Fusion protein comprising IL-12 and anti-FAP antibody, and its use

Abstract

The present invention provides a bispecific antibody comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to a FAP. The bispecific antibody exhibits an IL-12-mediated anti-cancer effect. In particular, when an anti-FAP antibody is realized as a single antibody, it specifically targets a FAP that is highly expressed in tumors, thereby enabling the specific localization of IL-12 at the tumor site, thereby enabling efficient cancer treatment. Therefore, the bispecific antibody can be used as a pharmaceutical composition for anti-cancer treatment and therefore has high industrial applicability.

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 Art

[0002] Interleukin-12 (IL-12) is a typical inflammatory cytokine and plays an extremely important 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 cytotoxic T lymphocytes, and inhibit angiogenesis. Generally, 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 establishment of humoral immunity. However, a drawback in the clinical application of IL-12 is that cytokine-related toxicity can occur systemically upon administration. These clinical results indicate 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 cancer and pancreatic cancer. Therefore, in recent years, interest in the development of immunotherapy targeting FAP-expressing cells has been rapidly increasing (Fang J. et al., Mol. Ther. Oncolytics., 3:16007, 2016).

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, the present inventors conducted research to develop a novel fusion protein having an anti-cancer effect. As a result, the present inventors found that a fusion protein containing 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 Problems

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

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

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

[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 present invention, there is provided a method for producing a fusion protein, the method comprising: i) culturing the transformed cell; and ii) recovering the fusion protein containing the first monomer and the second monomer.

[0013] In yet another aspect of the present invention, there is provided a method for treating or preventing cancer, the method 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 FAP.

[0014] In yet another aspect of the present invention, there is provided the 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.

[0015] In yet another aspect of the present invention, there is provided the 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 manufacturing a medicament for treating cancer. [[Effect of the Invention]]

[0016] A fusion protein comprising IL-12 and an antigen-binding site that specifically binds to FAP may exhibit an anti-cancer effect by IL-12. In addition, when the anti-FAP antibody is realized by one antibody, it has been found that IL-12 specifically accumulates in tumors having high FAP expression, thereby reducing systemic toxicity and exhibiting a tumor-specific anti-cancer effect. That is, by specifically targeting FAP highly expressed in tumors and specifically localizing IL-12 to the tumor site, cancer can be efficiently treated. [[Brief Description of the Drawings]]

[0017]

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Modes for Carrying Out the Invention

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

[0019] In one embodiment, 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 FAP.

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

[0021] IL-12 or a variant thereof As used herein, the term "IL-12" refers to 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 effects 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 function similar or identical to that of the wild-type IL-12 protein. Specifically, the amino acid sequences of the p35 and p40 subunits that make up 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, IL-12 or a 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 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, Linker(1) may be a peptide linker, o may be 0 or 1] and may be a fusion protein containing the same.

[0026] One embodiment of IL-12 or its variant may have a structure in which IL-12B or its variant, a peptide linker, and IL-12A or its variant are sequentially linked from the N-terminus. In addition, one embodiment of IL-12 or its variant may have a structure in which IL-12A or its variant, a peptide linker, and IL-12B or its variant are sequentially linked from the N-terminus.

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

[0028] The amino acid sequence of IL-12A may be the one described in GenBank accession number AAK84425 (see GenBank accession number AAA39292 for the amino acid sequence of mouse p35). In addition, 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 described in GenBank accession number AF404773 (see GenBank accession number M86672 for the mouse sequence).

[0029] As used herein, the term "variant" of IL-12A includes an amino acid sequence having a function similar or identical to that of the wild-type IL-12A protein. Specifically, the "variant" of IL-12A means that the amino acid sequence of IL-12A has substitutions, insertions, or deletions compared to the wild-type. Specifically, the 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 specifically, the variant of IL-12A may have the amino acid sequence of SEQ ID NO: 75.

[0030] In addition, 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, the 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. IL-12B binds to the 35 kDa subunit (p35) of IL-12 via a disulfide bond to form IL-12, and also binds to the p19 subunit of IL-23 to form IL-23. On the other hand, IL-12B is also called natural killer cell stimulatory factor 2.

[0032] The amino acid sequence of "IL-12B" may be the one described in GenBank accession number AAD56386 (for the amino acid sequence of mouse p40, see GenBank accession number AAA39296). In addition, 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 described in GenBank accession number AF180563 (for the mouse sequence, see GenBank accession number M86671).

[0033] In addition, the IL-12B used in one embodiment of the present invention may be human IL-12B. Specifically, IL-12B may be the amino acid sequence of SEQ ID NO: 74. In one embodiment, the 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 having a function similar or identical to that of the wild-type IL-12B protein. Specifically, the "variant" of IL-12B means that the amino acid sequence of IL-12B has substitutions, insertions or deletions compared to the wild-type.

[0035] Specifically, the IL-12B variant may have a deletion of amino acids 1 to 22 of SEQ ID NO: 121. More specifically, the IL-12B variant may have 1 to 8 amino acids substituted in the sequence of SEQ ID NO: 74. In addition, the IL-12B variant may have a deletion of amino acids 1 to 22 of SEQ ID NO: 123. More specifically, the IL-12B variant may have up to 1 to 8 amino acids substituted in the sequence of SEQ ID NO: 86.

[0036] The variant of IL-12B (p40) may have amino acids involved in the heparin binding of IL-12 substituted.

[0037] In one embodiment, the variant of IL-12B (p40) may be in a form where the lysine (K) binding to heparin in IL-12B is substituted with another amino acid. Specifically, the variant of IL-12B (p40) may be in a form where lysines at positions 258, 260, 263, and 264 of SEQ ID NO: 74 are substituted with other amino acids. Specifically, the variant of IL-12B (p40) may include 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. Specifically, the variant of IL-12B (p40) may include an amino acid sequence obtained by the substitutions of K258A and K263A in the amino acid sequence of SEQ ID NO: 74, or may further include the mutations of 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 in a form in which the binding of arginine (R) or lysine (K) to heparin in IL-12B is replaced with another amino acid. Specifically, the variant of IL-12B(p40) may be in a form in which the 254th arginine and / or the 255th, 256th, and 260th lysines of SEQ ID NO: 86 are replaced with other amino acids. Specifically, the variant of IL-12B(p40) may include 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. Specifically, the variant of IL-12B(p40) may include an amino acid sequence obtained by the substitutions of R254A and K260A in the amino acid sequence of SEQ ID NO: 86, or may further include the mutations of K255A and / or K256A in the sequence of SEQ ID NO: 86.

[0039] In one embodiment, the variant of IL-12B(p40) may include 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 the amino acid sequence consisting of V-A1-A2-Q-A3-K * -A4-A5-A6-A7-K * -A8] and may consist of. A1 is R or Q, A2 is V, A or I, A3 is G or R * and 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 with a "]" may be substituted with a non-polar amino acid residue selected from the group consisting of A, G, I, L, M, F, P, and V, but is not limited thereto.

[0041] In one embodiment, a variant of IL-12B(p40) may include substitutions of amino acids involved in heparin binding in order to maintain the killing effect on cancer cells while reducing the cytokine-related toxicity of IL-12. FAP and an antigen-binding site that specifically binds 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 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, FAP is a homodimer containing two N-glycosylated subunits having a C-terminal extracellular domain where the enzyme catalytic domain is located, and the glycosylated form of FAP has both prolyl dipeptidyl peptidase and gelatinase activities.

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

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

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

[0046] As used herein, the term "specifically binds" refers to a binding that is clearly distinct from non-specific interactions. Specific binding can be determined by competition with a control molecule similar to the target 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. The antigen-binding site is a group of molecules, such as amino acids or sugar side chains, that usually have specific structural and specific charge characteristics. An antigen-binding site that specifically binds to FAP can be a general term for molecules capable of specifically antigen-antibody binding to FAP.

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

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

[0050] Specifically, the antigen-binding site may be an antibody or its fragment that specifically binds to FAP.

[0051] As used herein, the term "antibody" refers to a molecule containing an antigen-binding site and an immunologically active fragment of an immunoglobulin molecule containing 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, for example, monospecific and bispecific, etc.), Fab fragments, F(ab’) fragments, disulfide-linked Fv (sdFv), anti-idiotype (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, such as F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv. Regardless of structure, the antibody fragment binds to the same antigen recognized by the intact antibody.

[0053] In one embodiment, the antigen-binding site that specifically binds to FAP may include a heavy-chain variable region comprising HCDR1 of SEQ ID NO: 96, HCDR2 of SEQ ID NO: 97, and HCDR3 of SEQ ID NO: 98; and a light-chain variable region comprising LCDR1 of SEQ ID NO: 99, LCDR2 of SEQ ID NO: 100, and LCDR3 of SEQ ID NO: 101. Here, in one embodiment, the antigen-binding site that specifically binds to 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] In addition, in one embodiment, the antigen-binding site that specifically binds to FAP may include 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, the antigen-binding site that specifically binds to 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 FAP may be an scFv. Here, the scFv may contain the heavy and light chain CDRs described above. In one embodiment, the scFv that specifically binds to FAP may have the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 84.

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

[0057] The anti-FAP antibody or a fragment thereof may include 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, as the antibody, an anti-FAP antibody or a fragment thereof disclosed in Korean Patent Application Publication No. KR20200037826A, US Patent Application Publication No. US2020-0385488A1, US Patent No. US3,924,445B2, US Patent No. US9,011,847B2, or US Patent Application Publication No. US2017-007716A1 can be used.

[0059] In one embodiment, the anti-FAP antibody may include the variable region of NG-641. Specifically, the antibody may include a heavy chain variable region containing 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 containing 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 include the heavy chain variable region of SEQ ID NO: 248 and the light chain variable region of SEQ ID NO: 249.

[0060] In one embodiment, the anti-FAP antibody may comprise the variable region of 28H1. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 133, HCDR2 of SEQ ID NO: 134, and HCDR3 of SEQ ID NO: 135, and a light chain variable region comprising LCDR1 of SEQ ID NO: 136, LCDR2 of SEQ ID NO: 137, and LCDR3 of SEQ ID NO: 138. Additionally, 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. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 139, HCDR2 of SEQ ID NO: 140, and HCDR3 of SEQ ID NO: 141, and a light chain variable region comprising LCDR1 of SEQ ID NO: 142, LCDR2 of SEQ ID NO: 143, and LCDR3 of SEQ ID NO: 144. Additionally, 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. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 145, HCDR2 of SEQ ID NO: 146, and HCDR3 of SEQ ID NO: 147, and a light chain variable region comprising LCDR1 of SEQ ID NO: 148, LCDR2 of SEQ ID NO: 149, and LCDR3 of SEQ ID NO: 150. Additionally, 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. Specifically, 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. Additionally, 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. Specifically, 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. Additionally, 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. Specifically, 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. Additionally, 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. Specifically, 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. Additionally, 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. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 175, HCDR2 of SEQ ID NO: 176, and HCDR3 of SEQ ID NO: 177, and a light chain variable region comprising LCDR1 of SEQ ID NO: 178, LCDR2 of SEQ ID NO: 179, and LCDR3 of SEQ ID NO: 180. Additionally, 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 include the variable region of 20G8. Specifically, the antibody may include a heavy chain variable region containing 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 containing LCDR1 of SEQ ID NO: 184, LCDR2 of SEQ ID NO: 185, and LCDR3 of SEQ ID NO: 186. Additionally, the anti-FAP antibody may include 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 include the variable region of 5B8. Specifically, the antibody may include a heavy chain variable region containing 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 containing LCDR1 of SEQ ID NO: 190, LCDR2 of SEQ ID NO: 191, and LCDR3 of SEQ ID NO: 192. Additionally, the anti-FAP antibody may include 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 include the variable region of 5F1. Specifically, the antibody may include a heavy chain variable region containing HCDR1 of SEQ ID NO: 193, HCDR2 of SEQ ID NO: 194, and HCDR3 of SEQ ID NO: 195, and a light chain variable region containing LCDR1 of SEQ ID NO: 196, LCDR2 of SEQ ID NO: 197, and LCDR3 of SEQ ID NO: 198. Additionally, the anti-FAP antibody may include 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 include the variable region of 14B3. Specifically, the antibody may include a heavy chain variable region containing HCDR1 of SEQ ID NO: 199, HCDR2 of SEQ ID NO: 200, and HCDR3 of SEQ ID NO: 201, and a light chain variable region containing LCDR1 of SEQ ID NO: 202, LCDR2 of SEQ ID NO: 203, and LCDR3 of SEQ ID NO: 204. Additionally, the anti-FAP antibody may include 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. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 205, HCDR2 of SEQ ID NO: 206, and HCDR3 of SEQ ID NO: 207, and a light chain variable region comprising LCDR1 of SEQ ID NO: 208, LCDR2 of SEQ ID NO: 209, and 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. Specifically, 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. Specifically, 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. Specifically, 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 include the variable region of 29B11. Specifically, the antibody may include a heavy chain variable region containing HCDR1 of SEQ ID NO: 229, HCDR2 of SEQ ID NO: 230, and HCDR3 of SEQ ID NO: 231, and a light chain variable region containing LCDR1 of SEQ ID NO: 232, LCDR2 of SEQ ID NO: 233, and LCDR3 of SEQ ID NO: 234. In addition, the anti-FAP antibody may include 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 include the variable region of O2D7. Specifically, the antibody may include a heavy chain variable region containing 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 containing 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 include 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 include the variable region of 23C10. Specifically, the antibody may include a heavy chain variable region containing HCDR1 of SEQ ID NO: 241, HCDR2 of SEQ ID NO: 242, and HCDR3 of SEQ ID NO: 243, and a light chain variable region containing LCDR1 of SEQ ID NO: 244, LCDR2 of SEQ ID NO: 245, and LCDR3 of SEQ ID NO: 246. In addition, the anti-FAP antibody may include 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. Specifically, the antibody may include the amino acid sequence of SEQ ID NO: 247.

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

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

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

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

[0084] The first monomer has the following structural formula (III) or (IV): N’-X-[Linker(2)]p-Fc region fragment or a variant thereof-[Linker(3)]q-(T)r-C’ (III) N’-(T)r-[Linker(2)]q-Fc region fragment or a variant thereof-[Linker(3)]p-X-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 structural formula (I) or (II), T may be an antigen-binding site that specifically binds to FAP, Linkers (2) and (3) may be peptide linkers, p, q, and r may each independently be 0 or 1] and 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 include 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, in Structural Formula (III), when r is 1, the first monomer may be in a form in which a monomer of an anti-FAP antibody is bound to IL-12 or a variant thereof.

[0088] Here, in Structural Formula (III), when r is 1, the first monomer may contain 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 contain 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 the following Structural Formulas (III') and (III"): N'-X-[Linker(2)]p-Fc region fragment or a 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 FAP and includes a variable region and a CH1 region, or a light chain region of the antibody, T" is a light chain region of an antibody that specifically binds to FAP, or a heavy chain region of the antibody that includes a variable region and a CH1 region, Here, T' and T" bind to each other to form a variable region of the antibody, and the variable region specifically binds to FAP, Linkers (2) to (3) are peptide linkers, p and q are each independently 0 or 1, N', X, and C' are as defined above] may be included. In one embodiment, when r is 1, Structural Formula (IV) is the following Structural Formulas (IV') and (IV"): N'-(T')-[Linker(2)]q-Fc region fragment or its variant-[Linker(3)]p-X-C' (IV'); and N'-(T")-C' (IV")[[]]END]] [In Structural Formulas (IV') and (IV"), T' is the heavy chain region of an antibody that specifically binds to FAP and contains a variable region and a CH1 region, or the light chain region of an antibody, T" is the light chain region of an antibody that specifically binds to FAP, or the heavy chain region of an antibody that contains a variable region and a CH1 region, Here, T' and T" bind to each other to form the variable region of an antibody, and the variable region specifically binds to FAP, Linker(2) to (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 contain 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 contain an antigen-binding site that specifically binds to FAP.

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

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

[0094] The second FAP binding site may be bound to the N-terminus or C-terminus of the second monomer. Specifically, the second FAP binding site may additionally be bound 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)]t-Q-[Linker(5)]u-Fc region fragment or its variant-[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 FAP, W may be an scFv that specifically binds to FAP; or IL-12 of structural formula (I) or (II) or its variant, Linker(4), (5) and (6) may each be a peptide linker, s, t, u, v and a may each independently be 0 or 1] and may include.

[0096] In one embodiment, structural formula (V) 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: 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 region fragment or its variant-[Linker(6)]p-(W)a-C’ (V’) N’-(R”)s-[Linker(4)]t-Q”-[Linker(7)]x-(W)b-C’ (V”) In Structural Formulas (V') and (V"), R' may be the heavy chain region of an antibody that specifically binds to FAP and includes a variable region and a CH1 region, or the light chain region of an antibody, R" may be the light chain region of an antibody that specifically binds to FAP, or the heavy chain region of an antibody that includes a variable region and a CH1 region, Here, R' and R" may bind to each other to form the variable region of an antibody, and the variable region specifically binds to FAP, Q' may be the heavy chain region of an antibody that specifically binds to FAP and includes a variable region and a CH1 region, or the light chain region of an antibody, Q" may be the light chain region of an antibody that specifically binds to FAP, or the heavy chain region of an antibody that includes a variable region and a CH1 region, Here, Q' and Q" may bind to each other to form the variable region of an antibody, and the variable region specifically binds to FAP, W may be an scFv that specifically binds to FAP; or IL-12 of Structural Formula (I) or (II) or a variant thereof, Linkers (4), (5), (6) and (7) may each be a peptide linker, s, t, u, x, a and b may each independently be 0 or 1] may be included.

[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 include 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 a 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 a physicochemical distance in a fusion protein or to connect domains. The linker may include the hinge region of an immunoglobulin.

[0103] Peptide linkers (1) to (7) refer to peptide linkers composed of amino acids. Specifically, peptide linker (2) or (5) may consist 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 (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.

[0104] In one embodiment, 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, 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. Additionally, the peptide linker (2) may contain at least one cysteine. Specifically, the peptide linker (2) may contain 1, 2, or 3 cysteines. Additionally, the peptide linker (2) may be derived from the hinge of an immunoglobulin and may further contain (G4S)n (where n is an integer from 1 to 10). Specifically, 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] Additionally, 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 contain (G4S)n (where n is an integer from 1 to 10). Here, in (G4S)n, n may each be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0107] Additionally, specifically, the peptide linker (2), (3), or (5) may contain SEQ ID NO: 93 or SEQ ID NO: 94.

[0108] In one embodiment of the present invention, the peptide linker (2) or (5) may be a peptide linker containing the amino acid sequence of SEQ ID NO: 118 or 120. Fc region or a fragment thereof Here, the above immunoglobulin fragment may be the Fc region of an immunoglobulin. The Fc region of an immunoglobulin can be a wild-type Fc domain and an Fc domain variant. Here, the Fc region may be the Fc region of IgG, IgA, IgE, IgD, or IgM. Specifically, 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 the wild-type Fc domain with respect to the 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 non-glycosylated Fc domains. The Fc domain or its variant can be configured to have an adjusted number of sialic acids, fucosylation, or glycosylation by host culture conditions or genetic manipulation.

[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 respective Fc regions of 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 substituted 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 include a knob structure or a hole structure.

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

[0114] The "knob" is constructed by replacing small amino acid side chains from the interface of the first polypeptide (e.g., the first CH3 domain of the first antibody heavy chain) with larger side chains (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A complementary "hole" of the same or similar size in the knob is created by replacing large amino acid side chains from the interface of the second polypeptide (e.g., the second CH3 domain of the second antibody heavy chain) with smaller side chains (e.g., alanine, serine, valine, or threonine). The knob and hole 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 a form in which the 146th, 148th, and 187th amino acids of the amino acid sequence of SEQ ID NO: 304 are replaced with other amino acids. Specifically, in one embodiment of the present invention, the knob structure or hole structure may be a form in which the amino acid sequence of SEQ ID NO: 304 is substituted with T146W, T146S, L148A, and Y187A. Specifically, the knob structure may be a form in which the amino acid sequence of SEQ ID NO: 304 is substituted with T146W, and the hole structure may be 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 a form in which the 152nd, 154th, and 193rd amino acids of the amino acid sequence of SEQ ID NO: 305 are substituted with other amino acids. Specifically, in one embodiment of the present invention, the knob structure or the hole structure may be a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152W or T152S, M154A, and Y193V. Specifically, the knob structure may be a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152W, and the hole structure may be 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 contain a DANG mutation or an NG mutation. Here, the "DANG mutation" refers to the D265A / N297G mutation for removing effector functions 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, downregulation of cell surface receptors (e.g., B cell receptor, BCR), etc. Generally, these effector functions require the binding of the Fc region to the binding domain (e.g., the antibody variable domain).

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

[0120] In one embodiment, the DANG mutation may be in a form in which the amino acids in human IgG1 of SEQ ID NO: 289 are substituted with D45A / N77G. Additionally, the DANG mutation may be in a form in which the amino acids in mouse IgG2a of SEQ ID NO: 291 are substituted with D51A / N83G. Structure of the fusion protein The fusion protein may be an antibody. Specifically, the antibody may be a heterodimeric antibody containing a knob-into-hole structure.

[0121] The fusion protein may include Structural Formula (III) and Structural Formula (V); or Structural Formula (IV) and Structural Formula (V). More specifically, one embodiment of the fusion protein may include a first monomer of (i) or (ii) below and a second monomer of (iii), (iv), (v) or (vi) below: Examples of the first monomer: (i) When r is 0, Structural Formula (III) (ii) When r is 1, Structural Formula (IV) Examples of the second monomer: (iii) When s, a and b are 0, Structural Formula (V’) and Structural Formula (V”) (iv) When s is 1 and a and b are 0, Structural Formula (V’) and Structural Formula (V”) (v) When s and b are 0 and a is 1, Structural Formula (V’) and 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 containing the above (i) and a second monomer containing the above (iii) (the 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. Specifically, 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 include a first monomer containing the above (i) and a second monomer containing the above (iv) (the second 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: 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. Specifically, the fusion protein may include 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 include a first monomer containing the above (i) and a second monomer containing the above (v) (the third 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: 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. Specifically, the fusion protein may include 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 include a first monomer containing the above (i) and a second monomer containing the above (vi) (the fourth 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: 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. Specifically, the fusion protein may include 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 containing the above (ii) and a second monomer containing the above (iii) (the 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. Specifically, 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 containing IL-12 or a variant thereof and an antigen-binding site that specifically binds to FAP may be a fusion protein dimer containing a monomer of structural formula (III) and a monomer of structural formula (V) (left in FIG. 34). Here, in structural formula (III), r is 1, and in structural formula (V), s, a, and t are 0. Here, structural formula (V) may include (V’) and (V”). Here, in (V’) and (V”), s, a, t, and b are 0. In addition, the Fc region of the dimer has a knob-into-hole structure in which different fusion proteins can bind to each other.

[0128] Specifically, 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: 18, SEQ ID NO: 19, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 298, and SEQ ID NO: 299. Specifically, the fusion protein may include 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, a 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 include (V') and (V"). Here, in structural formula (V), s and t are 0, and a is 1. In addition, in (V') and (V"), s, t, and b are 0, and a is 1. In addition, 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] Specifically, the fusion protein may include 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. Specifically, the fusion protein may include 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, a 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 in FIG. 34). Here, in structural formula (III), r is 1.

[0132] Specifically, the fusion protein may include SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 302, or SEQ ID NO: 303.

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

[0134] In one embodiment, the polypeptide of the first monomer comprises 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 with 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 polypeptide of the second monomer 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 with 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 has 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 with 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 a 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 in the host cell and then cleaved. Specifically, the signal sequence is an amino acid sequence that initiates the transport of a protein through the endoplasmic reticulum (ER) membrane.

[0139] Signal sequences are well known in the art for their properties. Such signal sequences typically contain 16 - 30 amino acid residues and may contain more or fewer amino acid residues than such amino acid residues. A typical signal peptide consists of three regions, namely, 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 immobilize the signal sequence during the 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 signal peptidase. Here, the signal sequence may be the signal sequence of tPa (tissue plasminogen activator), HSV gD (signal sequence of herpes simplex virus glycoprotein D), or the secretory signal sequence of growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells including mammals and the like can be used. In addition, a wild-type signal sequence can be used, or a signal sequence substituted with codons having a high expression frequency in the host cell can be used.

[0141] Vector having a polynucleotide In another aspect of the present invention, a vector containing a polynucleotide is provided.

[0142] The vector can be introduced into a host cell, recombined with the genome of the host cell, and inserted. Alternatively, the vector is understood as a nucleic acid means containing a polynucleotide sequence that can replicate autonomously as an episome. The vector includes 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] Specifically, the vectors can include plasmid DNA, phage DNA, etc.; and commercially developed plasmids (such as pUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from Escherichia coli (such as pYG601BR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (such as pUB110, pTP5, etc.), plasmids derived from yeast (such as YEp13, YEp24, YCp50, etc.), phage DNA (such as Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (such as retrovirus, adenovirus, vaccinia virus, etc.), insect virus vectors (such as baculovirus, etc.). Since vectors show different expression levels and modifications of proteins by host cells, it is preferable to select and use the most suitable host cell for the purpose.

[0144] As used herein, the terms "gene expression" or "expression" of a target protein are 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 can be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may contain a human cytomegalovirus (CMV) promoter for promoting continuous transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence for enhancing the stability level of post-transcriptional RNA. Transformed cells expressing a fusion protein In another aspect of the present invention, transformed cells into which a vector has been introduced are provided.

[0145] The host cell of the transformed cell can 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, Escherichia coli can be used. In addition, as an example of a eukaryotic cell, yeast can 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. can be used. However, mammalian cells are not limited to these, and any cell known to those skilled in the art that can be used as a mammalian host cell can be used.

[0146] In addition, for the introduction of the expression vector into the host cell, methods such as CaCl2 precipitation, the Hanahan method with enhanced efficiency by using reducing agents such as dimethyl sulfoxide (DMSO) in CaCl2 precipitation, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, transformation mediated by Agrobacterium, transformation using PEG, dextran sulfate, lipofectamine, or transformation mediated by drying / inhibition can be used.

[0147] As described above, for the optimization of the properties of the fusion protein as a therapeutic agent or for any other purpose, the glycosylation pattern (e.g., sialic acid, fucosylation, glycosylation) of the fusion protein can be adjusted by manipulating the glycosylation-related genes possessed by the host cell by methods known to those skilled in the art. Method for producing a fusion protein In another aspect of the present invention, a method for producing a fusion protein is provided, which includes: i) culturing the transformed cell; and ii) recovering the fusion protein containing the first monomer and the second monomer.

[0148] The method of culturing the transformed cells can be carried out using methods well known in the art. Specifically, the culture can be carried out in a batch process or continuously in a fed-batch or repeated fed-batch process. Use of the fusion protein In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, which contains 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 lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0150] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, 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 the tumors of the present invention, the active ingredient can be contained in any amount (effective amount) according to the use, dosage form, formulation purpose, etc., as long as the active ingredient can exhibit the activity of treating the tumor or, in particular, the therapeutic effect against cancer. The normal 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 the active ingredient that can induce the effect of improving or treating the disease state, in particular, the effect of improving or treating the cancer state. Such an effective amount can be determined experimentally within the scope of the common knowledge of those skilled in the art.

[0151] As used herein, the term "treatment" can be used to mean both therapeutic treatment and prophylactic treatment. Here, prophylaxis can be used to mean that the condition or disease of the subject is reduced or alleviated. In one embodiment, the term "treatment" includes both the application or any form of administration for treating a disease in a mammal, including a human. In addition, the term "treatment" includes inhibiting or delaying the progression of a disease, restoring or repairing a reduced or lost function such that the disease is partially or completely alleviated; stimulating an inefficient process; or reducing a severe 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 be due to enhanced pharmacokinetic parameters and can be measured by comparing parameters such as clearance rate and the treatment or amelioration of tumors in test animals or human subjects.

[0153] As used herein, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition effective to prevent or treat a disease in question, which is sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment and which does not cause side effects. The level of the effective amount can be determined by factors including the health of the patient, the type and severity of the disease, the activity of the drug, the sensitivity of the patient to the drug, the mode of administration, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, the formulation or drugs used concomitantly, and other factors well known in the medical arts. In one embodiment, a therapeutically effective amount refers to an amount of a drug effective to treat cancer.

[0154] Here, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any carrier as long as it is a non-toxic substance suitable for delivery to the patient. Distilled water, alcohol, fats, waxes, and inert solids can be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) can also be contained in the pharmaceutical composition.

[0155] Specifically, by including a pharmaceutically acceptable carrier in addition to the active ingredient, the pharmaceutical composition can be prepared into a parenteral formulation using conventional methods known in the art according to the administration route. Here, the term "pharmaceutically acceptable" means that the carrier does not inhibit the activity of the active ingredient and at the same time does not have greater toxicity than the subject to which it is applied (formulated) can tolerate.

[0156] When the pharmaceutical composition is prepared into a parenteral formulation, the parenteral formulation can be made into a preparation in the form of an injection, a transdermal patch, a nasal inhalant, or a suppository using a suitable carrier according to methods known in the art. When made into an injection, polyols such as sterile water, ethanol, glycerol or propylene glycol, or mixtures thereof can be used as a suitable carrier, and isotonic solutions such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, and 5% dextrose etc. can preferably be used. The formulation of the pharmaceutical composition is known in the art, and specifically, reference can be made to Remington’s Pharmaceutical Sciences (19th Edition, 1995) etc. This document is considered to be part of this specification.

[0157] The preferred dosage of the pharmaceutical composition can be in the range of 0.01 μg / kg to 10 g / kg, or 0.01 mg / kg to 1 g / kg per day, depending on the patient's condition, weight, gender, age, severity of the patient, and administration route. The dosage can be administered once a day or divided into several times a day. Such dosages should not be considered to limit 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 present invention, there is provided the use of a fusion protein comprising a first monomer containing IL-12 or a variant thereof and a second monomer containing an antigen-binding site that specifically binds to FAP for treating cancer.

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

[0161] In yet another aspect of the present invention, there is provided a method for treating or preventing cancer, the method comprising the step of administering to a subject a fusion protein comprising a first monomer containing IL-12 or a variant thereof and a second monomer containing an antigen-binding site that specifically binds to 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 route of administration, dosage, and frequency of administration of the fusion protein or fusion protein dimer may vary depending on the condition of the patient and the presence or absence of side effects. Therefore, the fusion protein or fusion protein dimer can be administered to the subject in various ways and amounts. The optimal method of administration, dosage, and frequency of administration can be selected by those skilled in the art within an appropriate range. In addition, the fusion protein or fusion protein dimer can be administered in combination with other drugs or bioactive substances known to have a therapeutic effect on the disease to be treated, or can be formulated in the form of a combination 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. Outline of anti-FAP / IL-12 IgG1 DANG, a human fusion protein

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

[0167] [seq2] is composed of a human anti-FAP light chain sequence.

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

[0169] [seq4] is the sequence in which the lysine (K) at the 2nd and 2nd amino acids 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 the human IgG1 Fc hole DANG. 58 nd and 2 63 nd amino acids, and is composed of the linker GGGGSGGGGSGGGGS, the human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and the human IgG1 Fc hole DANG.

[0170] [seq5] consists of a sequence in which the lysine (K) at the 2nd, 2nd, 2nd, 2nd, 2nd, and 2nd amino acids 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 the human IgG1 Fc whole DANG. 58 The 2nd 60 The 2nd 63 The 2nd and 64 The sequence in which the 2nd amino acid, lysine (K), is mutated to alanine (A), the linker GGGGSGGGGSGGGGS, the human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and the human IgG1 Fc whole DANG.

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

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

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

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

[0175] [seq10] consists of the human anti-FAP heavy chain sequence, the human IgG1 Fc whole DANG, the linker GGGGSGGGGSGGGGS, the human IL-12 p40 (beta) region sequence, the linker GGGGSGGGGSGGGGS, and the human IL-12 p35 (alpha) region sequence.

[0176] [seq11] is composed of a human anti-FAP heavy chain sequence, a human IgG1 Fc whole DANG, a linker GGGGSGGGGSGGGGS, a human IL-12 p40 (beta) region sequence containing mutations of four amino acids involved in heparin binding, a linker GGGGSGGGGSGGGGS, and a human IL-12 p35 (alpha) region sequence.

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

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

[0179] [seq14] is composed of a human anti-FAP heavy chain variable region sequence and a human IgG1 Fc DANG.

[0180] [seq15] is composed of a human Il-12 p40 (beta) region sequence containing mutations of four amino acids involved in heparin binding, a linker GGGGSGGGGSGGGGS, a human IL-12 p35 (alpha) region sequence, a linker GGGGSGGGGS, and a human IgG1 Fc DANG.

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

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

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

[0184] [seq293] consists of a human IL-12 p40 (beta) region sequence containing mutations (4) of amino acids involved in heparin binding, linker GGGGSGGGGSGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and human IgG1 Fc DANG, linker GGGGSGGGGSGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

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

[0186] [seq295] consists of a human anti-FAP heavy chain sequence, human IgG1 Fc DANG, linker GGGGSGGGGSGGGGS, a human IL-12 p40 (beta) region sequence containing mutations (4) of amino acids involved in heparin binding, linker GGGGSGGGGSGGGGS, and human IL-12 p35 (alpha) region sequence.

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

[0188] [seq297] consists of a human IL-12 p40 (beta) region sequence containing mutations (4) of amino acids involved in heparin binding, linker GGGGSGGGGSGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and human IgG1 Fc DANG, linker GGGGSGGGGSGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and 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 in which a human anti-FAP sequence targeting human FAP and a human IL-12 sequence containing mutations (2) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function has been removed.

[0191] T1.03 (seq1, seq2, seq5) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP and a human IL-12 sequence containing mutations (4) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function has been removed.

[0192] T1.04 (seq3, seq6, seq7) is a bispecific antibody in which a human anti-FAP sequence of the double variable domain immunoglobulin (DVD-Ig) type 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 DVD-Ig type human anti-FAP sequence and a human IL-12 sequence containing mutations (4) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function is removed.

[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 anti-FAP sequence and a human IL-12 sequence form a knob-into-hole structure and the effector function is removed.

[0195] T1.07 (seq2, seq5, seq8) is a bispecific antibody in which a scFv of a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the heavy chain, and a human anti-FAP sequence and a human IL-12 sequence containing mutations (4) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function is removed.

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

[0197] T1.09 (seq1, seq5, seq9) is a bispecific antibody in which a scFv of a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the light chain, and a human anti-FAP sequence and a human IL-12 sequence containing mutations (4) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function is removed.

[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 with a human IL-12 sequence linked to the C-terminus of the heavy chain form a knob-into-hole structure.

[0199] T1.11 (seq1, seq2, seq11) is an antibody in which a human anti-FAP sequence targeting human FAP and a human anti-FAP sequence with a mutation (4) of an amino acid involved in heparin binding in the human IL-12 sequence linked to the C-terminus of the heavy chain form a knob-into-hole structure.

[0200] T1.12 (seq1, seq2, seq12) is an antibody in which the effector function of the knob-into-hole structure having only the human anti-FAP sequence targeting human FAP is removed.

[0201] T1.13 (seq3, seq13) is an antibody in which the effector function of the knob-into-hole structure having only the human IL-12 sequence is removed.

[0202] T1.14 (seq4, seq13) is an antibody in which the effector function of the knob-into-hole structure having only the human IL-12 sequence containing a mutation (2) of an amino acid involved in heparin binding is removed.

[0203] T1.15 (seq5, seq13) is an antibody in which the effector function of the knob-into-hole structure having only the human IL-12 sequence containing a mutation (4) of an amino acid involved in heparin binding is removed.

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

[0205] T1.17 (seq15) is an Fc fusion protein having only the human IL-12 sequence containing a mutation (4) of an amino acid 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 in which a human anti-CD20 sequence targeting human CD20 and a human IL-12 sequence containing mutations (two) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function has been removed.

[0208] T1.20 (seq5, seq16, seq17) is a bispecific antibody in which a human anti-CD20 sequence targeting human CD20 and a human IL-12 sequence containing mutations (four) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function has been removed.

[0209] T1.21 (seq12, seq16, seq17) is an antibody with a knob-into-hole structure having only a human anti-CD20 sequence targeting human CD20 and the effector function has been removed.

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

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

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

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

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

[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 IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0219] [seq4]hu scIL-12 mut1-hu IgG1 Fc whole DANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASKREAKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0220] [seq5]hu scIL-12 mut2-hu IgG1 Fc whole 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 knob 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 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

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

[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 whole DANG-anti-hu FAP scFv

[0235] [seq294]Anti-hu FAP HCP hu IgG1 Fc DANG-hu scIL-12 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0236] [seq295]Anti-hu FAP HC hu IgG1 Fc DANG-hu scIL-12 mut2 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[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. Outline 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 mouse IgG2a Fc, the effector function is removed by the DANG mutation (D265A, N297G), and the knob structure is formed by the T321W mutation.

[0242] [seq19] is composed of a mouse anti-FAP light chain sequence.

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

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

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

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

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

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

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

[0250] [seq27] consists of the mouse anti-FAP heavy chain sequence, the mouse IgG2a Fc hall DANG, the linker GGGGSGGGGSGGGGS, the p40 (beta) region sequence of mouse IL-12, the linker GGGGSGGGGSGGGGS, and the mouse IL-12 p35 (alpha) region sequence.

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

[0252] [seq29] consists of only a mouse IgG2a Fc whole DANG.

[0253] [seq30] consists of only a mouse IgG2a Fc knob DANG.

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

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

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

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

[0258] [seq35] consists of only a mouse IgG2a Fc DANG.

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

[0260] [seq299] is composed of a mouse IL-12 p40 (beta) region sequence containing mutations (4) of amino acids involved in heparin binding, linker GGGGSGGGGSGGGGS, a mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and a mouse IgG2a Fc hall DANG, linker GGGGSGGGGSGGGGS, a mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and a mouse anti-FAP light chain variable region sequence.

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

[0262] [seq301] is composed of a mouse anti-FAP heavy chain sequence, a mouse IgG1 Fc DANG, linker GGGGSGGGGSGGGGS, a mouse IL-12 p40 (beta) region sequence containing mutations (4) of amino acids involved in heparin binding, linker GGGGSGGGGSGGGGS, and a mouse IL-12 p35 (alpha) region sequence.

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

[0264] [seq303] is composed of a mouse IL-12 p40 (beta) region sequence containing mutations (4) of amino acids involved in heparin binding, a linker GGGGSGGGGSGGGGS, a mouse IL-12 p35 (alpha) region sequence, a linker GGGGSGGGGS, and a mouse IgG2a Fc DANG, a linker GGGGSGGGGSGGGGS, a mouse anti-FAP heavy chain variable region sequence, a linker GGGGSGGGGSGGGGSGGGGS, and a 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 is removed.

[0266] T1.02m (seq18, seq19, seq21) is a bispecific antibody in which a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing mutations (2) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function is removed.

[0267] T1.03m (seq18, seq19, seq22) is a bispecific antibody in which a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing mutations (4) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function is removed.

[0268] T1.04m (seq20, seq23, seq24) is a bispecific antibody in which a mouse anti-FAP sequence targeting mouse fibroblast activation protein (FAP) of the double variable domain immunoglobulin (DVD-Ig) type and a mouse IL-12 sequence form a knob-into-hole structure and the effector function has been removed.

[0269] T1.05m (seq22, seq23, seq24) is a bispecific antibody in which a mouse anti-FAP sequence targeting mouse FAP of the DVD-Ig type and a mouse IL-12 sequence containing mutations (four) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function has been removed.

[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 and a mouse IL-12 sequence form a knob-into-hole structure 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 a mouse anti-FAP sequence targeting mouse FAP at the C-terminus of the heavy chain and a mouse IL-12 sequence containing mutations (four) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function has been removed.

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

[0273] T1.09m (seq18, seq22, seq26) is a bispecific antibody in which a mouse anti-FAP sequence targeting mouse FAP and containing an scFv of the mouse anti-FAP sequence at the C-terminus of the light chain forms a knob-into-hole structure with a mouse IL-12 sequence containing mutations of amino acids involved in heparin binding (4), and the effector function has been removed.

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

[0275] T1.11m (seq18, seq19, seq28) is an antibody in which a mouse anti-FAP sequence targeting mouse FAP and a mouse anti-FAP sequence containing mutations of amino acids involved in heparin binding (4) and linked to the C-terminus of the heavy chain form a knob-into-hole structure.

[0276] T1.12m (seq18, seq19, seq29) is an antibody with the effector function removed in a knob-into-hole structure having only a mouse anti-FAP sequence targeting mouse FAP.

[0277] T1.13m (seq20, seq30) is an antibody with the effector function removed in a knob-into-hole structure having only a mouse IL-12 sequence.

[0278] T1.14m (seq21, seq30) is an antibody with the effector function removed in a knob-into-hole structure having only a mouse IL-12 sequence containing mutations of amino acids involved in heparin binding (2).

[0279] T1.15m (seq22, seq30) is an antibody with the effector function of the knob-into-hole structure removed, having only the mouse IL-12 sequence containing mutations (4) of amino acids involved in heparin binding.

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

[0281] T1.17m (seq32) is an Fc fusion protein dimer having only the mouse IL-12 sequence containing mutations (4) of amino acids involved in heparin binding.

[0282] T1.18m (seq20, seq33, seq34) is a bispecific antibody in which the mouse anti-CD20 (18B12) sequence targeting mouse CD20 and the mouse IL-12 sequence form a knob-into-hole structure and the effector function is removed.

[0283] T1.19m (seq21, seq33, seq34) is a bispecific antibody in which the mouse anti-CD20 (18B12) sequence targeting mouse CD20 and the mouse IL-12 sequence containing mutations (2) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function is removed.

[0284] T1.20m (seq22, seq33, seq34) is a bispecific antibody in which the mouse anti-CD20 sequence targeting mouse CD20 and the mouse IL-12 sequence containing mutations (4) of amino acids involved in heparin binding form a knob-into-hole structure and the effector function is removed.

[0285] T1.21m (seq29, seq33, seq34) is an antibody with the effector function of the knob-into-hole structure removed, having only the mouse anti-CD20 sequence targeting mouse CD20.

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

[0287] T1.23m (seq18, seq19, seq298) is a bispecific antibody in which the mouse anti-FAP sequence and the mouse IL-12 sequence with the scFv of the mouse anti-FAP sequence linked to 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 in which the mouse anti-FAP heavy chain sequence and the mouse IL-12 sequence containing mutations (4) of amino acids involved in heparin binding with the scFv of the mouse anti-FAP sequence linked to the C-terminus form a knob-into-hole and the effector function has been removed.

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

[0290] T1.26m (seq19, seq301) is an Fc fusion protein dimer containing the mouse anti-FAP sequence with the mouse IL-12 sequence containing mutations (4) of amino acids involved in heparin binding linked to the C-terminus of the heavy chain.

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

[0292] T1.28m (seq303) is an Fc fusion protein dimer composed of the mouse IL-12 sequence containing mutations (4) of amino acids involved in heparin binding with the scFv of the mouse anti-FAP sequence linked to the C-terminus.

[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 MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0296] [seq21] mu scIL-12 mut1-mu IgG2a Fc whole DANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRAKEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0297] [seq22] mu scIL-12 mut2-mu IgG2a Fc whole 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] Anti-mu FAP HC mu IgG2a Fc whole DANG-mu scIL-12 mut2 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

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

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

[0306] [seq31] Anti-mu FAP HC mu 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 whole 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 fragment was amplified by PCR, and the PCR product was purified by gel. The pTT5 vector was cleaved with restriction enzymes EcoRI and BamHI, and then the gel was purified. Each PCR product and the linear vector were ligated using an In-Fusion kit. The generated vector was transformed into ECOS101 DH5α competent cells, and the cells were cultured on a 2×YT agar plate containing 100 μg / ml ampicillin. All operation processes were carried out according to standard transformation protocols. Positive recombinants were identified by colony PCR, and sequence verification sequencing was performed on the recombinant plasmid. A single colony was selected, and the seed culture was inoculated into 5 ml of 2×YT medium containing 100 μg / ml ampicillin. It was cultured with shaking at 37°C for 8 hours.

[0321] Thereafter, the seed culture was diluted at a ratio of 1:1,000 in 200 ml of selective 2×YT medium. It was cultured with shaking at 37°C for 16 hours. The bacterial cells were collected by centrifugation at 4°C and 4,700 rpm for 10 minutes. The bacterial pellet was resuspended in 12 ml of RES-EF buffer. Thereafter, 12 ml of LYS-EF buffer was added, and the sealed tube was vigorously inverted to mix completely, and then incubated at room temperature for 5 minutes. 12 ml of NEU-EF buffer was added to the lysate, and it was vigorously inverted to mix completely and rapidly.

[0322] Before injecting the lysate into the NucleoBond (trademark) Xtra column filter, the lysate tube was inverted three times to prepare a uniform suspension of the precipitate and prevent the filter from clogging. Then, the NucleoBond (trademark) Xtra column filter and the NucleoBond (trademark) Xtra column were washed with 10 ml of filter wash buffer FIL-EF. The NucleoBond (trademark) Xtra column filter was removed by pulling out or inverting the column. The NucleoBond (trademark) Xtra column was washed with 90 ml of wash buffer ENDO.

[0323] The NucleoBond (trademark) Xtra column was washed with 45 ml of wash buffer WASH-EF. The plasmid DNA was eluted with 15 ml of elution buffer ELU. The eluate was collected in a 50 ml centrifuge tube. 10.5 ml of isopropanol was added at room temperature to precipitate the eluted plasmid DNA. 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). Then, the DNA pellet was dissolved in 1,000 μl of H2O.

[0325] Production Example 2. Cell Transfection and Protein Expression Production Example 2.1. Cell Transfection The materials and reagents used are shown in Table 5 below.

[0326]

Table 5

[0327] The 293F seed strain containing complete medium was maintained in an incubator shaker at 130 rpm, 37 °C, and 8% CO2. 0.3 - 0.4 × 10 6Cultured at a concentration of cells / ml and the medium was changed every 2 - 3 days. 24 hours before transfection, a new passage number of 293F cells was adjusted to 2.6×10 6 cells / ml. The prepared 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 6 cells / ml using fresh medium. It was carried out in a total volume of 1 L in a 3 L shaker flask. 0.4 mg of HC and 0.6 mg of LC plasmid 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 immediately mixed. Then, it was cultured at room temperature for 15 minutes. The DNA-PEI mixture was added to 293F cells prepared at 2.6×10 6 cells / ml. Then, the cells were continuously cultured in an incubator shaker at 130 rpm, 37 °C, and 8% CO2 for 24 hours. 24 hours after transfection, 10% peptone was added to 1 / 20 of the culture solution to make the final concentration 0.5%. Then, the cells were continuously cultured in an incubator shaker at 130 rpm, 37 °C, and 8% CO2. The cell density / survival rate was measured and recorded daily for 2 - 5 days after transfection. The cells were harvested for purification 7 days after transfection or when the cell survival rate was less than 70%.

[0329] Production Example 2.2. Protein Purification The reagents, buffer compositions, and equipment used for protein purification are shown in Tables 6 - 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, 4°C for 20 minutes. Then, the supernatant was filtered through a Sartopore2 filter. The clarified supernatant was loaded onto a 5-ml MabSelect Sure column equilibrated with Buffer A. Then, the column was washed with Buffer A until the A280 absorbance reached the baseline. The column was washed with 10 CV of Buffer B. The column was washed with 10 CV of Buffer A. The bound protein was eluted with 6 CV of Buffer C, and 1 / 6 volume of Buffer D was added to neutralize the eluted substance. SDS-PAGE and SEC-HPLC analyses were performed. Then, the protein was purified using an HIC column. Next, the protein was dialyzed against Buffer E at 4°C overnight. The supernatant was loaded onto an HIC column equilibrated with Buffer E. Then, the column was washed with Buffer E until the A280 absorbance reached the baseline. The bound protein was eluted by gradient elution (10 CV of Buffer F 0% - 40%). The bound protein was eluted with 2 CV of 100% Buffer F, and SDS-PAGE analysis was performed.

[0334] The purified proteins were pooled, and then the protein was dialyzed against the final buffer at 4°C overnight. Then, SDS-PAGE and SEC-HPLC analyses were performed.

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

[0336] Production Example 3. Improvement in bispecific antibody production Production Example 3.1 Identification of improvement in the production of a fusion protein containing human IL-12 Changes in the production of human anti-FAP / IL-12 bispecific antibodies due to human IL-12 mutations are shown in Table 9 below. Parentheses indicate the scale of protein production, with the unit being L. The number before the parentheses is the production amount per liter obtained by dividing the amount of purified protein by the production scale.

[0337] [Table 9]

[0338] As shown in Table 9 above, as the number of mutations in the amino acids involved in heparin binding in the human IL-12 p40 (beta) region increased, the amount of protein purified through a Protein A column (MabSelect Sure column) increased. 58 The second and 63 second bispecific antibody T1.02 in which the lysine (K) of the amino acid was mutated to alanine (A) was found to have improved production compared to T1.01. 58 The second, 60 second, 63 second and 64 second bispecific antibody T1.03 in which the lysine (K) of the amino acid was mutated to alanine (A) was found to have approximately 2.5-fold improved production compared to T1.01.

[0339] Production Example 3.2. Identification of Improvement in the Production of a Fusion Protein Containing Mouse IL-12 Table 10 below shows changes in the production of mouse anti-FAP / IL-12 bispecific antibodies 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 mouse 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 describing the total protein production amount on a production scale. The bispecific antibody T1.02m in which the lysine (K) at the 277th and 282nd amino acids was mutated to alanine (A), and the bispecific antibody T1.03m in which the arginine (R) at the 276th amino acid was mutated to alanine (A), and the lysine (K) at the 277th, 278th and 282nd amino acids was mutated to alanine (A) were found to have gradually improved protein production compared to T1.01m and T1.02m. Furthermore, it was found that the production of T1.03m using the CHO cell line was improved by about 13 times compared to the production using the HEK293 cell line. Production Example 4. Cell line and cell culture The HEK-Blue IL-12 cell line, which is a human embryonic kidney fibroblast HEK293 cell transformed with the IL-12 receptor gene and the STAT4-inducible SEAP reporter gene, 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] The human embryonic kidney fibroblast cell line HEK293, the mouse colon cancer cell line CT26-WT, the mouse melanoma cell line B16F10 and the mouse fibroblast cell line NIH3T3 were obtained from ATCC (American Type Culture Collection, Manasas, VA, USA). HEK293 cells, B16F10 cells 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) overexpressing mouse FAP (fibroblast activation protein alpha) in CT26-WT cells, B16F10 cells, and NIH3T3 cells were prepared using a lentivirus capable of delivering the 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] Production Example 5. Isolation and activation of human immune cells Blood packs were obtained from the Korean Red Cross with approval from the Institutional Review Board (IRB), and peripheral blood mononuclear cells (PBMCs) were isolated and frozen. The thawed PBMCs were subjected to positive selection, and human NK cells were isolated using an Easy sep (Stem cell, Vancuver, BC, CA) kit. The isolated NK cells were activated and maintained in RPMI-1640 (GIBCO) containing 50 IU / ml recombinant human IL-2 (rhIL-2, R&D systems, Minnesota, USA) and 10% FBS.

[0346] The thawed PBMCs were subjected to negative selection, and human T cells were isolated using an 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 the fusion protein Example 1.1. Identification of binding of the fusion protein 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 the 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 on the CM5 chip immobilized with the anti-human IgG (Fc) antibody. Recombinant human FAP was diluted to 200 nM in 1×HBS-EP+ buffer and serially diluted. The diluted recombinant human FAP was reacted at a rate of 30 μl / min. The association and dissociation times were 180 seconds and 400 seconds, respectively. After dissociation, stabilization was performed for 60 seconds, and then regeneration was performed with a 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 can 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 the 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.

[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 serially diluted. The diluted recombinant human FAP was reacted at a rate of 30 μl / min. The association and dissociation times were 180 seconds and 400 seconds, respectively. After dissociation, stabilization was performed for 60 seconds, and then regeneration was performed with a 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min for 30 seconds.

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

[0353]

Table 11

[0354]

Table 12

[0355] Example 1.2. Identification of the 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 the 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 on the CM5 chip immobilized with the anti-mouse IgG (Fc) antibody. 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. The association and dissociation times were 180 seconds and 400 seconds, respectively. After dissociation, stabilization was performed for 60 seconds, and then regeneration was performed with a 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min for 30 seconds.

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

[0358] Example 1.3. Identification of Binding to FAP-Expressing Cells The degree of binding of T1.12, T1.01, T1.02 and T1.03 to HEK293 and HEK293-hFAP cells was identified.

[0359] Specifically, cell lines were suspended in 1×10 5 cells / 100 μl of FACS buffer and prepared by treatment with 1 μg each of T1.12, T1.01, T1.02 and T1.03. The cells were washed twice with FACS buffer. The cells were stained with an anti-human IgG antibody (Biolegend). The negative control was stained with the anti-human IgG antibody (Biolegend) only. The expression rate of the stained cells was measured using a BD LSR and analyzed using FlowJo software.

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

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

[0362] Specifically, cell lines were prepared by suspending them in FACS buffer at 1×10 5 cells / 100 μl and treating them with 1 μg of each of T1.12m, T1.01m, T1.02m and T1.03m. The cells were washed twice with FACS buffer. The cells were stained with anti-mouse IgG2a antibody (Biolegend). The negative control was stained with anti-mouse IgG2a antibody (Biolegend) only. The expression rate of the stained cells was measured using a BD LSR and analyzed using FlowJo software.

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

[0364] Example 1.4. Identification of the Binding of the Fusion Protein to Recombinant Human IL-12 Receptor Considering that the IL-12 of the fusion protein must bind to the IL-12 receptor for its action, it was found that T1.01, T1.02, T1.03 and T1.12 bind to recombinant human IL-12 receptor.

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

[0366] (i) T1.01, T1.02, T1.03 and T1.12 were suspended at 5 μg / ml and diluted by serial dilution. 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 a washing solution and then treated with 1 nM of IL-12 receptor beta 1-biotin (IL-12Rβ1-biotin; Acrobiosystems, Newark, USA) for 2 hours.

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

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

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

[0372] As a result, as shown in Figure 17, 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. T1.12 was used as a control.

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

[0374] Specifically, the HEK-Blue IL-12 cell line, which is an IL-12 recognizing cell, was diluted to 2.8×10 5 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. The HEK-Blue IL-12 cells were treated with the diluted recombinant human IL-12, T1.01m, T1.02m, T1.03m and T1.12m. After 24 hours of treatment, the cell supernatant was collected and mixed with QUANTI-Blue Solution (Invivogen) in a 96-well plate. The reporter expression level was measured at 620 nm using a spectrophotometer (Thermo Fisher).

[0375] As a result, as shown in Figure 18, in the case of the IL-12, T1.01m, T1.02m and T1.03m treatment groups, it was identified by the absorbance at 620 nm that IL-12 was recognized and the reporter gene was expressed by inducing phosphorylation of STAT4. In contrast, it was identified that the control T1.12m did not recognize IL-12 when the 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 signal transduction ability in human T cells To identify whether IL-12 initiates signal transduction by inducing phosphorylation of STAT4, a signal transduction mediator, when IL-12 binds to the IL-12 receptor of 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 100 ng / ml and diluted by serial dilution. Human T cells activated with anti-CD3 (OKT3, Invitrogen) were treated with the diluted recombinant human IL-12, T1.01, T1.02, T1.03, and T1.12. After 2 hours of treatment, the 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 680 nm and an emission wavelength of 615 nm.

[0378] As a result, as shown in Figure 19, 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. In contrast, phosphorylated STAT4 was not detected in the group treated with the control T1.12.

[0379] These results indicate that the IL-12 mutation attenuated the T cell signal transduction ability.

[0380] Example 2.3. Identification of Changes in Signal Transduction Ability of Fusion Proteins by Low Molecular Weight Heparin in Human T Cells Next, using human T cells activated with anti-CD3 (OKT3, Invitrogen) and AlphaLISA SureFire Ultra p-STAT4 (Tyr693) assay kit, the enhancement of the signaling ability of the low molecular weight heparin-treated IL-12 mutant (IL-12 mut1) fusion protein was identified.

[0381] Specifically, the signaling ability of low molecular weight heparin (LMWH) in human T cells was identified by observing whether the induction of phosphorylation of STAT4, a signal transduction mediator, was attenuated when the attenuated IL-12 mut2 bound to the IL-12 receptor of human T cells.

[0382] Specifically, recombinant human IL-12, T1.01, T1.02, and T1.03 were suspended at 100 ng / ml and diluted by serial dilution. Activated human T cells were treated with the diluted recombinant human IL-12, T1.01, T1.02, and T1.03 together with low molecular weight heparin. After 2 hours of treatment, the 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 680 nm and an emission wavelength of 615 nm.

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

[0384] These results indicate that when IL-12 mutant (IL-12 mut2) was added to the existing IL-12 mutant (IL-12 mut1), the degree of enhancement of the signaling ability 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 Protein Example 3.1. Enzyme Immunoassay for Cytokine Measurement For cytokine measurement, the supernatant stored at -20°C was subjected to an enzyme immunoassay (ELISA, R&D systems) in the following steps: (i) The capture antibody was diluted according to the certificate of analysis (CoA) and coated on a 96-well plate for 24 hours.

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

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

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

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

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

[0391] (vii) The wells were treated with the 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) secretion, a major immune response, occurs when human T cells are treated with IL-12, 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 was evaluated.

[0393] Specifically, human T cells activated with anti-CD3 (OKT3, Invitrogen) were diluted to 5×10 5 cells / ml and dispensed into a 96-well plate at 200 μl / well. 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, the cell supernatant was collected and the samples were stored at -80°C.

[0394] As a result, as shown in Figure 21, the samples treated with recombinant human IL-12 had an average of 1,640 pg / ml of IFN-γ, the samples treated with T1.01 had an average of 1,115 pg / ml of IFN-γ, the samples treated with T1.02 had an average of 845 pg / ml of IFN-γ, and the samples treated with T1.03 had an average of 607 pg / ml of IFN-γ. 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 ability in human NK cells Considering that IFN-γ (interferon-gamma) secretion, a major immune response, occurs when human NK cells are treated with IL-12, the inventors evaluated the IFN-γ secretion ability when human NK cells activated with recombinant human IL-2 (R&D systems) were treated with recombinant human IL-12, T1.12, T1.01, T1.02, and T1.03.

[0397] Specifically, human NK cells activated with recombinant human IL-2 (R&D systems) were diluted to 5×10 5 cells / ml and dispensed into a 96-well plate at 200 μl / well. 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, the cell supernatant was collected and the samples were stored at -80°C.

[0398] As a result, as shown in Figure 22, the sample treated with recombinant human IL-12 had an average of 180 pg / ml of IFN-γ, the sample treated with T1.01 had an average of 205 pg / ml of IFN-γ, the sample treated with T1.02 had an average of 133 pg / ml of IFN-γ, and the sample treated with T1.03 had an average of 80 pg / ml of IFN-γ. In contrast, the sample treated with T1.12 as a control was 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 the anti-cancer effect of the fusion protein in a colorectal cancer animal model Example 4.1. Evaluation of the anti-cancer effect in a colorectal cancer animal model by co-injection of FAP-expressing fibroblasts The fusion protein is in the form of a bispecific 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, after overexpressing mouse FAP in mouse fibroblast cell line NIH3T3 cells, the tumor growth inhibitory ability was evaluated in a tumor animal model co-injected with mouse colorectal cancer cell line CT26 at a ratio of 1:1.

[0400] Using CT26, a mouse colorectal cancer cell line, and NIH-3T3, a mouse fibroblast cell line, an FAP-expressing tumor animal model was prepared. Next, NIH-3T3 was prepared as an FAP-expressing cell line.

[0401] Specifically, mouse FAP overexpressed in cultured NIH-3T3 cells and CT26 cells was 1×10 6Resuspended in Hank's balanced salt solution (HBSS, Gibco) at a concentration of cells / 50 μl, the cells were mixed at a ratio of 1:1. Next, using a 1 cc syringe (25G), 100 μl per mouse was transplanted into the subcutaneous region of the right flank of 6-week-old BALB / c mice to obtain an animal model with simultaneous injection of FAP-expressing fibroblasts. Tumor size was measured using a digital Vernier caliper by measuring the short and long axes of the tumor, and the tumor size was measured twice a week using the calculation formula of (short axis, mm) 2 ×(long axis, mm)×0.5.

[0402] Next, in the evaluation of the efficacy of the drug, when the tumor size reached 100 mm 3 , the mice were randomly divided into groups. Each experimental group consisted of 10 mice. As a control, 100 μl of each 50 μg of T1.24m was intraperitoneally administered 3 times at 3-day intervals. In the test groups, in order to evaluate the tumor growth inhibitory ability, 100 μl of each 10 μg and 50 μg of T1.02m, and 50 μg of T1.01m were intraperitoneally administered 3 times at 3-day intervals, respectively.

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

[0404] Furthermore, as shown in Figure 24, 24 days after administration, complete responders were observed in the groups administered T1.01m and T1.02m compared with the control.

[0405] These results indicate that the fusion protein of one embodiment has excellent anti-cancer effects in a colorectal cancer animal model.

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

[0407] To prepare a mouse FAP-expressing tumor animal model, overexpression of FAP was induced in mouse colorectal cancer cell line CT26. Specifically, FAP-overexpressing CT26 cells during culture were resuspended in Hank's balanced salt solution (HBSS, Gibco), and then using a 1 cc syringe (25G), 100 μl of 1×10 6 tumor cells were transplanted into the subcutaneous region on the left dorsal side of 6-week-old BALB / c mice to obtain a mouse FAP-expressing colorectal cancer animal model. The tumor size was measured using a digital Vernier caliper by measuring the short axis and long axis of the tumor, and the tumor size was measured twice a week using the calculation formula of (short axis, mm) 2 ×(long axis, mm)×0.5.

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

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

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

[0411] Example 4.3. Evaluation of Growth Inhibition of Recurrent Solid Cancer in Mice with Complete Response by Fusion Protein To evaluate whether tumor recurrence can be inhibited by inducing a long-term immune response after fusion protein treatment, tumor re-challenge was performed in subjects who achieved complete response by administering T1.01m to a tumor model in which fibroblasts NIH-3T3 and CT26 cells were co-injected. BALB / c mice without treatment history were used as control mice.

[0412] 4T1 cells were subcutaneously injected into the left flank of control mice and mice that achieved complete response by administration of T1.01m, and CT26 cells were subcutaneously injected into the right flank to obtain a tumor recurrence model.

[0413] Specifically, in a model in which fibroblasts NIH-3T3 overexpressing FAP and CT26 cells were co-injected, a tumor recurrence model was induced in mice that showed complete response by administration of T1.01m. CT26 cells and 4T1 cells were each suspended in Hank's balanced salt solution (HBSS, Gibco) at a concentration of 1×10 6 cells / 100 μl, and CT26 cells were transplanted into the subcutaneous region of the right flank of mice using a 1 cc syringe (25G). Tumor formation and growth were evaluated by subcutaneous transplantation of 4T1 cells into the left flank of the same mice.

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

[0415] Example 4.4. Evaluation of the decrease in activity due to amino acid mutations of the fusion protein To reduce the in vivo toxicity caused by the overactivation of the immune system by IL-12, the inventors used an IL-12 sequence structure containing mutations in the amino acid sequence that binds to heparin to block the binding to the IL-12 receptor, thereby inducing a decrease in activity, which was identified using a tumor animal model.

[0416] Among the anti-FAP antibodies, T1.02m and T1.03m in which the amino acids involved in heparin binding were 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 mutations (two) in the amino acids involved in heparin binding, together with the anti-FAP sequence structure. T1.03m is a fusion protein having an IL-12 sequence structure containing mutations (four) in the amino acids involved in heparin binding, together with the anti-FAP sequence structure.

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

[0418] Specifically, when the tumor size in the model reached 100 mm 3 the mice were randomly divided into groups. Each experimental group consisted of 8 animals, and 100 μl of phosphate-buffered saline (PBS) was administered once by tail vein injection as a control, and each of T1.01m, T1.02m and T1.03m was administered intravenously once at a dose of 10 μg.

[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 excellent tumor growth inhibitory ability 15 days after administration as compared with the control.

[0420] Furthermore, in the group administered with T1.03m having IL-12-containing mutations (4) of amino acids involved in heparin binding together with the mouse anti-FAP sequence, it was found that the tumor growth inhibitory ability was reduced as compared with the group administered with T1.01m. These results indicate that in the group administered with T1.03m having a mouse IL-12 sequence structure containing mutations (4) of amino acids involved in heparin binding, the IL-12 activity was reduced as compared with other experimental groups and the control.

[0421] Example 4.5. Evaluation of the anti-cancer effect upon single administration of the IL-12 amino acid mutant fusion protein To evaluate the anti-cancer effect of the fusion protein with reduced IL-12 activity due to the sequence structure containing mutations (4) of amino acids involved in heparin binding, the tumor growth inhibitory ability was evaluated 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 abdomen of 6-week-old BALB / c mice, and when the tumor size of the animal model reached 70 to 100 mm 3 the mice were randomly divided into groups and administered. PBS was administered as a control, and 100 μl of T1.03m was intravenously administered once at doses of 2 μg, 10 μg, and 100 μg as a test group. The tumor size was measured by the same method as in Example 4.1.

[0423] As a result, as shown in Fig. 29, it was observed that the groups administered with 2 μg, 10 μg, and 100 μg of T1.03m had a high tumor growth inhibitory ability as compared with the control. No change was observed in body weight during the test period. These results indicate that T1.03m with reduced IL-12 activity due to the sequence structure containing mutations (4) of amino acids involved in heparin binding has an excellent anti-cancer effect at a certain concentration or higher.

[0424] Example 5. Evaluation of the anti-cancer effect of the fusion protein in a melanoma animal model To identify the anti-cancer effect by the immunostimulation of the fusion protein, mouse FAP was overexpressed in the mouse melanoma cell line B16F10, which is known to have little immune cell infiltration, and then transplanted into C57BL / 6J mice to evaluate the tumor growth inhibitory ability.

[0425] Specifically, the cultured cells were resuspended in Hank's balanced salt solution (HBSS, Gibco), and then 100 μl of 1 × 10 6 tumor cells were transplanted into the subcutaneous region of the left flank of 6-week-old C57BL / 6J mice per mouse.

[0426] Next, in the evaluation of the drug efficacy, when the tumor size reached 70 - 100 mm 3 , the mice were randomly divided into groups. PBS was administered as a control, and T1.03m was intravenously administered once at doses of 10 μg and 100 μg as a test group.

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

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

[0429] Specifically, the NIH-3T3 cell line and LLC1 cells that overexpress FAP were resuspended in Hank's balanced salt solution (HBSS, Gibco) at a concentration of 1×10 6 cells / 50 μl, the cells were mixed at a ratio of 1:1, and then 100 μl of each mouse was transplanted into the subcutaneous area of the right flank of 6-week-old C57BL / 6J mice using a 1 cc syringe (25G).

[0430] Next, in the evaluation of the efficacy of the drug, when the tumor size reached 70-100 mm 3 the mice were randomly divided into groups. PBS was administered as a control, and T1.03m was intravenously administered 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 10 μg or 100 μg of T1.03m had excellent tumor growth inhibitory ability compared to the control. No change in body weight was observed during the test period. These results indicate that T1.03m, which reduced IL-12 activity due to the sequence structure containing mutations (4) of amino acids involved in heparin binding, recognized the target factor in the tumor microenvironment and thus had an excellent anti-cancer effect even in a lung cancer animal model.

[0432] Example 6.2. Evaluation of the cancer targeting effect in a lung cancer animal model by co-injection of a fusion protein To compare the anti-cancer effects of cancer targeting of a bispecific antibody-type fusion protein in which an anti-FAP sequence structure and an IL-12 sequence structure form a knob-in-hole structure, the tumor growth inhibitory ability was identified 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 ratio of 1:1.

[0433] Specifically, in the evaluation of the efficacy of the drug, when the tumor size was 70-100 mm 3When the mice reached [a certain condition], they were randomly divided into groups. As a control, PBS was intraperitoneally administered 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 inhibitory ability. T1.16m and T1.17m, which are dimers of anti-FAP and IL-12, were intraperitoneally administered twice a week at a dose of 0.01 μg each, and T1.03m, which is a monomeric fusion protein, was intraperitoneally administered twice a week at a dose of 0.02 μg to observe the tumor growth inhibitory ability.

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

Claims

1. A first monomer comprising a variant of IL-12, and a second monomer comprising an antigen-binding site that specifically binds to FAP (fibroblast activation protein alpha), wherein the fusion protein comprises: the variant of IL-12 comprises variants of IL-12A (p35) and IL-12B (p40); the variant of IL-12B (p40) comprises an amino acid sequence comprising substitutions of K258A and K263A, or substitutions of K258A, K260A, K263A, and K264A in the amino acid sequence of SEQ ID NO: 74, a fusion protein.

2. The fusion protein according to claim 1, wherein the variant of IL-12B (p40) comprises an amino acid sequence comprising substitutions of K258A and K263A in the amino acid sequence of SEQ ID NO:

74.

3. The variant of IL-12 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 the IL-12A, Z is the variant of IL-12B, the Linker(1) is a peptide linker, o is 0 or 1] The fusion protein according to claim 1, comprising.

4. The fusion protein according to claim 1, wherein the variant of IL-12B (p40) comprises an amino acid sequence comprising substitutions of K258A, K260A, K263A, and K264A in the amino acid sequence of SEQ ID NO:

74.

5. The fusion protein according to claim 1, wherein the variant of IL-12B (p40) comprises the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO:

79.

6. The fusion protein according to claim 1, wherein the IL-12A (p35) comprises the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO:

87.

7. The fusion protein according to claim 1, wherein the variant of IL-12 comprises the amino acid sequence of SEQ ID NO: 76 or SEQ ID NO:

78.

8. The antigen-binding site that specifically binds to FAP is a heavy chain variable region comprising HCDR1 of SEQ ID NO: 96, HCDR2 of SEQ ID NO: 97, and HCDR3 of SEQ ID NO: 98, and a light chain variable region comprising LCDR1 of SEQ ID NO: 99, LCDR2 of SEQ ID NO: 100, and LCDR3 of SEQ ID NO: 101; 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; 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; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 133, HCDR2 of SEQ ID NO: 134, and HCDR3 of SEQ ID NO: 135, and a light chain variable region comprising LCDR1 of SEQ ID NO: 136, LCDR2 of SEQ ID NO: 137, and LCDR3 of SEQ ID NO: 138; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 139, HCDR2 of SEQ ID NO: 140, and HCDR3 of SEQ ID NO: 141, and a light chain variable region comprising LCDR1 of SEQ ID NO: 142, LCDR2 of SEQ ID NO: 143, and LCDR3 of SEQ ID NO: 144; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 145, HCDR2 of SEQ ID NO: 146, and HCDR3 of SEQ ID NO: 147, and a light chain variable region comprising LCDR1 of SEQ ID NO: 148, LCDR2 of SEQ ID NO: 149, and LCDR3 of SEQ ID NO: 150; 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; 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; 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; 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; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 175, HCDR2 of SEQ ID NO: 176, and HCDR3 of SEQ ID NO: 177, and a light chain variable region comprising LCDR1 of SEQ ID NO: 178, LCDR2 of SEQ ID NO: 179, and LCDR3 of SEQ ID NO: 180; 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; 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; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 193, HCDR2 of SEQ ID NO: 194, and HCDR3 of SEQ ID NO: 195, and a light chain variable region comprising LCDR1 of SEQ ID NO: 196, LCDR2 of SEQ ID NO: 197, and LCDR3 of SEQ ID NO: 198; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 199, HCDR2 of SEQ ID NO: 200, and HCDR3 of SEQ ID NO: 201, and a light chain variable region comprising LCDR1 of SEQ ID NO: 202, LCDR2 of SEQ ID NO: 203, and LCDR3 of SEQ ID NO: 204; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 205, HCDR2 of SEQ ID NO: 206, and HCDR3 of SEQ ID NO: 207, and a light chain variable region comprising LCDR1 of SEQ ID NO: 208, LCDR2 of SEQ ID NO: 209, and LCDR3 of SEQ ID NO: 210; 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; 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; 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; A heavy chain variable region comprising HCDR1 of SEQ ID NO: 229, HCDR2 of SEQ ID NO: 230, and HCDR3 of SEQ ID NO: 231, and a light chain variable region comprising LCDR1 of SEQ ID NO: 232, LCDR2 of SEQ ID NO: 233, and LCDR3 of SEQ ID NO: 234; 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; or A heavy chain variable region comprising HCDR1 of SEQ ID NO: 241, HCDR2 of SEQ ID NO: 242, and HCDR3 of SEQ ID NO: 243, and a light chain variable region comprising LCDR1 of SEQ ID NO: 244, LCDR2 of SEQ ID NO: 245, and LCDR3 of SEQ ID NO: 246 The fusion protein according to claim 1, comprising.

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

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

11. The first monomer has the following structural formula (III) or (IV): N'-X-[Linker(2)]p-Fc region fragment or its variant -[Linker(3)]q-(T)r-C' (III) N'-(T)r-[Linker(2)]q-Fc region fragment or its variant -[Linker(3)]p-X-C' (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 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 according to claim 3, comprising

12. wherein the linker is (G ４ S) ｎ linker, and n is any one of integers from 1 to 10, the fusion protein according to claim 3 or 11.

13. The fusion protein according to claim 11, wherein the Fc region of the first monomer is derived from human IgG1 or mouse IgG2a.

14. The fusion protein according to claim 11, wherein the Fc of the first monomer comprises a knob structure or a hole structure.

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

16. The fusion protein according to claim 15, wherein the additionally bound antigen-binding site that specifically binds to FAP is bound to the N-terminus or C-terminus of the second monomer.

17. The second monomer has the following structural formula (V): N'-(R)s-[Linker(4)]t-Q-[Linker(5)]u-Fc region fragment or its variant -[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 a variant of said IL-12 of structural formula (I) or (II), said linkers (4)-(6) are peptide linkers, s, t, u, v and a are each independently 0 or 1] The fusion protein according to claim 3, comprising

18. said structural formula (V) is the following structural formulas (V') and (V"): N'-(R')s-[linker (4)]t-Q'-[linker (5)]u-Fc region fragment or a variant thereof-[linker (6)]p-(W)a-C' (V') N'-(R")s-[linker (4)]t-Q"-[linker (7)]x-(W)b-C' (V")[[]END] [In said structural formulas (V') and (V"), R' is the heavy chain region of an antibody that specifically binds to FAP, comprising a variable region and a CH1 region, or the light chain region of said antibody, R" is the light chain region of an antibody that specifically binds to FAP, or the heavy chain region of said antibody, comprising a variable region and a CH1 region, wherein R' and R" bind to each other to form the variable region of said antibody, and said variable region specifically binds to FAP, Q' is the heavy chain region of an antibody that specifically binds to FAP, comprising a variable region and a CH1 region, or the light chain region of said antibody, Q" is the light chain region of an antibody that specifically binds to FAP, or the heavy chain region of said antibody, comprising a variable region and a CH1 region, wherein Q' and Q" bind to each other to form the variable region of said antibody, and said variable region specifically binds to FAP, R' and Q' are each the heavy chain region of said antibody, comprising a variable region and a CH1 region, or the light chain region of said antibody, R" and Q" are each the light chain region of said antibody, or the heavy chain region of said antibody, comprising a variable region and a CH1 region, wherein R' and R"; and Q' and Q" bind to each other to form the variable region of said antibody, and said variable region specifically binds to FAP, W is an scFv that specifically binds to FAP, said linkers (4), (5) and (7) are peptide linkers, s, t, u, x, a and b are each independently 0 or 1] The fusion protein according to claim 17, comprising

19. The fusion protein according to claim 11, comprising Structural formula (III) and the following 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 the following Structural formula (V) and the following Structural formula (V). N'-(R)s-[Linker(4)]t-Q-[Linker(5)]u-Fc region fragment or a variant thereof-[Linker(6)]v-(W)a-C' (V) [In the above 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 a scFv that specifically binds to FAP; or a variant of the above IL-12 of Structural formula (I) or (II), The above Linkers (4) to (6) are peptide linkers, s, t, u, v, and a are each independently 0 or 1]

20. The fusion protein according to 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.

21. The fusion protein according to claim 1, wherein the Fc of the second monomer comprises a knob structure or a hole structure.

22. A pharmaceutical composition for preventing or treating cancer, comprising the fusion protein according to any one of claims 1 to 21 as an active ingredient.

23. The pharmaceutical composition according to claim 22, 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 lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

24. A polynucleotide encoding the first monomer according to claim 1.

25. A vector comprising the polynucleotide according to claim 24.

26. A transformed cell into which the vector according to claim 25 has been introduced.

27. A method for producing a fusion protein, i) culturing the transformed cell according to claim 26; ii) recovering the fusion protein according to claim 1 A method comprising. **Claim 28** Use of the fusion protein according to claim 1 for the manufacture of a medicament for treating cancer.

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