Fusion protein comprising il-12 and Anti-FAP antibody and use thereof

Abstract

To provide novel fusion proteins having anticancer effects.SOLUTION: Provided is a fusion protein comprising a first monomer comprising IL-12 or variant thereof and a second monomer comprising an antigen binding site that specifically binds to FAP. The fusion protein exhibits anticancer effect due to the IL-12. In particular, when the anti-FAP binding site is formed into one antibody, the fusion protein targets FAP highly expressed on tumor so that the fusion protein is capable of efficiently treating tumor by localizing IL-12 specifically at the tumor site. Thus the fusion protein has high industrial applicability.SELECTED DRAWING: Figure 33

Description

[Technical field]

[0001] The present invention relates to a novel fusion protein comprising IL-12 or a variant thereof and an anti-FAP antibody, and an anti-cancer pharmaceutical composition comprising the fusion protein. [Background technology]

[0002] Interleukin-12 (IL-12) is a representative inflammatory cytokine and plays a crucial role in the effective induction of cellular immune responses. IL-12 is a heterodimeric protein containing 40 kDa (p40) and 35 kDa (p35) subunits linked by disulfide bonds, and is produced by activated macrophages, monocytes, dendritic cells, and activated B lymphocytes. IL-12 can enhance helper T1 cell immunity, increase the cytotoxicity of cellular T lymphocytes, and inhibit angiogenesis. In general, IL-12 activates IFN-γ production in T cells and NK cells.

[0003] Local expression of IL-12 makes tumor cells sensitive to T cell-mediated cytotoxicity, resulting in tumor growth inhibition and the establishment of humoral immunity. However, the drawback of the clinical application of IL-12 is that upon administration, cytokine-related toxicity may occur throughout the body. These clinical results show the limitations of IL-12 as a single therapeutic agent for treating cancer.

[0004] On the other hand, fibroblast activation protein alpha (FAP) is a gelatinase expressed in activated fibroblasts. FAP is known to be expressed in more than 90% of cancer-associated fibroblasts in various human carcinomas, including prostate and pancreatic cancer. Therefore, in recent years, interest in developing immunotherapy targeting FAP-expressing cells has rapidly increased (Fang J. et al., Mol. Ther. Oncolytics., 3:16007, 2016). Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the present inventors have conducted research to develop a novel fusion protein having anti-cancer effects. As a result, the present inventors have found that a fusion protein comprising IL-12 and an anti-FAP antibody has enhanced anti-cancer activity while reducing systemic toxicity, thereby completing the present invention. [Means for solving the problem]

[0006] In one aspect of the invention, there is provided a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to a FAP.

[0007] In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising the fusion protein as an active ingredient.

[0008] In another aspect of the invention, a polynucleotide encoding a first monomer is provided.

[0009] In another aspect of the invention, a polynucleotide encoding a second monomer is provided.

[0010] In another aspect of the invention, a vector comprising the polynucleotide is provided.

[0011] In another aspect of the present invention, there is provided a transformed cell into which the vector has been introduced.

[0012] In another aspect of the invention, there is provided a method for producing a fusion protein, the method comprising the steps of i) culturing a transformed cell; and ii) recovering a fusion protein comprising a first monomer and a second monomer.

[0013] In yet another aspect of the invention, there is provided a method for treating or preventing cancer, comprising administering to a subject a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to a FAP.

[0014] In yet another aspect of the invention, there is provided a use of a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to a FAP, for treating cancer.

[0015] In yet another aspect of the present invention, there is provided a use of a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen-binding site that specifically binds to a FAP, for the manufacture of a medicament for treating cancer. Effect of the Invention

[0016] Fusion proteins that contain IL-12 and an antigen binding site that specifically binds to FAP can show anti-cancer effects by IL-12.In addition, when anti-FAP antibody is realized by one antibody, it has been found that IL-12 specifically accumulates in tumors with high FAP expression, thereby reducing systemic toxicity and showing tumor-specific anti-cancer effects.In other words, cancer can be effectively treated by specifically targeting FAP that is highly expressed in tumors and specifically localizing IL-12 at tumor sites. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the results obtained by identifying T1.01, T1.02, T1.03, T1.04, T1.05, T1.06 and T1.07 according to one embodiment using SDS-PAGE. [Diagram 2]FIG. 1 shows the results obtained by identifying T1.08, T1.09, T1.10, T1.11, T1.12, T1.13, T1.14 and T1.15 according to one embodiment using SDS-PAGE. [Diagram 3] FIG. 1 shows the results obtained by identifying T1.16, T1.17, T1.18, T1.19 and T1.21 according to one embodiment using SDS-PAGE. [Figure 4] FIG. 1 shows the results obtained by identifying T1.01m, T1.02m, T1.03m, T1.04m, T1.05m, T1.06m and T1.07m according to one embodiment using SDS-PAGE. [Diagram 5] FIG. 1 shows the results obtained by identifying T1.08m, T1.09m, T1.10m, T1.11m, T1.12m, T1.13m and T1.15m according to one embodiment using SDS-PAGE. [Figure 6] FIG. 1 shows the results obtained by identifying T1.16m, T1.17m, T1.18m, T1.19m, T1.20m, T1.21m and T1.22m according to one embodiment using SDS-PAGE. [Figure 7] 1 is a graph showing the binding affinity of T1.01 and T1.02 to recombinant human FAP (rhFAP) by surface plasmon resonance analysis. [Figure 8] 1 is a graph showing the binding affinity of T1.03 to recombinant human FAP by surface plasmon resonance analysis. [Figure 9] 1 is a graph showing the binding affinity of T1.04 and T1.05 to recombinant human FAP by surface plasmon resonance analysis. [Figure 10] 1 is a graph showing the binding affinity of T1.06 and T1.07 to recombinant human FAP by surface plasmon resonance analysis. [Figure 11] 1 is a graph showing the binding affinity of T1.08 and T1.09 to recombinant human FAP by surface plasmon resonance analysis. [Figure 12]1 is a graph showing the binding affinity of T1.10 and T1.11 to recombinant human FAP by surface plasmon resonance analysis. [Figure 13] 1 is a graph showing the binding affinity of T1.01m and T1.02m to recombinant mouse FAP by surface plasmon resonance analysis. [Figure 14] 1 is a graph showing the binding affinity of T1.03m to recombinant mouse FAP by surface plasmon resonance analysis. [Figure 15] 1 is a graph showing the binding affinity of T1.12, T1.01, T1.02 and T1.03 to HEK293-hFAP cells and HEK293 cells by flow cytometry. [Figure 16] 1 is a graph showing the binding affinity of T1.12m, T1.01m, T1.02m and T1.03m to B16F10-mFAP cells and B16F10 cells by flow cytometry. [Figure 17] 1 is a graph showing the binding affinity of T1.01, T1.02, T1.03 and T1.12 to IL-12 receptor β1 by enzyme-linked immunosorbent assay. [Figure 18] 1 is a graph showing the results obtained by identifying the IL-12 activity of T1.01m, T1.02m, T1.03m and T1.12m via absorbance in HEK Blue reporter cells. [Figure 19] 1 is a graph showing IL-12 activity of T1.01, T1.02, T1.03 and T1.12 through the signaling capacity of human T cells. [Figure 20] FIG. 1 is a graph showing IL-12 activity of T1.01, T1.02 and T1.03 in the presence of low molecular weight heparin (LMWH) through the signaling capacity of human T cells. [Figure 21] Graph showing results obtained by measuring the ability of human T cells to secrete the cytokine IFN-γ by T1.12, T1.01, T1.02 and T1.03. [Figure 22]Graph showing results obtained by measuring the ability of human NK cells to secrete the cytokine IFN-γ by T1.12, T1.01, T1.02 and T1.03. [Diagram 23] This is a graph showing the tumor growth inhibition ability of T1.01m and T1.02m by measuring the change in tumor size in a mouse tumor model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26. [Figure 24] This graph shows the results obtained by measuring the tumor size for each subject to identify the tumor growth inhibitory ability of T1.01m and T1.02m in a mouse tumor model in which mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26 were co-injected. [Diagram 25] This is a graph showing the tumor growth inhibition ability of T1.01m and T1.02m administered at each concentration in a mouse tumor model transplanted with the mouse FAP-overexpressing colon cancer cell line CT26, by measuring the change in tumor size for each subject. [Figure 26] This is a graph showing the change in CT26 tumor size after establishing a tumor re-induction model by transplanting CT26 cells and 4T1 cells into mice, in which a complete response was achieved by T1.01m treatment in a mouse tumor model in which mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26 were co-injected. [Figure 27] This is a graph showing the change in 4T1 tumor size after establishing a tumor re-induction model by transplanting CT26 cells and 4T1 cells into mice, in which a complete response was achieved by T1.01m treatment in a mouse tumor model in which mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26 were co-injected. [Figure 28] This is a graph showing the tumor growth inhibition ability of T1.01m, T1.02m and T1.03m administered to each subject by measuring the tumor size of each subject in a mouse model implanted with mouse FAP-overexpressing CT26 cells. [Figure 29]This is a graph showing the tumor growth inhibition ability for each subject by administering T1.03m at each concentration, by measuring the change in tumor size for each subject in a mouse model implanted with mouse FAP-overexpressing CT26 cells. [Diagram 30] This is a graph showing the tumor growth inhibition ability of T1.03m administered at each concentration by measuring the change in tumor size for each subject in a mouse model implanted with mouse FAP-overexpressing B16F10 cells. [Diagram 31] This is a graph showing the tumor growth inhibition ability of T1.03m administered at each concentration by measuring the change in tumor size for each subject in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and lung cancer cell line LLC1. [Diagram 32] This graph shows the results obtained by measuring tumor size 10 days after administration of T1.16m, T1.17m, T1.03m, or a combination of T1.16m and T1.17m in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and lung cancer cell line LLC1. [Diagram 33] Schematic diagrams of an anti-FAP / IL-12 fusion protein (first from the left; in the figure, the left is an anti-FAP monomer and the right is a monomer containing IL-12), an anti-FAP / IL-12 fusion protein with dual variable domains (second), an anti-FAP / IL-12 dual antibody to which a FAP-specific single-chain variable fragment (scFv) is attached (third and fourth), and an anti-FAP / IL-12 fusion protein in which IL-12 is attached to the C-terminus of the Fc (fifth). [Diagram 34] Schematic diagrams of a FAP-specific Fab / FAP-specific single-chain variable fragment (scFv) and IL-12 fusion protein (left), a fusion protein dimer comprising a FAP-specific Fab and IL-12 (center), and a fusion protein dimer comprising a FAP-specific single-chain variable fragment (scFv) and IL-12 (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Fusion protein comprising IL-12 and anti-FAP antibody In one aspect of the present invention, a fusion protein is provided that comprises IL-12 or a variant thereof and an antigen-binding site that specifically binds to FAP (fibroblast activation protein alpha). In particular, the fusion protein may comprise an immunoglobulin Fc region. Here, the IL-12 or a variant thereof may be a variant having low heparin-binding ability.

[0019] In one embodiment, a fusion protein is provided that comprises a first monomer that comprises IL-12 or a variant thereof and a second monomer that comprises an antigen binding site that specifically binds to a FAP.

[0020] In another embodiment, a fusion protein dimer is provided comprising IL-12 or a variant thereof and an antigen binding site that specifically binds to a FAP.

[0021] IL-12 or its variants As used herein, the term "IL-12" is a heterodimeric cytokine composed of p35 and p40 subunits encoded by two separate genes, IL-12A and IL-12B, respectively. IL-12 is produced by antigen-presenting cells such as macrophages and binds to receptors on the cell surface of activated T cells and NK cells. IL-12 promotes the proliferation of T cells and NK cells and enhances the cytotoxic effect of T cells, NK cells and macrophages. In addition, IL-12 induces the production of IFN-γ, TNF-α and GM-CSF, and acts to induce the activation of Th1 cells. On the other hand, IL-12 binds to the IL-12 receptor, a heterodimeric receptor formed by IL-12Rβ1 and IL-12Rβ2.

[0022] As used herein, the term "variant" of IL-12 includes an amino acid sequence having a similar or identical function to that of the wild-type IL-12 protein. In particular, the amino acid sequences of the p35 and p40 subunits constituting IL-12 may have substitutions, insertions or deletions compared to the wild-type.

[0023] IL-12 or a variant thereof may include IL-12A(p35) or a variant thereof, and IL-12B(p40) or a variant thereof.

[0024] In addition, IL-12 or a variant thereof may include a structure in which IL-12A or a variant thereof; and IL-12B or a variant thereof are linked by a peptide linker.

[0025] In one embodiment, the IL-12 or variant thereof has the following structural formula (I) or (II): N'-Y-[linker (1)]oZ-C' (I) N'-Z-[Linker (1)]oY-C' (II) [In structural formulas (I) and (II), N' may be the N-terminus of the fusion protein; C' may be the C-terminus of the fusion protein; Y may be IL-12A or a variant thereof; Z may be IL-12B or a variant thereof; The linker (1) may be a peptide linker, o may be 0 or 1. It may also be a fusion protein comprising:

[0026] One embodiment of IL-12 or a variant thereof may have a structure in which IL-12B or a variant thereof, a peptide linker, and IL-12A or a variant thereof are bound in this order from the N-terminus. Additionally, one embodiment of IL-12 or a variant thereof may have a structure in which IL-12A or a variant thereof, a peptide linker, and IL-12B or a variant thereof are bound in this order from the N-terminus.

[0027] As used herein, the term "IL-12A" refers to the 35 kDa subunit (p35) of IL-12, which binds via a disulfide bond to the 40 kDa subunit (p40) of IL-12 to produce the active cytokine.

[0028] The amino acid sequence of IL-12A may be that set forth in GenBank Accession No. AAK84425 (for the amino acid sequence of mouse p35, see GenBank Accession No. AAA39292). Additionally, the IL-12A(p35) gene is a sequence encoding the IL-12A subunit, and may be a nucleotide sequence corresponding to the coding sequence (CDS) in the sequence set forth in GenBank Accession No. AF404773 (for the mouse sequence, see GenBank Accession No. M86672).

[0029] As used herein, the term "variant" of IL-12A includes an amino acid sequence having a similar or identical function to that of the wild-type IL-12A protein. In particular, a "variant" of IL-12A means that the amino acid sequence of IL-12A has a substitution, insertion or deletion compared to the wild-type. In particular, an IL-12A variant may be a fragment of IL-12A, and may have an amino acid sequence in which amino acids 1 to 22 of SEQ ID NO: 122 are deleted. More particularly, an IL-12A variant may have the amino acid sequence of SEQ ID NO: 75.

[0030] Additionally, the IL-12A used in one embodiment of the present invention may be the amino acid sequence of SEQ ID NO: 75 as human IL-12A. In one embodiment, mouse IL-12A may be the amino acid sequence of SEQ ID NO: 87.

[0031] As used herein, the term "IL-12B" refers to the 40 kDa subunit (p40) of IL-12, which binds to the 35 kDa subunit (p35) of IL-12 via a disulfide bond to form IL-12, and to the subunit p19 of IL-23 to form IL-23. On the other hand, IL-12B is also called natural killer cell stimulating factor 2.

[0032] The amino acid sequence of "IL-12B" may be that set forth in GenBank Accession No. AAD56386 (for the amino acid sequence of mouse p40, see GenBank Accession No. AAA39296). Additionally, the IL-12B(p40) gene is a sequence encoding the IL-12B subunit, and may be a nucleotide sequence corresponding to the coding sequence (CDS) in the sequence set forth in GenBank Accession No. AF180563 (for the mouse sequence, see GenBank Accession No. M86671).

[0033] Additionally, the IL-12B used in one embodiment of the invention may be human IL-12B, in particular, the IL-12B may have the amino acid sequence of SEQ ID NO: 74. In one embodiment, mouse IL-12B may have the amino acid sequence of SEQ ID NO: 86.

[0034] As used herein, the term "variant" of IL-12B includes an amino acid sequence that has a similar or identical function to that of the wild-type IL-12B protein. In particular, a "variant" of IL-12B means that the amino acid sequence of IL-12B has substitutions, insertions, or deletions compared to the wild-type.

[0035] In particular, the IL-12B variant may be one in which the 1st to 22nd amino acids of SEQ ID NO: 121 have been deleted. More particularly, the IL-12B variant may be one in which 1 to 8 amino acids have been substituted in the sequence of SEQ ID NO: 74. In addition, the IL-12B variant may be one in which the 1st to 22nd amino acids of SEQ ID NO: 123 have been deleted. More particularly, the IL-12B variant may be one in which up to 1 to 8 amino acids have been substituted in the sequence of SEQ ID NO: 86.

[0036] The variant of IL-12B(p40) may have a substitution in an amino acid involved in heparin binding of IL-12.

[0037] In one embodiment, the variant of IL-12B(p40) may be a form in which the lysine (K) binding to heparin in IL-12B is replaced by another amino acid. In particular, the variant of IL-12B(p40) may be a form in which the lysines at positions 258, 260, 263 and 264 of SEQ ID NO: 74 are replaced by other amino acids. In particular, the variant of IL-12B(p40) may comprise an amino acid sequence obtained by at least one substitution selected from the group consisting of K258A, K260A, K263A and K264A in the amino acid sequence of SEQ ID NO: 74. In particular, the variant of IL-12B(p40) may comprise an amino acid sequence obtained by substitution of K258A and K263A in the amino acid sequence of SEQ ID NO: 74 or may further comprise the mutations K260A and / or K264A in the amino acid sequence of SEQ ID NO: 74.

[0038] In one embodiment, the variant of IL-12B(p40) may be a form in which the arginine (R) or lysine (K) binding to heparin in IL-12B is replaced with another amino acid. In particular, the variant of IL-12B(p40) may be a form in which the arginine at position 254 and / or the lysines at positions 255, 256, and 260 of SEQ ID NO: 86 are replaced with other amino acids. In particular, the variant of IL-12B(p40) may comprise an amino acid sequence obtained by at least one substitution selected from the group consisting of R254A, K255A, K256A, and K260A in the amino acid sequence of SEQ ID NO: 86. In particular, the variant of IL-12B(p40) may comprise an amino acid sequence obtained by substitution of R254A and K260A in the amino acid sequence of SEQ ID NO:86 or may further comprise the mutations K255A and / or K256A in the sequence of SEQ ID NO:86.

[0039] In one embodiment, a variant of IL-12B(p40) may comprise the amino acid sequence of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:89 or SEQ ID NO:91.

[0040] In one embodiment, the human IL-12B variant has the following structural formula A: [Structural formula A] X1-L-X2 wherein X1 is the amino acid sequence of SEQ ID NO: 125; X2 is the amino acid sequence of SEQ ID NO: 126, L is V-A1-A2-Q-A3-K * -A4-A5-A6-A7-K * -A8] It may consist of. A1 is R or Q, A2 is V, A or I, and A3 is G or R. * A4 is S, N or K * and A5 is K * , N or E, A6 is R or K, A7 is E, M or T, and A8 is K * Or E. " *At least one amino acid residue marked with " may be substituted with a nonpolar amino acid residue selected from the group consisting of, but not limited to, A, G, I, L, M, F, P, and V.

[0041] In one embodiment, variants of IL-12B(p40) may contain substitutions of amino acids involved in heparin binding to reduce the cytokine-associated toxicity of IL-12 while maintaining its killing effect on cancer cells. FAP and antigen-binding sites that specifically bind to FAP As used herein, the term "FAP (fibroblast activation protein)" refers to fibroblast activation protein, a protein that is expressed on the cell surface and is presented in the environment of tumor cells of various tumor types or in tumor cells of various tumor types. FAP is known to be selectively overexpressed on the surface of cancer-associated fibroblasts (CAFs) in the tumor microenvironment.

[0042] In addition, FAPs are homodimers containing two N-glycosylated subunits with a C-terminal extracellular domain in which the enzyme catalytic domain is located, and the glycosylated forms of FAPs have both post-prolyl dipeptidyl peptidase and gelatinase activities.

[0043] On the other hand, FAP is a prolyl endopeptidase, a membrane-bound gelatinase of about 170 kDa. Prolyl endopeptidase FAP can recognize and cleave a specific amino acid sequence. Therefore, FAP is also called FAPα, separase, or circulating antiplasmin cleavage enzyme.

[0044] The amino acid sequence of FAP may be that set forth in GenBank Accession No. AAC51668, and human FAP was first identified in cultured fibroblasts by use of monoclonal antibody (mAb) F19 (see International Patent Application Publication No. WO93 / 05804). FAP is expressed in many cancers, and due to its restricted expression in normal tissues, it serves as a promising antigenic target for imaging, diagnosis and treatment of various carcinomas.

[0045] FAP includes any variant, isoform, and species homolog of human FAP expressed by cells naturally or in cells transfected with FAP genes. The antibodies of the present invention can bind to FAPs present on the membrane or cell surface of cancer cells (tumor cells or cells in the tumor stroma) and can trigger ADCC or other effector-mediated killing of cancer cells. In addition, the antibodies of the present invention can be used to block FAP enzymatic activity (e.g., serine peptidase, gelatinase, collagenase activity), FAP-mediated ECM degradation, and FAP-mediated cell invasion or migration.

[0046] As used herein, the term "specifically binds" refers to binding that is distinct from non-specific interactions. Specific binding can be determined by competition between the target and a similar control molecule that does not have binding activity.

[0047] As used herein, the term "antigen-binding site (epitope)" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody. An antigen-binding site is a group of molecules that usually have specific structural and charge characteristics, such as amino acids or sugar side chains. An antigen-binding site that specifically binds to a FAP can be a collective term for molecules capable of antigen-antibody binding specifically to a FAP.

[0048] In addition, the antibody or fragment thereof can be used in any form as long as it contains an antigen-binding domain capable of specifically binding to a FAP.

[0049] The antigen-binding site may contain other amino acids that are not directly involved in binding or whose effects are blocked by amino acid residues in the antigen-binding site.

[0050] In particular, the antigen-binding site may be an antibody or a fragment thereof that specifically binds to the FAP.

[0051] As used herein, the term "antibody" refers to a molecule that contains an antigen-binding site and an immunologically active fragment of an immunoglobulin molecule that contains an antigen-binding site. The immunoglobulin molecule may be an immunoglobulin molecule of IgG, IgE, IgM, IgD, IgA, IgY or a subclass thereof. Antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFv (including, e.g., monospecific and bispecific antibodies), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of these antibodies.

[0052] As used herein, the term "antibody fragment" can be a portion of an antibody, e.g., F(ab')2, F(ab)2, Fab', Fab, Fv, scFv. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.

[0053] In one embodiment, an antigen-binding site that specifically binds to a FAP may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 96, an HCDR2 of SEQ ID NO: 97, and an HCDR3 of SEQ ID NO: 98; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 99, an LCDR2 of SEQ ID NO: 100, and an LCDR3 of SEQ ID NO: 101. Here, in one embodiment, an antigen-binding site that specifically binds to a FAP may have a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 103.

[0054] Additionally, in one embodiment, an antigen-binding site that specifically binds to a FAP may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 104, HCDR2 of SEQ ID NO: 105, and HCDR3 of SEQ ID NO: 106; and a light chain variable region comprising LCDR1 of SEQ ID NO: 107, LCDR2 of SEQ ID NO: 108, and LCDR3 of SEQ ID NO: 109. Here, in one embodiment, an antigen-binding site that specifically binds to a FAP may have a heavy chain variable region of SEQ ID NO: 110 and a light chain variable region of SEQ ID NO: 111.

[0055] In one embodiment, the antigen-binding site that specifically binds to the FAP may be an scFv, where the scFv may comprise the heavy and light chain CDRs described above. In one embodiment, the scFv that specifically binds to the FAP may have the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 84.

[0056] In addition, an antigen-binding site that specifically binds to a FAP can include a known anti-FAP antibody or a fragment thereof. An anti-FAP antibody or a fragment thereof can refer to, without limitation, an antibody known to those skilled in the art.

[0057] The anti-FAP antibody or fragment thereof may comprise at least one variable region selected from the group consisting of NG-641, AMG-506 / MP-0310, 28H1 of RG-7827, 4B9 of RG-7827, OMTX-705, 3F2, 4G8, 3D9, 4B3, 19G1, 20G8, 5B8, 5F1, 14B3, 16F1, 16F8, O3C9, 29B11, O2D7, and 23C10.

[0058] In another example, the antibody may be an anti-FAP antibody or a fragment thereof disclosed in Korean Patent Application Publication No. KR20200037826A, U.S. Patent Application Publication No. US2020-0385488A1, U.S. Patent No. US3,924,445B2, U.S. Patent No. US9,011,847B2, or U.S. Patent Application Publication No. US2017-007716A1.

[0059] In one embodiment, the anti-FAP antibody may comprise the variable region of NG-641. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 127, HCDR2 of SEQ ID NO: 128, and HCDR3 of SEQ ID NO: 129, and a light chain variable region comprising LCDR1 of SEQ ID NO: 130, LCDR2 of SEQ ID NO: 131, and LCDR3 of SEQ ID NO: 132. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 248, and a light chain variable region of SEQ ID NO: 249.

[0060] In one embodiment, the anti-FAP antibody may comprise the variable region of 28H1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 133, an HCDR2 of SEQ ID NO: 134, and an HCDR3 of SEQ ID NO: 135, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 136, an LCDR2 of SEQ ID NO: 137, and an LCDR3 of SEQ ID NO: 138. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 250, and a light chain variable region of SEQ ID NO: 251.

[0061] In one embodiment, the anti-FAP antibody may comprise the variable region of 4B9. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 139, an HCDR2 of SEQ ID NO: 140, and an HCDR3 of SEQ ID NO: 141, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 142, an LCDR2 of SEQ ID NO: 143, and an LCDR3 of SEQ ID NO: 144. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 252, and a light chain variable region of SEQ ID NO: 253.

[0062] In one embodiment, the anti-FAP antibody may comprise the variable region of OMTX-705. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 145, an HCDR2 of SEQ ID NO: 146, and an HCDR3 of SEQ ID NO: 147, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 148, an LCDR2 of SEQ ID NO: 149, and an LCDR3 of SEQ ID NO: 150. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 254, and a light chain variable region of SEQ ID NO: 255.

[0063] In one embodiment, the anti-FAP antibody may comprise the variable region of 3F2. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 151, HCDR2 of SEQ ID NO: 152, and HCDR3 of SEQ ID NO: 153, and a light chain variable region comprising LCDR1 of SEQ ID NO: 154, LCDR2 of SEQ ID NO: 155, and LCDR3 of SEQ ID NO: 156. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 256, and a light chain variable region of SEQ ID NO: 257.

[0064] In one embodiment, the anti-FAP antibody may comprise the variable region of 4G8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 157, HCDR2 of SEQ ID NO: 158, and HCDR3 of SEQ ID NO: 159, and a light chain variable region comprising LCDR1 of SEQ ID NO: 160, LCDR2 of SEQ ID NO: 161, and LCDR3 of SEQ ID NO: 162. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 258, and a light chain variable region of SEQ ID NO: 259.

[0065] In one embodiment, the anti-FAP antibody may comprise the variable region of 3D9. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 163, HCDR2 of SEQ ID NO: 164, and HCDR3 of SEQ ID NO: 165, and a light chain variable region comprising LCDR1 of SEQ ID NO: 166, LCDR2 of SEQ ID NO: 167, and LCDR3 of SEQ ID NO: 168. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 260, and a light chain variable region of SEQ ID NO: 261.

[0066] In one embodiment, the anti-FAP antibody may comprise the variable region of 4B3. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 169, HCDR2 of SEQ ID NO: 170, and HCDR3 of SEQ ID NO: 171, and a light chain variable region comprising LCDR1 of SEQ ID NO: 172, LCDR2 of SEQ ID NO: 173, and LCDR3 of SEQ ID NO: 174. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 262, and a light chain variable region of SEQ ID NO: 263.

[0067] In one embodiment, the anti-FAP antibody may comprise the variable region of 19G1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 175, an HCDR2 of SEQ ID NO: 176, and an HCDR3 of SEQ ID NO: 177, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 178, an LCDR2 of SEQ ID NO: 179, and an LCDR3 of SEQ ID NO: 180. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 264, and a light chain variable region of SEQ ID NO: 265.

[0068] In one embodiment, the anti-FAP antibody may comprise the variable region of 20G8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 181, HCDR2 of SEQ ID NO: 182, and HCDR3 of SEQ ID NO: 183, and a light chain variable region comprising LCDR1 of SEQ ID NO: 184, LCDR2 of SEQ ID NO: 185, and LCDR3 of SEQ ID NO: 186. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 266, and a light chain variable region of SEQ ID NO: 267.

[0069] In one embodiment, the anti-FAP antibody may comprise the variable region of 5B8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 187, HCDR2 of SEQ ID NO: 188, and HCDR3 of SEQ ID NO: 189, and a light chain variable region comprising LCDR1 of SEQ ID NO: 190, LCDR2 of SEQ ID NO: 191, and LCDR3 of SEQ ID NO: 192. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 268, and a light chain variable region of SEQ ID NO: 269.

[0070] In one embodiment, the anti-FAP antibody may comprise the variable region of 5F1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 193, an HCDR2 of SEQ ID NO: 194, and an HCDR3 of SEQ ID NO: 195, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 196, an LCDR2 of SEQ ID NO: 197, and an LCDR3 of SEQ ID NO: 198. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 270, and a light chain variable region of SEQ ID NO: 271.

[0071] In one embodiment, the anti-FAP antibody may comprise the variable region of 14B3. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 199, an HCDR2 of SEQ ID NO: 200, and an HCDR3 of SEQ ID NO: 201, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 202, an LCDR2 of SEQ ID NO: 203, and an LCDR3 of SEQ ID NO: 204. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 272, and a light chain variable region of SEQ ID NO: 273.

[0072] In one embodiment, the anti-FAP antibody may comprise the variable region of 16F1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:205, an HCDR2 of SEQ ID NO:206, and an HCDR3 of SEQ ID NO:207, and a light chain variable region comprising an LCDR1 of SEQ ID NO:208, an LCDR2 of SEQ ID NO:209, and an LCDR3 of SEQ ID NO:210. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:274, and a light chain variable region of SEQ ID NO:275.

[0073] In one embodiment, the anti-FAP antibody may comprise the variable region of 16F8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO:211, HCDR2 of SEQ ID NO:212, and HCDR3 of SEQ ID NO:213, and a light chain variable region comprising LCDR1 of SEQ ID NO:214, LCDR2 of SEQ ID NO:215, and LCDR3 of SEQ ID NO:216. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:276, and a light chain variable region of SEQ ID NO:277.

[0074] In one embodiment, the anti-FAP antibody may comprise the variable region of O3C9. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO:217, HCDR2 of SEQ ID NO:218, and HCDR3 of SEQ ID NO:219, and a light chain variable region comprising LCDR1 of SEQ ID NO:220, LCDR2 of SEQ ID NO:221, and LCDR3 of SEQ ID NO:222. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:278, and a light chain variable region of SEQ ID NO:279.

[0075] In one embodiment, the anti-FAP antibody may comprise the variable region of 22A3. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 223, HCDR2 of SEQ ID NO: 224, and HCDR3 of SEQ ID NO: 225, and a light chain variable region comprising LCDR1 of SEQ ID NO: 226, LCDR2 of SEQ ID NO: 227, and LCDR3 of SEQ ID NO: 228. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 280, and a light chain variable region of SEQ ID NO: 281.

[0076] In one embodiment, the anti-FAP antibody may comprise the variable region of 29B11. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:229, an HCDR2 of SEQ ID NO:230, and an HCDR3 of SEQ ID NO:231, and a light chain variable region comprising an LCDR1 of SEQ ID NO:232, an LCDR2 of SEQ ID NO:233, and an LCDR3 of SEQ ID NO:234. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:282, and a light chain variable region of SEQ ID NO:283.

[0077] In one embodiment, the anti-FAP antibody may comprise the variable region of O2D7. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 235, HCDR2 of SEQ ID NO: 236, and HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising LCDR1 of SEQ ID NO: 238, LCDR2 of SEQ ID NO: 239, and LCDR3 of SEQ ID NO: 240. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 284, and a light chain variable region of SEQ ID NO: 285.

[0078] In one embodiment, the anti-FAP antibody may comprise the variable region of 23C10. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:241, an HCDR2 of SEQ ID NO:242, and an HCDR3 of SEQ ID NO:243, and a light chain variable region comprising an LCDR1 of SEQ ID NO:244, an LCDR2 of SEQ ID NO:245, and an LCDR3 of SEQ ID NO:246. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:286, and a light chain variable region of SEQ ID NO:287.

[0079] In one embodiment, the anti-FAP antibody may be AMG-506 / MP-0310. In particular, the antibody may comprise the amino acid sequence of SEQ ID NO:247.

[0080] Any anti-FAP antibody can be used as long as it can specifically bind to FAP present in cancer cells and induce the death of the cancer cells. Structure of the first monomer The first monomer comprises IL-12 or a variant thereof.

[0081] The first monomer may further comprise an antigen binding site that specifically binds to the FAP.

[0082] In one embodiment, the first monomer may comprise IL-12 or a variant thereof and an Fc region fragment or a variant thereof.

[0083] In one embodiment, the first monomer may comprise IL-12 or a variant thereof, an Fc region fragment or a variant thereof, and an antigen binding site that specifically binds to a FAP.

[0084] The first monomer has the following structural formula (III) or (IV): N'-X-[linker (2)]p-Fc region fragment or variant thereof-[linker (3)]q-(T)r-C' (III) N'-(T)r-[linker (2)]q-Fc region fragment or variant thereof-[linker (3)]pX-C' (IV) [In structural formulas (III) and (IV), N' may be the N-terminus of the fusion protein; C' may be the C-terminus of the fusion protein; X may be of structural formula (I) or (II), T can be an antigen-binding site that specifically binds to a FAP, The linkers (2) and (3) may be peptide linkers; p, q and r may each independently be 0 or 1. may include.

[0085] In one embodiment, when r is 0, the first monomer may be in the form of a fusion protein in which IL-12 or a variant thereof and an Fc region fragment or a variant thereof are linked by a peptide linker.

[0086] Here, the first monomer may comprise the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:32.

[0087] Here, when r is 1 in structural formula (III), the first monomer may be in a form in which an anti-FAP antibody monomer is bound to IL-12 or a variant thereof.

[0088] Here, in structural formula (III), when r is 1, the first monomer may include the amino acid sequence of SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:302, or SEQ ID NO:303.

[0089] Here, in structural formula (IV), when r is 1, the first monomer may comprise the amino acid sequence of SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300, or SEQ ID NO:301.

[0090] In one embodiment, when r is 1, structural formula (III) is represented by the following structural formulas (III') and (III"): N'-X-[linker (2)]p-Fc region fragment or variant thereof-[linker (3)]q-(T')-C' (III') N'-(T")-C' (III") [In structural formulas (III') and (III"), T' is a heavy chain region of an antibody that specifically binds to a FAP, or a light chain region of an antibody, comprising a variable region and a CH1 region; T" is a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including the variable region and the CH1 region; wherein T' and T" bind to each other to form a variable region of an antibody, the variable region specifically binds to a FAP; The linkers (2) and (3) are peptide linkers, p and q are each independently 0 or 1; N', X and C' are as defined above. may include. In one embodiment, when r is 1, structural formula (IV) has the following structural formulas (IV') and (IV"): N'-(T')-[linker (2)]q-Fc region fragment or variant thereof-[linker (3)]pX-C' (IV'); and N'-(T")-C' (IV") [In structural formulas (IV') and (IV"), T' is a heavy chain region of an antibody that specifically binds to a FAP, or a light chain region of an antibody, comprising a variable region and a CH1 region; T" is a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including the variable region and the CH1 region; wherein T' and T" bind to each other to form a variable region of an antibody, the variable region specifically binds to a FAP; The linkers (2) and (3) are peptide linkers, p and q are each independently 0 or 1; N', X and C' are as defined above. may include.

[0091] Here, the first monomer may comprise the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:2; SEQ ID NO:11 and SEQ ID NO:2; SEQ ID NO:27 and SEQ ID NO:19; SEQ ID NO:28 and SEQ ID NO:19; SEQ ID NO:294 and SEQ ID NO:2; SEQ ID NO:295 and SEQ ID NO:2; SEQ ID NO:300 and SEQ ID NO:19; or SEQ ID NO:301 and SEQ ID NO:19. Structure of the second monomer The second monomer may further comprise an antigen binding site that specifically binds to the FAP.

[0092] In one embodiment, the second monomer may comprise a first FAP binding site and a second FAP binding site.

[0093] The antigen-binding site that specifically binds to a FAP may be a Fab, scFv, Fv, or fragments thereof, where a first FAP-binding site may be a Fab and a second FAP-binding site may be an Fv or scFv.

[0094] The second FAP binding site may be attached to the N-terminus or C-terminus of the second monomer. In particular, the second FAP binding site may additionally be attached to the C-terminus of the heavy chain, the C-terminus of the light chain, or the N-terminus of the variable region.

[0095] The second monomer has the following structural formula (V): N'-(R)s-[linker (4)]tQ-[linker (5)]u-Fc region fragment or variant thereof-[linker (6)]v-(W)a-C' (V) [In structural formula (V), N' may be the N-terminus of the fusion protein; C' may be the C-terminus of the fusion protein; R and Q may be antigen binding sites that specifically bind to the FAP; W may be an scFv that specifically binds to a FAP; or IL-12 of structural formula (I) or (II) or a variant thereof, each of the linkers (4), (5) and (6) may be a peptide linker; s, t, u, v and a may each independently be 0 or 1. may include.

[0096] In one embodiment, structural formula (V) may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300, and SEQ ID NO:301.

[0097] In one embodiment, structural formula (V) has the following structural formulas (V') and (V"): N'-(R')s-[linker (4)]t-Q'-[linker (5)]u-Fc domain fragment or variant thereof-[linker (6)]p-(W)a-C' (V') N'-(R")s-[linker (4)]tQ"-[linker (7)]x-(W)b-C' (V") [In structural formulas (V') and (V"), R' may be a heavy chain region of an antibody that specifically binds to a FAP, including a variable region and a CH1 region, or a light chain region of an antibody, R″ can be a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including the variable region and the CH1 region; wherein R′ and R″ may be combined together to form a variable region of an antibody, the variable region specifically binding to a FAP; Q' can be a heavy chain region of an antibody that specifically binds to a FAP, including a variable region and a CH1 region, or a light chain region of an antibody, Q″ can be a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including a variable region and a CH1 region; wherein Q′ and Q″ may be linked together to form a variable region of an antibody, the variable region specifically binding to a FAP; W may be an scFv that specifically binds to a FAP; or IL-12 of structural formula (I) or (II) or a variant thereof, each of the linkers (4), (5), (6) and (7) may be a peptide linker; s, t, u, x, a, and b may each independently be 0 or 1. may include.

[0098] In one embodiment, structural formula (V') may be SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300, or SEQ ID NO:301.

[0099] In one embodiment, structural formula (V") may be SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:24, or SEQ ID NO:26.

[0100] In one embodiment, the second monomer may comprise a heavy chain consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain consisting of the amino acid sequence of SEQ ID NO: 103; or a heavy chain consisting of the amino acid sequence of SEQ ID NO: 110 and a light chain consisting of the amino acid sequence of SEQ ID NO: 111.

[0101] In addition, when W is an scFv, W may have the amino acid sequence of SEQ ID NO:72 or SEQ ID NO:84.

[0102] Peptide Linker As used herein, the term "peptide linker" refers to a peptide used to provide physicochemical distance or connect domains in a fusion protein. The linker may include an immunoglobulin hinge region.

[0103] The peptide linkers (1) to (7) refer to peptide linkers composed of amino acids. In particular, the peptide linkers (2) and (5) may be composed of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, 10 to 60 consecutive amino acids, or 12 to 50 amino acids. The peptide linker may include (G4S)n (wherein n is an integer of 1 to 10). Here, in (G4S)n, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively.

[0104] In one embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO:93, SEQ ID NO:94 or SEQ ID NO:95.

[0105] The peptide linker (2) or (5) may consist of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, or 10 to 60 consecutive amino acids, or 12 to 50 amino acids. In one embodiment, the peptide linker (2) may consist of 30 amino acids. In addition, the peptide linker (2) may contain at least one cysteine. In particular, the peptide linker (2) may contain one, two or three cysteines. In addition, the peptide linker (2) may be derived from an immunoglobulin hinge and may further contain (G4S)n (wherein n is an integer of 1 to 10). In particular, the peptide linker (2) may contain a hinge region consisting of any one of the amino acid sequences of SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120.

[0106] In addition, the peptide linkers (3), (4), (6) and (7) may consist of 1 to 30 consecutive amino acids, 5 to 20 consecutive amino acids, or 10 to 15 consecutive amino acids. The peptide linker may comprise (G4S)n, where n is an integer from 1 to 10. Here, in (G4S)n, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, respectively.

[0107] In addition, in particular, the peptide linker (2), (3) or (5) may comprise SEQ ID NO:93 or SEQ ID NO:94.

[0108] In one embodiment of the invention, the peptide linker (2) or (5) may be a peptide linker comprising the amino acid sequence of SEQ ID NO: 118 or 120. Fc region or a fragment thereof Here, the immunoglobulin fragment may be an Fc region of an immunoglobulin. The Fc region of an immunoglobulin may be a wild-type Fc domain or an Fc domain variant. Here, the Fc region may be an Fc region of IgG, IgA, IgE, IgD or IgM. In particular, the Fc region may be derived from IgG1 or IgG2a.

[0109] As used herein, the term "Fc domain variant" may refer to a form that differs from a wild-type Fc domain in terms of glycosylation pattern, has higher glycosylation compared to the wild-type Fc domain, has lower glycosylation compared to the wild-type Fc domain, or has a deglycosylated form. In addition, "Fc domain variant" includes a non-glycosylated Fc domain. The Fc domain or its variants may be configured to have a regulated number of sialic acids, fucosylation, or glycosylation by host culture conditions or genetic engineering.

[0110] In addition, the glycosylation of the Fc domain of an immunoglobulin can be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a mixed form of the Fc region of each of the immunoglobulins IgG, IgA, IgE, IgD, or IgM. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are replaced with other amino acids.

[0111] The "amino acid" introduced by substitution and / or addition may be any one selected from the group consisting of lysine (K), alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0112] In addition, the Fc region may comprise a knob or hole structure.

[0113] As used herein, the term "knobs-into-holes" is a design strategy for producing antibodies that specifically bind to different regions, such as bispecific, multispecific, or heterodimeric antibodies. In general, this technique involves introducing a knob at the interface of a first polypeptide (e.g., a first CH3 domain of a first antibody heavy chain) and a corresponding hole at the interface of a second polypeptide (e.g., a second CH3 domain of a second antibody heavy chain) such that the knob can be positioned within the hole to promote heterodimer formation and prevent homodimer formation.

[0114] A "knob" is constructed by replacing a small amino acid side chain from the interface of a first polypeptide (e.g., the first CH3 domain of a first antibody heavy chain) with a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A complementary "hole" of the same or similar size in the knob is created by replacing a large amino acid side chain from the interface of a second polypeptide (e.g., the second CH3 domain of a second antibody heavy chain) with a smaller side chain (e.g., alanine, serine, valine, or threonine). Knobs and holes can be created by altering the nucleic acid encoding the polypeptide, for example by site-directed mutagenesis or peptide synthesis.

[0115] In one embodiment, the knob structure of IgG1 may be SEQ ID NO: 13 or 288, and the hole structure may be SEQ ID NO: 12 or 289. The knob structure or hole structure may be in a form in which the amino acids at positions 146, 148, and 187 of the amino acid sequence of SEQ ID NO: 304 are substituted with other amino acids. In particular, in one embodiment of the present invention, the knob structure or hole structure may be in a form in which the amino acid sequence of SEQ ID NO: 304 is substituted with T146W, T146S, L148A, and Y187A. In particular, the knob structure may be in a form in which the amino acid sequence of SEQ ID NO: 304 is substituted with T146W, and the hole structure may be in a form in which the amino acid sequence of SEQ ID NO: 304 is substituted with T146S, L148A, and Y148V.

[0116] On the other hand, in one embodiment of the present invention, the knob structure of IgG2a may be SEQ ID NO: 30 or 290, and the hole structure may be SEQ ID NO: 29 or 291. The knob structure or the hole structure may be in a form in which the amino acids at positions 152, 154, and 193 of the amino acid sequence of SEQ ID NO: 305 are substituted with other amino acids. In particular, in one embodiment of the present invention, the knob structure or the hole structure may be in a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152W or T152S, M154A, and Y193V. In particular, the knob structure may be in a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152W, and the hole structure may be in a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152S, M154A, and Y193V.

[0117] In one embodiment, the Fc domain variant may comprise a DANG mutation or an NG mutation, where "DANG mutation" refers to the D265A / N297G mutation to eliminate effector function in human IgG1 or mouse IgG2a.

[0118] Effector functions mediated by the Fc region in IgG molecules include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), etc. Generally, these effector functions require association of the Fc region with a binding domain (e.g., an antibody variable domain).

[0119] Effector functions can be altered by substitutions in the amino acid sequence of the non-mutated Fc region, and Fc regions with altered effector functions can be designed, for example, by modifying C1q binding and / or FcR binding, thereby altering CDC and / or ADCC activity. Thus, "DANG mutation" means that the effector functions mediated by the Fc region are removed from the IgG molecule, such that undesired effector functions do not arise during antibody production.

[0120] In one embodiment, the DANG mutation may be in the form of a D45A / N77G substitution of amino acids in human IgG1 of SEQ ID NO: 289. Additionally, the DANG mutation may be in the form of a D51A / N83G substitution of amino acids in mouse IgG2a of SEQ ID NO: 291. Fusion protein structure The fusion protein may be an antibody. In particular, the antibody may be a heterodimeric antibody that includes a knob-into-hole structure.

[0121] The fusion protein can comprise structural formula (III) and structural formula (V); or structural formula (IV) and structural formula (V). More specifically, one embodiment of the fusion protein may comprise a first monomer of (i) or (ii) below, and a second monomer of (iii), (iv), (v) or (vi) below: Example of the first monomer: (i) When r is 0, the structural formula (III) (ii) When r is 1, the structural formula (IV) Examples of second monomers: (iii) When s, a, and b are 0, the structural formula (V') and the structural formula (V") are (iv) When s is 1 and a and b are 0, the structural formula (V') and the structural formula (V") (v) When s and b are 0 and a is 1, the structural formula (V') and the structural formula (V") (vi) When b is 1, and s and a are 0, Structural Formula (V') and Structural Formula (V").

[0122] In one embodiment, the fusion protein may include a first monomer including the above (i) and a second monomer including the above (iii) (first from the left in FIG. 33). Here, the fusion protein may include at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22. In detail, the fusion protein may include SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5; SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:21; or SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:22.

[0123] In one embodiment, the fusion protein may comprise a first monomer comprising the above (i) and a second monomer comprising the above (iv) (second from the left in FIG. 33). Here, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24. In particular, the fusion protein may comprise SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:20, SEQ ID NO:23 and SEQ ID NO:24; or SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0124] In one embodiment, the fusion protein may comprise a first monomer comprising the above (i) and a second monomer comprising the above (v) (third from the left in FIG. 33). Here, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:25. In particular, the fusion protein may comprise SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:8; SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8; SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:25; or SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:25.

[0125] In one embodiment, the fusion protein may comprise a first monomer comprising the above (i) and a second monomer comprising the above (vi) (fourth from the left in FIG. 33). Here, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:26. In particular, the fusion protein may comprise SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:9; SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:9; SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:26; or SEQ ID NO:18, SEQ ID NO:22 and SEQ ID NO:26.

[0126] In one embodiment, the fusion protein may include a first monomer including the above (ii) and a second monomer including the above (iii) (fifth from the left in FIG. 33). Here, the fusion protein may include at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:27, and SEQ ID NO:28. In detail, the fusion protein may include SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:10; SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:11; SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:27; or SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:28.

[0127] In addition, in one embodiment, the fusion protein dimer comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to a FAP may be a fusion protein dimer comprising a monomer of structural formula (III) and a monomer of structural formula (V) (left side of FIG. 34 ), where r in structural formula (III) is 1, and s, a, and t in structural formula (V) are 0. Here, structural formula (V) may comprise (V′) and (V″), where s, a, t, and b in (V′) and (V″) are 0. In addition, the Fc region of the dimer has a knobs-into-holes structure that allows different fusion proteins to bind to each other.

[0128] In particular, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:298 and SEQ ID NO:299. In particular, the fusion protein may comprise SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:292; SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:293; SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:298; or SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:299.

[0129] In addition, the fusion protein dimer comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to FAP may be a fusion protein dimer comprising the structure of structure (V) (center of FIG. 34 ). Here, the fusion protein dimer may comprise (V') and (V"), where in structural formula (V), s and t are 0, and a is 1. Additionally, in (V') and (V"), s, t and b are 0, and a is 1. Additionally, the fusion protein dimer may be a fusion protein dimer comprising the structure of structural formula (IV). Here, in structural formula (IV), r is 1.

[0130] In particular, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:19, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300 and SEQ ID NO:301. In particular, the fusion protein may comprise SEQ ID NO:2 and SEQ ID NO:294; SEQ ID NO:2 and SEQ ID NO:295; SEQ ID NO:19 and SEQ ID NO:300; or SEQ ID NO:19 and SEQ ID NO:301.

[0131] In one embodiment, the fusion protein dimer comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to FAP may be a fusion protein dimer comprising the structure of structural formula (III) (right side of FIG. 34), where r in structural formula (III) is 1.

[0132] In particular, the fusion protein may comprise SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:302 or SEQ ID NO:303.

[0133] The multispecific fusion protein herein may be in a chemically modified form. In one embodiment, the fusion protein can be chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage and / or binding to cellular ligands or other proteins. Many of these chemical modifications can be performed by known techniques. Polynucleotides encoding fusion proteins In another aspect of the invention, there is provided a polynucleotide encoding the first monomer or a polynucleotide encoding the second monomer.

[0134] In one embodiment, the first monomeric polypeptide may comprise an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, or SEQ ID NO:303.

[0135] In one embodiment, the polynucleotide encoding the first monomer may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:67.

[0136] In one embodiment, the second monomeric polypeptide may comprise an amino acid sequence having about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300, or SEQ ID NO:301.

[0137] In one embodiment, the polynucleotide encoding the second monomer may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:66.

[0138] The polynucleotide may further comprise a nucleic acid encoding a signal sequence or leader sequence. As used herein, the term "signal sequence" refers to a signal peptide that directs the secretion of a target protein. The signal peptide is translated and then cleaved in the host cell. In particular, a signal sequence is an amino acid sequence that initiates the transport of a protein across the endoplasmic reticulum (ER) membrane.

[0139] Signal sequences are well known in the art for their characteristics. Such signal sequences typically contain 16-30 amino acid residues, but may contain more or fewer than these amino acid residues. A typical signal peptide is composed of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4-12 hydrophobic residues that anchor the signal sequence during transport of the immature polypeptide through the membrane lipid bilayer.

[0140] After initiation, the signal sequence is cleaved in the lumen of the ER by a cellular enzyme commonly known as a signal peptidase. Here, the signal sequence may be a secretory signal sequence of tPa (tissue plasminogen activator), HSV gDs (signal sequence of herpes simplex virus glycoprotein D), or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells, including mammals, can be used. In addition, a wild-type signal sequence can be used, or a signal sequence substituted with a codon having a high expression frequency in a host cell can be used.

[0141] Vectors Containing Polynucleotides In another aspect of the invention, a vector comprising the polynucleotide is provided.

[0142] A vector can be introduced into a host cell and recombine and insert into the genome of the host cell. Alternatively, a vector is understood to be a nucleic acid vehicle that contains a polynucleotide sequence that can replicate autonomously as an episome. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0143] In particular, the vector may include plasmid DNA, phage DNA, etc.; and commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.), and insect virus vectors (baculovirus, etc.). Since vectors show different protein expression levels and modifications depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.

[0144] As used herein, the term "gene expression" or "expression" of a target protein is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or a fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may contain a human cytomegalovirus (CMV) promoter to promote continuous transcription of the target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase the level of stability of the RNA after transcription. Transformed cells expressing the fusion protein In another aspect of the present invention, there is provided a transformed cell into which the vector has been introduced.

[0145] The host cell of the transformed cell may include, but is not limited to, prokaryotic cells, eukaryotic cells, and cells of mammalian, plant, insect, fungal, or bacterial origin. As an example of a prokaryotic cell, E. coli may be used. In addition, as an example of a eukaryotic cell, yeast may be used. In addition, for mammalian cells, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, HEK293T cells, etc. may be used. However, the mammalian cells are not limited thereto, and any cell known to those skilled in the art to be usable as a mammalian host cell may be used.

[0146] Additionally, CaCl2 precipitation, the Hanahan method with its increased efficiency due to the use of a reducing agent such as dimethylsulfoxide (DMSO) in CaCl2 precipitation, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, transformation using PEG, dextran sulfate, lipofectamine, or desiccation / inhibition mediated transformation can be used to introduce an expression vector into a host cell.

[0147] As described above, in order to optimize the properties of the fusion protein as a therapeutic agent or for any other purpose, the glycosylation pattern of the fusion protein (e.g., sialic acid, fucosylation, glycosylation) can be adjusted by manipulating glycosylation-related genes carried by the host cell by methods known to those skilled in the art. Methods for Producing Fusion Proteins In another aspect of the invention, there is provided a method for producing a fusion protein, the method comprising the steps of i) culturing a transformed cell; and ii) recovering a fusion protein comprising a first monomer and a second monomer.

[0148] The method of culturing the transformed cells can be carried out using methods well known in the art. In particular, the culturing can be carried out in a batch process or continuously in a fed-batch or repeated fed-batch process. Use of fusion proteins In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising the fusion protein as an active ingredient.

[0149] Here, the cancer may be any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0150] The preferred dose of the pharmaceutical composition varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing tumors of the present invention, the active ingredient can be contained in any amount (effective amount) depending on the use, dosage form, purpose of formulation, etc., as long as the active ingredient can exhibit activity to treat tumors or, in particular, therapeutic effect against cancer. The usual effective amount of the active ingredient is determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, the term "effective amount" refers to the amount of active ingredient capable of inducing an effect of improving or treating the condition of a disease, in particular, an effect of improving or treating the condition of a cancer. Such an effective amount can be determined experimentally within the scope of common sense of a person skilled in the art.

[0151] As used herein, the term "treatment" can be used to mean both therapeutic and prophylactic treatment. Here, prevention can be used to mean that a condition or disease in a subject is alleviated or ameliorated. In one embodiment, the term "treatment" includes both application or any form of administration to treat a disease in a mammal, including a human. In addition, the term "treatment" includes inhibiting or slowing the progression of a disease, restoring or repairing a decreased or lost function such that the disease is partially or completely alleviated; stimulating an inefficient process; or reducing the severity of the disease.

[0152] Pharmacokinetic parameters such as bioavailability and fundamental parameters such as clearance rate can also affect efficacy. Thus, "enhanced efficacy" (e.g., improved efficacy) can result from enhanced pharmacokinetic parameters and enhanced efficacy, which can be measured by comparing parameters such as clearance rate and tumor treatment or improvement in test animals or human subjects.

[0153] As used herein, the term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount of a compound or composition effective to prevent or treat the disease in question, sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatments and without causing side effects. The level of the effective amount can be determined by factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the mode of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, co-administered or co-used drugs, and other factors well known in the medical art. In one embodiment, a therapeutically effective amount refers to an amount of drug effective to treat cancer.

[0154] Here, the pharmaceutical composition may further include a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fat, wax, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0155] In particular, by including a pharma- ceutical composition in addition to the active ingredient, a pharma- ceutical composition can be prepared into a parenteral formulation by using a conventional method known in the art depending on the administration route. Here, the term "pharma- ceutical acceptable" means that the carrier does not inhibit the activity of the active ingredient and at the same time does not have a toxicity greater than that which can be accommodated by the subject to which it is applied (prescribed).

[0156] When the pharmaceutical composition is prepared into a parenteral preparation, the parenteral preparation can be prepared in the form of injection, transdermal patch, nasal inhalant, or suppository using a suitable carrier according to a method known in the art.When it is made into an injection, sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof can be used as a suitable carrier, and isotonic liquid such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, and 5% dextrose can be preferably used.The formulation of pharmaceutical compositions is known in the art, and can be referred to Remington's Pharmaceutical Sciences (19th edition, 1995) for details.The same document is considered to be a part of this specification.

[0157] The preferred dose of the pharmaceutical composition may range from 0.01 μg / kg to 10 g / kg, or from 0.01 mg / kg to 1 g / kg per day, depending on the patient's condition, body weight, sex, age, severity of the patient, and route of administration. The dose can be administered once a day or divided into several doses per day. Such doses should not be considered as limiting the scope of the present invention in any way.

[0158] The subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, with humans being particularly preferred. In addition to the active ingredient, the pharmaceutical composition of the present application may further contain any compound or natural extract known to have a therapeutic effect on tumors.

[0159] In another aspect of the invention, there is provided a use of a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to a FAP, for treating cancer.

[0160] In another aspect of the invention, there is provided a use of a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen-binding site that specifically binds to a FAP, for the manufacture of a medicament for treating cancer.

[0161] In yet another aspect of the invention, there is provided a method for treating or preventing cancer, comprising administering to a subject a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to a FAP.

[0162] Here, the subject may be a subject suffering from cancer. In addition, the subject may be a mammal, preferably a human.

[0163] The administration route, dosage, and administration frequency of the fusion protein or fusion protein dimer may vary depending on the patient's condition and the presence or absence of side effects, so the fusion protein or fusion protein dimer can be administered to the subject in various ways and amounts.The optimal administration method, dosage, and administration frequency can be selected within an appropriate range by those skilled in the art.In addition, the fusion protein or fusion protein dimer can be administered in combination with other drugs or bioactive substances known to have therapeutic effects on the disease to be treated, or can be formulated in the form of a combined preparation with other drugs. EXAMPLES

[0164] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to the examples.

[0165] Preparation Example 1. Overview of anti-FAP / IL-12 IgG1 DANG, human fusion protein

[0166] [Table 1] [seq1] consists of the human anti-FAP heavy chain variable region sequence and human IgG1 Fc, with the effector function eliminated by DANG mutations (D265A, N297G) and the knob structure formed by the T366W mutation.

[0167] [seq2] consists of the human anti-FAP light chain sequence.

[0168] [seq3] consists of human IL-12 p40 (beta) region sequence and linker GGGGSGGGGSGGGGS, human interleukin-12 p35 (alpha) region sequence and linker GGGGSGGGGS, and human IgG1 Fc, with effector function eliminated by DANG mutations (D265A, N297G) and hole structure formed by T366S, L368A and Y407V mutations.

[0169] [seq4] consists of a sequence in which the lysine (K) at positions 280 and 285, which are involved in heparin binding in the human IL-12 p40 (beta) region, is mutated to alanine (A), the linker GGGGSGGGSGGGGGS, the human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and human IgG1 Fc hole DANG.

[0170] [seq5] consists of a sequence in which the lysine (K) at amino acids 280, 282, 285, and 286 involved in heparin binding in the human IL-12 p40 (beta) region is mutated to alanine (A), the linker GGGGSGGGGSGGGGS, the human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and human IgG1 Fc hole DANG.

[0171] [seq6] consists of the human anti-FAP heavy chain variable region sequence, linker GGGGSGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, and human IgG1 Fc knob DANG.

[0172] [seq7] consists of the human anti-FAP light chain variable region sequence, linker GGGGSGGGSGGGGGS, and the human anti-FAP light chain sequence.

[0173] [seq8] consists of the human anti-FAP heavy chain variable region sequence, human IgG1 Fc knob DANG, linker GGGGSGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0174] [seq9] consists of a human anti-FAP light chain sequence, a linker GGGGSGGGSGGGGGS, a human anti-FAP heavy chain variable region sequence, a linker GGGGSGGGGSGGGGSGGGGS, and a human anti-FAP light chain variable region sequence.

[0175] [seq10] is composed of a human anti-FAP heavy chain sequence, a human IgG1 Fc hole DANG, a linker GGGSGGGGSGGGGGS, a p40 (beta) region sequence of human IL-12, a linker GGGGSGGGGSGGGGS, and a human IL-12 p35 (alpha) region sequence.

[0176] [seq11] consists of the human anti-FAP heavy chain sequence, human IgG1 Fc hole DANG, linker GGGSGGGGSGGGGGS, human IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, and human IL-12 p35 (alpha) domain sequence.

[0177] [seq12] is composed only of human IgG1 Fc hole DANG.

[0178] [seq13] is composed only of human IgG1 Fc knob DANG.

[0179] [seq14] consists of the human anti-FAP heavy chain variable region sequence and human IgG1 Fc DANG.

[0180] [seq15] consists of the p40 (beta) domain sequence of human IL-12 containing four amino acid mutations involved in heparin binding, the linker GGGGSGGGSGGGGGS, the human IL-12 p35 (alpha) domain sequence, the linker GGGGSGGGGS, and human IgG1 Fc DANG.

[0181] [seq16] is composed of the heavy chain sequence of a human anti-CD20 antibody and a human IgG1 Fc knob DANG.

[0182] [seq17] is the light chain sequence of a human anti-CD20 antibody.

[0183] [seq292] is composed of human IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and human IgG1 Fc hole DANG, linker GGGGSGGGGSGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0184] [seq293] is composed of human IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and human IgG1 Fc hole DANG, linker GGGSGGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0185] [seq294] is composed of human anti-FAP heavy chain sequence, human IgG1 Fc DANG, linker GGGSGGGGSGGGGGS, human IL-12 p40 (beta) domain sequence, linker GGGGSGGGGSGGGGS, and human IL-12 p35 (alpha) domain sequence.

[0186] [seq295] consists of human anti-FAP heavy chain sequence, human IgG1 Fc DANG, linker GGGGSGGGSGGGGS, human IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGGSGGGSGGGGGS, and human IL-12 p35 (alpha) domain sequence.

[0187] [seq296] is composed of human IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS and human IgG1 Fc DANG, linker GGGSGGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0188] [seq297] is composed of human IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, the linker GGGGSGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and human IgG1 Fc DANG, the linker GGGSGGGGSGGGGGS, the human anti-FAP heavy chain variable region sequence, the linker GGGGSGGGGSGGGGSGGGGS, and the human anti-FAP light chain variable region sequence.

[0189] T1.01 (seq1, seq2, seq3) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP and a human IL-12 sequence form a knob-into-hole structure and the effector function has been removed.

[0190] T1.02 (seq1, seq2, seq4) is a bispecific antibody that consists of a human anti-FAP sequence targeting human FAP and a human IL-12 sequence containing two amino acid mutations involved in heparin binding, forming a knob-into-hole structure and eliminating the effector function.

[0191] T1.03 (seq1, seq2, seq5) is a bispecific antibody that consists of a human anti-FAP sequence targeting human FAP and a human IL-12 sequence containing four amino acid mutations involved in heparin binding, forming a knob-into-hole structure and eliminating the effector function.

[0192] T1.04 (seq3, seq6, seq7) is a bispecific antibody in which a dual variable domain immunoglobulin (DVD-Ig) type human anti-FAP sequence and a human IL-12 sequence form a knob-into-hole structure and the effector function has been removed.

[0193] T1.05 (seq5, seq6, seq7) is a bispecific antibody in which a knob-into-hole structure is formed between a DVD-Ig type human anti-FAP sequence and a human IL-12 sequence containing four amino acid mutations involved in heparin binding, and the effector function is eliminated.

[0194] T1.06 (seq2, seq3, seq8) is a bispecific antibody in which a single-chain variable fragment (scFv) of a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the heavy chain and a human IL-12 sequence form a knob-into-hole structure, and the effector function has been removed.

[0195] T1.07 (seq2, seq5, seq8) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the heavy chain and a human IL-12 sequence containing four amino acid mutations involved in heparin binding form a knob-into-hole structure, thereby eliminating the effector function.

[0196] T1.08 (seq1, seq3, seq9) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the light chain and a human IL-12 sequence form a knob-into-hole structure, and the effector function has been removed.

[0197] T1.09 (seq1, seq5, seq9) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the light chain and a human IL-12 sequence containing four amino acid mutations involved in heparin binding form a knob-into-hole structure, thereby eliminating the effector function.

[0198] T1.10 (seq1, seq2, seq10) is an antibody in which a human anti-FAP sequence targeting human FAP and a human anti-FAP sequence in which a human IL-12 sequence is linked to the C-terminus of the heavy chain form a knob-into-hole structure.

[0199] T1.11 (seq1, seq2, seq11) is an antibody that forms a knob-into-hole structure with a human anti-FAP sequence that targets human FAP and a human IL-12 sequence containing four amino acid mutations involved in heparin binding linked to the C-terminus of the heavy chain.

[0200] T1.12 (seq1, seq2, seq12) is a knobs-into-hole effector function-deleted antibody that has only the human anti-FAP sequence targeting human FAP.

[0201] T1.13 (seq3, seq13) is a knobs-into-hole effector function-deleted antibody containing only the human IL-12 sequence.

[0202] T1.14 (seq4, seq13) is a knobs-into-hole effector function-deleted antibody that contains only human IL-12 sequence containing two amino acid mutations involved in heparin binding.

[0203] T1.15 (seq5, seq13) is a knob-into-hole effector function-deleted antibody that contains only human IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0204] T1.16 (seq2, seq14) is a human anti-FAP antibody that targets human FAP.

[0205] T1.17(seq15) is an Fc fusion protein that contains only the human IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0206] T1.18 (seq3, seq16, seq17) is a bispecific antibody in which a human anti-CD20 sequence targeting human CD20 and a human IL-12 sequence form a knob-into-hole structure and the effector function has been removed.

[0207] T1.19 (seq4, seq16, seq17) is a bispecific antibody that consists of a human anti-CD20 sequence that targets human CD20 and a human IL-12 sequence that contains two amino acid mutations involved in heparin binding, forming a knobs-into-hole structure and eliminating the effector function.

[0208] T1.20 (seq5, seq16, seq17) is a bispecific antibody that consists of a human anti-CD20 sequence that targets human CD20 and a human IL-12 sequence that contains four amino acid mutations involved in heparin binding, forming a knobs-into-hole structure and eliminating the effector function.

[0209] T1.21 (seq12, seq16, seq17) is a knobs-into-hole effector function-deleted antibody that has only the human anti-CD20 sequence to target human CD20.

[0210] T1.23 (seq1, seq2, seq292) is a bispecific antibody in which a human anti-FAP sequence and a human IL-12 sequence to which the scFv of the human anti-FAP sequence is linked at the C-terminus form a knob-into-hole structure, and the effector function has been removed.

[0211] T1.24 (seq1, seq2, seq293) is a bispecific antibody in which a human anti-FAP heavy chain sequence and a human IL-12 sequence containing four amino acid mutations involved in heparin binding are linked to the C-terminus of the scFv of the human anti-FAP sequence, forming a knobs-into-hole structure and removing the effector function.

[0212] T1.25 (seq2, seq294) is an Fc fusion protein dimer containing a human anti-FAP sequence linked to the C-terminus of the heavy chain with a human IL-12 sequence.

[0213] T1.26 (seq2, seq295) is an Fc fusion protein dimer containing a human anti-FAP sequence linked to the C-terminus of the heavy chain with a human IL-12 sequence containing amino acid mutations (four) involved in heparin binding.

[0214] T1.27(seq296) ​​is an Fc fusion protein dimer containing human IL-12 sequences linked to the C-terminus of a scFv of human anti-FAP sequences.

[0215] T1.28 (seq297) is an Fc fusion protein dimer composed of a human IL-12 sequence containing four amino acid mutations involved in heparin binding linked to the C-terminus of a scFv of a human anti-FAP sequence.

[0216] [seq1] Anti-hu FAP HC hu IgG1 Fc knob DANG QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0217] [seq2] anti - hu FAP LC DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0218] [seq3] hu scIL - 12 - hu IgG1 Fc whole DANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEIDITKDK TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVLFPPKPKDTLMISRTPE VTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK

[0219] [seq4]hu scIL-12 mut1-hu IgG1 FcホールDANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASKREAKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEIDKDK TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVLFPPKPKDTLMISRTPE VTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK

[0220] [seq5]hu scIL-12 mut2-hu IgG1 FcホールDANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0221] [seq6](Anti - hu FAP VH)2 - hu IgG1 Fc Nob DANG QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0222] [seq7](Anti - hu FAP VL)2 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0223] [seq8] Anti-hu FAP HC hu IgG1 Fc nob DANG - Anti-hu FAP scFv QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK

[0224] [seq9] anti-hu FAP LC - anti-hu FAP scFv DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK

[0225] [seq10]Anti - hu FAP HCP hu IgG1 Fc whole DANG - hu scIL - 12 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGKSKREKDRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0226] [seq11]Anti-hu FAP HC hu IgG1 Fc whole DANG-hu scIL-12 mut2 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0227] [seq12]hu IgG1 Fc whole DANG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0228] [seq13]hu IgG1 Fc knob DANG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0229] [seq14]Anti - hu FAP HC hu IgG1 Fc DANG QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0230] [seq15]hu scIL-12 mut2-hu IgG1 Fc DANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0231] [seq16] Anti - hu CD20 HC hu IgG1 Fc Nob DANG QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0232] [seq17] Anti - hu CD20 LC QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0233] [seq292] hu scIL - 12 - hu IgG1 Fc whole DANG - anti - hu FAP scFv

[0234] [seq293]hu scIL-12 mut2-hu IgG1 Fc Other-chain hu FAP scFv

[0235] [seq294]Anti-hu FAP HCP hu IgG1 Fc DANG-hu scIL-12 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGKSKREKDRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0236] [seq295]Anti-hu FAP HC hu IgG1 Fc DANG-hu scIL-12 mut2 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0237] [seq296]hu scIL-12-hu IgG1 Fc DANG-anti-hu FAP scFv

[0238] [seq297] hu scIL-12 mut2-hu IgG1 Fc DANG-anti-hu FAP scFv

[0239] Preparation Example 2. Overview of anti-FAP / IL-12 IgG2a DANG, mouse fusion protein

[0240] [Table 2]

[0241] [seq18] is composed of a mouse anti-FAP heavy chain variable region sequence and a mouse IgG2a Fc, with effector function eliminated by DANG mutations (D265A, N297G) and the knob structure formed by a T321W mutation.

[0242] [seq19] consists of the mouse anti-FAP light chain sequence.

[0243] [seq20] consists of mouse IL-12 p40 (beta) region sequence, linker GGGGSGGGGSGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc, with effector function eliminated by DANG mutations and hole structure formed by T321S, M323A, and Y362V mutations.

[0244] [seq21] consists of a sequence in which the lysine (K) at amino acids 277 and 282 involved in heparin binding in the mouse IL-12 p40 (beta) region is mutated to alanine (A), the linker GGGGSGGGSGGGGGS, the mouse IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and mouse IgG2a Fc hole DANG.

[0245] [seq22] consists of a sequence in which the arginine (R) at amino acid 276, which is involved in heparin binding in the mouse IL-12 p40 (beta) region, and the lysines (K) at amino acids 277, 278, and 282 are mutated to alanine (A), the linker GGGGSGGGSGGGGGS, the mouse IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and mouse IgG2a Fc hole DANG.

[0246] [seq23] consists of the mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, and mouse IgG2a Fc knob DANG.

[0247] [seq24] consists of the mouse anti-FAP light chain variable region sequence, the linker GGGGSGGGSGGGGGS, and the mouse anti-FAP light chain sequence.

[0248] [seq25] consists of the mouse anti-FAP heavy chain variable region sequence, mouse IgG2a Fc knob DANG, linker GGGGSGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0249] [seq26] consists of the mouse anti-FAP light chain sequence, linker GGGGSGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0250] [seq27] is composed of mouse anti-FAP heavy chain sequence, mouse IgG2a Fc hole DANG, linker GGGGSGGGSGGGGGS, mouse IL-12 p40 (beta) domain sequence, linker GGGGSGGGSGGGGGS, and mouse IL-12 p35 (alpha) domain sequence.

[0251] [seq28] consists of mouse anti-FAP heavy chain sequence, mouse IgG2a Fc hole DANG, linker GGGGSGGGGSGGGGS, mouse IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, and mouse IL-12 p35 (alpha) domain sequence.

[0252] [seq29] is composed only of mouse IgG2a Fc hole DANG.

[0253] [seq30] is composed only of mouse IgG2a Fc knob DANG.

[0254] [seq31] is composed of mouse anti-FAP heavy chain variable region and mouse IgG2a Fc DANG.

[0255] [seq32] consists of the p40 (beta) domain sequence of mouse IL-12 containing four amino acid mutations involved in heparin binding, the linker GGGGSGGGSGGGGGS, the mouse IL-12 p35 (alpha) domain sequence, the linker GGGGSGGGGS, and mouse IgG2a Fc DANG.

[0256] [seq33] consists of the heavy chain variable region sequence of the mouse anti-CD20 antibody 18B12 and the mouse IgG2a knob DANG.

[0257] [seq34] consists of the light chain sequence of the mouse anti-CD20 antibody 18B12.

[0258] [seq35] is composed only of mouse IgG2a Fc DANG.

[0259] [seq298] is composed of mouse IL-12 p40 (beta) region sequence, linker GGGSGGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS and mouse IgG2a Fc hole DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0260] [seq299] is composed of mouse IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc hole DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0261] [seq300] consists of mouse anti-FAP heavy chain sequence, mouse IgG2a Fc DANG, linker GGGGSGGGSGGGGGS, mouse IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, and mouse IL-12 p35 (alpha).

[0262] [seq301] consists of mouse anti-FAP heavy chain sequence, mouse IgG1 Fc DANG, linker GGGGSGGGSGGGGGS, mouse IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGGSGGGSGGGGGS, and mouse IL-12 p35 (alpha) domain sequence.

[0263] [seq302] is composed of mouse IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0264] [seq303] is composed of mouse IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0265] T1.01m (seq18, seq19, seq20) is a bispecific antibody in which a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence form a knob-into-hole structure and the effector function has been removed.

[0266] T1.02m (seq18, seq19, seq21) is a bispecific antibody that consists of a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing two amino acid mutations involved in heparin binding, forming a knob-into-hole structure and eliminating the effector function.

[0267] T1.03m (seq18, seq19, seq22) is a bispecific antibody consisting of a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, forming a knob-into-hole structure and eliminating the effector function.

[0268] T1.04m (seq20, seq23, seq24) is a bispecific antibody that combines a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence in a knob-into-hole structure and has removed effector functions.

[0269] T1.05m (seq22, seq23, seq24) is a bispecific antibody that has a knob-into-hole structure formed between a mouse anti-FAP sequence targeting mouse FAP of the DVD-Ig type and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, and has no effector function.

[0270] T1.06m (seq19, seq20, seq25) is a bispecific antibody in which a mouse anti-FAP sequence containing a single-chain variable fragment (scFv) of a mouse anti-FAP sequence targeting mouse FAP at the C-terminus of the heavy chain forms a knob-into-hole structure with a mouse IL-12 sequence, and the effector function has been removed.

[0271] T1.07m (seq19, seq22, seq25) is a bispecific antibody in which a mouse anti-FAP sequence containing an scFv of the mouse anti-FAP sequence targeting mouse FAP at the C-terminus of the heavy chain and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding form a knob-into-hole structure, thereby eliminating the effector function.

[0272] T1.08m (seq18, seq20, seq26) is a bispecific antibody in which the mouse anti-FAP sequence, which targets mouse FAP and contains an scFv at the C-terminus of the light chain, forms a knob-into-hole structure with the mouse IL-12 sequence, and the effector function has been removed.

[0273] T1.09m (seq18, seq22, seq26) is a bispecific antibody in which a mouse anti-FAP sequence containing an scFv of the mouse anti-FAP sequence targeting mouse FAP at the C-terminus of the light chain and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding form a knob-into-hole structure, thereby eliminating the effector function.

[0274] T1.10m (seq18, seq19, seq27) is an antibody that forms a knob-into-hole structure with a mouse anti-FAP sequence targeting mouse FAP and a mouse anti-FAP sequence in which a mouse IL-12 sequence is linked to the C-terminus of the heavy chain.

[0275] T1.11m (seq18, seq19, seq28) is an antibody in which a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding are linked to the C-terminus of the heavy chain, forming a knob-into-hole structure.

[0276] T1.12m (seq18, seq19, seq29) is a knobs-into-hole effector function-deleted antibody that has only the mouse anti-FAP sequence to target mouse FAP.

[0277] T1.13m (seq20, seq30) is a knobs-into-hole effector function-deleted antibody that contains only the mouse IL-12 sequence.

[0278] T1.14m (seq21, seq30) is a knobs-into-hole effector function ablated antibody that contains only the murine IL-12 sequence containing two amino acid mutations involved in heparin binding.

[0279] T1.15m (seq22, seq30) is a knobs-into-hole effector function-deleted antibody that contains only the mouse IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0280] T1.16m (seq19, seq31) is a mouse anti-FAP antibody that targets mouse FAP.

[0281] T1.17m (seq32) is an Fc fusion protein dimer that contains only the mouse IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0282] T1.18m (seq20, seq33, seq34) is a bispecific antibody that consists of a mouse anti-CD20 (18B12) sequence targeting mouse CD20 and a mouse IL-12 sequence forming a knob-into-hole structure and has deleted effector functions.

[0283] T1.19m (seq21, seq33, seq34) is a bispecific antibody that consists of a mouse anti-CD20 (18B12) sequence targeting mouse CD20 and a mouse IL-12 sequence containing two amino acid mutations involved in heparin binding, forming a knobs-into-hole structure and eliminating effector function.

[0284] T1.20m (seq22, seq33, seq34) is a bispecific antibody that consists of a mouse anti-CD20 sequence targeting mouse CD20 and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, forming a knobs-into-hole structure and eliminating effector function.

[0285] T1.21m (seq29, seq33, seq34) is a knobs-into-hole effector-function-deleted antibody that has only the mouse anti-CD20 sequence to target mouse CD20.

[0286] T1.22m(seq35) is a protein that contains only the mouse Fc sequence with effector functions removed.

[0287] T1.23m (seq18, seq19, seq298) is a bispecific antibody in which a mouse anti-FAP sequence and a mouse IL-12 sequence to which the scFv of the mouse anti-FAP sequence is linked at the C-terminus form a knob-into-hole structure, and the effector function has been removed.

[0288] T1.24m (seq18, seq19, seq299) is a bispecific antibody consisting of a mouse anti-FAP heavy chain sequence and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, linked to the C-terminus of an scFv of the mouse anti-FAP sequence, forming a knob-into-hole and abolishing effector functions.

[0289] T1.25m (seq19, seq300) is an Fc fusion protein dimer containing a mouse anti-FAP sequence linked to the C-terminus of the heavy chain with a mouse IL-12 sequence.

[0290] T1.26m (seq19, seq301) is an Fc fusion protein dimer containing a mouse anti-FAP sequence linked to the C-terminus of the heavy chain with a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0291] T1.27m (seq302) is an Fc fusion protein dimer containing mouse IL-12 sequences linked to the C-terminus of a scFv of mouse anti-FAP sequence.

[0292] T1.28m (seq303) is an Fc fusion protein dimer composed of a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding linked to the C-terminus of a scFv of a mouse anti-FAP sequence.

[0293] [seq18]Anti-mu FAP HC mu IgG2a Fc knob DANG QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0294] [seq19] Anti-mu FAP LC QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWK IDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0295] [seq20] mu scIL-12-mu IgG2a Fc whole DANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELEAR QQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGGSGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPKIKDVLMISLS PIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFCKKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKFSRTPGK

[0296] [seq21]mu scIL-12 mut1-mu IgG2a FcホールDANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELEAR QQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRAKEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGGSGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPKIKDVLMISLS PIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFCKKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKFSRTPGK

[0297] [seq22]mu scIL-12 mut2-mu IgG2a FcホールDANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0298] [seq23] (Anti-mu FAP VH)2, mu IgG2a Fc nob DANG QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0299] [seq24](Anti-mu FAP VL)2 QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0300] [seq25]Anti-mu FAP HC mu IgG2a Fc knob DANG - Anti-mu FAP scFv QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0301] [seq26]Anti-mu FAP LC - Anti-mu FAP scFv QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0302] [seq27] Anti-mu FAP HC mu IgG2a Fc whole DANG-mu scIL-12 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0303] [seq28]mut FAP HC mu IgG2a Fc-mut DANG-mu scIL-12 mut2 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0304] [seq29]murine IgG2a Fc whole DANG EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0305] [seq30]murine IgG2a Fc knob DANG EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0306] [seq31]Anti-mu FAP HC murine IgG2a Fc DANG QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0307] [seq32]mu scIL-12 mut2-mu IgG2a Fc DANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0308] [seq33] Anti-mu CD20 HC mu IgG2a Fc nob DANG QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPGQGLEWIGVIDPSDNYTKYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYFCAREGYYGSSPWFAYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0309] [seq34]Anti-mu CD20 LC QIVMSQSPAILSASPGEKVTMTCRARSSVSYIHWYQQKPGSSPKPWIYATSNLASGVPGRFSGSGSGTSYSLTITRVEAEDAATYYCQQWSSKPPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0310] [seq35]mu IgG2a Fc DANG EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0311] [seq298] mu scIL-12-mu IgG2a Fc whole DANG-anti-mu FAP scFv

[0312] [seq299]mu scIL-12 mut2-mu IgG2a Fc hole DANG-anti-mu FAP scFv

[0313] [seq300]Anti-mu FAP HC mu IgG2a Fc DANG-mu scIL-12 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0314] [seq301]Anti-mu FAP HC mu IgG2a Fc DANG-mu scIL-12 mut2 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0315] [seq302]mu scIL-12-mu IgG2a Fc DANG-anti-mu FAP scFv

[0316] [seq303] mu scIL-12 mut2-mu IgG2a Fc DANG-anti-mu FAP scFv

[0317] Production Example 1. Production of fusion protein The reagents and equipment are described in Tables 3 and 4 below.

[0318] [Table 3]

[0319] [Table 4]

[0320] The synthesized DNA fragments were amplified by PCR, and the PCR products were gel purified. The pTT5 vector was cleaved by restriction enzymes EcoRI and BamHI, and then gel purified. Each PCR product and the linearized vector were ligated using the In-Fusion kit. The resulting vector was transformed into ECOS101 DH5α competent cells, and the cells were cultured on 2xYT agar plates containing 100 μg / ml ampicillin. All the operation processes were performed according to the standard transformation protocol. The positive recombinants were identified by colony PCR, and sequence verification sequencing was performed on the recombinant plasmids. A single colony was selected, and the seed culture was inoculated into 5 ml of 2xYT medium containing 100 μg / ml ampicillin. The culture was shaken at 37°C for 8 hours.

[0321] The seed culture was then diluted in 200 ml of selective 2xYT medium at a ratio of 1:1,000. The culture was incubated at 37°C for 16 hours with shaking. The bacterial cells were harvested by centrifugation at 4,700 rpm for 10 minutes at 4°C. The bacterial pellet was resuspended in 12 ml of RES-EF buffer. Then, 12 ml of LYS-EF buffer was added and the sealed tube was mixed thoroughly by vigorously inverting, and then incubated at room temperature for 5 minutes. 12 ml of NEU-EF buffer was added to the lysate and mixed thoroughly and quickly by vigorously inverting.

[0322] Prior to pouring the lysate into the NucleoBond™ Xtra column filter, the lysate tube was inverted three times to prepare a uniform suspension of the precipitate and to prevent clogging of the filter. The NucleoBond™ Xtra column filter and NucleoBond™ Xtra column were then washed with 10 ml of filter wash buffer FIL-EF. The NucleoBond™ Xtra column filter was removed by pulling and inverting the column. The NucleoBond™ Xtra column was washed with 90 ml of wash buffer ENDO.

[0323] The NuCryobond™ Xtra column was washed with 45 ml of wash buffer WASH-EF. Plasmid DNA was eluted with 15 ml of elution buffer ELU. The eluate was collected in a 50 ml centrifuge tube. The eluted plasmid DNA was precipitated by adding 10.5 ml of isopropanol at room temperature. After vortexing, the mixture was left for 2 minutes.

[0324] Then, 5 ml of 70% ethanol was added to the pellet. The ethanol was carefully and completely removed from the tube using a pipette tip. The pellet was dried at room temperature (20°C). The DNA pellet was then dissolved in 1,000 μl of H2O.

[0325] Preparation Example 2. Cell transfection and protein expression Preparation example 2.1. Cell transfection The materials and reagents used are shown in Table 5 below.

[0326] [Table 5]

[0327] The 293F seed stock containing complete medium was maintained in an incubator shaker at 130 rpm, 37 °C, and 8% CO2. 6The cells were cultured at a concentration of 2.6 × 10 cells / ml and the medium was changed every 2–3 days. 24 h before transfection, a new passage number of 293F cells was added to each well. 6 The cells were cultured in an incubator shaker at 130 rpm, 37°C, and 8% CO2. On the day of transfection, the cell density was adjusted to 5.0 × 10 using fresh medium. 6 The transfection reagent was adjusted to 1 L cells / ml. The transfection was performed in a 3 L shaker flask with a total volume of 1 L. 0.4 mg of HC and 0.6 mg of LC plasmids were diluted in 50 ml of OPTI MEM I and filtered through a 0.22 μm filter. Then, 2 mg of PEI was diluted in 50 ml of OPTI MEM I to prepare the transfection reagent.

[0328] The diluted PEI was added to the DNA mixture and mixed immediately, then incubated at room temperature for 15 min. The DNA-PEI mixture was diluted to 2.6 × 10 6 The transfection was added to 293F cells prepared at 1000 rpm, 37°C, and 8% CO2. The cells were then continuously cultured for 24 hours in an incubator shaker at 130 rpm, 37°C, and 8% CO2. 24 hours after transfection, 10% peptone was added to 1 / 20 of the culture solution to a final concentration of 0.5%. The cells were then continuously cultured in an incubator shaker at 130 rpm, 37°C, and 8% CO2. Cell density / viability was measured and recorded daily for 2-5 days after transfection. Cells were harvested for purification 7 days after transfection or when cell viability was less than 70%.

[0329] Example 2.2. Protein purification The reagents, buffer compositions, and equipment used in protein purification are shown in Tables 6 to 8 below.

[0330] [Table 6]

[0331] [Table 7]

[0332] [Table 8]

[0333] The protein was purified using a Mabselect Sure column. Specifically, the supernatant was collected by centrifugation at 2,000×g at 4°C for 20 min. The supernatant was then filtered through a Sartopore2 filter. The clarified supernatant was loaded onto a 5 ml MabSelect Sure column equilibrated with Buffer A. The column was then washed with Buffer A until the A280 absorbance reached baseline. The column was washed with 10 CV Buffer B. The column was washed with 10 CV Buffer A. The bound protein was eluted with 6 CV Buffer C, and 1 / 6 volume of Buffer D was added to neutralize the eluted material. SDS-PAGE and SEC-HPLC analysis were performed. The protein was then purified using a HIC column. The protein was then dialyzed against Buffer E overnight at 4°C. The supernatant was loaded onto a HIC column equilibrated with Buffer E. The column was then washed with Buffer E until the A280 absorbance reached baseline. Bound proteins were eluted by gradient elution (10 CV Buffer F 0% to 40%). Bound proteins were eluted with 2 CV 100% Buffer F and subjected to SDS-PAGE analysis.

[0334] The purified proteins were pooled and then the proteins were dialyzed overnight against the final buffer at 4° C., followed by SDS-PAGE and SEC-HPLC analysis.

[0335] As a result, as shown in FIG. 1 to FIG. 6, it was found that the human protein and mouse protein of one embodiment were purified.

[0336] Preparation Example 3. Improved bispecific antibody production Preparation 3.1 Identification of improvements in the production of fusion proteins containing human IL-12 The change in the production of human anti-FAP / IL-12 bispecific antibody due to human IL-12 mutation is shown in Table 9 below. The brackets indicate the production scale of the protein in units of L. The number before the brackets is the production amount per liter, calculated by dividing the amount of purified protein by the production scale.

[0337] [Table 9]

[0338] As shown in Table 9 above, the amount of protein purified through a Protein A column (MabSelect Sure column) increased with increasing number of mutations in the amino acids involved in heparin binding in the human IL-12 p40 (beta) region. Bispecific antibody T1.02, in which the lysines (K) at amino acids 280 and 285 were mutated to alanines (A), was found to have improved production compared to T1.01. Bispecific antibody T1.03, in which the lysines (K) at amino acids 280, 282, 285, and 286 were mutated to alanines (A), was found to have approximately 2.5-fold improved production compared to T1.01.

[0339] Preparation Example 3.2. Identification of improvements in the production of fusion proteins containing mouse IL-12 Table 10 below shows the change in mouse anti-FAP / IL-12 bispecific antibody production due to mouse IL-12 mutations.

[0340] [Table 10]

[0341] As shown in Table 10 above, as the number of mutations in the amino acids involved in heparin binding in the murine IL-12 p40(beta) region increased, the amount of protein purified through a Protein A column (MabSelect Sure column) increased.

[0342] In each table, the amount of protein after passing through the Protein A column was compared by plotting the total protein production at production scale. Bispecific antibody T1.02m, in which the lysine (K) at amino acid positions 277 and 282 was mutated to alanine (A), and bispecific antibody T1.03m, in which the arginine (R) at amino acid position 276 was mutated to alanine (A) and the lysine (K) at amino acid positions 277, 278, and 282 was mutated to alanine (A), were found to have gradually improved protein production compared to T1.01m and T1.02m. Furthermore, production of T1.03m using a CHO cell line was found to be approximately 13-fold improved compared to production using a HEK293 cell line. Production Example 4. Cell lines and cell line culture HEK-Blue IL-12 cell line, which was transfected with IL-12 receptor gene and STAT4-inducible SEAP reporter gene into human embryonic kidney fibroblast HEK293 cells, was obtained from Invivogen (San Diego, USA). HEK-Blue IL-12 cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO), 100 μg / ml normocin, and 1×HEK-Blue selection.

[0343] Human embryonic kidney fibroblast cell line HEK293, mouse colon cancer cell line CT26-WT, mouse melanoma cell line B16F10 and mouse fibroblast cell line NIH3T3 were obtained from ATCC (American Type Culture Collection, Manasas, VA, USA). HEK293, B16F10 and NIH3T3 cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO). A cell line (HEK293-hFAP) in which human FAP (fibroblast activation protein alpha) was overexpressed in HEK293 cells was prepared using a lentivirus capable of delivering the human FAP gene. HEK293-hFAP cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO) and 5 μg / ml puromycin.

[0344] Cell lines (CT26-mFAP, B16F10-mFAP, and NIH3T3-mFAP) in which mouse FAP (fibroblast activation protein alpha) was overexpressed in CT26-WT, B16F10, and NIH3T3 cells were prepared using lentivirus capable of delivering mouse FAP gene. CT26-mFAP cells were maintained in RPMI-1640 (GIBCO) containing 10% FBS (GIBCO) and 10 μg / ml puromycin. B16F10-mFAP cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO) and 10 μg / ml puromycin. NIH3T3-mFAP cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO) and 5 μg / ml puromycin.

[0345] Example 5. Isolation and activation of human immune cells Blood packs were obtained from the Korean Red Cross with Institutional Review Board (IRB) approval, and peripheral blood mononuclear cells (PBMCs) were isolated and frozen. Thawed PBMCs were subjected to positive selection, and human NK cells were isolated using the Easy sep (Stem cell, Vancouver, BC, CA) kit. The isolated NK cells were activated and maintained in RPMI-1640 (GIBCO) and 10% FBS containing 50 IU / ml recombinant human IL-2 (rhIL-2, R&D systems, Minnesota, USA).

[0346] Thawed PBMCs were subjected to negative selection and human T cells were isolated using the Easy sep (Stem cell) kit. Human T cells were cultured on plates coated with 1 μg / ml anti-CD3 (OKT3, Invitrogen) and activated for 72 hours. Human T cells were maintained in RPMI-1640 (GIBCO) containing 10% FBS (GIBCO).

[0347] Example 1. Identification of binding affinity of fusion proteins Example 1.1. Identification of Binding of Fusion Proteins to Recombinant Human FAP The binding of T1.01, T1.02 and T1.03 to recombinant human FAP was identified by surface plasmon resonance (SPR).

[0348] Specifically, the surface of a CM5 chip was activated with a 1:1 mixture of 50 nM NHS (N-hydroxysuccinimide) and 200 nM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 25 μg / ml of anti-human IgG (Fc) antibody was immobilized at a rate of 10 μl / min for 400 seconds. The remaining active ester groups were blocked with 1 M ethanolamine. T1.01, T1.02 and T1.03 were diluted to 2 μg / ml and reacted with anti-human IgG (Fc) antibody on the immobilized CM5 chip. Recombinant human FAP was diluted to 200 nM in 1×HBS-EP+ buffer and diluted by serial dilution. The diluted recombinant human FAP was reacted at a rate of 30 μl / min. The association and dissociation times were 180 and 400 seconds, respectively. After dissociation, stabilization was performed for 60 seconds, followed by regeneration with 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min for 30 seconds.

[0349] As a result, as shown in Figures 7 and 8, it was found that T1.01, T1.02 and T1.03 could specifically bind to recombinant human FAP.

[0350] The binding of T1.04, T1.05, T1.06, T1.07, T1.08, T1.09, T1.10 and T1.11 to recombinant human FAP was identified by surface plasmon resonance (SPR). The surface of a CM5 chip was activated with a 1:1 mixture of 50 nM NHS (N-hydroxysuccinimide) and 200 nM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 25 μg / ml of anti-human IgG (Fc) antibody was immobilized at a rate of 10 μl / min for 400 s. The remaining active ester groups were blocked with 1 M ethanolamine.

[0351] Specifically, human proteins T1.04, T1.05, T1.06, T1.07, T1.08, T1.09, T1.10, and T1.11 were diluted to 1 μg / ml, 1 μg / ml, 1 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.5 μg / ml, 0.5 μg / ml, and 1 μg / ml, respectively, and reacted on an anti-human IgG (Fc) antibody-immobilized CM5 chip. Recombinant human FAP was diluted to 25 nM or 50 nM in 1×HBS-EP+ buffer and diluted by serial dilution. The diluted recombinant human FAP was reacted at a rate of 30 μl / min. Association and dissociation times were 180 s and 400 s, respectively. After dissociation, stabilization was performed for 60 s, and then regeneration was performed for 30 s with 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min.

[0352] As a result, as shown in Table 11 and Figures 9 and 10, it was found that T1.04, T1.05, T1.06 and T1.07 can bind to recombinant human FAP and specifically target FAP. Furthermore, as shown in Table 12 and Figures 11 and 12, it was found that T1.08, T1.09, T1.10 and T1.11 can bind to recombinant human FAP and specifically target FAP.

[0353] [Table 11]

[0354] [Table 12]

[0355] Example 1.2. Identification of binding of mouse proteins to recombinant mouse FAP The binding of T1.01m, T1.02m and T1.03m to recombinant mouse FAP was identified by surface plasmon resonance (SPR).

[0356] The surface of a CM5 chip was activated with a 1:1 mixture of 50 nM NHS (N-hydroxysuccinimide) and 200 nM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 25 μg / ml of anti-mouse IgG (Fc) antibody was immobilized at a rate of 10 μl / min for 400 seconds. The remaining active ester groups were blocked with 1 M ethanolamine. T1.01m, T1.02m, and T1.03m were diluted to 2 μg / ml and reacted with anti-mouse IgG (Fc) antibody on the immobilized CM5 chip. Recombinant mouse FAP was diluted to 200 nM in 1×HBS-EP+ buffer and serially diluted. The diluted recombinant mouse FAP was reacted at a rate of 30 μl / min. Association and dissociation times were 180 and 400 seconds, respectively. After dissociation, stabilization was performed for 60 seconds, followed by regeneration with 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min for 30 seconds.

[0357] As a result, as shown in Figures 13 and 14, it was found that T1.01m, T1.02m and T1.03m could bind to recombinant mouse FAP and specifically target FAP.

[0358] Example 1.3. Identification of binding to FAP-expressing cells The extent of binding of T1.12, T1.01, T1.02 and T1.03 to HEK293 and HEK293-hFAP cells was determined.

[0359] Specifically, the cell line was cultured at 1 × 10 5 Cells were prepared by suspending in FACS buffer at 100 μl per cell and treating with 1 μg each of T1.12, T1.01, T1.02 and T1.03. Cells were washed twice with FACS buffer. Cells were stained with anti-human IgG antibody (Biolegend). Negative control was stained with anti-human IgG antibody (Biolegend) only. The expression rate of stained cells was measured using BD LSR and analyzed using FlowJo software.

[0360] As a result, as shown in Figure 15, T1.12, T1.01, T1.02 and T1.03 were found to bind to the human FAP-expressing cell line by 99% or more. These results indicate that the fusion protein of one embodiment can bind to the human FAP-expressing cell line and specifically target the FAP.

[0361] Furthermore, the extent of binding of T1.12m, T1.01m, T1.02m and T1.03m to B16F10 and B16F10-mFAP cells was determined.

[0362] Specifically, the cell line was cultured at 1 × 10 5 Cells were prepared by suspending in FACS buffer at 100 μl per cell and treated with 1 μg each of T1.12m, T1.01m, T1.02m and T1.03m. Cells were washed twice with FACS buffer. Cells were stained with anti-mouse IgG2a antibody (Biolegend). Negative controls were stained with anti-mouse IgG2a antibody (Biolegend) alone. The expression rate of stained cells was measured using a BD LSR and analyzed using FlowJo software.

[0363] As a result, as shown in Figure 16, T1.12m, T1.01m, T1.02m and T1.03m were found to bind to the FAP-expressing cell line by 99% or more. These results indicate that the fusion protein of one embodiment can bind to the mouse FAP-expressing cell line and specifically target FAP.

[0364] Example 1.4. Identification of fusion protein binding to recombinant human IL-12 receptor Considering that the fusion protein IL-12 must bind to the IL-12 receptor for action, T1.01, T1.02, T1.03 and T1.12 were found to bind to the recombinant human IL-12 receptor.

[0365] T1.01, T1.02 and T1.03 were immobilized on a plate and recombinant human IL-12 receptor protein conjugated with horseradish peroxidase (HRP) was bound to them. Specifically, the following steps were performed:

[0366] (i) T1.01, T1.02, T1.03 and T1.12 were suspended at 5 μg / ml and serially diluted. The diluted T1.01, T1.02, T1.03 and T1.12 were aliquoted into 96-well immunoplates and treated for 24 hours.

[0367] (ii) The wells were washed with a washing solution and blocked with a 1% BSA (bovine serum albumin, Sigma) solution for 1 hour.

[0368] (iii) The wells were washed with washing solution and then treated with 1 nM IL-12 receptor beta 1-biotin (IL-12Rβ1-biotin; Acrobiosystems, Newark, USA) for 2 h.

[0369] (iv) The wells were washed with washing solution and then treated with streptavidin-HRP for 20 minutes.

[0370] (v) The wells were washed with wash solution and then treated with substrate solution for 20 minutes.

[0371] (vi) The wells were treated with stop solution and the absorbance was measured at 450 nm.

[0372] As a result, it was found that T1.01, T1.02 and T1.03 bind to the recombinant human IL-12 receptor protein in a concentration-dependent manner, as shown in Figure 17. T1.12 was used as a control.

[0373] Example 2. Identification of the ability of fusion proteins to induce signal transduction Example 2.1. Identification of signal transduction capabilities in IL-12-recognizing cells Considering that IL-12 is known to initiate signal transduction by inducing phosphorylation of the signal transduction mediator STAT4 (signal transducer and activator of transcription 4), the signal transduction ability of an embodiment fusion protein in IL-12 recognizing cells expressing the IL-12 receptor when IL-12 binds to the IL-12 receptor was evaluated.

[0374] Specifically, 2.8 × 10 HEK-Blue IL-12 cell line, which is an IL-12-recognizing cell line, was used. 5 The cells were diluted to 1000 cells / ml and dispensed at 180 μl / well. Recombinant human IL-12, T1.01m, T1.02m, T1.03m and T1.12m were suspended at 4 nM and diluted by serial dilution. HEK-Blue IL-12 cells were treated with diluted recombinant human IL-12, T1.01m, T1.02m, T1.03m and T1.12m. After 24 h of treatment, cell supernatants were collected and mixed with QUANTI-Blue Solution (Invivogen) in a 96-well plate. Reporter expression levels were measured at 620 nm using a spectrophotometer (Thermo Fisher).

[0375] As a result, as shown in Figure 18, in the case of IL-12, T1.01m, T1.02m and T1.03m treated groups, IL-12 was recognized and STAT4 was induced to phosphorylate and express the reporter gene, as identified by absorbance at 620 nm. In contrast, it was identified that the control T1.12m did not recognize IL-12 when IL-12-recognizing cells were treated with T1.12m. These results indicate that the fusion protein of one embodiment specifically binds to the IL-12 receptor to induce signal transduction.

[0376] Example 2.2. Identification of signaling capabilities in human T cells To identify whether IL-12 initiates signal transduction by inducing phosphorylation of the signal transduction mediator STAT4 when it binds to the IL-12 receptor on human T cells, human T cells activated with anti-CD3 (OKT3, Invitrogen) were analyzed using the AlphaLISA SureFire Ultra p-STAT4 (Tyr693) Assay Kit (PerkinElmer, Massachusetts, USA).

[0377] Specifically, human proteins IL-12, T1.01, T1.02, T1.03 and T1.12 were suspended at 100ng / ml and diluted by serial dilution. Human T cells activated with anti-CD3 (OKT3, Invitrogen) were treated with diluted recombinant human IL-12, T1.01, T1.02, T1.03 and T1.12. After 2 hours of treatment, cells were lysed and treated with a reagent (PerkinElmer) to detect p-STAT4 (phosphorylated STAT4). p-STAT4 was detected using a spectrophotometer at an excitation wavelength of 680nm and an emission wavelength of 615nm.

[0378] As a result, the half maximal effective concentration (EC50) of the group treated with recombinant human IL-12 was 8.945 pM, the EC50 of the group treated with T1.01 was 14.48 pM, the EC50 of the group treated with T1.02 was 56.51 pM, and the EC50 of the group treated with T1.03 was 147.2 pM, as shown in Figure 19. In contrast, no phosphorylated STAT4 was detected in the control group treated with T1.12.

[0379] These results indicate that IL-12 mutations attenuated T cell signaling capacity.

[0380] Example 2.3. Identification of changes in signaling capacity of fusion proteins by low molecular weight heparin in human T cells Next, we identified the enhancement of the signaling capacity of IL-12 mutant (IL-12 mut1) fusion protein by low molecular weight heparin using human T cells activated with anti-CD3 (OKT3, Invitrogen) and the AlphaLISA SureFire Ultra p-STAT4 (Tyr693) assay kit.

[0381] In particular, the signaling capacity in human T cells in the presence or absence of low molecular weight heparin (LMWH) was identified by observing whether induction of phosphorylation of the signaling mediator STAT4 was attenuated when attenuated IL-12 mut2 bound to the IL-12 receptor in human T cells.

[0382] Specifically, recombinant human IL-12, T1.01, T1.02 and T1.03 were suspended at 100ng / ml and diluted by serial dilution. Activated human T cells were treated with diluted recombinant human IL-12, T1.01, T1.02 and T1.03 together with low molecular weight heparin. After 2 hours of treatment, cells were lysed and treated with a reagent (PerkinElmer) for detecting p-STAT4. p-STAT4 was detected using a spectrophotometer at an excitation wavelength of 680nm and an emission wavelength of 615nm.

[0383] As a result, as shown in FIG. 20, the half maximal effective concentration (EC50) of the group treated with T1.01 and low molecular weight heparin was increased by 9.4-fold compared to the group treated with T1.01, the half maximal effective concentration (EC50) of the group treated with T1.02 and low molecular weight heparin was increased by 5.5-fold compared to the group treated with T1.02, and the half maximal effective concentration (EC50) of the group treated with T1.03 and low molecular weight heparin was increased by 4-fold compared to the group treated with T1.03.

[0384] These results indicate that when the IL-12 mutation (IL-12 mut2) was added to a pre-existing IL-12 mutation (IL-12 mut1), the degree of enhancement of signaling capacity by the addition of low molecular weight heparin was reduced and IL-12 was effectively attenuated.

[0385] Example 3. Identification of cytokine secretion ability of fusion proteins Example 3.1. Enzyme Immunoassays for Measuring Cytokines For cytokine measurements, supernatants stored at -20°C were subjected to enzyme-linked immunosorbent assay (ELISA, R&D systems) using the following steps: (i) Capture antibodies were diluted according to the Certificate of Analysis (CoA) and coated onto 96-well plates for 24 hours.

[0386] (ii) The wells were washed with a washing solution and blocked with a 1% BSA (bovine serum albumin, Sigma) solution for 1 hour.

[0387] (iii) The wells were washed with a washing solution and then treated with the samples for 2 hours.

[0388] (iv) The wells were washed with a washing solution, then the detection antibody was diluted according to the certificate of analysis and aliquoted into the wells, and then the wells were treated with it for 2 hours.

[0389] (v) The wells were washed with washing solution and then treated with streptavidin-HRP for 20 minutes.

[0390] (vi) The wells were washed with washing solution and then treated with substrate solution for 20 minutes.

[0391] (vii) The wells were treated with stop solution and the absorbance was measured at 450 nm.

[0392] Example 3.2. Identification of cytokine secretion ability in human T cells Considering that IFN-γ (interferon-gamma), a major immune response, is secreted when human T cells are treated with IL-12, we evaluated the IFN-γ secretion ability of human T cells when treated with recombinant human IL-12, T1.12, T1.01, T1.02, and T1.03.

[0393] Specifically, 5 × 10 human T cells activated with anti-CD3 (OKT3, Invitrogen) were 5 Cells / ml were diluted and dispensed at 200 μl / well into 96-well plates. Recombinant human IL-12, T1.12, T1.01, T1.02 and T1.03 were diluted to 10 nM and dispensed at 20 μl / well. After 48 hours of treatment, cell supernatants were collected and samples were stored at -80°C.

[0394] As a result, it was found that the samples treated with recombinant human IL-12 had an average of 1,640 pg / ml IFN-γ, the samples treated with T1.01 had an average of 1,115 pg / ml IFN-γ, the samples treated with T1.02 had an average of 845 pg / ml IFN-γ, and the samples treated with T1.03 had an average of 607 pg / ml IFN-γ, as shown in Figure 21. In contrast, the samples treated with T1.12 as a control were found to have no IFN-γ.

[0395] These results indicate that T1.01, T1.02 and T1.03 act on human T cells to induce IFN-γ secretion.

[0396] Example 3.3. Identification of cytokine secretion capacity in human NK cells Considering that IFN-γ (interferon-gamma), a major immune response, is secreted when human NK cells are treated with IL-12, the inventors evaluated the IFN-γ secretion ability of human NK cells activated with recombinant human IL-2 (R&D systems) when treated with recombinant human IL-12, T1.12, T1.01, T1.02 and T1.03.

[0397] Specifically, 5 × 10 human NK cells activated with recombinant human IL-2 (R&D Systems) were 5 Cells / ml were diluted and dispensed at 200 μl / well into 96-well plates. Recombinant human IL-12, T1.12, T1.01, T1.02 and T1.03 were diluted to 10 nM and dispensed at 20 μl / well. After 24 hours of treatment, cell supernatants were harvested and samples were stored at -80°C.

[0398] As a result, as shown in Figure 22, it was found that the samples treated with recombinant human IL-12 had an average of 180 pg / ml of IFN-γ, the samples treated with T1.01 had an average of 205 pg / ml of IFN-γ, the samples treated with T1.02 had an average of 133 pg / ml of IFN-γ, and the samples treated with T1.03 had an average of 80 pg / ml of IFN-γ. In contrast, the samples treated with T1.12 as a control were found to have no IFN-γ. These results indicate that T1.01, T1.02 and T1.03 act on human NK cells to induce IFN-γ secretion.

[0399] Example 4. Evaluation of anticancer effect of fusion protein in colon cancer animal model Example 4.1. Evaluation of anti-cancer effects in a colon cancer animal model by co-injection of FAP-expressing fibroblasts The fusion protein is in the form of a double antibody having an anti-FAP sequence structure and an IL-12 sequence structure. Considering that FAP is overexpressed in fibroblasts present in many tumor microenvironments, mouse FAP was overexpressed in mouse fibroblast cell line NIH3T3 cells, and then the tumor growth inhibitory ability was evaluated in a tumor animal model in which the cells were co-injected 1:1 with the mouse colon cancer cell line CT26.

[0400] We prepared FAP-expressing tumor animal models using mouse colon cancer cell line CT26 and mouse fibroblast cell line NIH-3T3. Next, we prepared NIH-3T3 as a FAP-expressing cell line.

[0401] Specifically, NIH-3T3 cells and CT26 cells overexpressing mouse FAP in culture were incubated at 1 × 10 6The cells were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) at a concentration of cells / 50 μl, mixed at a ratio of 1:1, and then implanted with 100 μl per mouse into the subcutaneous region of the right flank of 6-week-old BALB / c mice using a 1 cc syringe (25G) to obtain FAP-expressing fibroblast co-injected animal models. Tumor size was measured using a digital Vernier caliper by measuring the short and long axes of the tumor, and tumor size was expressed as (short axis, mm). 2 Measurements were taken twice weekly using the calculation formula: × (long axis, mm) × 0.5.

[0402] Next, the efficacy of the drug was evaluated in patients with tumors 100 mm 3 When the tumor growth rate reached 100 μl, the mice were randomly divided into groups. Each experimental group consisted of 10 mice, and as a control, 100 μl of 50 μg T1.24m was intraperitoneally administered three times at 3-day intervals. In the test groups, 10 μg and 50 μg T1.02m and 50 μg T1.01m were intraperitoneally administered three times at 3-day intervals to evaluate the tumor growth inhibition ability.

[0403] As a result, as shown in Figure 23, when comparing the tumor size on day 14, the groups administered 10μg and 50μg of T1.02m were found to have superior tumor growth inhibition abilities of 93.2% and 92.7%, respectively, compared to the group administered 50μg of T1.24m as a control. Furthermore, it was observed that the group administered 50μg of T1.01m had 91.9% tumor growth inhibition compared to the group administered 50μg of T1.24m as a control.

[0404] Furthermore, as shown in FIG. 24, 24 days after treatment, complete response subjects were observed in the groups receiving T1.01m and T1.02m compared to the control.

[0405] These results demonstrate that the fusion protein of one embodiment has excellent anti-cancer effects in an animal model of colon cancer.

[0406] Example 4.2. Evaluation of anticancer effect in FAP-expressing colon cancer animal model In the case of colorectal cancer, considering the high expression of FAP in the tumor cells themselves, to evaluate the anticancer effect, the tumor growth inhibition ability was evaluated in a tumor model in which overexpression of mouse FAP was induced in the mouse colon cancer cell line CT26 cells.

[0407] To prepare a mouse FAP-expressing tumor animal model, FAP overexpression was induced in the mouse colon cancer cell line CT26. Specifically, FAP-overexpressing CT26 cells in culture were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) and then injected into the left dorsal subcutaneous region of 6-week-old BALB / c mice using 1 cc syringe (25G) at 100 μl of 1 × 10 cells / mouse. 6 Tumor cells were transplanted to obtain a mouse FAP-expressing colon cancer animal model. Tumor size was measured using a digital Vernier caliper by measuring the short and long axes of the tumor, and tumor size was expressed as (short axis, mm). 2 Measurements were taken twice weekly using the calculation formula: × (long axis, mm) × 0.5.

[0408] Next, to evaluate the efficacy of the drug, 3 When the tumor growth rate reached 100 μl, the mice were randomly divided into groups. Each experimental group consisted of 8 animals, and 100 μl of 50 μg T1.24m was administered intraperitoneally 4 times at 3-day intervals as a control. In the test groups, 100 μl of 10 μg and 100 μg T1.01m, and 2 μg, 10 μg and 100 μg T1.02m were administered intraperitoneally 4 times at 3-day intervals to evaluate the tumor growth inhibition ability.

[0409] As a result, as shown in Figure 25, the groups administered 10μg and 100μg of T1.01m were found to have 84.9% and 85.6% higher tumor growth inhibition abilities, respectively, compared to the group administered 50μg of T1.24m as a control. Furthermore, it was observed that the groups administered T1.02m had 80.1%, 90.5% and 89.8% higher tumor growth inhibition abilities per dose, compared to the group administered T1.24m as a control.

[0410] Furthermore, 30 days after administration, each administration group showed complete response (CR) subjects along with tumor growth inhibition, and no change in body weight was observed in response to administration. These results indicate that the fusion protein of one embodiment has excellent anti-cancer effects in a colon cancer animal model.

[0411] Example 4.3. Evaluation of fusion protein inhibition of recurrent solid tumor growth in complete response mice To evaluate whether tumor recurrence could be inhibited by inducing a long-term immune response after fusion protein treatment, subjects who achieved a complete response were rechallenged with tumors by administering T1.01m to a tumor model co-injected with fibroblast NIH-3T3 and CT26 cells. Naive BALB / c mice served as control mice.

[0412] Control mice and mice that achieved a complete response by administration of T1.01m were subcutaneously injected with 4T1 cells in the left flank and with CT26 cells in the right flank to obtain tumor recurrence models.

[0413] Specifically, in a model in which FAP-overexpressing fibroblast NIH-3T3 cells and CT26 cells were co-injected, a tumor recurrence model was induced in mice that showed a complete response to T1.01m administration. CT26 cells and 4T1 cells were co-injected at 1 × 10 6 Suspended in Hank's Balanced Salt Solution (HBSS, Gibco) at a concentration of cells / 100 μl, CT26 cells were implanted into the subcutaneous region of the right flank of mice using a 1 cc syringe (25G). Tumor formation and growth were assessed by subcutaneous implantation of 4T1 cells into the left flank of the same mice.

[0414] As a result, it was found that both the transplanted 4T1 tumor and the CT26 tumor grew in the control mice without any prior treatment, whereas in the mice that achieved a complete response after administration of T1.01m, the 4T1 tumor grew but the growth of the CT26 tumor was inhibited, as shown in Figures 26 and 27. These results indicate that the fusion protein of one embodiment can inhibit tumor recurrence.

[0415] Example 4.4. Evaluation of reduction in activity due to IL-12 amino acid mutations in fusion proteins To reduce in vivo toxicity due to excessive activation of the immune system by IL-12, the present inventors used an IL-12 sequence structure containing a mutation in the amino acid sequence that binds to heparin to induce a decrease in activity by blocking binding to the IL-12 receptor, which was identified by a tumor animal model.

[0416] Among the anti-FAP antibodies, T1.02m and T1.03m, in which the amino acids involved in heparin binding are mutated, were compared with T1.01m, and phosphate buffered saline (PBS) was used as a control. Next, T1.02m has a mouse IL-12 sequence structure containing two amino acid mutations involved in heparin binding together with the anti-FAP sequence structure. T1.03m is a fusion protein having an IL-12 sequence structure containing four amino acid mutations involved in heparin binding together with the anti-FAP sequence structure.

[0417] To evaluate the change in IL-12 activity depending on the presence and degree of amino acid mutation, 5 × 10 5 FAP-overexpressing mouse colon cancer CT26 was prepared by resuspending in PBS at a concentration of cells / 100 μl, and the tumor growth inhibition ability was evaluated in a tumor animal model prepared by transplanting the cells into 5-week-old female BALB / c mice.

[0418] Specifically, the tumor size in the model was 100 mm 3 When the mice reached the target concentration, they were randomly divided into groups, each consisting of eight animals, that received a single tail vein injection of 100 μl phosphate buffered saline (PBS) for the control, and a single intravenous injection of T1.01m, T1.02m, and T1.03m at a dose of 10 μg each.

[0419] As a result, as shown in FIG. 28, it was found that all of the T1.01m, T1.02m and T1.03m administration groups had superior tumor growth inhibition abilities compared to the control 15 days after administration.

[0420] Furthermore, the group administered T1.03m, which has four IL-12-containing mutations of amino acids involved in heparin binding together with the mouse anti-FAP sequence, was found to have reduced tumor growth inhibition ability compared to the group administered T1.01m. These results indicate that the group administered T1.03m, which has a mouse IL-12 sequence structure containing four amino acid mutations involved in heparin binding, had reduced IL-12 activity compared to the other experimental groups and controls.

[0421] Example 4.5. Evaluation of anti-cancer effect of single administration of IL-12 amino acid mutant fusion protein To evaluate the anticancer effect of the fusion protein, which has reduced IL-12 activity due to a sequence structure containing four amino acid mutations involved in heparin binding, we evaluated its tumor growth inhibition ability in a tumor model transplanted with the FAP-overexpressing mouse colon cancer cell line CT26.

[0422] Specifically, tumor cells were transplanted into the right dorsal flank of 6-week-old BALB / c mice, and the tumor size of the animal model was 70 to 100 mm. 3 When the tumor size reached 10 μg, the mice were randomly divided into groups and administered. PBS was administered as a control, and 100 μl of T1.03m was administered intravenously once at doses of 2 μg, 10 μg, and 100 μg as a test group. The tumor size was measured in the same manner as in Example 4.1.

[0423] As a result, as shown in Figure 29, it was observed that the groups administered 2 μg, 10 μg and 100 μg of T1.03m had a higher tumor growth inhibition ability compared to the control. No change in body weight was observed during the test period. These results indicate that T1.03m, which has reduced IL-12 activity due to its sequence structure containing four amino acid mutations involved in heparin binding, has an excellent anti-cancer effect above a certain concentration.

[0424] Example 5. Evaluation of the anti-cancer effect of the fusion protein in a melanoma animal model To identify the anti-cancer effect of the fusion protein through immune activation, we overexpressed mouse FAP in the mouse melanoma cell line B16F10, which is known to have low immune cell infiltration, and then implanted it into C57BL / 6J mice to evaluate its ability to inhibit tumor growth.

[0425] Specifically, the cultured cells were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) and then injected into the subcutaneous region of the left flank of 6-week-old C57BL / 6J mice at 100 μl of 1 × 10 cells / mouse. 6 Tumor cells were implanted.

[0426] Next, to evaluate the efficacy of the drug, the tumor size was 70-100 mm 3 When the mice reached the threshold of 100 μg / kg, they were randomly divided into groups: PBS was administered as a control group, and T1.03m was administered intravenously once at doses of 10 μg and 100 μg as a test group.

[0427] As a result, as shown in Figure 30, it was observed that both groups administered with 10 μg and 100 μg of T1.03m had high tumor growth inhibition ability compared to the control. No change in body weight was observed during the test period. These results indicate that T1.03m, which has reduced IL-12 activity due to its sequence structure containing four amino acid mutations involved in heparin binding, has excellent anti-cancer effect even in melanoma animal models.

[0428] Example 6. Evaluation of the anticancer effect of the fusion protein in an animal model of lung cancer Example 6.1. Evaluation of the anti-cancer effects of IL-12 amino acid mutant fusion proteins in lung cancer animal models Considering that FAP is overexpressed in fibroblasts present in the tumor microenvironment, we overexpressed FAP in the mouse fibroblast cell line NIH-3T3 and then co-injected it with the mouse lung cancer cell line LLC1 at a 1:1 ratio to prepare a tumor animal model and evaluate its tumor growth inhibition ability.

[0429] Specifically, FAP-overexpressing NIH-3T3 cell line and LLC1 cells were cultured at 1 × 10 6 The cells were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) at a concentration of cells / 50 μl, mixed at a 1:1 ratio, and then 100 μl per mouse was implanted into the subcutaneous region of the right flank of 6-week-old C57BL / 6J mice using a 1 cc syringe (25G).

[0430] Next, to evaluate the efficacy of the drug, the tumor size was 70-100 mm 3 When the mice reached the threshold of 100 μg / kg, they were randomly divided into groups: PBS was administered as a control group, and T1.03m was administered intravenously once at doses of 10 μg and 100 μg as a test group.

[0431] As a result, as shown in Figure 31, it was observed that both groups administered with 10μg or 100μg of T1.03m had superior tumor growth inhibition ability compared to the control. No change in body weight was observed during the study period. These results indicate that T1.03m, which has reduced IL-12 activity due to its sequence structure containing four amino acid mutations involved in heparin binding, recognizes target factors in the tumor microenvironment, thereby having superior anti-cancer effect even in lung cancer animal models.

[0432] Example 6.2. Evaluation of cancer targeting effects in lung cancer animal models by co-injection of fusion proteins To compare the anti-cancer effects of the dual antibody fusion protein in which the anti-FAP sequence structure and the IL-12 sequence structure form a knob-in-hole structure, we identified the tumor growth inhibition ability in an animal model in which NIH-3T3 overexpressing FAP, a mouse fibroblast cell line, and LLC1, a mouse lung cancer cell line, were co-injected at a 1:1 ratio.

[0433] Specifically, the efficacy of the drug was evaluated in patients with tumors between 70 and 100 mm 3When the tumor growth rate reached 100%, the mice were randomly divided into groups. As a control, PBS was administered intraperitoneally twice a week. The groups administered T1.16m or T1.17m alone, the group administered T1.16m and T1.17m in combination (T1.16m+T1.17m), and the group administered T1.03m were used as test groups to evaluate the tumor growth inhibition ability. T1.16m and T1.17m, which are dimers of anti-FAP and IL-12, were administered intraperitoneally twice a week at a dose of 0.01 μg, respectively, and T1.03m, a monomeric fusion protein, was administered intraperitoneally twice a week at a dose of 0.02 μg to observe the tumor growth inhibition ability.

[0434] As a result, as shown in Figure 32, it was observed that the group administered with T1.03m had superior tumor growth inhibition ability at 10 days after administration compared to the control. Comparing the group administered with T1.16m or T1.17m alone and each combination administration group, it was found that the group administered with T1.03m alone had extremely good tumor growth inhibition ability. These results indicate that the synergistic effect of the fusion protein of one embodiment is significant when only FAP is targeted or when compared with IL-12 without a cancer target.

Claims

1. a first monomer comprising IL-12 or a variant thereof; a second monomer that contains an antigen binding site that specifically binds to FAP (fibroblast activation protein alpha); A fusion protein comprising:

2. The fusion protein of claim 1, wherein the IL-12 or variant thereof comprises IL-12A (p35) or a variant thereof; and IL-12B (p40) or a variant thereof.

3. The IL-12 or the variant thereof has the following structural formula (I) or (II): N'-Y-[linker (1)]o-Z-C' (I) N'-Z-[linker (1)]o-Y-C' (II) [In the structural formulas (I) and (II), N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; Y is said IL-12A or said variant thereof, Z is said IL-12B or said variant thereof; The linker (1) is a peptide linker, o is 0 or 1. The fusion protein of claim 2 .

4. 3. The fusion protein of claim 2, wherein the variant of IL-12B(p40) comprises an amino acid sequence obtained by at least one substitution selected from the group consisting of K258A, K260A, K263A, and K264A in the amino acid sequence of SEQ ID NO:

74.

5. The fusion protein of claim 2, wherein the IL-12B(p40) comprises the amino acid sequence of SEQ ID NO:74 or SEQ ID NO:

86.

6. 5. The fusion protein of claim 4, wherein the variant of IL-12B(p40) comprises the amino acid sequence of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:89 or SEQ ID NO:

91.

7. The fusion protein of claim 2, wherein the IL-12A(p35) comprises the amino acid sequence of SEQ ID NO:75 or SEQ ID NO:

87.

8. 2. The fusion protein of claim 1, wherein the IL-12 comprises the amino acid sequence of SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:88 or SEQ ID NO:

90.

9. The antigen-binding site that specifically binds to FAP is a heavy chain variable region comprising an HCDR1 of SEQ ID NO:96, an HCDR2 of SEQ ID NO:97, and an HCDR3 of SEQ ID NO:98, and a light chain variable region comprising an LCDR1 of SEQ ID NO:99, an LCDR2 of SEQ ID NO:100, and an LCDR3 of SEQ ID NO:101; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 104, an HCDR2 of SEQ ID NO: 105, and an HCDR3 of SEQ ID NO: 106, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 107, an LCDR2 of SEQ ID NO: 108, and an LCDR3 of SEQ ID NO: 109; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 127, an HCDR2 of SEQ ID NO: 128, and an HCDR3 of SEQ ID NO: 129, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 130, an LCDR2 of SEQ ID NO: 131, and an LCDR3 of SEQ ID NO: 132; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 133, an HCDR2 of SEQ ID NO: 134, and an HCDR3 of SEQ ID NO: 135, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 136, an LCDR2 of SEQ ID NO: 137, and an LCDR3 of SEQ ID NO: 138; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 139, an HCDR2 of SEQ ID NO: 140, and an HCDR3 of SEQ ID NO: 141, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 142, an LCDR2 of SEQ ID NO: 143, and an LCDR3 of SEQ ID NO: 144; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 145, an HCDR2 of SEQ ID NO: 146, and an HCDR3 of SEQ ID NO: 147, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 148, an LCDR2 of SEQ ID NO: 149, and an LCDR3 of SEQ ID NO: 150; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 151, an HCDR2 of SEQ ID NO: 152, and an HCDR3 of SEQ ID NO: 153, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 154, an LCDR2 of SEQ ID NO: 155, and an LCDR3 of SEQ ID NO: 156; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 157, an HCDR2 of SEQ ID NO: 158, and an HCDR3 of SEQ ID NO: 159, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 160, an LCDR2 of SEQ ID NO: 161, and an LCDR3 of SEQ ID NO: 162; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 163, an HCDR2 of SEQ ID NO: 164, and an HCDR3 of SEQ ID NO: 165, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 166, an LCDR2 of SEQ ID NO: 167, and an LCDR3 of SEQ ID NO: 168; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 169, an HCDR2 of SEQ ID NO: 170, and an HCDR3 of SEQ ID NO: 171, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 172, an LCDR2 of SEQ ID NO: 173, and an LCDR3 of SEQ ID NO: 174; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 175, an HCDR2 of SEQ ID NO: 176, and an HCDR3 of SEQ ID NO: 177, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 178, an LCDR2 of SEQ ID NO: 179, and an LCDR3 of SEQ ID NO: 180; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 181, an HCDR2 of SEQ ID NO: 182, and an HCDR3 of SEQ ID NO: 183, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 184, an LCDR2 of SEQ ID NO: 185, and an LCDR3 of SEQ ID NO: 186; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 187, an HCDR2 of SEQ ID NO: 188, and an HCDR3 of SEQ ID NO: 189, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 190, an LCDR2 of SEQ ID NO: 191, and an LCDR3 of SEQ ID NO: 192; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 193, an HCDR2 of SEQ ID NO: 194, and an HCDR3 of SEQ ID NO: 195, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 196, an LCDR2 of SEQ ID NO: 197, and an LCDR3 of SEQ ID NO: 198; a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 199, an HCDR2 of SEQ ID NO: 200, and an HCDR3 of SEQ ID NO: 201, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 202, an LCDR2 of SEQ ID NO: 203, and an LCDR3 of SEQ ID NO: 204; a heavy chain variable region comprising an HCDR1 of SEQ ID NO:205, an HCDR2 of SEQ ID NO:206, and an HCDR3 of SEQ ID NO:207, and a light chain variable region comprising an LCDR1 of SEQ ID NO:208, an LCDR2 of SEQ ID NO:209, and an LCDR3 of SEQ ID NO:210; a heavy chain variable region comprising an HCDR1 of SEQ ID NO:211, an HCDR2 of SEQ ID NO:212, and an HCDR3 of SEQ ID NO:213, and a light chain variable region comprising an LCDR1 of SEQ ID NO:214, an LCDR2 of SEQ ID NO:215, and an LCDR3 of SEQ ID NO:216; a heavy chain variable region comprising an HCDR1 of SEQ ID NO:217, an HCDR2 of SEQ ID NO:218, and an HCDR3 of SEQ ID NO:219, and a light chain variable region comprising an LCDR1 of SEQ ID NO:220, an LCDR2 of SEQ ID NO:221, and an LCDR3 of SEQ ID NO:222; a heavy chain variable region comprising an HCDR1 of SEQ ID NO:223, an HCDR2 of SEQ ID NO:224, and an HCDR3 of SEQ ID NO:225, and a light chain variable region comprising an LCDR1 of SEQ ID NO:226, an LCDR2 of SEQ ID NO:227, and an LCDR3 of SEQ ID NO:228; a heavy chain variable region comprising an HCDR1 of SEQ ID NO:229, an HCDR2 of SEQ ID NO:230, and an HCDR3 of SEQ ID NO:231, and a light chain variable region comprising an LCDR1 of SEQ ID NO:232, an LCDR2 of SEQ ID NO:233, and an LCDR3 of SEQ ID NO:234; a heavy chain variable region comprising an HCDR1 of SEQ ID NO:235, an HCDR2 of SEQ ID NO:236, and an HCDR3 of SEQ ID NO:237, and a light chain variable region comprising an LCDR1 of SEQ ID NO:238, an LCDR2 of SEQ ID NO:239, and an LCDR3 of SEQ ID NO:240; or a heavy chain variable region comprising an HCDR1 of SEQ ID NO:241, an HCDR2 of SEQ ID NO:242, and an HCDR3 of SEQ ID NO:243, and a light chain variable region comprising an LCDR1 of SEQ ID NO:244, an LCDR2 of SEQ ID NO:245, and an LCDR3 of SEQ ID NO:246; The fusion protein of claim 1 , comprising:

10. The antigen-binding site that specifically binds to FAP is A heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 103; A heavy chain variable region of SEQ ID NO: 110 and a light chain variable region of SEQ ID NO: 111; A heavy chain variable region of SEQ ID NO:248 and a light chain variable region of SEQ ID NO:249; A heavy chain variable region of SEQ ID NO:250 and a light chain variable region of SEQ ID NO:251; A heavy chain variable region of SEQ ID NO: 252 and a light chain variable region of SEQ ID NO: 253; A heavy chain variable region of SEQ ID NO: 254 and a light chain variable region of SEQ ID NO: 255; A heavy chain variable region of SEQ ID NO:256 and a light chain variable region of SEQ ID NO:257; A heavy chain variable region of SEQ ID NO:258 and a light chain variable region of SEQ ID NO:259; A heavy chain variable region of SEQ ID NO:260 and a light chain variable region of SEQ ID NO:261; A heavy chain variable region of SEQ ID NO: 262 and a light chain variable region of SEQ ID NO: 263; A heavy chain variable region of SEQ ID NO: 264 and a light chain variable region of SEQ ID NO: 265; A heavy chain variable region of SEQ ID NO: 266 and a light chain variable region of SEQ ID NO: 267; A heavy chain variable region of SEQ ID NO:268 and a light chain variable region of SEQ ID NO:269; A heavy chain variable region of SEQ ID NO:270 and a light chain variable region of SEQ ID NO:271; A heavy chain variable region of SEQ ID NO:272 and a light chain variable region of SEQ ID NO:273; A heavy chain variable region of SEQ ID NO:274 and a light chain variable region of SEQ ID NO:275; A heavy chain variable region of SEQ ID NO:276 and a light chain variable region of SEQ ID NO:277; A heavy chain variable region of SEQ ID NO:278 and a light chain variable region of SEQ ID NO:279; A heavy chain variable region of SEQ ID NO:280 and a light chain variable region of SEQ ID NO:281; A heavy chain variable region of SEQ ID NO:282 and a light chain variable region of SEQ ID NO:283; A heavy chain variable region of SEQ ID NO:284 and a light chain variable region of SEQ ID NO:285; A heavy chain variable region of SEQ ID NO:286 and a light chain variable region of SEQ ID NO:287; scFv of SEQ ID NO: 72; or scFv of SEQ ID NO: 84 The fusion protein of claim 1 , comprising:

11. The fusion protein of claim 1 , wherein the first monomer further comprises an antigen binding site that specifically binds to FAP.

12. The first monomer has the following structural formula (III) or (IV): N'-X-[linker (2)]p-Fc region fragment or variant thereof-[linker (3)]q-(T)r-C' (III) N'-(T)r-[linker (2)]q-Fc region fragment or variant thereof-[linker (3)]p-XC' (IV) [In the structural formulas (III) and (IV), N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is represented by the structural formula (I) or (II); T is the antigen-binding site that specifically binds to FAP, The linkers (2) and (3) are peptide linkers, p, q and r are each independently 0 or 1. The fusion protein of claim 3 .

13. The linker is ４ S) ｎ The fusion protein according to claim 3 or 12, comprising a linker, wherein n is any one of integers from 1 to 10.

14. The fusion protein of claim 12, wherein the Fc region of the first monomer is derived from human IgG1 or mouse IgG2a.

15. The fusion protein of claim 12 , wherein the Fc of the first monomer comprises a knob or hole structure.

16. The fusion protein of claim 1 , wherein the second monomer further comprises an antigen binding site that specifically binds to FAP.

17. 17. The fusion protein of claim 16, wherein the antigen binding site that specifically binds to FAP is a Fab, scFv, Fv, or fragment thereof.

18. 17. The fusion protein of claim 16, wherein the additionally attached antigen binding site that specifically binds to FAP is attached to the N-terminus or C-terminus of the second monomer.

19. The second monomer has the following structural formula (V): N'-(R)s-[linker (4)]t-Q-[linker (5)]u-Fc region fragment or variant thereof-[linker (6)]v-(W)a-C' (V) [In the structural formula (V), N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; R is the antigen-binding site that specifically binds to FAP, Q is the antigen-binding site that specifically binds to FAP, W is an scFv that specifically binds to FAP; or the IL-12 of structural formula (I) or (II) or the variant thereof, The linkers (4) to (6) are peptide linkers, s, t, u and v are each independently 0 or 1. The fusion protein of claim 3 .

20. The structural formula (V) is represented by the following structural formulas (V') and (V"): N'-(R')s-[linker (4)]t-Q'-[linker (5)]u-Fc region fragment or variant thereof-[linker (6)]p-(W)a-C'(V') N'-(R'')s-[linker (4)]t-Q''-[linker (7)]x-(W)b-C'(V'') [In the structural formulas (V') and (V"), R' is a heavy chain region of an antibody that specifically binds to FAP, comprising a variable region and a CH1 region, or a light chain region of said antibody; R″ is a light chain region of an antibody that specifically binds to FAP, or a heavy chain region of said antibody, including the variable region and the CH1 region; wherein R′ and R″ combine together to form a variable region of the antibody, the variable region specifically binds to FAP; Q' is a heavy chain region of an antibody that specifically binds to FAP, comprising a variable region and a CH1 region, or a light chain region of said antibody; Q″ is a light chain region of an antibody that specifically binds to FAP, or a heavy chain region of said antibody, including the variable region and the CH1 region; wherein Q′ and Q″ bind to each other to form a variable region of the antibody, the variable region specifically binds to FAP; R' and Q' are each a heavy chain region of the antibody, including a variable region and a CH1 region, or a light chain region of the antibody; R″ and Q″ are each a light chain region of the antibody, or a heavy chain region of the antibody, including a variable region and a CH1 region; wherein R′ and R″; and Q′ and Q″ combine together to form a variable region of the antibody, said variable region specifically binds to FAP; W is an scFv that specifically binds to FAP, the linkers (4), (5) and (7) are peptide linkers; s, t, u, x, a and b are each independently 0 or 1.

20. The fusion protein of claim 19, comprising:

21. The fusion protein of claim 1, comprising structural formula (III) and structural formula (V); structural formula (IV) and structural formula (V); structural formula (III) and structural formula (III); structural formula (IV) and structural formula (IV); or structural formula (V) and structural formula (V).

22. The fusion protein of claim 1, wherein the second monomer comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain consisting of the amino acid sequence of SEQ ID NO: 103; or a heavy chain consisting of the amino acid sequence of SEQ ID NO: 110 and a light chain consisting of the amino acid sequence of SEQ ID NO:

111.

23. The fusion protein of claim 2 , wherein the Fc of the second monomer comprises a knob or hole structure.

24. A pharmaceutical composition for preventing or treating cancer, comprising the fusion protein according to any one of claims 1 to 23 as an active ingredient.

25. 25. The pharmaceutical composition of claim 24, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

26. A polynucleotide encoding the first monomer of claim 1.

27. A vector comprising the polynucleotide of claim 26.

28. A polynucleotide encoding the second monomer of claim 1.

29. A vector comprising the polynucleotide of claim 28.

30. A transformed cell into which the vector according to claim 27 or 29 has been introduced.

31. 1. A method for producing a fusion protein, comprising: i) culturing the transformed cell according to claim 30; ii) recovering the fusion protein comprising the first monomer and the second monomer; The method includes:

32. 1. A method for treating or preventing cancer, comprising: administering to the subject a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to FAP. The method includes:

33. Use of a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to FAP for treating cancer.

34. 2. Use of a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen-binding site that specifically binds to FAP for the manufacture of a medicament for treating cancer.

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