using the same method as in Example 5, with LIC401 used as a positive control. As shown in Figure 25 , the stimulatory activity of LIC422 and LIC423 cleaved by urokinase was partially restored compared to that of the cleaved proteins, but was weaker than that of the positive control. This demonstrates that Fab can mask IL-12 activity, similar to the steric hindrance created by the Fc hinge. Retaining a single Fab fusion after cleavage maintains targeting and prolongs the half-life of the cytokine, resulting in a more sustained therapeutic effect.

[0390] Example 19: Preparation of PDL1-Fab-double (single / non-) cleaved-IL-12-Fc (LIC424, LIC425, LIC426 and LIC427) fusion protein

[0391] LIC424 fusion protein, LIC425 fusion protein, LIC426 fusion protein, and LIC427 fusion protein were prepared using the same method as in Example 1, wherein DNA fragments were prepared by chemical gene synthesis and PCR technology and plasmids were constructed. After expression and purification, the amino acid sequences of the LIC425 fusion protein were shown in SEQ ID NO: 44 and SEQ ID NO: 40, the amino acid sequences of the LIC425 fusion protein were shown in SEQ ID NO: 43 and SEQ ID NO: 45, the amino acid sequences of the LIC426 fusion protein were shown in SEQ ID NO: 43 and SEQ ID NO: 44, and the amino acid sequences of the LIC427 fusion protein were shown in SEQ ID NO: 40 and SEQ ID NO: 45. As shown in Figure 1, the VL and VH (single Fab) fragments of the LIC424, LIC425, LIC426, and LIC427 fusion proteins are located at the N-terminus, and the Fc is located at the C-terminus. They are connected to IL-12P35 and IL-12P40 via a cleavable linker (or non-cleavable linker). The disulfide bonds between the VL and VH and the disulfide bond between the Fc-hinge form an upper and lower "lock" to mask the activity of IL-12. The Protein A affinity chromatography elution peak of the prepared LIC425 fusion protein is shown in Figure 26. A UV absorption peak exceeding 300 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0392] Example 20: Preparation of LIC425 fusion protein and in vitro enzymatic cleavage

[0393] The fusion protein LIC425 prepared in Example 19 and urokinase uPA were added to an EP tube at a mass ratio of 20:1 (10 μg LIC425 and 0.5 μg urokinase). The volume was made up to 20 μL with PBS. A non-lysed protein group and a UPA group were set up as controls. The mass of the control group and the corresponding components of the lysis system were kept consistent. Lysis was carried out at 37°C for 12 h, and cleavage was verified by SDS-PAGE. The results, as shown in Figure 27, show that under these conditions, LIC425 can be cleaved by urokinase by over 85%. The theoretical molecular weights of the Y-IL12-uPA-VH single chain are 87.2 kDa, and the Y-IL12-uPA-VL single chain are 73.8 kDa. The cleaved fragments, VL-CL, are 24.3-25.1 kDa, P35-Fc-Knob, 48.7-49.6 kDa, VH-CH1, 22.9-23.8 kDa, and P40-Fc-Hole, are 62.5-63.4 kDa. SDS-PAGE results show that the molecular weights of the uncleaved LIC425 fusion protein and the fragments after cleavage are consistent with the theoretical molecular weights. Because the purchased uPA is pharmaceutical urokinase and contains a large amount of albumin, the bands in the figure are relatively dense.

[0394] Example 21: Preparation of LIC425 fusion protein and in vitro activity assay

[0395] The in vitro activity of the LIC425 fusion protein prepared in Example 19 was determined using the same method as in Example 5, and LIC401 was used as a positive control. The results are shown in FIG28 . The stimulatory activity of the positive control LIC401 on splenocytes EC 50 The stimulatory activity of LIC425 without urokinase cleavage was weak, and the EC 50 greater than 400 pM, which is higher than the EC 50 The value is large. This indicates that the upper and lower "Lock" formed by the disulfide bond between VL and VH and the disulfide bond between Fc-Hinge has a better masking effect on IL-12 activity than the masking by the steric hindrance of the two Fabs. The activity of LIC425 can be almost completely restored after cleavage by UPA, and its EC 50 The heterologous molecule IL-12 located in the middle of the fusion protein has high masking activity, good activity recovery after cleavage, and retains Fc fusion after cleavage, which can prolong the half-life of the cytokine.

[0396] Example 22: ELISA determination of affinity of PDL1 monoclonal antibody or LIC425 for PDL1 antigen

[0397] The affinity of the single Fab in the LIC425 fusion protein prepared in Example 19 for the antigen was determined using the same method as in Example 9, with PDL1 monoclonal antibody used as a positive control. The results, shown in Figure 29, show that the affinity Kd value of the PDL1 monoclonal antibody for the PDL1 antigen was 0.1 nM, while the affinity Kd value of the fusion protein with IL-12 for the PD-L1 antigen was 0.17 nM, with no significant difference between the two. This demonstrates that the single Fab format can still enable the LIC425 fusion protein to have comparable targeting ability to PDL1.

[0398] Example 23: ELISA determination of tissue targeting of LIC425 fusion protein

[0399] 6-8 weeks old C57BL / 6N mice were subcutaneously inoculated with B16F10 melanoma and the tumors were grown to 800 mm. 3 At 3:10 pM, a single tail vein injection of 3 mg / kg of the LIC425 fusion protein prepared in Example 19 was performed. 12 hours later, blood was collected from the heart, the abdominal vena cava was cut, and 40 mL of PBS was perfused into the heart until the lungs were cleaned and turned pale, and the blood color of the liver faded (yellowish). The heart, liver, spleen, lung, kidney, and tumor were removed and weighed. 50-100 mg was then cut to prepare a 10% tissue homogenate. Three mice were fed a total of 3 samples, and the blood concentration of each tissue was measured by ELISA. The results, as shown in Figure 30, show that the in vivo tissue distribution of LIC425 exhibits certain specificity, with low levels in the heart, lungs, and kidneys. It has strong tumor targeting and is mainly distributed in tumor tissues, but there is also a small amount of enrichment in the liver and spleen. Specifically, the concentration of LIC45 in tumors is 6.56 times that in the heart, 1.30 times that in the liver, 3.08 times that in the spleen, 6.86 times that in the lungs, and 11.9 times that in the kidneys. In summary, the IL-12 fused to the PDL1 antibody Fab, namely the LIC425 molecule, has good tumor targeting ability, which is conducive to the development of anti-tumor efficacy.

[0400] Example 24: Preparation of PD1-Fab-bilobate-IL-12-Fc (LIC425-1) fusion protein

[0401] The VL and VH of the PDL1 antibody in the LIC425 fusion protein prepared in Example 19 were replaced with the VL and VH of the PD1 antibody, and the cleavable linker for the urokinase substrate in LIC425 was replaced with the cleavable linker for the matrix metalloproteinase substrate, thereby generating a new fusion protein, LIC425-1. The LIC425-1 fusion protein was prepared using the same method as in Example 1, wherein DNA fragments encoding the amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68 were prepared by chemical gene synthesis and PCR techniques, and plasmids were constructed. LIC425-1 is identical to the schematic diagram of the LIC425 fusion protein shown in FIG1 , wherein the VL and VH (single Fab) fragments are located at the N-terminus, the Fc is located at the C-terminus, and the VL and VH fragments are connected to IL-12P35 and IL-12P40 via a cleavable linker. The disulfide bond between the VL and VH fragments and the disulfide bond between the Fc and the hinge form a "lock" to mask the activity of IL-12. The Protein A affinity chromatography elution peak of the prepared fusion protein is shown in Figure 31. A UV absorption peak exceeding 300 mAU can be seen, indicating that the target protein was eluted by the elution buffer.

[0402] Example 25: In vivo safety evaluation of LIC425-1 fusion protein

[0403] The results of the in vitro LIC425 cell activity assay in Example 21 showed that IL-12 activity was attenuated in this structure, suggesting that LIC425-like molecules may have relatively low peripheral immune stimulatory activity and systemic toxic side effects. Therefore, normal C57BL / 6 mice were used to evaluate the in vivo safety of LIC425-1, one of the LIC425-like molecules prepared in Example 24, compared to the positive drug LIC401. The specific steps are as follows:

[0404] In this example, C57BL / 6N mice were selected and divided into four groups (4 mice per group) according to Figure 32. The four groups of mice were administered with the drug via the tail vein on Day 1 and Day 4 (see Figure 32 for the type of administration, dosage, and route of administration for each group), and the weight of the mice was monitored. As shown in Figure 32, LIC401, administered twice at a dose of 0.1 mg / kg, induced severe weight loss, slowed movement, and frizzy hair in the mice. However, LIC425 had no effect on mouse weight at a dose of 0.5 mg / kg, and only induced weight loss at a dose of 1 mg / kg, with the magnitude of the decrease being less than that observed with the 0.1 mg / kg dose of the LIC401 fusion protein.

[0405] In order to further explore the intrinsic mechanism of the improved safety of LIC425-1, mice were killed on Day 6, and spleens and peripheral blood were collected for analysis. The spleen stimulation results are shown in Figure 33. The spleen weight of the LIC401 group was significantly higher than that of the PBS group, while the spleen weight of the LIC425-1 group was significantly lower than that of the positive drug LIC401 group. This indicates that the single Fab segment of the fused PD1 antibody can mask the immunostimulatory activity of IL-12 to a large extent in the periphery. Correspondingly, the flow cytometry analysis results show that in peripheral blood, 0.5 mg / kg and 1 mg / kg of LIC425 molecules have a significant effect on CD8 + The stimulation of T and NK cells was significantly lower than that of 0.1 mg / kg group of IL12-Fc or LIC401. + The stimulation of T cells was also significantly weaker than that of 0.1 mg / kg of LIC401.

[0406] Example 26: Activity Assay of PDL1-Fab-Single-IL-12-Fc (LIC426, LIC427) Fusion Protein

[0407] The in vitro activity of the LIC426 and LIC427 fusion proteins prepared in Example 19 was determined using the same method as in Example 5, with LIC401 used as a positive control. As shown in Figure 36 , compared to the positive control LIC401, the LIC426 and LIC427 fusion proteins had weaker stimulatory activity on splenocytes, but their activity was restored after cleavage with urokinase.

[0408] Example 27: Preparation of PDL1-(Fab)2-double (single) cleft-IL-12 tandem-Fc (LIC430 and LIC431) protein

[0409] LIC430 and LIC431 fusion proteins were prepared using the same method as in Example 1. DNA fragments were prepared and plasmids constructed using chemical gene synthesis and PCR techniques. The amino acid sequences of the LIC430 fusion protein and the LIC431 fusion protein, respectively, were expressed and purified. The amino acid sequences are shown in SEQ ID NOs: 9 and 48, and the amino acid sequences of the LIC431 fusion protein are shown in SEQ ID NOs: 9, 49, and 50. As shown in Figure 1 , the LIC430 and LIC431 fusion proteins have a Fab fragment at the N-terminus and an Fc fragment at the C-terminus. These fragments are linked to IL-12P70 via a cleavable linker (or non-cleavable linker), resulting in one LIC430 molecule containing two IL-12P70 molecules. The elution peak of the prepared LIC430 fusion protein after Protein A affinity chromatography is shown in Figure 37 . A UV absorbance peak exceeding 500 mAU is observed, indicating that the target protein was eluted by the elution buffer.

[0410] Example 28: Activity determination of LIC430 and control fusion proteins

[0411] The in vitro activity of the LIC430 and single-cleaved LIC431 fusion proteins prepared in Example 27 was determined using the same method as in Example 5, with LIC401 used as a positive control. As shown in Figure 38 , compared to the positive control LIC401, the LIC430 and LIC431 fusion proteins showed weaker stimulatory activity on splenocytes. After cleavage by urokinase, LIC430 activity was somewhat restored, but still lower than that of monovalent IL-12. This suggests that heterologous IL-12 (IL-12 subunits in parallel) is more active than homologous IL-12 (IL-12 subunits in series).

[0412] Example 29: Preparation of PDL1-Fab-double (single) cleft-IL-12 tandem-Fc (LIC433, LIC434 and LIC435) protein

[0413] LIC433, LIC434, and LIC435 fusion proteins were prepared using the same method as in Example 1. DNA fragments were prepared and plasmids constructed using chemical gene synthesis and PCR. The amino acid sequences of the LIC433 fusion proteins, the LIC434 fusion proteins, and the LIC435 fusion proteins were expressed and purified, as shown in SEQ ID NOs:53 and 50. The amino acid sequences of the LIC434 fusion proteins are shown in SEQ ID NOs:53 and 52, and the amino acid sequences of the LIC435 fusion proteins are shown in SEQ ID NOs:51 and 50. As shown in Figure 1 , the LIC433, LIC434, and LIC435 fusion proteins have a Fab fragment at the N-terminus and an Fc fragment at the C-terminus, connected to IL-12P70 via a cleavable linker. Thus, one LIC433 molecule contains two IL-12P70 molecules. The elution peak of the prepared LIC433 fusion proteins by Protein A affinity chromatography is shown in Figure 39 . A UV absorption peak exceeding 500 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0414] Example 30: Activity determination of LIC433 and control fusion proteins

[0415] The in vitro activity of the LIC433, LIC434, and LIC435 fusion proteins prepared in Example 29, along with the single-chain cleavable linker, was determined using the same method as in Example 5, with LIC401 used as a positive control. As shown in Figure 40 , the LIC433 fusion protein exhibited weaker stimulatory activity on splenocytes compared to the positive control, LIC401. While activity was partially restored after cleavage by urokinase, it was still lower than that of monovalent IL12.

[0416] Example 31: Preparation of LIC436 fusion protein

[0417] The LIC436 fusion protein was prepared using the same method as in Example 1. DNA fragments encoding the amino acid sequences set forth in SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 54 were prepared by chemical gene synthesis and PCR, and plasmids were constructed. As shown in Figure 1 , the PDL1 monoclonal antibody was directly linked to IL-12P70 via a non-cleavable linker, with the P40 subunit of IL-12P70 positioned externally. The protein A affinity chromatography elution peak of the prepared fusion protein is shown in Figure 41 . A UV absorption peak exceeding 1000 mAU was observed, indicating that the target protein was eluted by the elution buffer.

[0418] Example 32: Preparation of LIC444 fusion protein

[0419] In LIC436, only the N-terminus-P35 subunit-P40 subunit-C-terminus of IL-12P70 was adjusted to the N-terminus-P40 subunit-P35 subunit-C-terminus, forming a new fusion protein, LIC444. DNA fragments encoding the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:69 were prepared by chemical gene synthesis and PCR techniques, and plasmids were constructed. As shown in Figure 1, the PDL1 monoclonal antibody is directly linked to IL-12P70 via a non-cleavable linker, with the P35 subunit of IL-12P70 external. The elution peak of the prepared fusion protein by Protein A affinity chromatography is shown in Figure 42. A UV absorbance peak exceeding 500 mAU is visible, indicating that the target protein is eluted by the elution buffer.

[0420] Example 33: Activity Assay of LIC436 and LIC444 Fusion Proteins

[0421] The in vitro activities of the LIC436 fusion proteins prepared in Example 31 and the LIC444 fusion proteins prepared in Example 32 were determined using the same method as in Example 5. The results are shown in FIG43 . Compared with the positive control LIC401, the stimulatory activity of the PDL1 monoclonal antibody-fused proteins LIC436 and LIC444 on splenocytes was slightly weaker, but the activities of LIC436 and LIC444 were comparable.

[0422] Example 34: Preparation of LIC438 and LIC441 fusion proteins and their activity assays

[0423] 1. LIC438 fusion protein and LIC441 fusion protein were prepared using the same method as in Example 1, wherein DNA fragments were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. The amino acid sequences of the LIC438 fusion protein obtained after expression and purification are shown in SEQ ID NOs: 56 and 57, and the amino acid sequences of the LIC441 fusion protein are shown in SEQ ID NOs: 59 and 61. As shown in Figure 1, in the structures of the LIC438 fusion protein and the LIC441 fusion protein, one chain of the PDL1 single-chain antibody is directly linked to the Fc via a non-cleavable linker, and the other chain of the PDL1 (i.e., LIC438) or PD1 (i.e., LIC441) single-chain antibody is linked to the Fc via IL-12P70. IL-12P70 is linked to the PDL1 single-chain antibody and the Fc via a non-cleavable linker, with IL-12P70 in the middle.

[0424] 2. The in vitro activity of the LIC438 and LIC441 fusion proteins was determined using the same method as in Example 5. The results, as shown in Figure 44, show that compared to the previously determined positive control LIC401, the fusion proteins LIC438 and LIC441 exhibited reduced stimulatory activity on splenocytes. The N-terminal masking of a single IL12-p70 molecule by the Fc was stronger than that achieved by two ScFvs with the same target. This molecular format can achieve IL12-p70 inactivation.

[0425] Example 35: Preparation of EGFR-bilobate-I tandem fusion protein (LIC445)

[0426] The LIC445 fusion protein was prepared using the same method as in Example 1, wherein DNA fragments encoding the amino acid sequences set forth in SEQ ID NO:74 and SEQ ID NO:73 were prepared by chemical gene synthesis and PCR techniques, and plasmids were constructed. As shown in Figure 1 , the heavy chain of the human EGFR monoclonal antibody was directly linked to human IL-12P70 via a cleavable linker. The Protein A affinity chromatography elution peak of the prepared fusion protein is shown in Figure 45 . A UV absorption peak exceeding 1000 mAU was observed, indicating that the target protein was eluted by the elution buffer.

[0427] Example 36: Activity determination of LIC445 fusion protein

[0428] The in vitro activity of the LIC445 fusion protein was determined using the same method as in Example 5. PBMCs were isolated from human whole blood by density gradient centrifugation. The cells were then treated with ACK lysis buffer and repeatedly washed with PBS. The cells were then resuspended in culture medium (RPMI 1640 + 10% FBS + 1% Pen / Strep + 4 mM Glutamax + 50 mM β-mercaptoethanol).

[0429] 1×10 cells were seeded per well in a 96-well plate. 5 Freshly treated PBMC cells were activated by adding PHA to a final concentration of 5 mg / ml. LIC445 prepared in Example 37 and cleaved LIC445 were then added in serial dilutions at varying concentrations and cultured for 72 hours at 37°C in a 5% CO2 incubator. Cell supernatants were collected and IFNγ production was measured using a human IFNγ ELISA kit. The results, as shown in Figure 46, show that human IL-12 fused to the C-terminus of the EGFR monoclonal antibody exhibited different activity before and after enzymatic cleavage, demonstrating that fusion of IL-12 to the C-terminus of the heavy chain of the EGFR antibody effectively masks its activity and could be used as a strategy for cytokine inactivation or prodrug preparation.

[0430] Example 37: Comparison of the activities of LIC407, LIC421, LIC425, LIC430, LIC433, LIC437, LIC438, and LIC441 fusion proteins

[0431] 1. The same method as in Example 1 was used to prepare a LIC437 fusion protein. The corresponding amino acid sequences of the LIC437 fusion proteins prepared by chemical gene synthesis and PCR techniques are shown in SEQ ID NO: 9, SEQ ID NO: 52, and SEQ ID NO: 55. As shown in FIG1 , in the LIC437 fusion protein, the Fab fragment is located at the N-terminus, the Fc is located at the C-terminus, the Fab of PDL1 on one chain is directly linked to the Fc via a non-cleavable linker, and the Fab and Fc of PDL1 on the other chain are linked via IL-12P70. IL-12P70 is linked to the Fab and Fc of PDL1 via a non-cleavable linker, with IL-12P70 located in the middle.

[0432] 2. The same method as in Example 5 was used to simultaneously determine the in vitro activities of LIC407 prepared in Example 9, LIC421 prepared in Example 16, LIC425 prepared in Example 19, LIC430 prepared in Example 27, LIC433 prepared in Example 29, LIC437 prepared in Example 39, LIC438 prepared in Example 34, and LIC441. LIC401 was used as a positive control to compare the effects of different molecular masking structures on IL-12 activity. The results, as shown in Figure 47 and Table 1, show that the LIC407 structure, located at the N-terminus of the IL12-P35 and IL12-P40 subunits, can shield IL-12 activity by more than 4000-fold. The disulfide bond between the VL and VH in a single Fab and the disulfide bond between the Fc-hinge form a "lock" structure. The shielding effect of LIC425 is stronger than that of the LIC421 molecule shielded by two Fabs and an Fc. Furthermore, the shielding effects of LIC425 and LIC421 are stronger than those of LIC433, indicating that the "sandwich" steric hindrance is more effective for shielding a single IL-12 in parallel than for two or more IL-12s in series. The shielding effect of LIC441 is greater than that of LIC437 and LIC438, indicating that Fc steric hindrance exceeds that of Fab and ScFv.

[0433] Table 1

[0434] Example 38: In vivo safety evaluation of LIC406, LIC407, LIC408, LIC409, and LIC425 fusion proteins

[0435] 3. Using the same method as in Example 25, the in vivo safety evaluation of LIC407 prepared in Example 9, LIC406, LIC408, and LIC409 prepared in Example 10, and the LIC425 fusion protein prepared in Example 19 was simultaneously conducted, with LIC401 used as a positive control. The results are shown in Figure 48 . The results showed that LIC401, administered twice at a dose of 0.1 mg / kg, induced severe weight loss, slowed movement, and frizzy hair in mice. LIC425, at a dose of 1 mg / kg, induced weight loss in mice, but the magnitude of the decrease was less than that observed with the 0.1 mg / kg dose of the LIC401 fusion protein. LIC406-LIC409, at a dose of 1 mg / kg, had no effect on mouse body weight.

[0436] The amino acid sequence or nucleotide sequence of the present invention is as follows:

[0437] Mouse IL-12-P35 subunit amino acid sequence SEQ ID No: 1:

[0438] Mouse IL-12-P40 subunit amino acid sequence SEQ ID No: 2:

[0439] Human IgG1-Fc-Knob-Mut amino acid sequence SEQ ID No: 3:

[0440] Human IgG1-Fc-Hole-Mut amino acid sequence SEQ ID No: 4:

[0441] The amino acid sequence of the non-cleavable linker 1 at the N-terminus of the IL12 subunit is SEQ ID No: 5:

[0442] The amino acid sequence of the non-cleavable linker 2 at the C-terminus of the IL12 subunit is SEQ ID No: 6:

[0443] Cleavable UPA substrate linker 3 amino acid sequence SEQ ID No: 7:

[0444] Cleavable MMP substrate linker 4 amino acid sequence SEQ ID No: 8:

[0445] Human PDL1 monoclonal antibody light chain amino acid sequence SEQ ID No: 9:

[0446] Human PDL1-IgG1 monoclonal antibody heavy chain (Mut) amino acid sequence SEQ ID No: 10:

[0447] Human PDL1-IgG1 monoclonal antibody heavy chain (Knob-Mut) amino acid sequence SEQ ID No: 11:

[0448] Human PDL1-IgG1 monoclonal antibody heavy chain (Hole-Mut) amino acid sequence SEQ ID No: 12:

[0449] Human PD1 monoclonal antibody light chain amino acid sequence SEQ ID No: 13:

[0450] Human PD1-IgG4 monoclonal antibody heavy chain amino acid sequence SEQ ID No: 14:

[0451] Human PD1-IgG4 monoclonal antibody heavy chain (Knob) amino acid sequence SEQ ID No: 15:

[0452] Human PD1-IgG4 monoclonal antibody heavy chain (Hole) amino acid sequence SEQ ID No: 16:

[0453] Human PDL1-ScFv amino acid sequence SEQ ID No: 17:

[0454] Human PD1-ScFv amino acid sequence SEQ ID No: 18:

[0455] Human PDL1-VH-CH1 amino acid sequence SEQ ID No: 19:

[0456] Human PD1-VH-CH1 amino acid sequence SEQ ID No: 20:

[0457] IF fusion protein LIC401: Chain 1 amino acid sequence SEQ ID No: 21 (SEQ ID No: 1 + SEQ ID No: 6 + SEQ ID No: 3), Chain 2 amino acid sequence SEQ ID No: 22 (SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 4):

[0458] FI fusion protein LIC402: Chain 1 amino acid sequence SEQ ID No: 23 (SEQ ID No: 3 + SEQ ID No: 5 + SEQ ID No: 1), Chain 2 amino acid sequence SEQ ID No: 24 (SEQ ID No: 4 + SEQ ID No: 5 + SEQ ID No: 2):

[0459] F-bilobate-I fusion protein LIC403: chain 1 amino acid sequence SEQ ID No: 25 (SEQ ID No: 3 + SEQ ID No: 7 + SEQ ID No: 1), chain 2 amino acid sequence SEQ ID No: 26 (SEQ ID No: 4 + SEQ ID No: 7 + SEQ ID No: 2):

[0460] F-right cleft-I fusion protein LIC404: chain 1 amino acid sequence is SEQ ID No: 23, chain 2 amino acid sequence is SEQ ID No: 26;

[0461] F-left cleft-I fusion protein LIC405: chain 1 amino acid sequence is SEQ ID No: 25, chain 2 amino acid sequence is SEQ ID No: 24;

[0462] PI fusion protein LIC406: Chain 1 amino acid sequence SEQ ID No: 27 (SEQ ID No: 11 + SEQ ID No: 5 + SEQ ID No: 1), Chain 2 amino acid sequence SEQ ID No: 28 (SEQ ID No: 12 + SEQ ID No: 5 + SEQ ID No: 2), Chain 3 amino acid sequence SEQ ID No: 9:

[0463] P-bilobate-I fusion protein LIC407: Chain 1 amino acid sequence SEQ ID No: 29 (SEQ ID No: 11 + SEQ ID No: 7 + SEQ ID No: 1), Chain 2 amino acid sequence SEQ ID No: 30 (SEQ ID No: 12 + SEQ ID No: 7 + SEQ ID No: 2), Chain 3 amino acid sequence SEQ ID No: 9:

[0464] P-right cleft-I fusion protein LIC408: chain 1 amino acid sequence is SEQ ID No: 27, chain 2 amino acid sequence is SEQ ID No: 30, chain 3 amino acid sequence is SEQ ID No: 9;

[0465] P-left cleft-I fusion protein LIC409: chain 1 amino acid sequence is SEQ ID No: 29, chain 2 amino acid sequence is SEQ ID No: 28, chain 3 amino acid sequence is SEQ ID No: 9;

[0466] IF-PDL1-ScFv fusion protein LIC410: Chain 1 amino acid sequence SEQ ID No: 31 (SEQ ID No: 21 + SEQ ID No: 17), Chain 2 amino acid sequence SEQ ID No: 32 (SEQ ID No: 22 + SEQ ID No: 17):

[0467] PDL1-ScFv-FI fusion protein LIC411: chain 1 amino acid sequence SEQ ID No: 33 (SEQ ID No: 17 + SEQ ID No: 23), chain 2 amino acid sequence SEQ ID No: 34 (SEQ ID No: 17 + SEQ ID No: 24);

[0468] PDL1-ScFv-F-bilobate-I fusion protein LIC412: chain 1 amino acid sequence SEQ ID No: 35 (SEQ ID No: 17 + SEQ ID No: 25), chain 2 amino acid sequence SEQ ID No: 36 (SEQ ID No: 17 + SEQ ID No: 26);

[0469] PDL1-ScFv-F-right cleft-I fusion protein LIC413: chain 1 amino acid sequence is SEQ ID No: 33, chain 2 amino acid sequence is SEQ ID No: 36;

[0470] PDL1-ScFv-F-Left Cleavage-I fusion protein LIC414: Chain 1 amino acid sequence is SEQ ID No: 35, and Chain 2 amino acid sequence is SEQ ID No: 34;

[0471] IF-PDL1-ScFv-PD1-ScFv fusion protein LIC415: Chain 1 amino acid sequence is SEQ ID No: 31, and Chain 2 amino acid sequence is SEQ ID No: 37 (SEQ ID No: 22 + SEQ ID No: 18):

[0472] PDL1-ScFv-PD1-ScFv-FI fusion protein LIC416: Chain 1 amino acid sequence is SEQ ID No: 33, and Chain 2 amino acid sequence is SEQ ID No: 38 (SEQ ID No: 18 + SEQ ID No: 24):

[0473] PDL1-ScFv-PD1-ScFv-F-double split-I fusion protein LIC417: chain 1 amino acid sequence SEQ ID No: 35, chain 2 amino acid sequence SEQ ID No: 39 (SEQ ID No: 18 + SEQ ID No: 26):

[0474] PDL1-ScFv-PD1-ScFv-F-right cleft-I fusion protein LIC418: chain 1 amino acid sequence is SEQ ID No: 33, chain 2 amino acid sequence is SEQ ID No: 39;

[0475] PDL1-ScFv-PD1-ScFv-F-Left Cleft-I fusion protein LIC419: Chain 1 amino acid sequence is SEQ ID No: 35, and Chain 2 amino acid sequence is SEQ ID No: 38;

[0476] PIF-T fusion protein LIC420: Chain 1 amino acid sequence SEQ ID No: 40 (SEQ ID No: 19 + SEQ ID No: 5 + SEQ ID No: 21), Chain 2 amino acid sequence SEQ ID No: 41: (SEQ ID No: 19 + SEQ ID No: 5 + SEQ ID No: 22), Chain 3 amino acid sequence is SEQ ID No: 9:

[0477] P-bilobate-IF-T fusion protein LIC421: Chain 1 amino acid sequence SEQ ID No: 42 (SEQ ID No: 19 + SEQ ID No: 7 + SEQ ID No: 21), Chain 2 amino acid sequence SEQ ID No: 43 (SEQ ID No: 19 + SEQ ID No: 7 + SEQ ID No: 22), Chain 3 amino acid sequence is SEQ ID No: 9:

[0478] P-right-cleaved-IF-T fusion protein LIC422: chain 1 amino acid sequence is SEQ ID No: 40, chain 2 amino acid sequence is SEQ ID No: 43, chain 3 amino acid sequence is SEQ ID No: 9;

[0479] P-left-cleaved-IF-T fusion protein LIC423: chain 1 amino acid sequence is SEQ ID No: 42, chain 2 amino acid sequence is SEQ ID No: 41, chain 3 amino acid sequence is SEQ ID No: 9;

[0480] PIF-γ fusion protein LIC424: Chain 1 amino acid sequence is SEQ ID No: 44 (SEQ ID No: 9 + SEQ ID No: 5 + SEQ ID No: 21), and Chain 2 amino acid sequence is SEQ ID No: 40:

[0481] P-bilobate-IF-γ fusion protein LIC425: Chain 1 amino acid sequence is SEQ ID No: 45 (SEQ ID No: 9 + SEQ ID No: 7 + SEQ ID No: 21), and Chain 2 amino acid sequence is SEQ ID No: 43:

[0482] P-right cleavage-IF-γ fusion protein LIC426: chain 1 amino acid sequence is SEQ ID No: 44, chain 2 amino acid sequence is SEQ ID No: 43;

[0483] P-left cleft-IF-γ fusion protein LIC427: chain 1 amino acid sequence is SEQ ID No: 45, chain 2 amino acid sequence is SEQ ID No: 40;

[0484] PI homodimeric fusion protein LIC428: Chain 1 amino acid sequence is SEQ ID No: 46 (SEQ ID No: 10 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2), and Chain 2 amino acid sequence is SEQ ID No: 9:

[0485] PIF-T homodimeric fusion protein LIC429: Chain 1 amino acid sequence is SEQ ID No: 47 (SEQ ID No: 19 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + PDL1 heavy chain Fc (Mut)), and Chain 2 amino acid sequence is SEQ ID No: 9:

[0486] P-bilobate-IF-T homodimeric fusion protein LIC430: Chain 1 amino acid sequence SEQ ID No: 48 (SEQ ID No: 19 + SEQ ID No: 7 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + PDL1 heavy chain Fc (Mut)), Chain 2 amino acid sequence SEQ ID No: 9:

[0487] P-single-split-IF-T homodimeric fusion protein LIC431: chain 1 amino acid sequence SEQ ID No:49 (SEQ ID No:19+SEQ ID No:5+SEQ ID No:1+SEQ ID No:75+SEQ ID No:2+SEQ ID No:6+SEQ ID No:3), chain 2 amino acid sequence SEQ ID No:50 (SEQ ID No:19+SEQ ID No:7+SEQ ID No:1+SEQ ID No:75+SEQ ID No:2+SEQ ID No:6+SEQ ID No:4), chain 3 amino acid sequence SEQ ID No:9:

[0488] PIF-γ homodimeric fusion protein LIC432: Chain 1 amino acid sequence SEQ ID No: 51 (SEQ ID No: 9 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 3), Chain 2 amino acid sequence SEQ ID No: 52 (SEQ ID No: 19 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 4):

[0489] P-bilobate-IF-γ homodimeric fusion protein LIC433: Chain 1 amino acid sequence is SEQ ID No: 53 (SEQ ID No: 9 + SEQ ID No: 7 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 3), and Chain 2 amino acid sequence is SEQ ID No: 50:

[0490] P-left split-IF-γ homodimeric fusion protein LIC434: chain 1 amino acid sequence is SEQ ID No: 53, chain 2 amino acid sequence is SEQ ID No: 52;

[0491] P-right split-IF-γ homodimeric fusion protein LIC435: chain 1 amino acid sequence is SEQ ID No: 51, chain 2 amino acid sequence is SEQ ID No: 50;

[0492] PI tandem unimolecular fusion protein LIC436: Chain 1 amino acid sequence is SEQ ID No: 54 (SEQ ID No: 11 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2), Chain 2 amino acid sequence is SEQ ID No: 12, and Chain 3 amino acid sequence is SEQ ID No: 9:

[0493] PIF-T tandem single cytokine fusion protein LIC437: Chain 1 amino acid sequence is SEQ ID No: 55 (SEQ ID No: 19 + SEQ ID No: 75 + SEQ ID No: 3), Chain 2 amino acid sequence is SEQ ID No: 52, and Chain 3 amino acid sequence is SEQ ID No: 9:

[0494] PDL1-ScFv-IF tandem single cytokine fusion protein LIC438: Chain 1 amino acid sequence SEQ ID No: 56 (SEQ ID No: 17 + SEQ ID No: 75 + SEQ ID No: 3), Chain 2 amino acid sequence SEQ ID No: 57 (SEQ ID No: 17 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 4):

[0495] PDL1-ScFv-PD1-ScFv-IF tandem single cytokine fusion protein LIC439: Chain 1 amino acid sequence is SEQ ID No: 56, and Chain 2 amino acid sequence is SEQ ID No: 58 (SEQ ID No: 18 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 4):

[0496] FI-PDL1-ScFv tandem single cytokine fusion protein LIC440: Chain 1 amino acid sequence SEQ ID No: 59 (SEQ ID No: 3 + SEQ ID No: 75 + SEQ ID No: 17), Chain 2 amino acid sequence SEQ ID No: 60 (SEQ ID No: 4 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 17):

[0497] FI-PDL1-ScFv-PD1-ScFv tandem single cytokine fusion protein LIC441: Chain 1 amino acid sequence is SEQ ID No: 59, and Chain 2 amino acid sequence is SEQ ID No: 61 (SEQ ID No: 4 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2 + SEQ ID No: 6 + SEQ ID No: 18):

[0498] PDL1-ScFv-FI tandem single cytokine fusion protein LIC442: Chain 1 amino acid sequence SEQ ID No: 62 (SEQ ID No: 17 + SEQ ID No: 3 + SEQ ID No: 5 + SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2), Chain 2 amino acid sequence SEQ ID No: 63 (SEQ ID No: 17 + SEQ ID No: 4):

[0499] PDL1-ScFv-PD1-ScFv-FI tandem single cytokine fusion protein LIC443: Chain 1 amino acid sequence is SEQ ID No: 62, and chain 2 amino acid sequence is SEQ ID No: 64 (SEQ ID No: 18 + SEQ ID No: 4):

[0500] F-bilobate-I fusion protein LIC403-1: Chain 1 amino acid sequence SEQ ID No: 65: (the cleavable linker is 16 amino acids less than that of LIC403), Chain 2 amino acid sequence SEQ ID No: 66: (the cleavable linker is 16 amino acids less than that of LIC403):

[0501] PD1 (Fab)-bilobate-IF-γ fusion protein LIC425-1: Chain 1 amino acid sequence SEQ ID No: 67 (SEQ ID No: 13 + SEQ ID No: 8 + SEQ ID No: 21), Chain 2 amino acid sequence SEQ ID No: 68 (SEQ ID No: 20 + SEQ ID No: 8 + SEQ ID No: 22):

[0502] PI tandem unimolecular fusion protein LIC444: Chain 1 amino acid sequence SEQ ID No: 69 (SEQ ID No: 11 + SEQ ID No: 5 + SEQ ID No: 2 + SEQ ID No: 75 + SEQ ID No: 1), Chain 2 amino acid sequence SEQ ID No: 12, Chain 3 amino acid sequence SEQ ID No: 9:

[0503] Human IL-12-P35 subunit amino acid sequence SEQ ID No: 70:

[0504] Human IL-12-P40 subunit amino acid sequence SEQ ID No: 71:

[0505] Human EGFR monoclonal antibody heavy chain amino acid sequence SEQ ID No: 72:

[0506] Human EGFR monoclonal antibody light chain amino acid sequence SEQ ID No: 73:

[0507] E-double split-I tandem fusion protein LIC445: chain 1 amino acid sequence SEQ ID No: 74 (SEQ ID No: 72 + SEQ ID No: 8 + SEQ ID No: 70 + SEQ ID No: 75 + SEQ ID No: 71), chain 2 amino acid sequence SEQ ID No: 73:

[0508] The connecting peptide between P35 and P40 is SEQ ID No: 75:

[0509] Mouse P35-connecting peptide-P40 SEQ ID No: 76 (SEQ ID No: 1 + SEQ ID No: 75 + SEQ ID No: 2):

[0510] Human P35-connecting peptide-P40 SEQ ID No: 77 (SEQ ID No: 70 + SEQ ID No: 75 + SEQ ID No: 71):

[0511] The amino acid sequence of the cleavable cathepsin substrate linker is SEQ ID No: 78:

[0512] The amino acid sequence of the cleavable protein cleaving enzyme substrate linker is SEQ ID No: 79:

[0513] The amino acid sequence of the cleavable legumin substrate linker is SEQ ID No: 80:

[0514] F-bilobate-I fusion protein LIC403-2: Chain 1 amino acid sequence SEQ ID No: 81: (the cleavable linker has 14 more amino acids than LIC403), Chain 2 amino acid sequence SEQ ID No: 82: (the cleavable linker has 14 more amino acids than LIC403):

[0515] Cleavable UPA substrate linker 3 amino acid sequence SEQ ID No: 83:

[0516] Cleavable UPA substrate linker 3 amino acid sequence SEQ ID No: 84:

[0517] The amino acid sequence of the non-cleavable linker 2 at the N-terminus of the IL12 subunit is SEQ ID No: 85:

[0518] FI fusion protein LIC402-1: Chain 1 amino acid sequence SEQ ID No: 86 (the non-cleavable linker has 21 more amino acids than LIC402), Chain 2 amino acid sequence SEQ ID No: 87 (the non-cleavable linker has 21 more amino acids than LIC402):

[0519] The amino acid sequence of the signal peptide of human EGFR monoclonal antibody is SEQ ID No: 88:

[0520] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0521] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that include: A first structural unit, wherein the first structural unit is selected from a first Fc fragment, or a first antibody or an antigen-binding fragment thereof, wherein the first antibody or the antigen-binding fragment thereof has tumor antigen or immune checkpoint binding activity; A second structural unit, wherein the second structural unit is selected from a cleavable linker or a non-cleavable linker, wherein the cleavable linker can be cleaved by a protease expressed in tumor tissue; a third structural unit comprising an IL-12 cytokine or a functional fragment thereof; The first structural unit is connected to one or more third structural units, and the third structural unit is connected to the C-end of the first structural unit through the second structural unit.

2. The fusion protein according to claim 1, characterized in that The fusion protein comprises one or more of the following conditions: 1) When the first structural unit is connected to one of the third structural units, the second structural unit is selected from a cleavable linker; 2) When the first structural unit is connected to a plurality of the third structural units, the second structural unit connected to the plurality of the third structural units contains at least one cleavable linker, and preferably, all the second structural units connected to the plurality of the third structural units are cleavable linkers; 3) The fusion protein does not include an antibody or antigen-binding fragment thereof to the IL-12 cytokine; 4) the first antibody or antigen-binding fragment thereof comprises a first full-length antibody, a first Fab fragment, a first ScFv fragment, or a first VHH fragment; 5) The tumor antigen or immune checkpoint is selected from at least one of Her2, EGFR, VEGF, VEGFR, Claudin 18.2, B7-H3, Nectin-4, MSLN, Trop2, Siglec-9, Siglec-15, PD-L1, PD-1, TIGIT, LAG3, TIM-3, CTLA-4, BTLA, and VISTA; 6) The IL-12 cytokine or its functional fragment includes at least one of the IL-12 P35 fragment or its functional fragment, and the IL-12 P40 fragment or its functional fragment; 7) The second structural unit is a cleavable linker; 8) the second structural unit is a non-cleavable linker; 9) The fusion protein comprises two monomers, and the two second structural units are a cleavable linker and a non-cleavable linker, respectively; 10) The proteases expressed in the tumor tissue include matrix metalloproteinases, serine proteases, or asparagine endopeptidases; 11) The cleavable linker comprises a protease substrate sequence and optionally a flexible peptide segment; 12) The flexible peptide segment includes at least one of (GS)n, (GGS)n, (GGSG)n, (GSSG)n, (GGGS)n, (GGGGS)n and (GSGGS)n, where n is any integer between 1 and 10; 13) The amino acid length of the flexible peptide segment is 1-60, preferably 5-30, 5-25 or 5-20; 14) The non-cleavable linker includes at least one of (GS)n, (GGS)n, (GGSG)n, (GSSG)n, (GGGS)n, (GGGGS)n and (GSGGS)n, where n is any integer between 1 and 10; 15) The amino acid length of the non-cleavable linker is 1 to 60, preferably 5 to 50, 5 to 40, 5 to 30 or 10 to 20.

3. The fusion protein according to claim 2, characterized in that The fusion protein comprises one or more of the following conditions: 1) The IL-12 cytokine or a functional fragment thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 2 and 70 to 71; 2) The IL-12 cytokine or its functional fragment comprises two subunit fragments: a P35 fragment of IL-12 or its functional fragment, and a P40 fragment of IL-12 or its functional fragment, and the third structural unit further comprises a non-cleavable connecting peptide, and the two subunit fragments are connected by the connecting peptide; 3) the protease substrate sequence is selected from QLLGFLTA, LSGRSDNH, GFFY, RQARAVGG or AANL; 4) The cleavable linker is a flexible peptide segment-protease substrate sequence, or a flexible peptide segment-protease substrate sequence-flexible peptide segment; 5) The amino acid length of the cleavable linker is 15 to 60, preferably 15 to 50, 15 to 45, or 15 to 40; 6) The non-cleavable linker has an amino acid sequence as shown in (GGGGS)n, where n is 3, 4, 5 or 6.

4. The fusion protein according to claim 3, characterized in that The fusion protein comprises one or more of the following conditions: 1) The IL-12 cytokine or its functional fragment comprises two subunit fragments: a P35 fragment of IL-12 or its functional fragment, and a P40 fragment of IL-12 or its functional fragment, wherein the C-terminus of one subunit fragment is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the other subunit fragment; 2) the connecting peptide comprises at least one of (GS)n, (GGS)n, (GGSG)n, (GSSG)n, (GGGS)n, (GGGGS)n, and (GSGGS)n, where n is any integer between 1 and 10; 3) The amino acid length of the connecting peptide is 1-60, preferably 10-40, 15-40; 4) the cleavable linker has an amino acid sequence as shown in SEQ ID NO: 7, 8, 78, 79, 80, 83 or 84; 5) The non-cleavable linker has an amino acid sequence as shown in SEQ ID NO: 5 or 6.

5. The fusion protein according to claim 3, characterized in that The fusion protein comprises one or more of the following conditions: 1) The connecting peptide has an amino acid sequence as shown in (GGGGS)n, where n is 3, 4, 5 or 6; 2) The third structural unit has an amino acid sequence as shown in SEQ ID NO: 1, 2, 70, 71, 76 or 77.

6. The fusion protein according to claim 2, characterized in that The fusion protein comprises one or more of the following conditions: 1) The first structural unit is a first ScFv fragment, wherein the C-terminus of the first structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the third structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit; 2) The first structural unit is a first Fc fragment or a first VHH fragment, the first Fc fragment or the first VHH fragment is connected to one or two third structural units, and each of the third structural units is connected to the first Fc fragment or the first VHH fragment through the second structural unit; The C-terminus of the first structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the third structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the second structural unit, and the C-terminus of the second structural unit is connected to the N-terminus of the first structural unit; 3) When the first Fc fragment or the first VHH fragment is connected to one of the third structural units, the IL-12 cytokine or its functional fragment in the third structural unit includes two subunit fragments: the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, preferably the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment; 4) When the first Fc fragment or the first VHH fragment is connected to two of the third structural units, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker; 5) When the first Fc fragment or the first VHH fragment is connected to two of the third structural units, the IL-12 cytokine or its functional fragment in the two third structural units is the same or different; 6) When the fusion protein consists of a first structural unit, a second structural unit, and a third structural unit, and the first structural unit is a first Fc fragment, the IL-12 cytokine or its functional fragment of the third structural unit includes two subunit fragments: a P35 fragment of IL-12 or its functional fragment, and a P40 fragment of IL-12 or its functional fragment; The two subunit fragments are respectively connected to the two fragments of the first Fc fragment.

7. The fusion protein according to claim 2, characterized in that The fusion protein comprises one or more of the following conditions: a) the first structural unit is a first full-length antibody, the first full-length antibody is connected to one or more third structural units, and each of the third structural units is connected to the first full-length antibody via the second structural unit; b) The first structural unit is a first Fab fragment, the first Fab fragment is connected to one or two third structural units, and each of the third structural units is connected to the C-terminus of the first Fab fragment through the second structural unit.

8. The fusion protein according to claim 7, characterized in that The fusion protein comprises one or more of the following conditions: a-1) the first full-length antibody is connected to one of the third structural units, and the third structural unit is connected to the C-terminus of the heavy chain of the first full-length antibody through the second structural unit; a-2) the first full-length antibody is connected to a plurality of the third structural units, and the plurality of the third structural units are respectively connected to the C-terminus of the heavy chain of the first full-length antibody through the second structural unit; a-3) the first full-length antibody is connected to two of the third structural units, and the two third structural units are respectively connected to the C-termini of the two heavy chains in the first full-length antibody through the second structural unit; a-4) When the first full-length antibody is linked to one of the third structural units, the IL-12 cytokine or its functional fragment in the third structural unit comprises two subunit fragments: the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, preferably the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment; a-5) when the first full-length antibody is linked to a plurality of the third structural units, the plurality of the second structural units are each independently selected from a cleavable linker or a non-cleavable linker, preferably all of which are cleavable linkers; a-6) when the first full-length antibody is connected to two third structural units, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker; a-7) when the first full-length antibody is connected to two third structural units, and the two third structural units are respectively connected to the C-termini of the two heavy chains in the first full-length antibody through the second structural unit, the IL-12 cytokines or functional fragments thereof in the two third structural units are the same or different, preferably, the IL-12 cytokines or functional fragments thereof in the two third structural units are the P35 fragment or functional fragment of IL-12, and the P40 fragment or functional fragment of IL-12, respectively; b-1) the first Fab fragment is connected to a third structural unit, and the third structural unit is connected to the C-terminus of the CH1 fragment or the C-terminus of the CL fragment in the first Fab fragment through the second structural unit; b-2) the first Fab fragment is connected to two of the third structural units, and the two third structural units are respectively connected to the C-terminus of the CH1 fragment and the C-terminus of the CL fragment in the first Fab fragment through the second structural unit; b-3) when the first Fab fragment is connected to one of the third structural units, the IL-12 cytokine or its functional fragment in the third structural unit comprises two subunit fragments: the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, preferably the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment; b-4) when the first Fab fragment is connected to two of the third structural units, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker; b-5) When the first Fab fragment is connected to two of the third structural units, the IL-12 cytokines or their functional fragments in the two third structural units are the same or different, and preferably, the IL-12 cytokines or their functional fragments in the two third structural units are the P35 fragment or its functional fragment of IL-12, and the P40 fragment or its functional fragment of IL-12, respectively.

9. The fusion protein according to claim 7, characterized in that When the first Fab fragment is connected to one of the third structural units, and the third structural unit is connected to the C-terminus of the CH1 fragment in the first Fab fragment through the second structural unit, the fusion protein includes two monomers; In the two monomers, the IL-12 cytokine or its functional fragment of the third structural unit is respectively the P35 fragment or its functional fragment of IL-12 and the P40 fragment or its functional fragment of IL-12, and the P35 fragment or its functional fragment of IL-12 and the P40 fragment or its functional fragment of IL-12 are connected by a disulfide bond.

10. The fusion protein according to any one of claims 2 to 9, characterized in that The fusion protein further includes a fourth structural unit, which is selected from a second Fc fragment, or a second antibody or its antigen-binding fragment, and the second antibody or its antigen-binding fragment has tumor antigen or immune checkpoint binding activity.

11. The fusion protein according to claim 10, characterized in that The fusion protein further comprises one or more of the following conditions: 1) the second antibody or antigen-binding fragment thereof comprises a second full-length antibody, a second Fab fragment, a second ScFv fragment, or a second VHH fragment; 2) the C-terminus of the fourth structural unit is connected to the N-terminus of the first structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the fourth structural unit; 3) The first structural unit is a first Fc fragment, and the fourth structural unit is selected from a second Fab fragment, a second ScFv fragment, or a second VHH fragment; or the first structural unit is selected from a first Fab fragment, a first ScFv fragment, or a first VHH fragment, and the fourth structural unit is a second Fc fragment, and the third structural unit is located between the first and fourth structural units; 4) the fusion protein further comprises a fifth structural unit, and the fourth structural unit is connected to the first structural unit or the third structural unit via the fifth structural unit; 5) The fifth structural unit is selected from a cleavable linker or a non-cleavable linker; 6) When the third structural unit is located between the first structural unit and the fourth structural unit, the third structural unit is connected to the first structural unit through the second structural unit, and the third structural unit is connected to the fourth structural unit through the fifth structural unit; 7) When the first structural unit is a first Fc fragment or a first full-length antibody, the fourth structural unit is selected from a second Fab fragment, a second ScFv fragment, or a second VHH fragment, and the C-terminus of the fourth structural unit is connected to the N-terminus of the first structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the fourth structural unit; preferably, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, and the C-terminus of the fifth structural unit is connected to the N-terminus of the first structural unit, or the C-terminus of the third structural unit is connected to the N-terminus of the fifth structural unit, and the C-terminus of the fifth structural unit is connected to the N-terminus of the fourth structural unit; 7) When the first structural unit is a first Fab fragment, a first ScFv fragment, or a first VHH fragment, the fourth structural unit is selected from a second Fc fragment, and the C-terminus of the third structural unit is connected to the N-terminus of the fourth structural unit; preferably, the C-terminus of the third structural unit is connected to the N-terminus of the fifth structural unit, and the C-terminus of the fifth structural unit is connected to the N-terminus of the fourth structural unit; 8) When the first structural unit is a first Fab fragment, a first ScFv fragment or a first VHH fragment, the fourth structural unit is selected from a second Fc fragment, and the N-terminus of each fragment in the fourth structural unit is connected to the C-terminus of the third structural unit.

12. The fusion protein according to claim 1, characterized in that The fusion protein comprises: The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 23, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 24; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 25, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 26; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 23, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 26; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 25, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 24; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 27, the amino acid sequence has a third peptide chain as shown in SEQ ID NO: 28, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO: 9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 29, the amino acid sequence has a third peptide chain as shown in SEQ ID NO: 30, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO: 9; An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 27, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 30, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 29, the amino acid sequence has a third peptide chain as shown in SEQ ID NO: 28, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO: 9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 34; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 36; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 36; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 34; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 38; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 39; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:33, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:39; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 38; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO:40, the amino acid sequence has a third peptide chain as shown in SEQ ID NO:41, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO:9; An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 42, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 43, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9; An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 40, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 43, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO:41, the amino acid sequence has a third peptide chain as shown in SEQ ID NO:42, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO:9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:44, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:40; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:45, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:43; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:44, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:43; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:45, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:40; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:46, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:47, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:48, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:9; An amino acid sequence having a first peptide chain as shown in SEQ ID NO: 9, an amino acid sequence having a second peptide chain as shown in SEQ ID NO: 49, an amino acid sequence having a third peptide chain as shown in SEQ ID NO: 50, and an amino acid sequence having a fourth peptide chain as shown in SEQ ID NO: 9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:51, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:52; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 53, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 50; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 53, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 52; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 51, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 50; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 54, the amino acid sequence has a third peptide chain as shown in SEQ ID NO: 12, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO: 9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO:55, the amino acid sequence has a third peptide chain as shown in SEQ ID NO:52, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO:9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 56, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 57; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 56, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 58; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:59, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:60; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:59, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:61; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 62, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 63; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 62, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 64; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 66, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 65; The amino acid sequence has a first peptide chain as shown in SEQ ID NO:81, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:82; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 67, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 68; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, the amino acid sequence has a second peptide chain as shown in SEQ ID NO: 69, the amino acid sequence has a third peptide chain as shown in SEQ ID NO: 12, and the amino acid sequence has a fourth peptide chain as shown in SEQ ID NO: 9; The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 74, and the amino acid sequence has a second peptide chain as shown in SEQ ID NO:

73.

13. A nucleic acid molecule, an expression vector and a recombinant cell, characterized in that: The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 12; The expression vector carries the above-mentioned nucleic acid molecule; The recombinant cell carries the aforementioned nucleic acid or the aforementioned expression vector; or the recombinant cell expresses the fusion protein according to any one of claims 1 to 12.

14. A fusion protein complex, characterized in that The invention comprises the fusion protein according to any one of claims 1 to 12.

15. An immunotherapy cell, characterized in that Expressing the fusion protein according to any one of claims 1 to 12.

16. A pharmaceutical composition, characterized in that The invention comprises the fusion protein according to any one of claims 1 to 12, the nucleic acid molecule, expression vector, recombinant cell according to claim 11, the fusion protein complex according to claim 14, or the immunotherapy cell according to claim 15, and optionally pharmaceutically acceptable excipients.

17. The fusion protein of any one of claims 1 to 12, the fusion protein complex of claim 14, the immunotherapy cell of claim 15, or the pharmaceutical composition of claim 16, for use in treating cancer, infectious diseases or autoimmune diseases.

18. The fusion protein according to any one of claims 1 to 12, the fusion protein complex according to claim 14, the immunotherapy cell according to claim 15, or the pharmaceutical composition according to claim 16 has the following uses: Treating cancer, infectious diseases or autoimmune diseases; and / or Preparation of medicaments for treating cancer, infectious diseases or autoimmune diseases.

19. A method for treating cancer, infectious disease or autoimmune disease, characterized in that: include: Administering a pharmaceutically acceptable dose of the fusion protein of any one of claims 1 to 12, the fusion protein complex of claim 14, the immunotherapeutic cell of claim 15, or the pharmaceutical composition of claim 16 to a subject.

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