IL-12 fusion protein with adjustable activity and application thereof

Abstract

The invention discloses an activity-adjustable IL-12 fusion protein and application 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, and the first antibody or the antigen binding fragment thereof has tumor antigen or immune checkpoint binding activity; the second structural unit is selected from a cleavable connexon or a non-cleavable connexon; the third structural unit comprises an IL-12 cytokine or a functional fragment thereof. The second structural unit mediates steric hindrance of the first structural unit to achieve a shielding activity effect on the IL-12 cytokines in the third structural unit, reduces toxic and side effects of the IL-12 cytokines in in-vivo application, has the advantage of high safety compared with wild IL-12, can be further fused with antibodies Fab, scFv, VHH, antigen-binding peptides or recombinant proteins, polypeptides and the like, and can be used for preparing anti-inflammatory drugs. The multifunctional fusion protein capable of being conditionally released and activated is obtained.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an IL-12 fusion protein with adjustable activity and its use, and more particularly 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 use in the preparation of drugs for treating or preventing cancer, infectious diseases or autoimmune diseases. Background Art

[0002] Interleukin 12 (IL-12) is a multifunctional cytokine encoded by two independent genes, IL-12A (p35) and IL-12B (p40), and forms an active p70 heterodimer bridged by two disulfide bonds. Activated dendritic cells (DCs), macrophages and neutrophils locally produce IL-12 during T cell priming and directly act on cytotoxic immune effector cells, including natural killer (NK) cells, natural killer T (NKT) cells and CD8+ T cells, to stimulate their proliferation and increase their cytotoxic function. IL-12 can also induce the differentiation of naive helper T cells into the Th1 phenotype and produce cytokines (mainly IFNγ), which itself has cell inhibitory / cytotoxic, anti-angiogenic, and can up-regulate the expression of MHC I and II on tumor cells to enhance recognition and lysis. Therefore, a lower concentration of IL-12 in clinical application also has strong stimulatory activity on immune cells in normal tissues, which is likely to cause serious systemic toxic side effects and has a narrow therapeutic window. In addition, IL-12 has a short serum half-life with rapid clearance and requires frequent administration to maintain a therapeutically relevant exposure.

[0003] Antibody-cytokine fusion proteins, also known as immunocytokines, can effectively achieve the enrichment of cytokines at the tumor site by utilizing the targeting property of antibodies, thereby reducing the dosage and drug toxicity. Currently, there are studies on 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 play an anti-tumor role, and the antibody can act as a "missile" carrying IL-12 to achieve the enrichment of IL-12 at the tumor site. However, due to the significant biological activity of IL-12, even a small off-target effect can bring strong toxic side effects, and the existing reported immunocytokines still cannot completely solve the problem of IL-12 systemic toxicity. Therefore, there is an urgent need to develop a drug product that can target and 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 at least 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 provides 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 its antigen-binding fragment, and the first antibody or its antigen-binding fragment has tumor antigen or immune checkpoint binding activity; the second structural unit is selected from a cleavable linker, or an uncleavable linker, and the cleavable linker can be cleaved by a protease expressed in tumor tissue; the third structural unit contains IL-12 cytokine or its functional fragment; wherein, one or more of the third structural units are connected to the first structural unit, 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 IL-12 cytokine or its functional fragment, and the second structural unit mediates the steric hindrance of the first structural unit to play a role in masking the activity of the IL-12 cytokine in the third structural unit. In particular, without the traditional affinity masking strategy, so the fusion protein does not need to contain any antibody or receptor of IL-12 cytokine, can reduce the toxic and side effects brought by IL-12 cytokine, and the fusion protein has the advantages of strong safety and simple structure.

[0006] In the 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 the third aspect of the present invention, 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, by using the expression vector of the present invention, the expression of the fusion protein described in the first aspect can be effectively achieved, and thus a large amount of the fusion protein can be obtained in vitro.

[0008] In the fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes: 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. Using this recombinant cell under suitable conditions, the fusion protein described in the first aspect can be effectively expressed in the cell.

[0009] In the fifth aspect of the present invention, the present invention provides a fusion protein complex. According to an embodiment of the present invention, the fusion protein complex comprises the fusion protein described in the first aspect. As previously known, the third structural unit of the fusion protein described in the first aspect contains the IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity on the IL-12 cytokine in the third structural unit. In particular, without the need for traditional affinity shielding strategies, the fusion protein can reduce the toxic and side effects brought by the IL-12 cytokine without containing any antibodies or receptors of the IL-12 cytokine, and 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 provides an immunotherapeutic cell. According to an embodiment of the present invention, the immunotherapeutic cell expresses the fusion protein described in the first aspect. As previously known, the third structural unit of the fusion protein described in the first aspect contains the IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity on the IL-12 cytokine in the third structural unit. In particular, without the need for traditional affinity shielding strategies, the fusion protein can reduce the toxic and side effects brought by the IL-12 cytokine without containing any antibodies or receptors of the IL-12 cytokine, and the fusion protein has advantages such as strong safety. Therefore, the immunotherapeutic cell containing the above 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 provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises 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 immunotherapeutic cell described in the sixth aspect. As previously known, the third structural unit of the fusion protein described in the first aspect contains the IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity on the IL-12 cytokine in the third structural unit. In particular, without the need for traditional affinity shielding strategies, the fusion protein can reduce the toxic and side effects brought by the IL-12 cytokine without containing any antibodies or receptors of the IL-12 cytokine, and the fusion protein has advantages such as strong safety. Therefore, the pharmaceutical composition containing the above fusion protein has advantages such as strong safety and can be used to treat cancer, infectious diseases or autoimmune diseases.

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

[0013] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or 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 be readily understood from the following description of embodiments in conjunction with the accompanying drawings, in which:

[0015] Figure 1Schematic diagram of the structure of the fusion protein of the present invention, where LIC401 is C-non-cleavable L-Fc with only Fc as the first structural unit; LIC402 is Fc-non-cleavable L-C with only Fc as the first structural unit; LIC403 is Fc-bis-cleavable L-C with only Fc as the first structural unit; LIC404 and LIC405 are Fc-mono-cleavable L-C with only Fc as the first structural unit; LIC406 is IgG-non-cleavable L-C with a monoclonal antibody as the first structural unit; LIC407 is IgG-bis-cleavable L-C with a monoclonal antibody as the first structural unit; LIC408 and LIC409 are IgG-mono-cleavable L-C with a monoclonal antibody as the first structural unit; LIC410 is C-non-cleavable L-Fc-ScFv with two identical ScFv-Fc as the first structural unit; LIC411 is ScFv-Fc-non-cleavable L-C with two identical ScFv-Fc as the first structural unit; LIC412 is ScFv-Fc-bis-cleavable L-C with two identical ScFv-Fc as the first structural unit; LIC413 and LIC414 are ScFv-Fc-mono-cleavable L-C with two identical ScFv-Fc as the first structural unit; LIC415 is C-non-cleavable L-Fc-ScFv with two different ScFv-Fc as the first structural unit; LIC416 is ScFv-Fc-non-cleavable L-C with two different ScFv-Fc as the first structural unit; LIC417 is ScFv-Fc-bis-cleavable L-C with two different ScFv-Fc as the first structural unit; LIC418 and LIC419 are ScFv-Fc-mono-cleavable L-C with two different ScFv-Fc as the first structural unit; LIC420 is (Fab)2-non-cleavable L-C-L-Fc with two Fabs as the first structural unit and Fc as the fourth structural unit; LIC421 is (Fab)2-bis-cleavable L-C-L-Fc with two Fabs as the first structural unit and Fc as the fourth structural unit; LIC422 and LIC423 are (Fab)2-mono-cleavable L-C-L-Fc with two Fabs as the first structural unit and Fc as the fourth structural unit; LIC424 is Fab-non-cleavable L-C-L-Fc with a single Fab as the first structural unit and Fc as the fourth structural unit; LIC425 is Fab-bis-cleavable L-C-L-Fc with a single Fab as the first structural unit and Fc as the fourth structural unit; LIC426 and LIC427 are Fab-mono-cleavable L-C-L-Fc with a single Fab as the first structural unit and Fc as the fourth structural unit; LIC428 is IgG-non-cleavable L-C2 with a monoclonal antibody as the first structural unit and containing two molecules of cytokine; LIC429 is (Fab)2-non-cleavable L-C2-L-Fc with two Fabs as the first structural unit, Fc as the fourth structural unit and containing two molecules of cytokine;LIC430 is (Fab)2-bis-cleavable L-C2-L-Fc with two Fabs as the first structural unit, Fc as the fourth structural unit, and containing two molecules of cytokine; LIC431 is (Fab)2-monocleavable L-C2-L-Fc with two Fabs as the first structural unit, Fc as the fourth structural unit, and containing two molecules of cytokine; LIC432 is Fab-non-cleavable L-C2-L-Fc with a single Fab as the first structural unit, Fc as the fourth structural unit, and containing two molecules of cytokine; LIC433 is Fab-bis-cleavable L-C2-L-Fc with a single Fab as the first structural unit, Fc as the fourth structural unit, and containing two molecules of cytokine; LIC434 and LIC435 are Fab-monocleavable L-C2-L-Fc with a single Fab as the first structural unit, Fc as the fourth structural unit, and containing two molecules of cytokine; LIC436 is IgG-non-cleavable L-C with a monoclonal antibody as the first structural unit and cytokine subunits in tandem; LIC437 is (Fab)2-non-cleavable L-C-L-Fc with two Fabs as the first structural unit, Fc as the fourth structural unit, and cytokine subunits in tandem; LIC438 is ScFv-non-cleavable L-C-L-Fc with two identical ScFvs as the first structural unit, Fc as the fourth structural unit, and cytokine subunits in tandem; LIC439 is ScFv-non-cleavable L-C-L-Fc with two different ScFvs as the first structural unit, Fc as the fourth structural unit, and cytokine subunits in tandem; LIC440 is Fc-non-cleavable L-C-L-ScFv with two identical ScFvs as the first structural unit, Fc as the fourth structural unit, and cytokine subunits in tandem; LIC441 is Fc-non-cleavable L-C-L-ScFv with two different ScFvs as the first structural unit, Fc as the fourth structural unit, and cytokine subunits in tandem; LIC442 is ScFv-Fc-non-cleavable L-C with two identical ScFv-Fcs as the first structural unit and cytokine subunits in tandem; LIC443 is ScFv-Fc-non-cleavable L-C with two different ScFv-Fcs as the first structural unit and cytokine subunits in tandem; LIC444 is IgG-non-cleavable L-C with a monoclonal antibody as the first structural unit and cytokine subunits in tandem but in a different order from LIC436; LIC445 is IgG-bis-cleavable L-C2 with a monoclonal antibody as the first structural unit and containing two molecules of cytokine. Among them, monocleavable L represents that only one chain in a fusion protein contains a cleavable linker; bis-cleavable L represents that two chains in a fusion protein contain cleavable linkers; C represents that a fusion protein contains 1 molecule of cytokine, and C2 represents that a fusion protein contains 2 molecules of cytokine; L represents an uncleavable linker at the C-terminus of the IL12 subunit.

[0016] Figure 2 It is the protein A affinity chromatography purification diagram of the LIC401 fusion protein.

[0017] Figure 3 It is the SDS-PAGE electrophoresis diagram of the purified LIC401 fusion protein. NR is the non-reduced sample, and R is the reduced sample. The mother liquor is the collected cell supernatant, NaOH is the impurities eluted by the NaOH solution after the chromatography, the flow-through is the flow-through liquid during the protein A affinity chromatography, and the elution is the LIC401 fusion protein.

[0018] Figure 4 It is the Western blot identification of the LIC401 fusion protein. The plasmid was transfected at a ratio of 1:1, and R is the reduced sample.

[0019] Figure 5 It is the protein A affinity chromatography purification diagram of the LIC402 fusion protein.

[0020] Figure 6 It is the Western blot identification of the LIC402 fusion protein. The plasmid was transfected at a ratio of 1:1, and R is the reduced sample.

[0021] Figure 7 It is the photo diagram of the stimulation of the mouse spleen by the LIC401 and LIC402 fusion proteins.

[0022] Figure 8 It is the statistical chart of the weights of the mouse spleens treated with PBS, LIC401, and LIC402 fusion proteins.

[0023] Figure 9 It is the protein A affinity chromatography purification diagram of the LIC403 fusion protein.

[0024] Figure 10 It is the SDS-PAGE electrophoresis diagram of the purified LIC403 fusion protein. NR is the non-reduced sample, and R is the reduced sample. The flow-through is the flow-through liquid during the protein A affinity chromatography, and the elution is the LIC403 fusion protein.

[0025] Figure 11 is the in vitro activity detection diagram of the purified LIC403 fusion protein, the in vivo activity detection diagrams of the LIC401, LIC402, LIC402-1, and LIC403 fusion proteins, and the diagram of the changes in the body weights of mice after administration of the LIC401, LIC402, LIC402-1, and LIC403 fusion proteins.

[0026] Figure 12 It is the protein A affinity chromatography purification diagram of the LIC404 fusion protein.

[0027] Figure 13It is the SDS-PAGE electrophoresis diagram of the purified LIC404 fusion protein. NR is the non-reduced sample, and R is the reduced sample. The flow-through is the liquid flowing through during the protein A affinity chromatography process, and the elution is the LIC404 fusion protein.

[0028] Figure 14 It is the protein A affinity chromatography purification diagram of the LIC405 fusion protein.

[0029] Figure 15 It is the diagram of the in vitro activity assay results of LIC404, LIC405 and the control fusion proteins.

[0030] Figure 16 It is the protein A affinity chromatography purification diagram of the LIC407 fusion protein.

[0031] Figure 17 It is the ELISA assay diagram of the affinity of the LIC407 fusion protein for the human PDL1 antigen.

[0032] Figure 18 It is the diagram of the in vitro activity assay of LIC407, LIC408, LIC409 fusion proteins, the diagram of the in vivo activity detection of LIC406-LIC409 and other fusion proteins, and the diagram of the body weight change of mice after administration of LIC406-LIC409 and other fusion proteins.

[0033] Figure 19 It is the protein A affinity chromatography purification diagram of the LIC412 fusion protein.

[0034] Figure 20 It is the diagram of the in vitro activity assay of LIC412 and the control fusion proteins.

[0035] Figure 21 It is the protein A affinity chromatography purification diagram of the LIC417 fusion protein.

[0036] Figure 22 It is the diagram of the in vitro activity assay of LIC417 and the control fusion proteins.

[0037] Figure 23 It is the protein A affinity chromatography purification diagram of the LIC421 fusion protein.

[0038] Figure 24 It is the diagram of the in vitro activity assay of the purified LIC421 fusion protein.

[0039] Figure 25 It is the diagram of the in vitro activity assay of the single-cleaved forms of LIC422 and LIC423 fusion proteins.

[0040] Figure 26It is the protein A affinity chromatography purification diagram of the LIC425 fusion protein.

[0041] Figure 27 It is the SDS-PAGE schematic diagram after in vitro enzymatic cleavage of the LIC425 fusion protein.

[0042] Figure 28 It is the in vitro activity assay diagram of the LIC425 fusion protein.

[0043] Figure 29 It is the ELISA assay diagram of the affinity of the LIC425 fusion protein for the human PDL1 antigen.

[0044] Figure 30 It is the ELISA assay diagram of the tissue distribution of the LIC425 fusion protein in tumor-bearing mice.

[0045] Figure 31 It is the protein A affinity chromatography purification diagram of the LIC425-1 fusion protein.

[0046] Figure 32 It is the diagram of the change in the body weight of mice after administration of the LIC425-1 and control fusion proteins.

[0047] Figure 33 It is the statistical chart of the spleen weight of terminal mice after administration of the LIC425-1 and control fusion proteins.

[0048] Figure 34 It is the result diagram of the counts of CD8+ T cells and NK cells in peripheral blood at the end point after administration of the LIC425-1 and control fusion proteins.

[0049] Figure 35 It is the schematic diagram of the proportion of CD8+ T cells in the spleen at the end point after administration of the LIC425-1 and control fusion proteins.

[0050] Figure 36 It is the in vitro activity assay diagram of the single-cleavage forms of the LIC426 and LIC427 fusion proteins.

[0051] Figure 37 It is the protein A affinity chromatography purification diagram of the LIC430 fusion protein.

[0052] Figure 38 It is the result diagram of the detection of the activity of promoting IFN-γ secretion by mouse spleen cells before and after uPA enzyme cleavage of the LIC430 fusion protein.

[0053] Figure 39 It is the protein A affinity chromatography purification diagram of the LIC433 fusion protein.

[0054] Figure 40It is a graph showing the in vitro activity assay of LIC433 and control fusion proteins.

[0055] Figure 41 It is a graph showing the protein A affinity chromatography purification of LIC436 fusion protein.

[0056] Figure 42 It is a graph showing the protein A affinity chromatography purification of LIC444 fusion protein.

[0057] Figure 43 It is a graph showing the results of detecting the activity of LIC436 and LIC444 fusion proteins in promoting mouse spleen cells to secrete IFN-γ.

[0058] Figure 44 It is a graph showing the results of in vitro stimulation of IFN-γ secretion by LIC438 and LIC441 fusion proteins.

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

[0060] Figure 46 It is a graph showing the results of in vitro stimulation of human PBMC by LIC445 fusion protein to secrete IFN-γ.

[0061] Figure 47 It is a graph showing the comparison results of the shielding effects of fusion proteins with different structures in Example 37 of the present invention.

[0062] Figure 48 It is a graph showing the changes in the body weights of mice after administration of fusion proteins with different structures in Example 38 of the present invention. Detailed implementation manners

[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 construed as limiting the present invention.

[0064] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0065] Detailed description of the present invention

[0066] Definitions and general terms

[0067] It should be noted that the endpoints and any values within the ranges disclosed in this document are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document. Where specific techniques or conditions are not indicated in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. Where the manufacturers of the reagents or instruments used are not indicated, they are all conventional products that can be obtained through commercial purchases.

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

[0069] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0070] In this document, the terms "optionally", "optional", or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

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

[0072] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used in this document have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0073] As used herein, the term "fusion protein" refers to a novel protein formed by the fusion of at least two proteins or polypeptides (such as a bioactive polypeptide and an effector molecule). Generally, such fusion can be achieved by techniques such as genetic engineering, chemical methods, or other appropriate methods. For example, the expression product after recombination of two genes obtained 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 multimer (such as a dimer). As used herein, it means that the first structural unit, the second structural unit and the third structural unit containing the IL-12 cytokine or its functional fragment are in a fused state.

[0074] The fusion protein described in the present invention is generally prepared by a biosynthesis method. According to the nucleotide sequence described in the present invention, those skilled in the art can conveniently obtain the encoding nucleic acid of the present invention by various known methods. These methods include, for example, but are not limited to: PCR, DNA artificial synthesis, etc. For specific methods, reference can be made to J. Sambrook, "Molecular Cloning: A Laboratory Manual". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by segmentally synthesizing the nucleotide sequence and then performing overlap extension PCR.

[0075] As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a light chain (L chain) and a heavy chain (H chain). Generally speaking, the heavy chain can be understood as the polypeptide chain with a larger molecular weight in the antibody, and the light chain is the polypeptide chain with a smaller molecular weight in the antibody. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The VH and VL regions can also be further divided into regions with high variability (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 by any specific method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as 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 an 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 that comprises a part or all of a full-length antibody and retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an antigen-binding portion. For example, the fragment may comprise a part or all of the antibody CDRs, and such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules, including the intact antibody, for binding to a given epitope. Antigen-binding fragments of an antibody can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. In some embodiments of the present invention, the antigen-binding fragment of the antibody is Fab, Fab’, (Fab’)2, Fv, scFv or VHH, or in the forms of Fab, Fab’, (Fab’)2, Fv, scFv, VHH and scFv, etc. 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 linked together by a disulfide bond. In the present invention, "F(ab’)2" is an antibody fragment with a molecular weight of approximately 100,000 Da obtained by digesting the lower part of the two disulfide bonds in the hinge region of an antibody molecule with pepsin and comprising two Fab regions linked at the hinge position. In the present invention, "Fab’" is an antibody fragment obtained by cleaving the disulfide bond in the hinge region of the above F(ab')2. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds an antigen with a reducing agent such as dithiothreitol. In the present invention, "Fv" is the smallest functional fragment in an antibody molecule that retains the antigen-binding site, consisting of the variable region of the light chain and the variable region of the heavy chain, which are bound together by non-covalent bonds. In the present invention, "scFv" refers to an antibody fragment that comprises the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an antibody. The size of scFv is generally 1 / 6 of that of a full-length antibody and preferably is an amino acid sequence of a single chain encoded by a single nucleotide chain. In the present invention, "VHH" is the single variable region of a heavy-chain antibody and has the ability to bind an antigen.

[0077] As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are all composed of at least two identical light chains and at least two identical heavy chains linked 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 text, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replicating after being inserted into a suitable host, which contains a nucleotide sequence capable of expressing a target protein and can transfer the nucleic acid molecule into host cells and / or between host cells. The expression vector may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for the transcription and / or translation of DNA or RNA. The expression vector also includes vectors having 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, by culturing a suitable host cell containing the expression vector, the expression vector can produce a desired expression product.

[0079] In this text, the term "recombinant cell" generally refers to a cell obtained by modifying or recombining the genetic material of a recipient cell (or host cell) using genetic engineering techniques or cell fusion techniques, and having unique traits with stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acid (such as 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] In this text, the term "pharmaceutical composition" generally refers to a drug in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining an active ingredient with a carrier constituting one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active fusion protein with a liquid carrier, a finely divided solid carrier, or both.

[0081] In this text, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. Except for the range where any conventional excipient is incompatible with the fusion protein of the present invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, their uses are also within the scope contemplated by the present invention.

[0082] As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by a suitable method. The fusion protein or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present invention is not limited to the exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection or subcutaneous injection.

[0083] As used herein, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic 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 adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, e.g., arresting the development of the disease; or (c) alleviating the disease, e.g., relieving symptoms associated with the disease. "Treatment" as used herein covers any administration of a fusion protein or pharmaceutical composition to an individual to treat, cure, relieve, improve, alleviate or inhibit a disease in the individual, including but not limited to administering a medicament containing the fusion protein described herein to an individual in need thereof. Detailed description of the cytokine fusion protein of the present invention and its uses

[0084] The present invention provides a fusion protein, nucleic acid molecule, expression vector, recombinant cell, fusion protein complex, immunotherapeutic cell and pharmaceutical composition containing the IL-12 cytokine and their uses in the preparation of medicaments for treating or preventing cancer, infectious diseases or autoimmune diseases, which will be described in detail below, respectively.

[0085] Fusion protein

[0086] In a first aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, 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, and the first antibody or an antigen-binding fragment thereof has tumor antigen or immune checkpoint binding activity; the second structural unit is selected from a cleavable linker, or an uncleavable 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, one or more of the third structural units are connected to the first structural unit, 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 on the IL-12 cytokine in the third structural unit. In particular, without the traditional affinity shielding strategy, the fusion protein does not need to contain any antibody or receptor of the IL-12 cytokine, and can reduce the toxic and side effects caused by the IL-12 cytokine. The fusion protein has the advantages of strong safety and the like.

[0087] 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 low biological activity. After the cleavable linker of the second structural unit is cleaved, the N-terminus or the N-terminus and C-terminus of the third structural unit are released, restoring the affinity for the receptor, and the biological activity is restored to a higher level, thereby reducing its toxic and side effects on non-target tissues while improving the therapeutic effect on the target.

[0088] In this article, the term "X1 is connected to the C-terminus of X3 through X2" means that the sequence is X3-X2-X1 from the N-terminus to the C-terminus. For example, "the third structural unit is connected to the C-terminus of the first structural unit through the second structural unit" means that the sequence is the first structural unit - the second structural unit - the third structural unit from the N-terminus to the C-terminus; the term "X1 is connected to the N-terminus of X3 through X2" means that the sequence is X1-X2-X3 from the N-terminus to the C-terminus. For example, "the third structural unit is connected to the N-terminus of the first structural unit through the second structural unit" means that the sequence is the third structural unit - the second structural unit - the first structural unit from the N-terminus to the C-terminus.

[0089] 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 mouse IL-12 cytokine or a human IL-12 cytokine. All structures of the present invention are applicable to mouse or human IL-12 cytokines, that is, the IL-12 cytokine is at least one of mouse or human.

[0090] 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.

[0091] 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 through the non-cleavable linker (cytokine C-terminus) of the second structural unit. Figure 1 As shown in LIC401, LIC410, and LIC415, from N-terminus to C-terminus, they include: A chain: P35 subunit, non-cleavable linker (cytokine C-terminus), antibody Knob sequence; B chain: P40 subunit, non-cleavable linker (cytokine C-terminus), antibody Hole sequence; wherein the targeting of the two antibody chains may be the same or different. In certain embodiments, different subunits of cytokines fused with Fc-Knob or Fc-Hole can be replaced with each other without affecting their structure and function. For example, the Fc-Knob of LIC401 is connected to the IL-12P35 subunit through a flexible linker, and the Fc-Hole is connected to the IL-12P40 subunit through a flexible linker. It is replaced by Fc-Knob connected to the IL-12P40 subunit through a flexible linker, and Fc-Hole connected to the IL-12P35 subunit through a flexible linker, and the activity of IL-12 remains unchanged. The present invention found that IL-12 located at the N-terminus of the fusion protein has a stronger activity and can stimulate the spleen of mice to enlarge significantly after being injected into the mice. Therefore, LIC401 can be used as a positive control molecule.

[0092] 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 non-cleavable linker (cytokine N-terminus) of the second structural unit. Figure 1In LIC402, LIC406, LIC411, LIC416, the fusion protein sequentially includes from the N-terminus to the C-terminus: Chain A: antibody Knob sequence, non-cleavable linker (N-terminus of cytokine), P35 subunit; Chain B: antibody Hole sequence, non-cleavable linker (N-terminus of cytokine), P40 subunit; wherein the targets of the two antibody chains can be the same or different. In some other embodiments, the fusion protein structure is as Figure 1 shown in LIC428, LIC436, LIC442, LIC443, LIC444, the fusion protein sequentially includes from the N-terminus to the C-terminus: antibody, non-cleavable linker (N-terminus of cytokine), P35 subunit - P40 subunit of IL-12; wherein, the fusion order of the P35 subunit - P40 subunit can be that the P35 subunit is at the N-terminus and the P40 subunit is at the C-terminus, or the P40 subunit is at the N-terminus and the P35 subunit is 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 only to the C-terminus of one Fc chain. It was found in this application that, compared with the fusion protein with IL-12 at the N-terminus, the activity of IL-12 at the C-terminus of the fusion protein is significantly lower. Secondly, the heterologous IL-12 form (such as LIC406) at the C-terminus has weaker activity than the homologous IL-12 form (such as LIC428).

[0093] According to an embodiment of the present invention, when one of the third structural units is connected to the first structural unit, the second structural unit is a cleavable linker.

[0094] According to an embodiment of the present invention, when multiple third structural units are connected to the first structural unit, at least one of the second structural units connected to the multiple third structural units contains a cleavable linker, preferably all of the second structural units connected to the multiple third structural units are cleavable linkers.

[0095] According to an embodiment of the present invention, when multiple third structural units are connected to the first structural unit, all of the second structural units are cleavable linkers.

[0096] 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 a second structural unit cleavable linker, and the fusion protein structure is as Figure 1 shown in LIC403, LIC407, LIC412, LIC417, the fusion protein sequentially includes from the N-terminus to the C-terminus: Chain A: antibody Knob sequence, cleavable linker, P35 subunit; Chain B: antibody Hole sequence, cleavable linker, P40 subunit; wherein the targets of the two antibody chains can be the same or different. In some other embodiments, the structure of the fusion protein is as Figure 1As shown in LIC445, the fusion protein sequentially includes, from the N-terminus to the C-terminus: an antibody, a cleavable linker, and an IL-12P35 subunit-P40 subunit fusion protein; wherein, the fusion order of the P35 subunit and the P40 subunit can be that the P35 subunit is at the N-terminus and the P40 subunit is at the C-terminus, or the P40 subunit is at the N-terminus and the P35 subunit is at the C-terminus; wherein, the targets of the two antibody chains can be the same or different. It is found in this application that the cleavable fusion protein with IL-12 at the C-terminus shows different activities before and after cleavage and has better safety.

[0097] According to an embodiment of the present invention, when a plurality of the third structural units are connected to the first structural unit, at least one of the plurality of the second structural units contains a cleavable linker.

[0098] 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 a non-cleavable linker (N-terminus of the cytokine) of the second structural unit. The first structural unit of the other chain is connected to the N-terminus of the third structural unit through a cleavable linker of the second structural unit. The structure of the fusion protein is as Figure 1 As shown in LIC404, LIC405, LIC408, LIC409, LIC413, LIC414, LIC418, LIC419, the fusion protein sequentially includes, from the N-terminus to the 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 antibody chains can be the same or different. It is found in this application that compared with the non-cleavable fusion protein with IL-12 at the C-terminus, the single-chain cleavable fusion protein with IL-12 at the C-terminus has stronger stimulating activity on lymphocytes after cleavage.

[0099] According to an embodiment of the present invention, the fusion protein does not include an antibody against the IL-12 cytokine or its antigen-binding fragment.

[0100] According to an embodiment of the present invention, the first antibody or its antigen-binding fragment includes a first full-length antibody, a first Fab fragment, a first ScFv fragment, or a first VHH fragment selected from the group.

[0101] 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, VISTA.

[0102] In the present invention, "EGFR" is the epidermal growth factor receptor. Its mutation or overexpression generally triggers tumors. After EGFR dimerization, it activates the downstream signaling pathway of cells and is related to the proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis of tumor cells. Antibodies or antigen-binding fragments that bind to EGFR can inhibit the activity of EGFR, prevent its phosphorylation, and then inhibit the activation of the downstream signaling pathway, achieving an anti-tumor effect.

[0103] In the present invention, "VEGF" is vascular endothelial growth factor, which can act on endothelial cells through a paracrine mechanism and plays an important role in promoting angiogenesis, inhibiting apoptosis of endothelial cells, and increasing vascular permeability. VEGF is overexpressed in almost 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 located on endothelial cells, thereby making VEGF lose its biological activity and reducing tumor angiogenesis, thus inhibiting tumor growth.

[0104] In the present invention, "Claudin 18.2" is the 18.2 isoform molecule of the Claudin family of tight junction proteins, which is a highly specific cell surface molecule that is only expressed on differentiated gastric mucosal epithelial cells in normal tissues and is highly expressed in solid tumors such as gastric cancer, pancreas, ovary, cholangiocarcinoma, and lung adenocarcinoma. Antibodies that bind to Claudin 18.2 can specifically recognize tumor cells, trigger antibody-dependent cell cytotoxicity (ADCC), complement-dependent cell cytotoxicity (CDC), apoptosis, and inhibit cell proliferation, and have a powerful ability to eliminate cancer cells and control the disease.

[0105] In the present invention, "PD-L1" is programmed death receptor-ligand 1, which is an immune checkpoint protein. Tumor cells overexpress PD-L1, continuously activating the PD-1 / PD-L1 signaling pathway to cause various immunosuppressions. Antibodies or antigen-binding fragments that bind to PD-L1, as blockers of immune checkpoint proteins, can inhibit the PD-1 / PD-L1 pathway, block the co-inhibitory function of CD80 and PD-L1, and are beneficial to fully activating T cell function and promoting cytokine production.

[0106] 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 surfaces of T cells, B cells, monocytes, and natural killer cells.

[0107] In the present invention, "TIGIT" refers to T cell immunoglobulin and ITIM domain-containing protein. Abnormal expression of TIGIT 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, immune cell functional exhaustion can be reversed, and an anti-tumor effect can be exerted. It is a new generation of immunotherapy target.

[0108] In the present invention, "LAG3" refers to lymphocyte activation gene-3, "TIM-3" refers to T cell immunoglobulin and mucin domain molecule 3, and "CTLA-4" refers to cytotoxic T lymphocyte-associated antigen-4. All three are involved in T cell immune regulation as immune inhibitory receptors.

[0109] According to the embodiments of the present invention, the IL-12 cytokine or its functional fragment includes at least one of the P35 fragment of IL-12 or its functional fragment and the P40 fragment of IL-12 or its functional fragment.

[0110] 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.

[0111] According to the embodiments of the present invention, the IL-12 cytokine or its functional fragment has an amino acid sequence as shown in any one of SEQ ID NO: 1-2 and 70-71.

[0112] According to the embodiments of the present invention, the IL-12 cytokine or its functional fragment 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. The third structural unit further includes an uncleavable linker peptide, and the two subunit fragments are connected by the linker peptide.

[0113] According to the embodiments of the present invention, the IL-12 cytokine or its functional fragment 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. The C-terminus of one subunit fragment is connected to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is connected to the N-terminus of the other subunit fragment.

[0114] According to an embodiment of the present invention, the linker 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.

[0115] In an alternative embodiment of the present invention, n is 2, 3, 4, 5, 6, 7, or 8.

[0116] According to an embodiment of the present invention, the linker peptide has an amino acid length of 1-60, such as 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 a range value between any two of these point values as the end point values.

[0117] According to an embodiment of the present invention, the linker peptide has an amino acid sequence as shown by (GGGGS)n, where n is 3, 4, 5, or 6.

[0118] According to an embodiment of the present invention, the linker peptide has an amino acid sequence as shown by any one of SEQ ID NO:75.

[0119] According to an embodiment of the present invention, the third structural unit has an amino acid sequence as shown by SEQ ID NO:1, 2, 70, 71, 76, or 77.

[0120] In an alternative embodiment of the present invention, the second structural unit is a cleavable linker.

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

[0122] In an alternative embodiment of the present invention, the fusion protein includes two monomers, and the two second structural units are a cleavable linker and a non-cleavable linker, respectively. In an alternative embodiment of the present invention, the fusion protein includes two monomers, and both of the two second structural units are cleavable linkers. In an alternative embodiment of the present invention, the fusion protein includes two monomers, and both of the two second structural units are non-cleavable linkers.

[0123] According to an embodiment of the present invention, the proteases expressed in the tumor tissue include matrix metalloproteinases, serine proteases, or asparaginyl endopeptidases.

[0124] According to an embodiment of the present invention, the cleavable linker includes a protease substrate sequence and optionally a flexible peptide segment.

[0125] 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.

[0126] According to an embodiment of the present invention, the amino acid length of the flexible peptide segment is 1 to 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 a range value between any two of these point values as endpoint values, such as 5 to 30, 5 to 25, or 5 to 20.

[0127] In some embodiments of the present invention, the second structural unit of the present application includes a peptide linker cleavable by matrix metalloproteinase. Peptide linkers cleavable by matrix metalloproteinase known in the art, such as the peptide linker disclosed in WO2019010219A, can be used in the present application, and the above-mentioned document is hereby incorporated herein by reference. In some embodiments, the amino acid sequence of the second structural unit includes the substrate QLLGFLTA of matrix metalloproteinases MMP-2 / 9 / 14.

[0128] In some embodiments of the present invention, the second structural unit of the present application includes a peptide linker cleavable by serine protease, such as urokinase and trypsin. Peptide linkers cleavable by 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.

[0129] In some embodiments of the present invention, the second structural unit of the present application includes a peptide linker cleavable by asparaginyl endopeptidase. Peptide linkers cleavable by asparaginyl 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 asparaginyl endopeptidase substrate AANL.

[0130] According to an embodiment of the present invention, the protease substrate sequence is selected from QLLGFLTA, LSGRSDNH, GFFY, RQARAVGG, or AANL.

[0131] 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.

[0132] According to an embodiment of the present invention, the cleavable linker has an amino acid length of 15 to 60, such as 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 a range value between any two of these point values as the end point values, for example, 15 to 50, 15 to 45 or 15 to 40.

[0133] In an alternative 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 (such as 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 alternative embodiment of the present invention, the amino acid sequence of the flexible peptide segment is GSSGG, GSSGGSGGSGGSG, GSSGGSGGSGGS, SSGGSGGSGGSG or GSSGGSGGSGGSGSSGGSGG.

[0134] 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.

[0135] 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.

[0136] According to an embodiment of the present invention, the non - cleavable linker has an amino acid length of 1 to 60, such as 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 a range value between any two of these point values as the end point values, for example, 5 to 50, 5 to 40, 5 to 30 or 10 to 20.

[0137] 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.

[0138] According to an embodiment of the present invention, the non-cleavable linker has the amino acid sequence shown in SEQ ID NO: 5 or 6.

[0139] 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.

[0140] 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.

[0141] According to an embodiment of the present invention, the first structural unit is a first Fc fragment or a first VHH fragment, one or two of the third structural units are connected to the first Fc fragment or the first VHH fragment, and each of the third structural units is connected to the first Fc fragment or the first VHH fragment through the second structural unit.

[0142] 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, 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.

[0143] 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.

[0144] According to an embodiment of the present invention, when one of the third structural units is connected to the first Fc fragment or the first VHH fragment, 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.

[0145] According to an embodiment of the present invention, when two of the third structural units are connected to the first Fc fragment or the first VHH fragment, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker.

[0146] 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.

[0147] According to an embodiment of the present invention, when two third structural units are linked to the first Fc fragment or the first VHH fragment, the IL-12 cytokine or its functional fragment in the two third structural units may be the same or different.

[0148] According to an embodiment of the present invention, the IL-12 cytokine or its functional fragment 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.

[0149] 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 the first Fc fragment, the IL-12 cytokine or its functional fragment of 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; the two subunit fragments are respectively linked to two fragments of the first Fc fragment.

[0150] According to an embodiment of the present invention, the first structural unit is a first full-length antibody, and the first full-length antibody is linked to one or more third structural units, and each third structural unit is linked to the first full-length antibody through the second structural unit.

[0151] According to an embodiment of the present invention, the first full-length antibody is linked to one third structural unit, and the third structural unit is linked to the C-terminus of the heavy chain in 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 linked to multiple third structural units, and multiple third structural units are respectively linked to the C-terminus of the heavy chain in 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 linked to two third structural units, and the two third structural units are respectively linked to the C-termini of two heavy chains in the first full-length antibody through the second structural unit.

[0154] According to an embodiment of the present invention, 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 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.

[0155] According to an embodiment of the present invention, when the first full-length antibody is linked to a plurality of the third structural units, the plurality of second structural units are each independently selected from a cleavable linker or a non-cleavable linker, preferably all are cleavable linkers.

[0156] According to an embodiment of the present invention, when the first full-length antibody is linked 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.

[0157] According to an embodiment of the present invention, the two second structural units are both cleavable linkers, both non-cleavable linkers, or one is a cleavable linker and the other is a non-cleavable linker.

[0158] According to an embodiment of the present invention, when the first full-length antibody is linked to two of the third structural units and the two third structural units are respectively linked to the C-termini of two heavy chains in the first full-length antibody through the second structural units, the IL-12 cytokine or its functional fragment in the two third structural units may be the same or different, preferably the IL-12 cytokine or its functional fragment 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.

[0159] According to an embodiment of the present invention, the first structural unit is a first Fab fragment, and the first Fab fragment is linked to one or two of the third structural units, and each of the third structural units is linked to the C-terminus of the first Fab fragment through the second structural unit.

[0160] According to an embodiment of the present invention, the first Fab fragment is linked to one of the third structural units, and the third structural unit is linked 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.

[0161] According to an embodiment of the present invention, the first Fab fragment is linked to two of the third structural units, and the two third structural units are respectively linked 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.

[0162] According to an embodiment of the present invention, when one third structural unit is linked to the first Fab fragment, 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.

[0163] According to an embodiment of the present invention, when two third structural units are linked to the first Fab fragment, the two second structural units are each independently selected from a cleavable linker or a non-cleavable linker.

[0164] 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.

[0165] According to an embodiment of the present invention, when two third structural units are linked to the full first Fab fragment, the IL-12 cytokine or its functional fragment in the two third structural units may be the same or different, preferably the IL-12 cytokine or its functional fragment 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.

[0166] According to an embodiment of the present invention, when one third structural unit is linked to the first Fab fragment and the third structural unit is linked to the C-terminus of the CH1 fragment in the first Fab fragment through the second structural unit, the fusion protein includes two monomers.

[0167] 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 are respectively the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, and the P35 fragment of IL-12 or its functional fragment and the P40 fragment of IL-12 or its functional fragment are linked by a disulfide bond.

[0168] 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.

[0169] In this article, without special instructions, the Fc fragment (e.g., the first Fc fragment and / or the second Fc fragment) includes CH2, CH3 region and optionally 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 the same or may not be completely the same, for example, to avoid mispairing, an Fc fragment containing a "Knob in Hole" mutation is used.

[0170] 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 the heavy chain biting to form a heterodimer. For example, Fc-Knob means that the CH3 of human IgG1-Fc is selected from S354C and T366W; Fc-Hole means that the CH3 of human IgG1-Fc is selected from T366S, L368A, Y407V, and optionally Y349C. In certain embodiments, different subunits of cytokines fused with Fc-Knob or Fc-Hole can be replaced with each other without affecting their structure and function. For example, LIC401 and LIC401's Fc-Knob are 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. When they are replaced with Fc-Knob connected to the IL-12P40 subunit via a flexible linker, and the Fc-Hole is connected to the IL-12P35 subunit via a flexible linker, the activity of IL-12 remains unchanged.

[0171] 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, so that the fusion protein weakens or even eliminates ADCC and / or CDC functions, thereby reducing nonspecific immune responses.

[0172] 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.

[0173] In an optional embodiment of the present invention, the amino acid sequence of the Fc-Knob fragment is shown as SEQ ID NO:3; the amino acid sequence of the Fc-Hole fragment is shown as SEQ ID NO:4.

[0174] In some embodiments of the present invention, the third structural unit is located at the N-terminus of the fusion protein, such as Figure 1as shown by LIC401, LIC410, and LIC415.

[0175] In some embodiments of the present invention, the third structural unit is located at the C-terminus of the fusion protein, such as Figure 1 shown by LIC402-409, LIC411-414, LIC416-419, LIC428, LIC436, LIC442-445.

[0176] 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, such as Figure 1 shown by LIC420-427, LIC429-435, LIC437-441.

[0177] According to an embodiment of the present invention, the second antibody or its antigen-binding fragment includes a second full-length antibody, a second Fab fragment, a second ScFv fragment, or a second VHH fragment.

[0178] 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, and the fourth structural unit is a second Fc fragment. The third structural unit is located between the first structural unit and the fourth structural unit. That is, the fusion protein sequentially includes a first structural unit, a third structural unit, and a fourth structural unit from the N-terminus to the C-terminus, thereby forming a "sandwich" structure.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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

[0183] According to an embodiment of the present invention, 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. That is, the fusion protein sequentially includes a first structural unit, a second structural unit, a third structural unit, a fifth structural unit, and a fourth structural unit from the N-terminus to the C-terminus, thereby forming a "sandwich" structure.

[0184] In an alternative embodiment of the present invention, in the above "sandwich" structure, for a dimeric fusion protein, at least one second structural unit is a cleavable linker.

[0185] 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 a non-cleavable linker (N-terminus of the cytokine) of the second structural unit, and at the same time, the fourth structural unit is connected to the C-terminus of the third structural unit through a non-cleavable linker (C-terminus of the cytokine) of the fifth structural unit. The fusion protein structure is as Figure 1 shown in LIC420, LIC424, LIC429, LIC432. The fusion protein sequentially includes from the N-terminus to the C-terminus: an antibody or antibody fragment such as Fab, a non-cleavable linker (N-terminus of the cytokine), a fusion protein of the P35 subunit or the P40 subunit or both, a non-cleavable linker (C-terminus of the cytokine), an antibody or antibody fragment such as Fc; the Fab segment can be two or one. The present invention finds that the masking ability of a single Fab at the N-terminus for the activity of IL-12 is stronger than the steric hindrance masking of two Fabs.

[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 through a cleavable linker of the second structural unit, and at the same time, the fourth structural unit is connected to the C-terminus of the third structural unit through a non-cleavable linker (C-terminus of the cytokine) of the fifth structural unit. The fusion protein structure is as Figure 1 shown in LIC421, LIC425, LIC430, LIC433. The fusion protein sequentially includes from the N-terminus to the 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 (C-terminus of the cytokine), an antibody or antibody fragment such as Fc; the Fab segment can be two or one. The present invention finds that after the cleavable linker of the second structural unit is cleaved by an enzyme, the activity of IL12 is greatly enhanced.

[0187] 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 a cleavable linker of the second structural unit, while the fourth structural unit is connected to the C-terminus of the third structural unit through a non-cleavable linker (C-terminus of the cytokine); the first structural unit of the other chain is connected to the N-terminus of the third structural unit through a non-cleavable linker (N-terminus of the cytokine) of the second structural unit, while the second fourth structural unit is connected to the C-terminus of the third structural unit through a non-cleavable linker (C-terminus of the cytokine) of the fifth structural unit. The fusion protein structure is as Figure 1 shown in LIC422, LIC423, LIC426, LIC427, LIC431, LIC434, LIC435. The fusion protein sequentially includes from the N-terminus to the C-terminus: an antibody or antibody fragment such as Fab, a cleavable (non-cleavable) linker, a fusion protein of the P35 subunit or the P40 subunit or both, a non-cleavable linker (C-terminus of the cytokine), and an antibody or antibody fragment such as Fc; where Fab can be two or one. The present invention discovers that after the cleavable linker of the second structural unit is cleaved by an enzyme, the IL12 activity is 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 a fragment of the fourth structural unit through a non-cleavable linker (cytokine subunit tandem) of the second structural unit; the first structural unit of the other chain is connected to the N-terminus of the third structural unit through a non-cleavable linker (N-terminus of the cytokine) of the second structural unit, and the other fragment of the fourth structural unit is connected to the C-terminus of the third structural unit through a non-cleavable linker (C-terminus of the cytokine) of the fifth structural unit. The fusion protein structure is as Figure 1 shown in LIC437, LIC438, LIC439, LIC440, LIC441. The fusion protein sequentially includes from the N-terminus to the C-terminus: Chain A: an antibody or antibody fragment, a non-cleavable linker (cytokine subunit tandem), an antibody or antibody fragment; Chain B: an antibody or antibody fragment, a non-cleavable linker (N-terminus of the cytokine subunit), a P35 subunit-P40 subunit fusion protein or a P40 subunit-P35 subunit fusion protein, a non-cleavable linker (C-terminus of the cytokine), and an antibody or antibody fragment. The targets of the antibody double strands can be the same or different.

[0189] 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, 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, 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.

[0190] 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.

[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 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.

[0192] According to an embodiment of the present invention, the fusion protein comprises:

[0193] a first peptide chain with an amino acid sequence as shown in SEQ ID NO: 23 and a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 24 (i.e., LIC402);

[0194] a first peptide chain with an amino acid sequence as shown in SEQ ID NO: 25 and a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 26 (i.e., LIC403);

[0195] a first peptide chain with an amino acid sequence as shown in SEQ ID NO: 23 and a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 26 (i.e., LIC404);

[0196] a first peptide chain with an amino acid sequence as shown in SEQ ID NO: 25 and a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 24 (i.e., LIC405);

[0197] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, a second peptide chain as shown in SEQ ID NO:27, a third peptide chain as shown in SEQ ID NO:28, and a fourth peptide chain as shown in SEQ ID NO:9 (i.e., LIC406);

[0198] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, a second peptide chain as shown in SEQ ID NO:29, a third peptide chain as shown in SEQ ID NO:30, and a fourth peptide chain as shown in SEQ ID NO:9 (i.e., LIC407);

[0199] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, a second peptide chain as shown in SEQ ID NO:27, a third peptide chain as shown in SEQ ID NO:30, and a fourth peptide chain as shown in SEQ ID NO:9 (i.e., LIC408);

[0200] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, a second peptide chain as shown in SEQ ID NO:29, a third peptide chain as shown in SEQ ID NO:28, and a fourth peptide chain as shown in SEQ ID NO:9 (i.e., LIC409);

[0201] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:33 and a second peptide chain as shown in SEQ ID NO:34 (i.e., LIC411);

[0202] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:35 and a second peptide chain as shown in SEQ ID NO:36 (i.e., LIC412);

[0203] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:33 and a second peptide chain as shown in SEQ ID NO:36 (i.e., LIC413);

[0204] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:35 and a second peptide chain as shown in SEQ ID NO:34 (i.e., LIC414);

[0205] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33 and a second peptide chain (i.e., LIC416) with an amino acid sequence as shown in SEQ ID NO: 38;

[0206] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35 and a second peptide chain (i.e., LIC417) with an amino acid sequence as shown in SEQ ID NO: 39;

[0207] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 33 and a second peptide chain (i.e., LIC418) with an amino acid sequence as shown in SEQ ID NO: 39;

[0208] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 35 and a second peptide chain (i.e., LIC419) with an amino acid sequence as shown in SEQ ID NO: 38;

[0209] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, a second peptide chain as shown in SEQ ID NO: 40, a third peptide chain as shown in SEQ ID NO: 41, and a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC420);

[0210] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, a second peptide chain as shown in SEQ ID NO: 42, a third peptide chain as shown in SEQ ID NO: 43, and a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC421);

[0211] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, a second peptide chain as shown in SEQ ID NO: 40, a third peptide chain as shown in SEQ ID NO: 43, and a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC422);

[0212] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, a second peptide chain as shown in SEQ ID NO: 41, a third peptide chain as shown in SEQ ID NO: 42, and a fourth peptide chain as shown in SEQ ID NO: 9 (i.e., LIC423);

[0213] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 44 and a second peptide chain (i.e., LIC424) with an amino acid sequence as shown in SEQ ID NO: 40;

[0214] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 45 and a second peptide chain (i.e., LIC425) with an amino acid sequence as shown in SEQ ID NO: 43;

[0215] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 44 and a second peptide chain (i.e., LIC426) with an amino acid sequence as shown in SEQ ID NO: 43;

[0216] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 45 and a second peptide chain (i.e., LIC427) with an amino acid sequence as shown in SEQ ID NO: 40;

[0217] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 46 and a second peptide chain (i.e., LIC428) with an amino acid sequence as shown in SEQ ID NO: 9;

[0218] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 47 and a second peptide chain (i.e., LIC429) with an amino acid sequence as shown in SEQ ID NO: 9;

[0219] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 48 and a second peptide chain (i.e., LIC430) with an amino acid sequence as shown in SEQ ID NO: 9;

[0220] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 49, a third peptide chain with an amino acid sequence as shown in SEQ ID NO: 50, and a fourth peptide chain with an amino acid sequence as shown in SEQ ID NO: 9 (i.e., LIC431);

[0221] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 51 and a second peptide chain (i.e., LIC432) with an amino acid sequence as shown in SEQ ID NO: 52;

[0222] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 53 and a second peptide chain (i.e., LIC433) with an amino acid sequence as shown in SEQ ID NO: 50;

[0223] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:53 and a second peptide chain (i.e., LIC434) with an amino acid sequence as shown in SEQ ID NO:52;

[0224] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:51 and a second peptide chain (i.e., LIC435) with an amino acid sequence as shown in SEQ ID NO:50;

[0225] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, a second peptide chain with an amino acid sequence as shown in SEQ ID NO:54, a third peptide chain with an amino acid sequence as shown in SEQ ID NO:12, and a fourth peptide chain with an amino acid sequence as shown in SEQ ID NO:9 (i.e., LIC436);

[0226] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:9, a second peptide chain with an amino acid sequence as shown in SEQ ID NO:55, a third peptide chain with an amino acid sequence as shown in SEQ ID NO:52, and a fourth peptide chain with an amino acid sequence as shown in SEQ ID NO:9 (i.e., LIC437);

[0227] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:56 and a second peptide chain (i.e., LIC438) with an amino acid sequence as shown in SEQ ID NO:57;

[0228] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:56 and a second peptide chain (i.e., LIC439) with an amino acid sequence as shown in SEQ ID NO:58;

[0229] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:59 and a second peptide chain (i.e., LIC440) with an amino acid sequence as shown in SEQ ID NO:60;

[0230] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:59 and a second peptide chain (i.e., LIC441) with an amino acid sequence as shown in SEQ ID NO:61;

[0231] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:62 and a second peptide chain (i.e., LIC442) with an amino acid sequence as shown in SEQ ID NO:63;

[0232] The amino acid sequence has a first peptide chain as shown in SEQ ID NO:62 and a second peptide chain (i.e., LIC443) with an amino acid sequence as shown in SEQ ID NO:64;

[0233] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 66 and a second peptide chain (i.e., LIC403-1) with an amino acid sequence as shown in SEQ ID NO: 65;

[0234] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 81 and a second peptide chain (i.e., LIC403-2) with an amino acid sequence as shown in SEQ ID NO: 82;

[0235] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 67 and a second peptide chain (i.e., LIC425-1) with an amino acid sequence as shown in SEQ ID NO: 68;

[0236] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 9, a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 69, a third peptide chain with an amino acid sequence as shown in SEQ ID NO: 12, and a fourth peptide chain with an amino acid sequence as shown in SEQ ID NO: 9 (i.e., LIC444);

[0237] The amino acid sequence has a first peptide chain as shown in SEQ ID NO: 74 and a second peptide chain (i.e., LIC445) with an amino acid sequence as shown in SEQ ID NO: 73.

[0238] That is to say, the connection order of each structural domain of the fusion protein of the present invention from the N-terminus to the C-terminus is any one selected from the following:

[0239] The third structural unit IL-12 cytokine or its functional fragment, the second structural unit non-cleavable linker (C-terminus of the cytokine), the first structural unit Fc-Knob (Hole) or Fc-Knob (Hole)+ScFv, and the two ScFv targets can be the same or different (such as Figure 1 shown in LIC401, LIC410, LIC415);

[0240] The first structural unit 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 non-cleavable linker (N-terminus of the cytokine), and the third structural unit IL-12 cytokine or its functional fragment (such as Figure 1 shown in LIC402, LIC406, LIC411, LIC416, LIC428, LIC436, LIC442, LIC443);

[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 cleavable linker, and the third structural unit is IL-12 cytokine or its functional fragment (such as Figure 1 shown in LIC403, LIC407, LIC412, LIC417 in

[0242] For one chain, 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 (N-terminus of the cytokine), and the third structural unit is IL-12 cytokine or its functional fragment; for the other chain, 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 cleavable linker, and the third structural unit is IL-12 cytokine or its functional fragment (such as Figure 1 shown in LIC404, LIC405, LIC408, LIC409, LIC413, LIC414, LIC418, LIC419 in

[0243] The first structural unit is Fab fragment (single or two), the second structural unit is a non-cleavable linker (N-terminus of the cytokine), the third structural unit is IL-12 cytokine or its functional fragment, the fifth structural unit is a non-cleavable linker (C-terminus of the cytokine), and the fourth structural unit is the N-terminus of the Fc segment (such as Figure 1 shown in LIC420, LIC424, LIC429, LIC432 in

[0244] The first structural unit is Fab fragment (single or two), the second structural unit is a cleavable linker, the third structural unit is IL-12 cytokine or its functional fragment, the fifth structural unit is a non-cleavable linker (C-terminus of the cytokine), and the fourth structural unit is the N-terminus of the Fc segment (such as Figure 1 shown in LIC421, LIC425, LIC430, LIC433 in

[0245] For one chain, it is Fab or VL and / or VH segment, the second structural unit is a cleavable linker, the third structural unit is IL-12 cytokine or its functional fragment, the second structural unit is a non-cleavable linker (C-terminus of the cytokine), and the first structural unit is the Fc segment; for the other chain, it is Fab or VL and / or VH segment, the second structural unit is a non-cleavable linker (C-terminus of the cytokine), and the third structural unit is IL-12 cytokine or its functional fragment (such as Figure 1LIC422, LIC423, LIC426, LIC427, LIC431, LIC434, LIC435 as shown);

[0246] One chain has a first structural unit of Fab or ScFv, a second structural unit of an uncleavable linker (tandem cytokine subunits), and a fragment of the fourth structural unit Fc; the other chain has a first structural unit of Fab or ScFv, a second structural unit of an uncleavable linker (N-terminus of cytokine), a third structural unit of IL-12 cytokine or its functional fragment, a fifth structural unit of an uncleavable linker (N-terminus of cytokine), and another fragment of the fourth structural unit Fc (such as Figure 1 as shown in LIC437, LIC438, LIC439); the two Fabs or ScFvs can be the same or different.

[0247] One chain has a fragment of the first structural unit Fc segment, a second structural unit of an uncleavable linker (tandem cytokine subunits), and a fourth structural unit of Fab or ScFv; the other chain has another fragment of the first structural unit Fc segment, a second structural unit of an uncleavable linker (N-terminus of cytokine), a third structural unit of IL-12 cytokine or its functional fragment, a fifth structural unit of an uncleavable linker (N-terminus of cytokine), and a fourth structural unit of Fab or ScFv (such as Figure 1 as shown in LIC440, LIC441); the two Fabs or ScFvs can be the same or different.

[0248] The fusion protein of the present invention can be selected from any of the following structures:

[0249] In an alternative 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 respectively, the fusion protein sequentially includes an Fc fragment, a cleavable linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (such as Figure 1 shown in LIC403). The present invention finds that Fc and the linker form steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the masking of the activity of LIC403 before cleavage and relatively low activity, while after cleavage, IL-12 is released, the steric hindrance is removed, and the activity is restored. This significantly improves the safety of LIC403 compared with the IL-12 monomer.

[0250] In an alternative 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. In an alternative embodiment of the present invention, the third structural unit is selected from the Fc fragment. The C-terminus of a 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 a cleavable linker and a non-cleavable linker respectively. Taking the first structural unit as the antibody Fc fragment, one of the second structural units as the cleavable linker and the other as the non-cleavable linker (the N-terminus of the cytokine), and the third structural units as IL-12P35 or IL-12P40 respectively, the fusion protein sequentially includes the Fc fragment, the cleavable (non-cleavable) linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (such as Figure 1 shown in LIC404 and LIC405). The present invention discovers that Fc and the linker form steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC404 and LIC405 being masked and relatively low before cleavage, while after cleavage, the single IL-12 subunit is released, the steric hindrance is partially removed, and the activity is partially restored. Compared with complete cleavage, the release of the single chain can have a longer half-life. This makes the safety of LIC404 and LIC405 significantly improved compared with the IL-12 monomer.

[0251] In an alternative 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 the cleavable linker, and the third structural units as IL-12P35 or IL-12P40 respectively, the fusion protein sequentially includes the full-length antibody, the cleavable linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (such as Figure 1 shown in LIC407). The present invention discovers that the full-length antibody and the linker form steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the activity of LIC407 being masked and relatively low before cleavage, while after cleavage, IL-12 is released, the steric hindrance is removed, and the activity is restored. This makes the safety of LIC407 significantly improved compared with the IL-12 monomer.

[0252] In an alternative 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. In an alternative embodiment of the present invention, the third structural unit is selected from full-length antibodies. The C-terminus of one CH3 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 the other CH3 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 a cleavable linker and a non-cleavable linker respectively. Taking the first structural unit as a tumor antigen-targeting or PD-L1 function-related full-length antibody, one of the second structural units as a cleavable linker and the other as a non-cleavable linker (N-terminus of the cytokine), and the third structural units as IL-12P35 or IL-12P40 respectively, the fusion protein sequentially includes a full-length antibody, a cleavable (non-cleavable) linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (such as Figure 1 shown in LIC408 and LIC409). The present invention discovers 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 masking of the activity of LIC408 and LIC409 before cleavage, which is relatively low. After cleavage, the single IL-12 subunit is released, the steric hindrance is partially removed, and the activity 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 with the IL-12 monomer.

[0253] In an alternative manner, the C-terminus of the fourth structural unit is connected to the N-terminus of the third structural unit, 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 alternative embodiment of the present invention, the third structural unit is selected from Fc fragments. In one chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the third structural unit, 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 the other chain, the C-terminus of the other CH3 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 both cleavable linkers. Taking the fourth structural unit as scFv, the first structural unit as an antibody Fc fragment, the second structural unit as a cleavable linker, and the third structural units as IL-12P35 or IL-12P40 respectively, the fusion protein sequentially includes scFv, Fc fragment, cleavable linker, IL-12P35 or IL-12P40 from the N-terminus to the C-terminus (such as Figure 1 shown in LIC412). The two scFvs can be different (such as Figure 1As shown in LIC417). The present invention discovers that the antibody scFv+Fc and the linker form steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the masking of the activity of LIC412 before cleavage, which is relatively low, while after cleavage, IL-12 is released, the steric hindrance is removed, and the activity is restored. This significantly improves the safety of LIC412 and LIC417 compared with the IL-12 monomer.

[0254] In an alternative manner, the C-terminus of the fourth structural unit is connected to the N-terminus of the third structural unit, 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 alternative embodiment of the present invention, the third structural unit is selected from the Fc fragment. In one chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the third structural unit, 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 the other chain, the C-terminus of another CH3 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 or a non-cleavable linker. Taking the fourth structural unit as scFv, the first structural unit as the antibody Fc fragment, the cleavable (non-cleavable) linker, IL-12P35 or IL-12P40 (such as Figure 1 As shown in LIC413 and LIC414). The two ScFvs can be different (such as Figure 1 As shown in LIC418 and LIC419). The present invention discovers that the antibody ScFv+Fc and the linker form steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the masking of the activity of LIC413, LIC414, LIC418, and LIC419 before cleavage, which is relatively low, while after cleavage, the single IL-12 subunit is released, the steric hindrance is partially removed, and the activity is partially restored. Compared with complete cleavage, the release of a single chain can have more stable targeting and a longer half-life. This significantly improves the targeting and safety of LIC413, LIC414, LIC418, and LIC419 compared with the IL-12 monomer.

[0255] In an alternative embodiment, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 alternative embodiment of the present invention, the third structural unit is selected from the Fc fragment. In one chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 the other chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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; both of the two second structural units are non-cleavable linkers, and both of the two fifth structural units are cleavable linkers. Taking the first structural unit as the Fc fragment, the second structural unit as the non-cleavable linker, the third structural unit as IL-12P35 or IL-12P40 respectively, the fourth structural unit as Fab (one or two), and the fifth structural unit as the cleavable linker as an example, the fusion protein sequentially comprises Fab, cleavable linker, IL-12P35 or IL-12P40, non-cleavable linker (C-terminus of cytokine), Fc (two Fabs as shown in Figure 1 LIC421, and a single Fab as shown in Figure 1 LIC425). The present invention discovers that the two Fabs and the linker form steric hindrance to IL-12, thus affecting the activity of IL-12, resulting in the masking of the activity of LIC421 before cleavage and relatively low activity. After cleavage, IL-12 is released, the steric hindrance is removed, and the activity is restored. The ability of the disulfide bond in a single Fab to mask is stronger than the steric hindrance of two Fabs, which significantly improves the safety of LIC421 and LIC425 compared with the IL-12 monomer.

[0256] In an alternative embodiment, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 alternative embodiment of the present invention, the third structural unit is selected from the Fc fragment. In one chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 the other chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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; the two second structural units are both 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 the Fc fragment, the second structural unit as the non-cleavable linker, the third structural unit as IL-12P35 or IL-12P40 respectively, the fourth structural unit as Fab (one or two), and the fifth structural unit as the cleavable linker as an example, the fusion protein sequentially includes Fab, the cleavable linker, IL-12P35 or IL-12P40, the non-cleavable linker (cytokine C-terminus), and Fc (two Fabs as shown by LIC422 and LIC423 in Figure 1 and a single Fab as shown by LIC426 and LIC427 in Figure 1 ). It is found in this application that the two Fabs and the linker form steric hindrance to IL-12, thus affecting the activity of IL-12. The ability of the disulfide bond in a single Fab to mask is stronger than the steric hindrance of two Fabs, resulting in the masking of the activity of LIC422, LIC423, LIC426, and LIC427 before cleavage, which is relatively low. After cleavage, a single IL-12 subunit is released, the steric hindrance is partially relieved, and the activity is partially 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 LIC422, LIC423, LIC426, and LIC427 compared with the IL-12 monomer.

[0257] In an alternative embodiment, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 alternative embodiment of the present invention, the third structural unit is selected from the Fc fragment. In one chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 the other chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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; both of the two second structural units are non-cleavable linkers, and both of the two fifth structural units are cleavable linkers. Taking the first structural unit as the Fc fragment, the second structural unit as the non-cleavable linker, the third structural unit as IL-12P70, the fourth structural unit as Fab (one or two), and the fifth structural unit as the cleavable linker as an example, the fusion protein sequentially includes Fab, cleavable linker, IL-12P70, non-cleavable linker (cytokine C-terminus), and Fc (two Fabs such as Figure 1 shown in LIC430, and a single Fab such as Figure 1 shown in LIC433). The present invention discovers that Fab and Fc form steric hindrance to IL-12, thereby affecting the activity of IL12, resulting in the masking of the activity of LIC430 before cleavage, which is relatively low, while after cleavage, IL12 is released, the steric hindrance is removed, 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 with the IL12 monomer.

[0258] In an alternative embodiment, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 alternative embodiment of the present invention, the third structural unit is selected from the Fc fragment. In one chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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 the other chain, the C-terminus of the fourth structural unit is connected to the N-terminus of the fifth structural unit, the C-terminus of the fifth structural unit is connected to the N-terminus of the third structural unit, 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; the two second structural units are both 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 the Fc fragment, the second structural unit as the non-cleavable linker, the third structural unit as IL-12P70, the fourth structural unit as Fab (one or two), and the fifth structural unit as the cleavable linker as an example, the fusion protein sequentially includes Fab, the cleavable linker, IL-12P70, the non-cleavable linker (cytokine C-terminus), and Fc (two Fabs such as Figure 1 shown in LIC431, and a single Fab such as Figure 1 shown in LIC434 and LIC435). The present invention discovers 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 and relatively low before cleavage. After cleavage, a single IL-12 subunit is released, and the steric hindrance is partially relieved, and the activity is partially 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 with the IL-12 monomer.

[0259] In an alternative embodiment, 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 an uncleavable linker, the third structural unit as IL-12P70, the fourth structural unit as Fab, ScFv or Fc, and the fifth structural unit as an uncleavable linker as an example, the fusion protein sequentially includes Fab or ScFv or Fc, an uncleavable linker (in series between cell subunits), Fc or Fab or ScFv from the N-terminus to the C-terminus of the A chain; the B chain sequentially includes Fab or ScFv or Fc, an uncleavable linker (at the N-terminus of the cytokine), IL-12P70, Fc or Fab or ScFv (such as Figure 1 shown in LIC437-LIC441). The present application has found that Fab and Fc form steric hindrance to IL-12, thereby affecting the activity of IL-12, resulting in the weakening of the IL-12 activity in LIC437-LIC441, and significantly improving the safety compared with the IL-12 monomer.

[0260] Nucleic acid molecules, expression vectors and recombinant cells

[0261] 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.

[0262] The nucleic acid of the present invention can be a DNA molecule or an RNA molecule, or a nucleic acid analogue. 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 has no size limitation unless otherwise specified. The nucleic acid molecule can also contain a promoter, and the promoter can be homologous or heterologous.

[0263] It should be noted that for the nucleic acid molecules mentioned herein, those skilled in the art should understand that it actually includes any one of the complementary double strands, or both. For convenience, in the present specification and claims, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the nucleic acid sequences in the present invention include DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0264] In a third aspect of the present invention, 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 the above nucleic acid molecule is ligated to the expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the expression vector itself or can be exogenous, that is, not from the expression vector itself. Of course, the nucleic acid molecule is operably linked to the control element.

[0265] As used herein, "operably linked" means that an exogenous gene is linked to an expression vector such that the control elements in the expression vector, such as transcriptional control sequences and translational control sequences, etc., can exert their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, plasmids, cosmids, viruses, phages, and other vectors commonly used in genetic engineering. In some embodiments, these vectors are suitable for transforming cells, eukaryotic cells such as fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a preferred embodiment, these vectors are suitable for stable transformation of bacterial cells, for example, to transcribe the nucleic acid molecule of the present invention.

[0266] According to some specific embodiments of the present invention, after the expression vector is introduced into a suitable recipient cell, under the mediation of a regulatory system, the expression of the fusion protein described in the first aspect can be effectively achieved, and thus a large amount of the fusion protein can be obtained in vitro.

[0267] In one embodiment, the expression vector may contain a marker gene and an origin of replication, a promoter, and a transcription termination signal that ensure replication in the selected host. Preferably, between the promoter and the termination signal, there is at least one restriction site capable of inserting the nucleic acid sequence / molecule to be expressed.

[0268] According to an embodiment of the present invention, the expression vector includes a eukaryotic expression vector or a prokaryotic expression vector selected therefrom.

[0269] In an alternative embodiment of the present invention, the expression vector is a plasmid expression vector, a viral expression vector, such as a lentiviral expression vector. Preferably, the expression vectors of the present application are selected from pET series expression vectors, pGEX series expression vectors, pcDNA series expression vectors, pCMV series expression vectors, and more preferably, the expression vector of the present application is the pMF09 expression vector.

[0270] In the fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment 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 use of this recombinant cell can effectively express the fusion protein described in the first aspect intracellularly.

[0271] According to some specific embodiments of the present invention, the recombinant cell can highly efficiently and abundantly express the fusion protein under suitable conditions. The fusion protein has stronger specificity, a longer half-life, and higher potency, with low toxic and side effects and higher safety.

[0272] It should be noted that "suitable conditions" refer to conditions suitable for the expression of the fusion protein described in the present invention. It is readily understood by those skilled in the art that the conditions suitable for the expression of the fusion protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, a healthy host cell state, a 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 most suitable conditions for the expression of the above-mentioned fusion protein according to the specific laboratory environment.

[0273] 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.

[0274] In one embodiment, the fusion protein is expressed in the recombinant cell, and then the fusion protein is separated and generally purified to a pharmaceutically acceptable purity. For protein expression, the nucleic acid encoding the protein is inserted into an expression vector by standard methods. Expression is carried out in a suitable stable host cell, and the protein is recovered from the cell (supernatant or lysed cell).

[0275] In another embodiment, the nucleic acid molecule of the present invention and / or the vector containing the nucleic acid molecule of the present application can be transduced, transformed or transfected, or otherwise introduced 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 cells of the present application can be human, yeast or fungal cells, such as Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK, ATCC CCL 10), Sertoli cells of baby mice, monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cells (HEK-293), monkey kidney cells (CVI, ATCC CCL-70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL-2), etc. Preferably, the host cell of the present invention is a human HEK293 cell.

[0276] The present invention also provides a method for preparing the fusion protein as described above, comprising the steps of: culturing the host cell of the present application under conditions suitable for expressing the fusion protein, and recovering the fusion protein from the cells or the supernatant of the cell culture. In one embodiment, the method for preparing the fusion protein of the present invention comprises the following steps: constructing an expression vector containing the coding gene of the fusion protein, constructing a host cell containing the expression vector by transient transfection of the host cell, culturing the host cell and collecting the cell supernatant, and purifying the fusion protein by affinity chromatography with Protein A / G as the packing material.

[0277] Suitable conditions for expressing the fusion protein should be known to those skilled in the art, and those skilled in the art can select a suitable culture medium according to experience and culture under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a further period of time. The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.

[0278] Methods for separating and purifying the fusion protein are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants, centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography and various other liquid chromatography techniques and combinations of these methods.

[0279] Fusion protein complex, immunotherapeutic cell and pharmaceutical composition

[0280] In a fifth aspect of the present invention, the present invention provides a fusion protein complex. According to an embodiment of the present invention, the fusion protein complex comprises the fusion protein described in the first aspect. As can be seen from the foregoing, the third structural unit of the fusion protein described in the first aspect contains the IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity on the IL-12 cytokine in the third structural unit. In particular, without the need for traditional affinity shielding strategies, the fusion protein can reduce the toxic and side effects brought by the IL-12 cytokine without containing any antibody or receptor of the IL-12 cytokine, and the fusion protein has advantages such as strong safety. Thus, the fusion protein complex of the present invention has advantages such as strong safety and can be used for treating cancer, infectious diseases or autoimmune diseases.

[0281] One embodiment of the present application relates to that the fusion protein complex can be a dimer protein formed by the binding of two different or identical fusion proteins; that is, the fusion protein complex can be a homodimer or a heterodimer. Each fusion protein in the fusion protein complex can be linked by an interchain bond formed between Fc fragments, preferably covalently linked by a disulfide bond formed between Fc fragments to form a dimer. In some embodiments, the fusion protein complex contains two fusion proteins of the present invention.

[0282] In a sixth aspect of the present invention, the present invention provides an immunotherapeutic cell. According to an embodiment of the present invention, the immunotherapeutic cell expresses the fusion protein described in the first aspect. As can be seen from the foregoing, the third structural unit of the fusion protein described in the first aspect contains the IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve a shielding activity on the IL-12 cytokine in the third structural unit. In particular, without the need for traditional affinity shielding strategies, the fusion protein can reduce the toxic and side effects brought by the IL-12 cytokine without containing any antibody or receptor of the IL-12 cytokine, and the fusion protein has advantages such as strong safety. Thus, the immunotherapeutic cell containing the above-mentioned fusion protein has advantages such as strong safety and can be used for treating cancer, infectious diseases or autoimmune diseases.

[0283] In the seventh aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises 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 immunotherapeutic cell described in the sixth aspect. As can be seen from the foregoing, the third structural unit of the fusion protein described in the first aspect contains the IL-12 cytokine, and the second structural unit mediates the steric hindrance of the first structural unit to achieve the masking activity of the IL-12 cytokine in the third structural unit. In particular, without the traditional affinity masking strategy, the fusion protein can reduce the toxic and side effects brought by the IL-12 cytokine without containing any antibody or receptor of the IL-12 cytokine, and the fusion protein has the advantages of strong safety. Therefore, the pharmaceutical composition containing the above fusion protein has the advantages of strong safety and can be used for treating cancer, infectious diseases or autoimmune diseases.

[0284] In some embodiments, the pharmaceutical composition of the present invention further comprises a monoclonal antibody targeting a tumor antigen or an immune checkpoint, such as antibodies against PD-1, PD-L1, LAG3, TIM-3, CTLA-4, Her2, EGFR, Claudin 18.2, VEGF, CD20, CD33, etc. The combination with the fusion protein of the present invention can play a synergistic anti-tumor effect and improve the disease treatment effect.

[0285] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Examples of suitable pharmaceutical excipients are well known in the art. The pharmaceutical composition containing such a carrier can be formulated by conventional methods well known in the art. In some embodiments, the pharmaceutical composition of the present application may further contain other active ingredients for treatment.

[0286] Use or treatment method

[0287] In the eighth aspect of the present invention, the present invention provides the use of the fusion protein described in the first aspect, the fusion protein complex described in the fifth aspect, the immunotherapeutic 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.

[0288] The above-mentioned fusion protein, fusion protein complex, immunotherapeutic cell, and pharmaceutical composition of the present invention can be administered in different ways, for example, by injecting (such as intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally) the pharmaceutical composition of the present invention. Preferably, the pharmaceutical composition of the present invention is in the form of a freeze-dried preparation or an aqueous solution. The clinical dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any patient depends on many factors, including the patient's physique, body surface area, age, the drug to be administered, gender, administration time and route, general health, and other drugs administered simultaneously. The fusion protein, fusion protein complex, immunotherapeutic cell, and 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, immunotherapeutic cell, and pharmaceutical composition of the present invention can also be directly administered to the target site, for example, by means of targeted drug delivery to internal or external target sites.

[0289] Among them, cancers include but are not limited to leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, 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 (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma-like tumor, pineal adenoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Infectious diseases include but are not limited to smallpox virus infection, HIV infection, bacterial infection, fungal infection, 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.

[0290] 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, which helps to improve the survival and activity of the cells at the tumor site, thereby improving the therapeutic effect during cell therapy for diseases.

[0291] In the eighth aspect of the present invention, 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 includes: administering a pharmaceutically acceptable dose of the fusion protein according to the first aspect, the fusion protein complex according to the fifth aspect, the immunotherapy cells according to the sixth aspect, or the pharmaceutical composition according to the seventh aspect to a subject.

[0292] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not indicated by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0293] Example 1: Preparation of IL-12-incleavable-Fc (LIC401) fusion protein

[0294] 1. Construction of an expression vector encoding the LIC401 fusion protein

[0295] 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 techniques. As Figure 1 shown, IL-12 in the LIC401 fusion protein is located at the N-terminus and is connected to Fc through an uncleavable linker. IL-12 contains the P35 subunit and the P40 subunit.

[0296] The obtained nucleic acid sequence was inserted between the Hind III and BamH I restriction sites of the pMF09 plasmid by homologous recombination. The homologous recombination product was transformed into the DH5α strain to obtain the expression vector pMF09 / LIC401. The strain with correct vector construction was verified by sequencing. After culturing to the logarithmic growth phase, glycerol with a final concentration of 20% was added and stored at -80 °C for long term.

[0297] 2. Plasmid amplification

[0298] Expand the culture volume of the bacteria expressing the expression vector pMF09 / LIC401 at an inoculation amount of 1:100 times, and use the E.Z.N.A Endo-free Plasmid Maxi Kit (product number: D6926-03) provided by OMEGA company to extract the plasmid by alkaline lysis method. After the plasmid is filtered through a sterile filter membrane, it is stored at -20 °C.

[0299] 3. Cell culture and transient transfection to express LIC401 fusion protein

[0300] 1) Cell preparation

[0301] Suspend and culture human HEK293E cells. The culture medium formulation is a 1:1 volume mixture of SFM4HEK293 Medium (Hyclone, USA) and Freestyle 293 Expression Medium (Invitrogen, USA), and then add fetal bovine serum with a final concentration of 2%. The suspension is passaged into fresh medium at a density of 1×10 6 / mL and suspended and cultured in a constant temperature shaker at 37 °C (5% CO2) at a rotation speed of 125 rpm.

[0302] One day before transfection, passage the cells into a 500 mL conical culture flask, and adjust the cell state and density to 4×10 6 / mL, and the culture volume is 200 mL.

[0303] When the cell density reaches 6×10 6 / mL and the cell viability is above 95%, perform transfection; take 100 mL of the culture solution, centrifuge to collect the cells, and wash them once with Freestyle293 medium. Resuspend the cells with 100 mL of Freestyle293 medium;

[0304] 2) Plasmid dilution

[0305] Take the plasmid at a mass of 0.5 μg plasmid / 10 6 cells, dilute it with Freestyle293 medium to 40 ng / μL, and mix well.

[0306]

[0307] 3) PEI encapsulation of plasmid

[0308] The mass ratio of plasmid:PEI is 1:5. Then take 1.5 mL of PEI (polyethyleneimine, transfection reagent, purchased from Polysciences company), add it to the diluted plasmid, mix well, and let it stand and encapsulate for 10 - 15 min. Control the time, and the mixture will become turbid.

[0309] 4) PEI transfection

[0310] Add the plasmid wrapped with PEI to the cells and place them in a shaker for culture.

[0311] 5) Rehydrate after 4 h

[0312] After 4 h, add an equal volume of 100 mL SFM4HEK293 medium, 1 mL VPA (200X, 750 mmol / L, final concentration 3.75 mmol / L), and 400 μL G418 (50 mg / mL).

[0313] 6) Rehydrate after 24 h

[0314] Add 200 μL anti-clumping (1000X) and 5.0 mL 20% TN1 (final concentration 0.5%).

[0315] 7) Monitor the change in cell viability. After culturing for 6 - 7 days until the cell viability drops below 50%, collect the cell culture supernatant for the next purification step.

[0316] 4. Purification of the LIC401 fusion protein

[0317] 1) Sample preparation: Centrifuge the cell suspension at 7000 rpm for 30 minutes, discard the precipitate, and filter the supernatant through a 0.45 μm filter membrane for later use.

[0318] 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.

[0319] 3) Use the peristaltic pump of the AKTA start protein purifier to load the sample. The flow rate is set according to the column volume, generally not exceeding 1 column volume per minute, and collect the flow-through.

[0320] 4) After the loading is completed, equilibrate the protein A column again with 10 column volumes of loading buffer.

[0321] 5) Elute with the elution buffer (citric acid - sodium citrate buffer, pH = 2.8). Collect the eluate in separate tubes, collect one tube per 1 mL, and observe the elution peak according to the UV absorption at 280 nm; Add an appropriate amount of 1 M Tris-HCl with pH 9.0 to the collected tubes of the elution peak to adjust the pH of the target protein solution to around 7.0 - 8.0; The protein A affinity chromatography purification diagram of the fusion protein LIC401 is as Figure 2 shown. It can be seen that there is a UV absorption peak exceeding 1000 mAU, indicating that the target protein is eluted by the elution buffer.

[0322] 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 in sequence, and finally submerge the column packing completely with ethanol;

[0323] 7) Detect the purity of each elution tube by SDS-PAGE, combine each elution tube, dissolve the protein in PBS after ultrafiltration and buffer replacement operations, and wait for the next step; The SDS-PAGE electrophoresis pattern of the fusion protein LIC401 after affinity chromatography purification is as Figure 3 shown. After affinity chromatography, the target protein is obtained. The molecular weight of the sample without reduction treatment is between 100 kDa and 150 kDa, and the molecular weight of the single chain of the sample after reduction treatment is between 45 kDa and 75 kDa. There are two bands, which are polypeptide chains with the amino acid sequences shown in SEQ ID NO:21 and SEQ ID NO:22 respectively.

[0324] 5. Verification of the expression of the LIC401 fusion protein by Western Blot

[0325] 1) Take an appropriate amount of the LIC401 fusion protein purified by affinity chromatography for SDS-PAGE. Electrophoresis conditions: 90 V, 20 min; 100 V, 70 min;

[0326] 2) Transfer the target protein to the PVDF membrane by wet transfer method. Transfer membrane conditions: 200 mA, 90 min;

[0327] 3) After the transfer is completed, block the PVDF membrane with 5% skim milk powder at room temperature for 2 h or at 4 °C overnight;

[0328] 4) Select goat anti-human heavy chain and light chain-HRP antibodies directly as the secondary antibody to verify the expression of the antibody fragment in the LIC401 fusion protein.

[0329] Incubation time: 1 h at room temperature;

[0330] 5) After washing the membrane and developing the color by ECL method, the results as shown in Figure 4 are obtained, which prove that the band of the LIC401 fusion protein without reduction treatment is at 100 - 150 kDa, and the molecular weights of the two chains of the fusion protein after reduction treatment are between 45 kDa and 75 kDa, which is consistent with the SDS-PAGE situation of the previous protein purification. And the binding of the secondary antibody to the antibody fragment of the LIC401 fusion protein in Western Blot determines the expression of this part.

[0331] Example 2: Preparation of Fc-non-cleavable-IL-12 (LIC402, LIC402-1) fusion protein

[0332] The LIC402 and LIC402-1 fusion proteins were prepared by the same method as in Example 1, in which DNA fragments encoding the amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 (LIC402), and the amino acid sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87 (LIC402-1) were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. As Figure 1 shown, the Fc fragment in the LIC402 fusion protein is located at the N-terminus and is linked to IL-12 by a non-cleavable linker. IL-12 consists of a P35 subunit and a P40 subunit. The Protein A affinity chromatography elution peak and Western Blot of the prepared fusion protein are shown in Figure 5 and 6 respectively. An ultraviolet absorption peak of more than 1000 mAU can be seen, indicating that the target protein was eluted by the elution buffer. The molecular weight of the reduced fusion protein is between 45 kDa and 75 kDa, showing two bands.

[0333] Example 3: Determination of the spleen-stimulating activity of LIC401 and LIC402 fusion proteins

[0334] Six- to eight-week-old female Balb / c mice were used to evaluate the spleen-stimulating activity of LIC401 prepared in Example 1 and LIC402 prepared in Example 2. The mice were intravenously injected with PBS or LIC401 (0.1 mg / kg) or LIC402 (0.1 mg / kg) on Day 1 / 4 / 7, and the mice were sacrificed on Day 9, and their spleens were isolated, observed and weighed. The results are shown in Figure 7 and 8 respectively. LIC401 significantly stimulated splenomegaly at a dose of 0.1 mg / kg, while the LIC402 fusion protein showed no obvious splenomegaly at a dose of 0.1 mg / kg. This result indicates that IL-12 located at the N-terminus of the fusion protein has strong biological activity, and the activity of IL12 located at the C-terminus of the fusion protein will be masked by the steric hindrance of Fc.

[0335] Example 4: Preparation of Fc-bisplit-IL-12 (LIC403) fusion protein

[0336] The LIC403 fusion protein, LIC403-1 fusion protein, and LIC403-2 fusion protein were prepared using the same method as in Example 1 (the only difference among the three proteins lies in the second structural unit). The DNA fragments were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. After the expression and purification steps, the amino acid sequences of the LIC403 fusion protein were as shown in SEQ ID NO:25 and SEQ ID NO:26, the amino acid sequences of the LIC403-1 fusion protein were as shown in SEQ ID NO:65 and SEQ ID NO:66, and the amino acid sequences of the LIC403-2 fusion protein were as shown in SEQ ID NO:81 and SEQ ID NO:82. As Figure 1 shown, in the LIC403 fusion protein, LIC403-1 fusion protein, and LIC403-2 fusion protein, the Fc fragment is located at the N-terminus and is linked to IL-12 through a cleavable linker. IL-12 contains the P35 subunit and the P40 subunit. It was found that all three fusion proteins were successfully prepared.

[0337] This example exemplarily shows the detection results of the LIC403 fusion protein. The Protein A affinity chromatography elution peak and SDS-PAGE of the prepared LIC403 fusion protein are respectively as Figure 9 、 10 shown. An ultraviolet absorption peak exceeding 1000 mAU can be seen, indicating that the target protein was eluted by the elution buffer. The molecular weight of the reduced fusion protein was between 45 kDa and 75 kDa, showing two bands.

[0338] Example 5: Activity detection and in vivo safety evaluation of LIC401-LIC403

[0339] 1. In vitro activity detection of the LIC403 fusion protein, LIC403-1 fusion protein, and LIC403-2 fusion protein

[0340] The fusion proteins LIC403, LIC403-1, or LIC403-2 prepared in Example 4 were respectively added to an EP tube at a mass ratio of 20:1 with urokinase uPA. After lysing in a 37°C water bath for 12 h, 3-fold serial dilutions were performed to obtain lysed LIC403.

[0341] Isolate the spleens of normal BALB / c mice and prepare single-cell suspensions. Specifically, dislocate the cervical vertebrae of the mice and soak them in an alcohol cup for 5 min; use autoclaved scissors and forceps to dissect the mice and remove the spleens; place 4 ml of sterile RPMI 1640 medium in a 35 mm culture dish; grind the spleens with the piston of a sterile syringe; centrifuge the obtained cell suspension and then resuspend it with the medium (RPMI 1640 + 10% FBS + 1% Pen / Strep + 4 mM Glutamax + 50 mM β-mercaptoethanol).

[0342] Inoculate 4×10 5 spleen cells into each well of a 96-well plate. Then add gradient-diluted LIC403 at different concentrations, lysed LIC403, and control fusion proteins (LIC401 prepared by the method of Example 1 and LIC402 prepared by the method of Example 2), and culture them in a 37 °C, 5% CO2 incubator for 60 hours. Place the 96-well plate in a centrifuge and centrifuge at 300×g for 10 min, collect the cell supernatant, and use a mouse IFNγ AlphaLISA detection kit to measure the production of IFNγ. The results show that the fusion proteins LIC403, LIC403-1, or LIC403-2 all have better safety and biological activity, and can significantly reduce systemic toxic side effects while anti-tumor.

[0343] This example exemplarily shows the detection results of the LIC403 fusion protein. As shown in Figure 11A , the stimulation activity EC 50 of the positive control LIC401 on spleen cells is 3.10 pM, and the negative control LIC402 has almost no stimulation activity. The stimulation activity of unlysed LIC403 is weak, at least 100 times weaker than that of the positive drug. After LIC403 is lysed, its EC 50 is 10.06 pM, almost returning to the level of LIC401. This suggests that when unlysed, the Fc fragment located at the N-terminus can mask the activity of the IL-12 cytokine, and the released IL-12 after linker lysis has high biological activity. Therefore, the LIC403 fusion protein has better safety and biological activity, and can significantly reduce systemic toxic side effects while anti-tumor.

[0344] 2. In vivo activity detection and in vivo safety evaluation of LIC401-LIC403

[0345] Perform the following tests on 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:

[0346] 2.1 Mouse colon cancer cells MC-38 were cultured and resuspended in PBS to 5×10 6 cells / ml. 100 μl of the cell suspension was subcutaneously inoculated into 6-8-week-old female C57BL / 6 mice at a dose of 100 μl per mouse. On the 9th day after tumor inoculation, the average tumor volume of the mice was approximately 75 mm 3 . The mice were randomly and evenly divided into 5 groups: IgG, LIC401-13.3 pmol, LIC402-13.3 pmol, LIC401-66.7 pmol, and LIC402-66.7 pmol, with 10 mice in each group. At the same time, on the 9th, 12th, and 19th days after tumor inoculation, the mice in each group were administered by tail vein injection. The tumor growth and body weight changes of the mice were continuously observed and recorded. The experiment ended when the tumor volume reached 2000 mm 3 .

[0347] The test results are shown in Figure 11B . In the MC38 model, in terms of safety, LIC401 showed strong toxicity. At a dose of 66.7 pmol, the body weight of the mice decreased by 22%, and 4 mice died after the second injection. At a dose of 13.3 pmol, the body weight decreased by about 5%. In the LIC402 group, there was no decrease in the body weight of the mice.

[0348] 2.2 Mouse colon cancer cells MC-38 were cultured and resuspended in PBS to 5×10 6 cells / ml. 100 μl of the cell suspension was subcutaneously inoculated into 6-8-week-old female C57BL / 6 mice at a dose of 100 μl per mouse. On the 9th day after tumor inoculation, the average tumor volume of the mice was approximately 86 mm 3 . The mice were randomly and evenly divided into 5 groups: IgG, LIC401-66.7 pmol, LIC402-66.7 pmol, LIC403-66.7 pmol, and LIC402-1-66.7 pmol, with 6 mice in each group. At the same time, on the 9th, 12th, and 15th days after tumor inoculation, the mice in each group were administered by tail vein injection. The tumor growth and body weight changes of the mice were continuously observed and recorded. When the average tumor volume of the IgG group reached 1000 mm 3 , the mice were sacrificed and the tumor weights were measured.

[0349] The test results are shown in Figure 11C . In the MC38 model, at a dose of 66.7 pmol, the body weight of LIC402-1 decreased by about 6%, while that of LIC403 decreased by only 4%. The tumor inhibition rate of LIC402-1 was 80%, while that of LIC403 was 99%. At a dose of 66.7 pmol, the tumor inhibition rate of LIC402-1 was higher than that of LIC402.

[0350] 2.3 Mouse prostate cancer cells RM-1 were cultured and resuspended in PBS to 5×10 6cells / ml, 100 μl per mouse were subcutaneously inoculated into 6 - 8 week old male C57BL / 6 mice. On the 10th day after tumor inoculation, the average tumor volume of the mice was approximately 100 mm 3 , and they were randomly and evenly divided into 4 groups: IgG, LIC401, LIC402, LIC403, with 10 mice in each group. The first intravenous injection was given via the tail vein, once every three days, for a total of 3 times. The doses of LIC401 - LIC403 were all 13.3 pmol. The tumor growth and body weight changes of the mice were continuously observed and recorded. When the tumor volume reached 2000 mm 3 , it was regarded as the survival endpoint.

[0351] The test results are shown in Figure 11D . In the RM - 1 model, LIC401 and LIC403 had stronger anti - tumor effects, and the tumor inhibition rates were 92% and 93% respectively. In terms of systemic toxicity, LIC401 caused a 5% decrease in the body weight of the mice, while there was no decrease in the body weight of the mice in the LIC402 and LIC403 groups.

[0352] Example 6: Preparation of Fc - single - split - IL - 12 (LIC404) fusion protein

[0353] The LIC404 fusion protein was prepared by the same method as in Example 1. Among them, DNA fragments encoding the amino acid sequences shown in SEQ ID NO:23 and SEQ ID NO:26 were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. As Figure 1 shown, the Fc fragment in the LIC404 fusion protein is located at the N - terminal. One chain is connected to IL - 12P35 through an uncleavable linker, and the other chain is connected to IL - 12P40 through a cleavable linker. The Protein A affinity chromatography elution peak and SDS - PAGE of the prepared fusion protein are shown in Figure 12 , 13 respectively. An ultraviolet absorption peak exceeding 1000 mAU can be seen, indicating that the target protein was eluted by the elution buffer. The molecular weight of the reduced fusion protein is between 45 kDa and 75 kDa, showing two bands.

[0354] Example 7: Preparation of Fc - single - split - IL - 12 (LIC405) fusion protein

[0355] The LIC405 fusion protein was prepared by the same method as in Example 1. Among them, DNA fragments encoding the amino acid sequences shown in SEQ ID NO:25 and SEQ ID NO:24 were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. As Figure 1As shown, the Fc fragment in the LIC405 fusion protein is located at the N-terminus. One chain is linked to IL-12P35 through a cleavable linker, and the other chain is linked to IL-12P40 through a non-cleavable linker. The Protein A affinity chromatography elution peak of the prepared fusion protein is as shown in Figure 14 As shown. An ultraviolet absorption peak exceeding 800 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0356] Example 8: In vitro activity detection of LIC404, LIC405 and control fusion proteins

[0357] The in vitro activities of the LIC404 fusion protein prepared in Example 6 and the LIC405 fusion protein prepared in Example 7 were measured using the same method as in Example 5, and LIC401 was used as a positive control. The results are as shown in Figure 15 As shown, the stimulation activity EC 50 of the positive control LIC401 on splenocytes was 2.14 pM. The stimulation activity of LIC404 without urokinase cleavage was weak, and the EC 50 was greater than 600 pM, at least 200-fold weaker than the positive drug. After single-chain cleavage of LIC404, its activity could be partially restored, and its calculated EC 50 was 55.27 pM. The activities of LIC405 and LIC404 were similar. After the single chain was cleaved by an enzyme, a subunit could be released, increasing the flexibility of the cytokine, and at the same time, the fusion of the Fc segment could be retained, with a significantly extended half-life compared to the IL-12 monomer.

[0358] Example 9: Preparation of PDL1 monoclonal antibody - double cleavage - IL-12 (LIC407) fusion protein

[0359] 1. LIC403 can be fused with antibodies targeting different antigens to obtain more bifunctional proteins, such as PDL1 monoclonal antibodies. The LIC407 fusion protein was prepared using the same method as in Example 1, in which DNA fragments encoding the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:29 and SEQ ID NO:30 were prepared by chemical gene synthesis and PCR technology and plasmids were constructed. As shown in Figure 1 As shown, the PDL1 antibody in the LIC407 fusion protein is located at the N-terminus and is linked to IL-12P35 and IL-12P40 through a cleavable linker. The Protein A affinity chromatography elution peak of the prepared fusion protein is as shown in Figure 16 . An ultraviolet absorption peak exceeding 1000 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0360] 2. ELISA was used to determine the affinity of PDL1 monoclonal antibody or LIC407 for PDL1 antigen

[0361] 1) Coated with human PD-L1 antigen, 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.

[0362] 2) Blocking: Discard the coating buffer and wash 4 times with PBST. Add 250 μL / well of blocking solution (5% BSA prepared with PBST) and incubate at room temperature for 2 h.

[0363] 3) Incubation with primary antibody: Discard the blocking solution and wash 4 times with PBST. Add the gradient-diluted PDL1 monoclonal antibody or LIC407 fusion protein to the wells at 100 μL / well and incubate at room temperature for 2 h.

[0364] 4) Incubation with secondary antibody: Discard the primary antibody and wash 4 times with PBST. Dilute the HRP-labeled secondary antibody (goat anti-human IgG (H+L)) with PBST at a ratio of 1:10000, add 100 μL / well to the wells, and incubate at room temperature for 1 h.

[0365] 5) Color development: Add 100 μL / well of TMB substrate solution and incubate at 37°C in the dark for 5 min. Add the stop solution (2M H2SO4) to terminate the reaction, 50 μL per well. Measure the absorbance at 450 nm. Process the data with GraphPad 8.0, as Figure 17 shown. It can be seen that after fusing the PDL1 monoclonal antibody with IL-12, it still retains a high affinity similar to that of the PDL1 monoclonal antibody for PD-L1.

[0366] Example 10: Preparation of PDL1 monoclonal antibody-non-cleavable-IL-12 (LIC406, LIC408, LIC409) fusion proteins

[0367] Prepare LIC406, LIC408, and LIC409 fusion proteins using the same method as in Example 1. DNA fragments are prepared by chemical gene synthesis and PCR technology and plasmids are constructed. After expression and purification, the corresponding amino acid sequences of LIC406 are as shown in SEQ ID NO 27, SEQ ID NO 28, and SEQ ID NO:9; the corresponding amino acid sequences of LIC408 are as shown in SEQ ID NO 27, SEQ ID NO 30, and SEQ ID NO:9; the corresponding amino acid sequences of LIC409 are as shown in SEQ ID NO 29, SEQ ID NO 28, and SEQ ID NO:9. As Figure 1As shown, 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 through an uncleavable linker. The PDL1 monoclonal antibody fragment in the LIC408 fusion protein is located at the N-terminus, one chain is linked to IL-12P35 through an uncleavable linker, and one chain is linked to IL-12P40 through a cleavable linker. The PDL1 monoclonal antibody fragment in the LIC409 fusion protein is located at the N-terminus, one chain is linked to IL-12P35 through a cleavable linker, and one chain is linked to IL-12P40 through an uncleavable linker.

[0368] Example 11: Activity Assay and In Vivo Safety Evaluation of LIC406, LIC407, LIC408 and LIC409 Fusion Proteins

[0369] 1. In Vitro Activity Assay of LIC406, LIC407, LIC408 and LIC409 Fusion Proteins

[0370] The in vitro activities of the LIC406, LIC407, LIC408 and LIC409 fusion proteins prepared in Example 9 or Example 10 were determined by the same method as in Example 5, and LIC401 was used as a positive control. The results are as Figure 18 shown. Compared with the positive control LIC401, the LIC407, LIC408 and LIC409 fusion proteins had weaker stimulating activities on splenocytes, and the EC 50 was greater than 400 pM. It was shown that when the PDL1 monoclonal antibody was located at the N-terminus of IL-12, it could mask the activity of IL-12. This form of molecule could also enhance the targeting of IL-12.

[0371] 2. In Vivo Activity Detection and In Vivo Safety Evaluation of LIC406, LIC407, LIC408 and LIC409

[0372] The fusion proteins LIC401 + αPDL1 prepared in Example 1 and the LIC406, LIC407, LIC408 and LIC409 prepared in Example 9 or Example 10 by the same method as in Example 5 were subjected to the following tests:

[0373] Mouse prostate cancer cells RM-1 were cultured and resuspended in PBS to 5×10 6 cells / ml, and 100 μl / rat was subcutaneously inoculated into 6-8-week-old male C57BL / 6 mice. On the 10th day after tumor inoculation, the average tumor volume of the mice was about 100 mm 3, randomly and evenly divided into 6 groups: IgG, LIC401 + αPDL1, LIC406, LIC407, LIC408, and LIC409, with 8 mice in each group. The first administration was performed 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. Continuously observe and record the tumor growth and body weight changes of the mice, and use the tumor volume reaching 2000 mm 3 as the survival endpoint.

[0374] The test results are shown in Figure 18 . From the perspective of reflecting the safety of each molecule from the body weight changes, LIC401 at a dose of 0.1 mg / kg caused a significant decrease in the body weight of mice by about 20%. LIC406 could significantly improve the safety of IL-12. By introducing different Linkers, the balance between safety and anti-tumor efficacy could be achieved. The body weight decrease of LIC407 was reduced to 10%, and LIC408 and LIC409 hardly caused a decrease in the body weight of mice.

[0375] Example 12: Preparation of PDL1-ScFv-Fc-bis(mono)split-IL-12 (LIC412, LIC413, and LIC414) fusion proteins

[0376] The LIC412 fusion protein, LIC413 fusion protein, and LIC414 fusion protein were prepared using the same method as in Example 1. Among them, DNA fragments were prepared by chemical gene synthesis and PCR technology and plasmids were constructed. After expression and purification, the amino acid sequences corresponding to the LIC412 fusion protein were as shown in SEQ ID NO:35 and SEQ ID NO:36, the amino acid sequences corresponding to the LIC413 fusion protein were as shown in SEQ ID NO:33 and SEQ ID NO:36, and the amino acid sequences corresponding to the LIC414 fusion protein were as shown in SEQ IDNO:35 and SEQ ID NO:34. As Figure 1 shown, the PDL1-ScFv-Fc fragment in the LIC412, LIC413, and LIC414 fusion proteins was located at the N-terminus and was connected to IL-12P35 and IL-12P40 through a cleavable linker (or non-cleavable linker). The Protein A affinity chromatography elution peak of the prepared LIC412 fusion protein was as Figure 19 shown. It can be seen that there was an ultraviolet absorption peak exceeding 1000 mAU, indicating that the target protein was eluted by the elution buffer.

[0377] Example 13: Activity determination of LIC412 and its control fusion proteins

[0378] The in vitro activities of the LIC412, single-cleaved form LIC413, and LIC414 fusion proteins prepared in Example 12 were determined using the same method as in Example 5, with LIC410 as the positive control and LIC411 as the negative control. The results are as Figure 20 shown. Compared with the positive control LIC410, the LIC412, LIC413, and LIC414 fusion proteins had weaker stimulatory activities on splenocytes, and their EC50 values were all significantly smaller than those of the positive drug. The activity of LIC412 was restored after cleavage by urokinase, indicating that the ScFv+Fc combination at the N-terminus of IL-12 could mask the activity of IL-12.

[0379] Example 14: Preparation of PD1 / PDL1-ScFv-Fc-bis(mono)-cleaved-IL-12 (LIC417, LIC418, and LIC419) fusion proteins

[0380] The LIC417 fusion protein, LIC418 fusion protein, and LIC419 fusion protein were prepared using the same method as in Example 1. DNA fragments were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. The LIC417 fusion protein obtained after expression and purification had corresponding amino acid sequences as shown in SEQ ID NO:35 and SEQ ID NO:39, the LIC418 fusion protein had corresponding amino acid sequences as shown in SEQ ID NO:33 and SEQ ID NO:39, and the LIC419 fusion protein had corresponding amino acid sequences as shown in SEQ ID NO:35 and SEQ ID NO:38. As Figure 1 shown, the PD1 / PDL1-ScFv-Fc fragment in the LIC417, LIC418, and LIC419 fusion proteins was located at the N-terminus and was linked to IL-12P35 and IL-12P40 through a cleavable linker (or non-cleavable linker). The Protein A affinity chromatography elution peak of the prepared LIC417 fusion protein is as Figure 21 shown. An ultraviolet absorption peak exceeding 1000 mAU can be seen, indicating that the target protein was eluted by the elution buffer.

[0381] Example 15: Activity determination of LIC417 and its control fusion proteins

[0382] The in vitro activities of the LIC417, single-cleaved form LIC418, and LIC419 fusion proteins prepared in Example 14 were determined using the same method as in Example 5, with LIC415 as the positive control and LIC416 as the negative control. The results are as Figure 22As shown, compared with the positive control LIC415, the stimulating activities of LIC417, LIC418, and LIC419 fusion proteins on splenocytes were weaker. This indicates that when ScFv+Fc with different targets is located at the N-terminus of IL-12, it can mask the activity of IL-12. This form of molecule can also enhance the targeting of IL-12.

[0383] Example 16: Preparation of PDL1-(Fab)2-bis(mono)cleaved-IL-12-Fc (LIC421, LIC422, and LIC423) fusion proteins

[0384] The LIC421 fusion protein, LIC422 fusion protein, and LIC423 fusion protein were prepared by the same method as in Example 1. DNA fragments were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. After expression and purification, the amino acid sequences corresponding to the LIC421 fusion protein are shown in SEQ ID NO:9, SEQ ID NO:42, and SEQ ID NO:43; the amino acid sequences corresponding to the LIC422 fusion protein are shown in SEQ ID NO:9, SEQ ID NO:40, and SEQ ID NO:43; the amino acid sequences corresponding to the LIC423 fusion protein are shown in SEQ ID NO:9, SEQ ID NO:42, and SEQ ID NO:41. As Figure 1 shown, in the LIC421 fusion protein, LIC422 fusion protein, and LIC423 fusion protein, the PDL1-Fab fragment is located at the N-terminus and the Fc is located at the C-terminus, and they are connected to IL-12P35 and IL-12P40 through a cleavable linker (or non-cleavable linker) in the middle. The Protein A affinity chromatography elution peak of the prepared LIC421 fusion protein is as Figure 23 shown. An ultraviolet absorption peak exceeding 500 mAU can be seen, indicating that the target protein was eluted by the elution buffer.

[0385] Example 17: Activity determination of PDL1-(Fab)2-bis-cleaved-IL-12-Fc (LIC421) fusion protein

[0386] The in vitro activity of the LIC421 fusion protein prepared in Example 16 was determined by the same method as in Example 5, with LIC401 as the positive control. The results are as Figure 24 shown. The EC50 of the stimulating activity of the positive control LIC401 on splenocytes was 2.2 pM. The stimulating activity of LIC421 without urokinase cleavage was weak, and the EC 50Greater than 400 pM, at least 200-fold weaker than the positive drug. It is proved that Fab can mask the activity of IL-12 just like the steric hindrance formed by the Hinge of Fc. The activity of LIC421 can be almost completely restored after cleavage by UPA, and its EC 50 is calculated to be 5.8 pM. The molecular form of IL-12 located in the middle of the fusion protein has a high masking activity, the activity is completely restored after cleavage, and the Fc fusion is retained after cleavage, which can prolong the half-life of cytokines and exert a more lasting therapeutic effect.

[0387] Example 18: Activity determination of PDL1-(Fab)2-single-cleaved-IL-12-Fc (LIC422, LIC423) fusion protein

[0388] The in vitro activities of the LIC422 and LIC423 fusion proteins prepared in Example 16 were determined by the same method as in Example 5, and LIC401 was used as a positive control. The results are as Figure 25 shown. The stimulating activities of LIC422 and LIC423 cleaved by urokinase were partially restored compared with those before cleavage, but were weaker than the positive drug. It is proved that Fab can mask the activity of IL-12 just like the steric hindrance formed by the Hinge of Fc. After cleavage, a single Fab fusion is retained, which still has targeting ability and can prolong the half-life of cytokines, and can exert a more lasting therapeutic effect.

[0389] Example 19: Preparation of PDL1-Fab-bis(single / non)-cleaved-IL-12-Fc (LIC424, LIC425, LIC426 and LIC427) fusion proteins

[0390] The LIC424 fusion protein, LIC425 fusion protein, LIC426 fusion protein, and LIC427 fusion protein were prepared by the same method as in Example 1. Among them, DNA fragments were prepared by chemical gene synthesis and PCR technology and plasmids were constructed. The amino acid sequences corresponding to the LIC425 fusion protein obtained by expression and purification are shown in SEQ ID NO:44 and SEQ ID NO:40, the amino acid sequences corresponding to the LIC425 fusion protein are shown in SEQ ID NO:43 and SEQ ID NO:45, the amino acid sequences corresponding to the LIC426 fusion protein are shown in SEQ ID NO:43 and SEQ ID NO:44, and the amino acid sequences corresponding to the LIC427 fusion protein are shown in SEQ ID NO:40 and SEQ ID NO:45. As Figure 1As shown, in the LIC424 fusion protein, LIC425 fusion protein, LIC426 fusion protein, and LIC427 fusion protein, the VL and VH (single Fab) fragments are located at the N-terminus, and the Fc is located at the C-terminus. They are connected to IL-12P35 and IL-12P40 through a cleavable linker (or non-cleavable linker) in the middle. The disulfide bond between VL and VH and the disulfide bond between Fc-Hinge can 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 as Figure 26 shown. An ultraviolet absorption peak exceeding 300 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0391] Example 20: In vitro enzymatic cleavage of the preparation of LIC425 fusion protein

[0392] The fusion protein LIC425 prepared in Example 19 and urokinase uPA were added to an EP tube at a mass ratio of 20:1. LIC425 was 10 μg and urokinase was 0.5 μg, and the volume was made up to 20 μL with PBS. A non-cleaved protein group and a UPA group were set as controls, and the corresponding components of the control group and the cleavage system had the same mass. Cleavage was carried out at 37 °C for 12 h, and SDS-PAGE was used to verify the cleavage situation. The results are as Figure 27 shown. Under this condition, LIC425 can be cleaved by urokinase by more than 85%. Theoretically, the Y-IL12-uPA-VH single chain is 87.2 kDa, the Y-IL12-uPA-VL single chain is 73.8 kDa, the cleaved fragment VL-CL is 24.3 - 25.1 KDa, P35-Fc-Knob is 48.7 - 49.6 KDa, VH-CH1 is 22.9 - 23.8 KDa, and P40-Fc-Hole is 62.5 - 63.4 KDa. The SDS-PAGE results show that the molecular weight of the non-enzymatically cleaved LIC425 fusion protein and the molecular weights of the fragments after enzymatic cleavage are consistent with the theory. Since the purchased uPA is medicinal urokinase and contains a large amount of albumin component, the bands in the figure are relatively thick.

[0393] Example 21: In vitro activity determination of the preparation of LIC425 fusion protein

[0394] The in vitro activity of the LIC425 fusion protein prepared in Example 19 was determined by the same method as in Example 5, and LIC401 was used as a positive control. The results are as Figure 28 shown. The stimulation activity EC 50 of the positive control LIC401 for splenocytes is 2.77 pM. The stimulation activity of non-urokinase-cleaved LIC425 is weak, and EC 50 is greater than 400 pM. This value is higher than the EC 50A large value indicates that the "Lock" formed by the disulfide bond between VL and VH and the disulfide bond between Fc-Hinge masks the activity of IL-12 better than the steric hindrance of the two Fabs. The activity of LIC425 can be almost completely restored after cleavage by UPA, and its EC 50 is 5.96 pM. The heterologous molecular form of IL-12 located in the middle of the fusion protein has a high masking activity, good activity recovery after cleavage, and retains the Fc fusion after cleavage, which can extend the half-life of cytokines.

[0395] Example 22: ELISA determination of the affinity of PDL1 monoclonal antibody or LIC425 for PDL1 antigen

[0396] The affinity of a single Fab in the LIC425 fusion protein prepared in Example 19 for the antigen was determined by the same method as in Example 9, and the PDL1 monoclonal antibody was used as a positive control. The results are as Figure 29 shown. The Kd value of the affinity between the PDL1 monoclonal antibody and the PDL1 antigen is 0.1 nM, and the Kd value of the affinity for the PD-L1 antigen after fusion with IL-12 is 0.17 nM. There is no significant difference between the two, indicating that the single Fab form can still endow the LIC425 fusion protein with a targeting ability comparable to that of PDL1.

[0397] Example 23: ELISA determination of the tissue targeting of the LIC425 fusion protein

[0398] B16F10 melanoma was subcutaneously inoculated into 6-8-week-old C57BL / 6N mice. When the tumor volume grew to 800 mm 3 , 3 mg / kg of the LIC425 fusion protein prepared in Example 19 was injected via the tail vein once. After 12 h, blood was collected from the heart, the abdominal vein was cut, and 40 mL of PBS was perfused through the heart until the lungs were washed white and the liver lost its blood color (turned yellow). The heart, liver, spleen, lungs, kidneys, and tumors were taken, weighed, and then 50-100 mg was cut to prepare 10% tissue homogenate. A total of 3 mice were used, and the blood drug concentration in each tissue was detected by ELISA. The results are as Figure 30 shown. The in vivo tissue distribution of LIC425 showed certain specificity, with low contents in the heart, lungs, and kidneys, strong tumor targeting, mainly distributed in tumor tissues, but also slightly enriched in the liver and spleen. Specifically, the concentration of LIC45 in the tumor 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. All in all, IL-12 fused with the PDL1 antibody Fab, namely the LIC425 molecule, has good tumor targeting ability, which is beneficial to the exertion of anti-tumor efficacy.

[0399] Example 24: Preparation of PD1-Fab-Double Split-IL-12-Fc (LIC425-1) Fusion Protein

[0400] Replace the VL and VH of the PDL1 antibody in the LIC425 fusion protein prepared in Example 19 with the VL and VH of the PD1 antibody, and replace the cleavable linker of the urokinase substrate in LIC425 with the cleavable linker of the matrix metalloproteinase substrate to regenerate a new fusion protein LIC425-1. The LIC425-1 fusion protein is prepared by the same method as in Example 1, in which DNA fragments encoding the amino acid sequences shown in SEQ ID NO: 67 and SEQ ID NO: 68 are prepared by chemical gene synthesis and PCR techniques and plasmids are constructed. LIC425-1 is as Figure 1 the same as the schematic diagram of the LIC425 fusion protein molecule shown in the figure, where the VL and VH (single Fab) fragments are located at the N-terminus, the Fc is located at the C-terminus, and they are connected to IL-12P35 and IL-12P40 through a cleavable linker in the middle. The disulfide bond between VL and VH and the disulfide bond between Fc-Hinge can be used to form an upper and lower "Lock" to mask the activity of IL-12. The Protein A affinity chromatography elution peak of the prepared fusion protein is as Figure 31 shown. It can be seen that there is an ultraviolet absorption peak exceeding 300 mAU, indicating that the target protein is eluted by the elution buffer.

[0401] Example 25: In Vivo Safety Evaluation of LIC425-1 Fusion Protein

[0402] The in vitro LIC425 cell activity experiment results in Example 21 showed that the activity of IL-12 in this structure was weakened, which suggested that LIC425-like molecules might have relatively low peripheral immune stimulation activity and systemic side effects. Therefore, normal C57BL / 6 mice were used to evaluate the in vivo safety of LIC425-1 prepared in Example 24, one of the LIC425-like molecules, compared with the positive drug LIC401. The specific steps are as follows:

[0403] In this example, C57BL / 6N mice were selected and divided into 4 groups with 4 mice in each group according to Figure 32 . The 4 groups of mice were administered by tail vein on Day 1 and Day 4 (see the administration type, dose, and administration method of each group in Figure 32 ), and the body weights of the mice were monitored. The body weight results are as Figure 32 shown. LIC401 was administered 2 times at a dose of 0.1 mg / kg, inducing severe weight loss, sluggish movement, and hair curling in mice. While LIC425 had no effect on the body weight of mice at a dose of 0.5 mg / kg and only induced weight loss in mice at a dose of 1 mg / kg, and the decrease amplitude was lower than that of the LIC401 fusion protein at a dose of 0.1 mg / kg.

[0404] To further explore the underlying mechanism of the safety improvement of LIC425-1, mice were sacrificed on Day 6, and spleens and peripheral blood were collected for analysis. The results of spleen stimulation are as Figure 33 shown. 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, indicating that the single Fab segment of the fused PD1 antibody can largely mask the immune-stimulating activity of IL-12 in the periphery. Correspondingly, the results of flow cytometry analysis showed that, as Figure 34 in peripheral blood, 0.5 mg / kg and 1 mg / kg of the LIC425 molecule significantly stimulated CD8 + T and NK cells less than the 0.1 mg / kg group of IL12-Fc, namely LIC401. Consistently, as Figure 35 in the spleen, the LIC425-1 molecule also significantly stimulated CD8 + T cells less strongly than the 0.1 mg / kg of LIC401.

[0405] Example 26: Activity determination of PDL1-Fab-single split-IL-12-Fc (LIC426, LIC427) fusion proteins

[0406] The in vitro activities of the LIC426 and LIC427 fusion proteins prepared in Example 19 were determined by the same method as in Example 5, and LIC401 was used as a positive control. The results are as Figure 36 shown. Compared with the positive control LIC401, the LIC426 and LIC427 fusion proteins had weaker stimulating activities on splenocytes, and their activities were restored after cleavage by urokinase.

[0407] Example 27: Preparation of PDL1-(Fab)2-bis (single) split-IL-12 tandem-Fc (LIC430 and LIC431) proteins

[0408] The LIC430 fusion protein and the LIC431 fusion protein were prepared by the same method as in Example 1. Among them, DNA fragments were prepared by chemical gene synthesis and PCR techniques and plasmids were constructed. After expression and purification, the amino acid sequences corresponding to the LIC430 fusion protein are shown in SEQ ID NO:9 and SEQ ID NO:48, and the amino acid sequences corresponding to the LIC431 fusion protein are shown in SEQ ID NO:9, SEQ ID NO:49 and SEQ ID NO:50. As Figure 1As shown, in the LIC430 fusion protein and the LIC431 fusion protein, the Fab fragment is located at the N-terminus, the Fc is located at the C-terminus, and they are connected to IL-12P70 through a cleavable linker (or non-cleavable linker) in the middle. One molecule of LIC430 contains two molecules of IL-12P70. The Protein A affinity chromatography elution peak of the prepared LIC430 fusion protein is as Figure 37 shown. An ultraviolet absorption peak exceeding 500 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0409] Example 28: Activity assay of LIC430 and control fusion proteins

[0410] The in vitro activities of LIC430 and the single-cleaved form LIC431 fusion protein prepared in Example 27 were measured using the same method as in Example 5, and LIC401 was used as a positive control. The results are as Figure 38 shown. Compared with the positive control LIC401, the LIC430 and LIC431 fusion proteins have weaker stimulating activities on splenocytes. After cleavage by urokinase, the activity of LIC430 is somewhat restored, but it is lower than that of monovalent IL12. It shows that the activity of heterologous IL12 (IL-12 subunits in parallel) is better than that of homologous IL-12 (IL-12 subunits in series).

[0411] Example 29: Preparation of PDL1-Fab-bis(mono)-cleaved-IL-12 tandem-Fc (LIC433, LIC434, and LIC435) proteins

[0412] The LIC433 fusion protein, LIC434 fusion protein, and LIC435 fusion protein were prepared using the same method as in Example 1. Among them, DNA fragments were prepared by chemical gene synthesis and PCR technology and plasmids were constructed. After expression and purification, the amino acid sequences corresponding to the LIC433 fusion protein are shown in SEQ ID NO:53 and SEQ ID NO:50, the amino acid sequences corresponding to the LIC434 fusion protein are shown in SEQ ID NO:53 and SEQ ID NO:52, and the amino acid sequences corresponding to the LIC435 fusion protein are shown in SEQ ID NO:51 and SEQ ID NO:50. As Figure 1 shown, in the LIC433 fusion protein, LIC434 fusion protein, and LIC435 fusion protein, the Fab fragment is located at the N-terminus, the Fc is located at the C-terminus, and they are connected to IL-12P70 through a cleavable linker in the middle. One molecule of LIC433 contains two molecules of IL-12P70. The Protein A affinity chromatography elution peak of the prepared LIC433 fusion protein is as Figure 39 shown. An ultraviolet absorption peak exceeding 500 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0413] Example 30: Activity Assay of LIC433 and Control Fusion Proteins

[0414] The in vitro activities of the LIC433, single-chain cleavable linker form LIC434, and LIC435 fusion proteins prepared in Example 29 were determined using the same method as in Example 5, with LIC401 as the positive control. The results are as Figure 40 shown. Compared with the positive control LIC401, the LIC433 fusion protein had weaker stimulating activity on splenocytes. After cleavage by urokinase, its activity was partially restored, but it was lower than that of the monovalent IL-12.

[0415] Example 31: Preparation of LIC436 Fusion Protein

[0416] The LIC436 fusion protein was prepared using the same method as in Example 1. DNA fragments encoding the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:54 were prepared by chemical gene synthesis and PCR techniques and used to construct plasmids. As Figure 1 shown, the PDL1 monoclonal antibody is directly linked to IL-12P70 through a non-cleavable linker, and the P40 subunit in IL-12P70 is on the outside. The Protein A affinity chromatography elution peak of the prepared fusion protein is as Figure 41 shown. An ultraviolet absorption peak exceeding 1000 mAU can be seen, indicating that the target protein was eluted by the elution buffer.

[0417] Example 32: Preparation of LIC444 Fusion Protein

[0418] Only the N-terminal -P35 subunit -P40 subunit -C-terminal in IL-12P70 of LIC436 was adjusted to N-terminal -P40 subunit -P35 subunit -C-terminal to form a new fusion protein LIC444. DNA fragments encoding the amino acid sequences shown in SEQID NO:9, SEQ ID NO:12, and SEQ ID NO:69 were prepared by chemical gene synthesis and PCR techniques and used to construct plasmids. As Figure 1 shown, the PDL1 monoclonal antibody is directly linked to IL-12P70 through a non-cleavable linker, and the P35 subunit in IL-12P70 is on the outside. The Protein A affinity chromatography elution peak of the prepared fusion protein is as Figure 42 shown. An ultraviolet absorption peak exceeding 500 mAU can be seen, indicating that the target protein was eluted by the elution buffer.

[0419] Example 33: Activity Assay of LIC436 and LIC444 Fusion Proteins

[0420] The same method as in Example 5 was used to determine the in vitro activity of the LIC436 prepared in Example 31 and the LIC444 fusion protein prepared in Example 32. The results are as follows Figure 43 shown. Compared with the positive control LIC401, the protein LIC436 and LIC444 fused with the PDL1 monoclonal antibody had slightly weaker stimulating activity on splenocytes, but the activities of LIC436 and LIC444 were equivalent.

[0421] Example 34: Preparation and Activity Determination of LIC438 and LIC441 Fusion Proteins

[0422] 1. The LIC438 fusion protein and the LIC441 fusion protein were prepared by the same method as in Example 1. Among them, DNA fragments were prepared by chemical gene synthesis and PCR technology and plasmids were constructed. After expression and purification, the amino acid sequences corresponding to the LIC438 fusion protein were as shown in SEQ ID NO:56 and SEQ ID NO:57, and the amino acid sequences corresponding to the LIC441 fusion protein were as shown in SEQ ID NO:59 and SEQ ID NO:61. As Figure 1 shown, in the structures of the LIC438 fusion protein and the LIC441 fusion protein, the PDL1 single-chain antibody of one chain was directly connected to Fc through an uncleavable linker, and the other chain of the PDL1 (i.e., LIC438) or PD1 (i.e., LIC441) single-chain antibody and Fc were connected through IL-12P70 in the middle. IL-12P70 was connected to the PDL1 single-chain antibody and Fc through an uncleavable linker, and IL-12P70 was in the middle.

[0423] 2. The same method as in Example 5 was used to determine the in vitro activity of the LIC438 and LIC441 fusion proteins. The results are as follows Figure 44 shown. Compared with the previously determined positive control LIC401, the stimulating activities of the fusion proteins LIC438 and LIC441 on splenocytes were weakened, and the masking of the single molecule IL12-P70 by Fc at the N-terminus was stronger than that by two ScFvs with the same target at the N-terminus. This molecular form can achieve the inactivation of IL12-P70.

[0424] Example 35: Preparation of EGFR-Bisplit-I Tandem Fusion Protein (LIC445) Fusion Protein

[0425] The LIC445 fusion protein was prepared by the same method as in Example 1. Among them, DNA fragments encoding the amino acid sequences shown in SEQ ID NO:74 and SEQ ID NO:73 were prepared by chemical gene synthesis and PCR technology and plasmids were constructed. As Figure 1As shown, the heavy chain of the human EGFR monoclonal antibody is directly linked to human IL-12P70 through a cleavable linker. The Protein A affinity chromatography elution peak of the prepared fusion protein is as shown in Figure 45 As shown. An ultraviolet absorption peak exceeding 1000 mAU can be seen, indicating that the target protein is eluted by the elution buffer.

[0426] Example 36: Activity assay of LIC445 fusion protein

[0427] The in vitro activity of the LIC445 fusion protein was determined using the same method as in Example 5. PBMCs of human whole blood were separated by density gradient centrifugation. The cells were then treated with ACK lysis buffer and washed repeatedly with PBS. The cells were resuspended in medium (RPMI 1640 + 10% FBS + 1% Pen / Strep + 4 mM Glutamax + 50 mM β-mercaptoethanol).

[0428] 1×10 5 freshly treated PBMC cells were seeded in each well of a 96-well plate. PHA was added at a final concentration of 5 mg / ml for activation, and then gradient-diluted different concentrations of LIC445 and cleaved LIC445 prepared in Example 37 were added. The cells were cultured in a 37°C, 5% CO2 incubator for 72 hours. The cell supernatant was collected, and the production of IFNγ was measured using a human IFNγ ELISA detection kit. The results are as shown in Figure 46 As shown, when human IL-12 was fused to the C-terminus of the EGFR monoclonal antibody, there were differences in activity before and after enzymatic cleavage, indicating that fusing IL-12 to the C-terminus of the heavy chain of the EGFR antibody can effectively mask its activity and can be used as a strategy for cytokine deactivation or prodrug preparation.

[0429] Example 37: Activity comparison of LIC407, LIC421, LIC425, LIC430, LIC433, LIC437, LIC438, LIC441 fusion proteins

[0430] 1. The LIC437 fusion protein was prepared using the same method as in Example 1. The corresponding amino acid sequence of the LIC437 fusion protein prepared by chemical gene synthesis and PCR technology is shown in SEQ ID NO:9, SEQ ID NO:52, and SEQ ID NO:55. As shown in Figure 1 As shown, in the LIC437 fusion protein, the Fab fragment is located at the N-terminus and the Fc is located at the C-terminus. The Fab of PDL1 of one chain is directly linked to the Fc through a non-cleavable linker. The Fab and Fc of PDL1 of the other chain are linked through IL-12P70 in the middle, and IL-12P70 is linked to the Fab and Fc of PDL1 through non-cleavable linkers.

[0431] 2. Using the same method as in Example 5, synchronously 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 and LIC441 prepared in Example 34, and use LIC401 as a positive control to compare the effects of different molecular masking structures on the activity of IL-12. The results are as Figure 47 shown in Table 1. The monoclonal antibody located at the N-terminus of the IL12-P35 and IL12-P40 subunits, i.e., the LIC407 structure, can have a strong masking effect on the activity of IL-12 by more than 4000 times; the disulfide bond between VL and VH in a single Fab and the disulfide bond between Fc-Hinge form an upper and lower "Lock" structure, and the masking effect of LIC425 is stronger than that of the LIC421 molecule masked by two Fabs and Fc. At the same time, the masking effects of LIC425 and LIC421 are stronger than that of LIC433, indicating that the steric hindrance of the "sandwich" type is better than that of two or more IL-12s in series for masking a single parallel IL-12. The masking effect is LIC441 > LIC437 > LIC438, indicating that the steric hindrance of Fc > Fab > ScFv.

[0432] Table 1

[0433] Molecule <![CDATA[EC 50 (pM)]]> Masking multiple LIC401 2.6 / LIC407 11050.0 4250.0 LIC421 4802.0 1846.9 LIC425 6830.0 2626.9 LIC430 127.1 48.9 LIC433 603.9 232.3 LIC437 525.6 202.2 LIC438 414.2 159.3 LIC441 691.4 265.9

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

[0435] 3. Using the same method as in Example 25, synchronously determine the in vivo safety evaluation of LIC407 prepared in Example 9, LIC406, LIC408, LIC409 prepared in Example 10, and LIC425 fusion protein prepared in Example 19, and use LIC401 as a positive control. The results are as Figure 48 shown. The results show that when LIC401 is administered twice at a dose of 0.1 mg / kg, it induces severe weight loss, slow movement, and curly hair in mice. While LIC425 induces weight loss in mice at a dose of 1 mg / kg, and the decrease amplitude is lower than that of the LIC401 fusion protein at a dose of 0.1 mg / kg. While LIC406-LIC409 has no effect on the body weight of mice at a dose of 1 mg / kg.

[0436] The amino acid sequences or nucleotide sequences involved in the present invention are as follows:

[0437] Amino acid sequence of mouse IL-12-P35 subunit SEQ ID No:1:

[0438] RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHK NESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLV AIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA;

[0439] Amino acid sequence of mouse IL-12-P40 subunit SEQ ID No:2:

[0440] MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS;

[0441] Amino acid sequence of human IgG1-Fc-Knob-Mut SEQ ID No:3:

[0442] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS;

[0443] Amino acid sequence of human IgG1-Fc-Hole-Mut SEQ ID No:4:

[0444] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS;

[0445] Amino acid sequence of non-cleavable linker 1 at the N-terminus of the IL12 subunit SEQ ID No:5:

[0446] GSSGGSGGSGGSG;

[0447] Amino acid sequence of non-cleavable linker 2 at the C-terminus of the IL12 subunit SEQ ID No:6:

[0448] GGGGSGGGGS;

[0449] Amino acid sequence of cleavable linker 3 for UPA substrate SEQ ID No:7:

[0450] GSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSG;

[0451] Amino acid sequence of cleavable linker 4 for MMP substrate SEQ ID No:8:

[0452] GSSGGSGGSGGSGQLLGFLTAGSSGGSGGSGGSG;

[0453] Amino acid sequence of the light chain of human PDL1 monoclonal antibody SEQ ID No:9:

[0454] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0455] Amino acid sequence SEQ ID No:10 of the heavy chain (Mut) of human PDL1-IgG1 monoclonal antibody:

[0456] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS;

[0457] Amino acid sequence SEQ ID No:11 of the heavy chain (Knob-Mut) of human PDL1-IgG1 monoclonal antibody:

[0458] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS;

[0459] Amino acid sequence of the heavy chain of human PDL1-IgG1 monoclonal antibody (Hole-Mut) SEQ ID No: 12:

[0460] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS;

[0461] Amino acid sequence of the light chain of human PD1 monoclonal antibody SEQ ID No:13:

[0462] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0463] Amino acid sequence of the heavy chain of human PD1-IgG4 monoclonal antibody SEQ ID No:14:

[0464] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0465] Amino acid sequence of the heavy chain (Knob) of human PD1-IgG4 monoclonal antibody SEQ ID No:15:

[0466] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0467] Amino acid sequence SEQ ID No:16 of the heavy chain (Hole) of human PD1-IgG4 monoclonal antibody

[0468] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0469] Amino acid sequence of human PDL1-ScFv SEQ ID No:17:

[0470] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK;

[0471] Amino acid sequence of human PD1-ScFv SEQ ID No:18:

[0472] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSGSSGGSGGSGGSGDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIK;

[0473] Amino acid sequence of human PDL1-VH-CH1 SEQ ID No:19:

[0474] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV;

[0475] Amino acid sequence of human PD1-VH-CH1 SEQ ID No:20:

[0476] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV;

[0477] IF fusion protein LIC401: Amino acid sequence of chain 1 SEQ ID No:21 (SEQ ID No:1 + SEQ ID No:6 + SEQ ID No:3), amino acid sequence of chain 2 SEQ ID No:22 (SEQ ID No:2 + SEQ ID No:6 + SEQ ID No:4):

[0478] RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:21);

[0479] MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:22);

[0480] FI fusion protein LIC402: Amino acid sequence of chain 1 SEQ ID No:23 (SEQ ID No:3 + SEQ ID No:5 + SEQ ID No:1), amino acid sequence of chain 2 SEQ ID No:24 (SEQ ID No:4 + SEQ ID No:5 + SEQ ID No:2):

[0481] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:23);

[0482] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:24);

[0483] F-Double split-I fusion protein LIC403: Amino acid sequence of chain 1 SEQ ID No:25 (SEQ ID No:3 + SEQ ID No:7 + SEQ ID No:1), amino acid sequence of chain 2 SEQ ID No:26 (SEQ ID No:4 + SEQ ID No:7 + SEQ ID No:2):

[0484] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:25);

[0485] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:26);

[0486] F - right cleavage - I fusion protein LIC404: Amino acid sequence of chain 1 is SEQ ID No:23, and amino acid sequence of chain 2 is SEQ ID No:26; F - left cleavage - I fusion protein LIC405: Amino acid sequence of chain 1 is SEQ ID No:25, and amino acid sequence of chain 2 is SEQ ID No:24;

[0487] PI fusion protein LIC406: Amino acid sequence of chain 1 is SEQ ID No:27 (SEQ ID No:11 + SEQ ID No:5 + SEQ ID No:1), amino acid sequence of chain 2 is SEQ ID No:28 (SEQ ID No:12 + SEQ ID No:5 + SEQ ID No:2), and amino acid sequence of chain 3 is SEQ ID No:9:

[0488] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:27);

[0489] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:28);

[0490] P-Bisplit-I fusion protein LIC407: Amino acid sequence of chain 1 SEQ ID No:29 (SEQ ID No:11 + SEQ ID No:7 + SEQ ID No:1), amino acid sequence of chain 2 SEQ ID No:30 (SEQ ID No:12 + SEQ ID No:7 + SEQ ID No:2), amino acid sequence of chain 3 is SEQ ID No:9:

[0491] VQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:29);

[0492] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:30);

[0493] P-right fissure-I fusion protein LIC408: The amino acid sequence of chain 1 is SEQ ID No: 27, the amino acid sequence of chain 2 is SEQ ID No: 30, and the amino acid sequence of chain 3 is SEQ ID No: 9;

[0494] P - Left Fissure - I Fusion Protein LIC409: Amino acid sequence of chain 1 is SEQ ID No: 29, amino acid sequence of chain 2 is SEQ ID No: 28, and amino acid sequence of chain 3 is SEQ ID No: 9;

[0495] IF - PDL1 - ScFv Fusion Protein LIC410: Amino acid sequence of chain 1 is SEQ ID No: 31 (SEQ ID No: 21 + SEQ ID No: 17), amino acid sequence of chain 2 is SEQ ID No: 32 (SEQ ID No: 22 + SEQ ID No: 17):

[0496] RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK(SEQ ID No: 31);

[0497] MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK (SEQ ID No:32);

[0498] PDL1-ScFv-FI fusion protein LIC411: Amino acid sequence of chain 1 SEQ ID No:33 (SEQ ID No:17 + SEQ ID No:23), amino acid sequence of chain 2 SEQ ID No:34 (SEQ ID No:17 + SEQ ID No:24);

[0499] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:33);

[0500] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:34);

[0501] PDL1-ScFv-F-Double split-I fusion protein LIC412: Amino acid sequence of chain 1 SEQ ID No:35 (SEQ ID No:17 + SEQ ID No:25), amino acid sequence of chain 2 SEQ ID No:36 (SEQ ID No:17 + SEQ ID No:26);

[0502] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:35);

[0503] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:36);

[0504] PDL1-ScFv-F-right cleavage-I fusion protein LIC413: Amino acid sequence of chain 1 is SEQ ID No:33, and amino acid sequence of chain 2 is SEQ ID No:36;

[0505] PDL1-ScFv-F-Left cleavage-I fusion protein LIC414: Amino acid sequence of chain 1 is SEQ ID No: 35, and amino acid sequence of chain 2 is SEQ ID No: 34;

[0506] IF-PDL1-ScFv-PD1-ScFv fusion protein LIC415: Amino acid sequence of chain 1 is SEQ ID No: 31, and amino acid sequence of chain 2 is SEQ ID No: 37 (SEQ ID No: 22 + SEQ ID No: 18):

[0507] MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKQVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSGSSGGSGGSGGSGDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIK (SEQ ID No:37);

[0508] PDL1-ScFv-PD1-ScFv-FI fusion protein LIC416: Amino acid sequence of chain 1 is SEQ ID No:33, and amino acid sequence of chain 2 is SEQ ID No:38 (SEQ ID No:18 + SEQ ID No:24):

[0509] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSGSSGGSGGSGGSGDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:38);

[0510] PDL1-ScFv-PD1-ScFv-F-Double split-I fusion protein LIC417: Amino acid sequence of chain 1 SEQ ID No:35, amino acid sequence of chain 2 SEQ ID No:39 (SEQ ID No:18 + SEQ ID No:26):

[0511] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSGSSGGSGGSGGSGDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:39);

[0512] PDL1-ScFv-PD1-ScFv-F-Right cleavage-I fusion protein LIC418: Amino acid sequence of chain 1 is SEQ ID No:33, and amino acid sequence of chain 2 is SEQ ID No:39;

[0513] PDL1-ScFv-PD1-ScFv-F-Left Cleavage-I Fusion Protein LIC419: Amino Acid Sequence of Chain 1 is SEQ ID No: 35, and Amino Acid Sequence of Chain 2 is SEQ ID No: 38;

[0514] PIF-T Fusion Protein LIC420: Amino Acid Sequence of Chain 1 is SEQ ID No: 40 (SEQ ID No: 19 + SEQ ID No: 5 + SEQ ID No: 21), Amino Acid Sequence of Chain 2 is SEQ ID No: 41: (SEQ ID No: 19 + SEQ ID No: 5 + SEQ ID No: 22), and Amino Acid Sequence of Chain 3 is SEQ ID No: 9:

[0515] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No: 40);

[0516] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:41);

[0517] P-Bisplit-IF-T fusion protein LIC421: Amino acid sequence of chain 1 SEQ ID No:42 (SEQ ID No:19 + SEQ ID No:7 + SEQ ID No:21), amino acid sequence of chain 2 SEQ ID No:43 (SEQ ID No:19 + SEQ ID No:7 + SEQ ID No:22), amino acid sequence of chain 3 is SEQ ID No:9:

[0518] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:42);

[0519] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:43);

[0520] P-Right-Fissure-IF-T fusion protein LIC422: Amino acid sequence of chain 1 is SEQ ID No:40, amino acid sequence of chain 2 is SEQ ID No:43, and amino acid sequence of chain 3 is SEQ ID No:9;

[0521] P - Left - Split - IF - T fusion protein LIC423: The amino acid sequence of chain 1 is SEQ ID No: 42, the amino acid sequence of chain 2 is SEQ ID No: 41, and the amino acid sequence of chain 3 is SEQ ID No: 9;

[0522] PIF - Y fusion protein LIC424: The amino acid sequence of chain 1 is SEQ ID No: 44 (SEQ ID No: 9 + SEQ ID No: 5 + SEQ ID No: 21), and the amino acid sequence of chain 2 is SEQ ID No: 40:

[0523] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No: 44);

[0524] P-Bifurcated-IF-Y Fusion Protein LIC425: Amino Acid Sequence of Chain 1 is SEQ ID No:45 (SEQ ID No:9 + SEQ ID No:7 + SEQ ID No:21), Amino Acid Sequence of Chain 2 is SEQ ID No:43:

[0525] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSSGGSGGSGGSGLSGRSDNHGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:45);

[0526] P-Right-Bifurcated-IF-Y Fusion Protein LIC426: Amino Acid Sequence of Chain 1 is SEQ ID No:44, Amino Acid Sequence of Chain 2 is SEQ ID No:43; P-Left-Bifurcated-IF-Y Fusion Protein LIC427: Amino Acid Sequence of Chain 1 is SEQ ID No:45, Amino Acid Sequence of Chain 2 is SEQ ID No:40;

[0527] PI Homodimeric Fusion Protein LIC428: Amino Acid Sequence of Chain 1 SEQ ID No:46 (SEQ ID No:10 + SEQ ID No:5 + SEQ ID No:1 + SEQ ID No:75 + SEQ ID No:2), Amino Acid Sequence of Chain 2 is SEQ ID No:9:

[0528] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ IDNo: 46);

[0529] PIF-T Homodimeric Fusion Protein LIC429: Amino Acid Sequence of Chain 1 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)), Amino Acid Sequence of Chain 2 i.e., SEQ ID No: 9:

[0530]

[0531] P-bisplit-IF-T Homodimeric Fusion Protein LIC430: Amino Acid Sequence of Chain 1 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)), Amino Acid Sequence of Chain 2 SEQ ID No:9:

[0532]

[0533] P - Monosplit - IF - T Homodimeric Fusion Protein LIC431: Amino Acid Sequence of Chain 1 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), Amino Acid Sequence of Chain 2 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), Amino Acid Sequence of Chain 3 SEQ ID No:9:

[0534]

[0535]

[0536] PIF-Y Homodimeric Fusion Protein LIC432: Amino Acid Sequence of Chain 1, 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), Amino Acid Sequence of Chain 2, 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):

[0537]

[0538]

[0539] P-bifurcated-IF-Y Homodimeric Fusion Protein LIC433: Amino Acid Sequence of Chain 1 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), Amino Acid Sequence of Chain 2 i.e., SEQ ID No:50:

[0540]

[0541] P - left cleavage - IF - Y homodimeric fusion protein LIC434: Amino acid sequence of chain 1 is SEQ ID No: 53, and amino acid sequence of chain 2 is SEQ ID No: 52;

[0542] P - right cleavage - IF - Y homodimeric fusion protein LIC435: Amino acid sequence of chain 1 is SEQ ID No: 51, and amino acid sequence of chain 2 is SEQ ID No: 50;

[0543] PI tandem single - molecule fusion protein LIC436: Amino acid sequence of chain 1 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), amino acid sequence of chain 2 is SEQ ID No: 12, and amino acid sequence of chain 3 is SEQ ID No: 9;

[0544] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ IDNo:54);

[0545] PIF-T tandem single-cell factor fusion protein LIC437: Amino acid sequence of chain 1 is SEQ ID No:55 (SEQ ID No:19 + SEQ ID No:75 + SEQ ID No:3), amino acid sequence of chain 2 is SEQ ID No:52, and amino acid sequence of chain 3 is SEQ ID No:9:

[0546] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No:55);

[0547] PDL1-ScFv-IF tandem single-cell factor fusion protein LIC438: Amino acid sequence of chain 1 is SEQ ID No:56 (SEQ ID No:17 + SEQ ID No:75 + SEQ ID No:3), amino acid sequence of chain 2 is 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):

[0548] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:56);

[0549]

[0550] PDL1-ScFv-PD1-ScFv-IF tandem single-cell cytokine fusion protein LIC439: Amino acid sequence of chain 1 is SEQ ID No:56, and amino acid sequence of chain 2 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):

[0551]

[0552] FI-PDL1-ScFv tandem single-cell factor fusion protein LIC440: Amino acid sequence of chain 1 SEQ ID No:59 (SEQ ID No:3 + SEQ ID No:75 + SEQ ID No:17), amino acid sequence of chain 2 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):

[0553] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK (SEQ ID No:59);

[0554]

[0555] FI-PDL1-ScFv-PD1-ScFv tandem single-cell factor fusion protein LIC441: Amino acid sequence of chain 1 is SEQ ID No: 59, and the amino acid sequence of chain 2 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):

[0556]

[0557] PDL1-ScFv-FI tandem single cell factor fusion protein LIC442: Amino acid sequence of chain 1 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), amino acid sequence of chain 2 SEQ ID No: 63 (SEQ ID No: 17 + SEQ ID No: 4):

[0558]

[0559] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGSSGGSGGSGGSGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No:63);

[0560] PDL1 - ScFv - PD1 - ScFv - FI tandem single - cell factor fusion protein LIC443: Amino acid sequence of chain 1 is SEQ ID No:62, and amino acid sequence of chain 2 is SEQ ID No:64 (SEQ ID No:18 + SEQ ID No:4):

[0561] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSGSSGGSGGSGGSGDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:64);

[0562] F-Double split-I fusion protein LIC403-1: Amino acid sequence of chain 1 SEQ ID No:65: (The cleavable linker has 16 fewer amino acids than LIC403), amino acid sequence of chain 2 SEQ ID No:66: (The cleavable linker has 16 fewer amino acids than LIC403):

[0563] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGLSGRSDNHGSGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:65);

[0564] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGLSGRSDNHGSGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:66);

[0565] PD1 (Fab)-bispecific-IF-Y fusion protein LIC425-1: Amino acid sequence of chain 1 SEQ ID No:67 (SEQ ID No:13 + SEQ ID No:8 + SEQ ID No:21), amino acid sequence of chain 2 SEQ ID No:68 (SEQ ID No:20 + SEQ ID No:8 + SEQ ID No:22):

[0566] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSSGGSGGSGGSGQLLGFLTAGSSGGSGGSGGSGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:67);

[0567] QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGSSGGSGGSGGSGQLLGFLTAGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No:68);

[0568] PI tandem single molecule fusion protein LIC444: Amino acid sequence of chain 1 is SEQ ID No:69 (SEQ ID No:11 + SEQ ID No:5 + SEQ ID No:2 + SEQ ID No:75 + SEQ ID No:1), amino acid sequence of chain 2 is SEQ ID No:12, and amino acid sequence of chain 3 is SEQ ID No:9:

[0569] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:69);

[0570] Amino acid sequence of human IL-12-P35 subunit SEQ ID No:70:

[0571] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPL ELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQ NMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS;

[0572] Amino acid sequence of human IL-12-P40 subunit SEQ ID No:71:

[0573] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS;

[0574] Amino acid sequence of the heavy chain of human EGFR monoclonal antibody SEQ ID No:72:

[0575] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0576] Amino acid sequence of the light chain of human EGFR monoclonal antibody SEQ ID No:73:

[0577] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0578] E-bifurcation-I tandem fusion protein LIC445: Amino acid sequence of chain 1 SEQ ID No:74 (SEQ ID No:72 + SEQ ID No:8 + SEQ ID No:70 + SEQ ID No:75 + SEQ ID No:71), amino acid sequence of chain 2 SEQ ID No:73:

[0579]

[0580] Linker peptide between P35 and P40, SEQ ID No:75:

[0581] GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID No:75);

[0582] Mouse P35-linker peptide-P40, SEQ ID No:76 (SEQ ID No:1 + SEQ ID No:75 + SEQ ID No:2):

[0583] RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS;

[0584] Human P35-linker peptide-P40, SEQ ID No:77 (SEQ ID No:70 + SEQ ID No:75 + SEQ ID No:71):

[0585] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSGGGGSGGGGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS;

[0586] Cleavable cathepsin substrate linker amino acid sequence SEQ ID No:78:

[0587] GSSGGSGGSGGSGFFYGSSGGSGGSGGSG;

[0588] Cleavable proteolytic enzyme substrate linker amino acid sequence SEQ ID No:79:

[0589] GSSGGSGGSGGSGRQARAVGGSSGGSGGSGGSG;

[0590] Cleavable legumain substrate linker amino acid sequence SEQ ID No:80:

[0591] GSSGGSGGSGGSGAANLGSSGGSGGSGGSG;

[0592] F-Bisplit-I Fusion Protein LIC403-2: Amino Acid Sequence of Chain 1 SEQ ID No:81: (The cleavable linker has 14 more amino acids than LIC403), Amino Acid Sequence of Chain 2 SEQ ID No:82: (The cleavable linker has 14 more amino acids than LIC403):

[0593] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGSSGGSGGLSGRSDNHGSSGGSGGSGGSGSSGGSGGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:81);

[0594] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGSSGGSGGLSGRSDNHGSSGGSGGSGGSGSSGGSGGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:82);

[0595] Cleavable UPA substrate linker 3 amino acid sequence SEQ ID No:83:

[0596] GSSGGLSGRSDNHGSGGS;

[0597] Cleavable UPA substrate linker 3 amino acid sequence SEQ ID No:84:

[0598] GSSGGSGGSGGSGSSGGSGGLSGRSDNHGSSGGSGGSGGSGSSGGSGG;

[0599] Non-cleavable linker 2 amino acid sequence at the N-terminus of the IL12 subunit SEQ ID No:85:

[0600] GSSGGSGGSGGSGGSSGGSGGSGGSGGSSGGSGG;

[0601] FI fusion protein LIC402-1: Amino acid sequence of chain 1 is SEQ ID No:86 (the non-cleavable linker has 21 more amino acids than LIC402), and the amino acid sequence of chain 2 is SEQ ID No:87 (the non-cleavable linker has 21 more amino acids than LIC402):

[0602] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGGSSGGSGGSGGSGGSSGGSGGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(SEQ ID No:86);

[0603] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGSGGSGGSGGSSGGSGGSGGSGGSSGGSGGMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(SEQ ID No:87);

[0604] Amino acid sequence of the signal peptide of the human EGFR monoclonal antibody SEQ ID No:88:

[0605] MGWSLILLFLVAVATRVHS。

[0606] In the description of this specification, the description referring to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection 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 a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0607] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to 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, and 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, wherein the third structural unit comprises 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 When the first structural unit is connected to one of the third structural units, the second structural unit is selected from a cleavable linker; Optionally, 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; Optionally, the fusion protein does not include an antibody or antigen-binding fragment thereof to the IL-12 cytokine; Optionally, 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; Optionally, 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; Optionally, 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; Optionally, 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; Optionally, the IL-12 cytokine or a functional fragment thereof comprises two subunit fragments, namely, a P35 fragment of IL-12 or a functional fragment thereof, and a P40 fragment of IL-12 or a functional fragment thereof, and the third structural unit further comprises a non-cleavable connecting peptide, and the two subunit fragments are connected by the connecting peptide; Optionally, the IL-12 cytokine or a functional fragment thereof comprises two subunit fragments, namely, a P35 fragment of IL-12 or a functional fragment thereof, and a P40 fragment of IL-12 or a functional fragment thereof, wherein 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; Optionally, 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; Optionally, the amino acid length of the connecting peptide is 1-60, preferably 10-40, 15-40; Optionally, the connecting peptide has an amino acid sequence as shown in (GGGGS)n, where n is 3, 4, 5 or 6; Optionally, the third structural unit has an amino acid sequence as shown in SEQ ID NO:1, 2, 70, 71, 76 or 77.

3. The fusion protein according to claim 1, characterized in that The second structural unit is a cleavable linker; Optionally, the second structural unit is a cleavable linker; Optionally, the fusion protein comprises two monomers, and the two second structural units are a cleavable linker and a non-cleavable linker, respectively; Optionally, the proteases expressed in the tumor tissue include matrix metalloproteinases, serine proteases, or asparagine endopeptidases; Optionally, the cleavable linker comprises a protease substrate sequence and optionally a flexible peptide segment; Optionally, 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; Optionally, the amino acid length of the flexible peptide segment is 1-60, preferably 5-30, 5-25 or 5-20; Optionally, the protease substrate sequence is selected from QLLGFLTA, LSGRSDNH, GFFY, RQARAVGG or AANL; Optionally, the cleavable linker is a flexible peptide segment-protease substrate sequence, or a flexible peptide segment-protease substrate sequence-flexible peptide segment; Optionally, the amino acid length of the cleavable linker is 15 to 60, preferably 15 to 50, 15 to 45 or 15 to 40; Optionally, the cleavable linker has an amino acid sequence as shown in SEQ ID NO: 7, 8, 78, 79, 80, 83 or 84; Optionally, the non-cleavable linker 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; Optionally, the non-cleavable linker has an amino acid length of 1 to 60, preferably 5 to 50, 5 to 40, 5 to 30 or 10 to 20; Optionally, the non-cleavable linker has an amino acid sequence as shown in (GGGGS)n, where n is 3, 4, 5 or 6; Optionally, the non-cleavable linker has an amino acid sequence as shown in SEQ ID NO: 5 or 6.

4. The fusion protein according to claim 1, characterized in that 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; Preferably, 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.

5. The fusion protein according to claim 1, characterized in that 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; Optionally, 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; Preferably, 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; Optionally, 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; Optionally, 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; Preferably, 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; Optionally, when the first Fc fragment or the first VHH fragment is connected to two of the third structural units, 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 respectively the P35 fragment of IL-12 or a functional fragment thereof, and the P40 fragment of IL-12 or a functional fragment thereof; Optionally, 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 a functional fragment thereof of the third structural unit includes two subunit fragments of a P35 fragment of IL-12 or a functional fragment thereof, and a P40 fragment of IL-12 or a functional fragment thereof; The two subunit fragments are respectively connected to the two fragments of the first Fc fragment.

6. The fusion protein according to claim 1, characterized in that 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; Optionally, 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 in the first full-length antibody through the second structural unit; Optionally, 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; Optionally, 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; Optionally, 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; Optionally, 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 of which are cleavable linkers; Optionally, when the first full-length antibody 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; Preferably, 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; Optionally, 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 of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, respectively.

7. The fusion protein according to claim 2, characterized in that 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; Optionally, 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 or the C-terminus of the CL fragment in the first Fab fragment through the second structural unit; Optionally, 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; Optionally, 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; Optionally, 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; Preferably, 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; Optionally, 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, preferably, the IL-12 cytokines or their functional fragments in the two third structural units are the P35 fragment of IL-12 or its functional fragment, and the P40 fragment of IL-12 or its functional fragment, respectively.

8. 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; Optionally, 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.

9. The fusion protein according to any one of claims 2 to 8, characterized in that The fusion protein further comprises a fourth structural unit, wherein the fourth structural unit is selected from a second Fc fragment, or a second antibody or an antigen-binding fragment thereof, wherein the second antibody or an antigen-binding fragment thereof has tumor antigen or immune checkpoint binding activity; Optionally, 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; Optionally, 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; Optionally, 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 structural unit and the fourth structural unit; Optionally, 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 through the fifth structural unit; Optionally, the fifth structural unit is selected from a cleavable linker or a non-cleavable linker; Optionally, 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; Optionally, 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; Optionally, 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; Optionally, 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.

10. 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; 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:30, 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: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; 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:42, the amino acid sequence has a third peptide chain as shown in SEQ ID NO:43, 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:40, the amino acid sequence has a third peptide chain as shown in SEQ ID NO:43, 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: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; 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:49, the amino acid sequence has a third peptide chain as shown in SEQ ID NO:50, 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: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.

11. A nucleic acid molecule, an expression vector, or a recombinant cell, characterized in that: Encoding the fusion protein according to any one of claims 1 to 10; The expression vector carries the above-mentioned nucleic acid molecule; The recombinant cell carries the above-mentioned nucleic acid or the above-mentioned expression vector; or the recombinant cell expresses the fusion protein according to any one of claims 1 to 10; Optionally, the expression vector is a eukaryotic expression vector, preferably, the expression vector is a lentiviral vector; Optionally, the recombinant cell is obtained by introducing the expression vector of claim 12 into a host cell.

12. A fusion protein complex, characterized in that: The invention comprises the fusion protein according to any one of claims 1 to 10.

13. An immunotherapy cell, characterized in that: Expressing the fusion protein according to any one of claims 1 to 10.

14. A pharmaceutical composition, characterized in that It comprises the fusion protein according to any one of claims 1 to 10, 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.

15. Use of the fusion protein of any one of claims 1 to 10, the fusion protein complex of claim 12, the immunotherapeutic cell of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a drug for treating cancer, infectious diseases or autoimmune diseases.

Images