A pharmaceutical composition for treating cancer, comprising a fusion protein comprising IL-12 and an anti-FAP antibody, and an anti-cancer agent.

Abstract

The present invention provides a combination therapeutic agent comprising IL-12 or a variant thereof, a fusion protein comprising an antigen-binding site that specifically binds to FAP, and an anti-cancer agent. The bispecific antibody exhibits an anti-cancer effect due to IL-12, and in particular, when an anti-FAP antibody is realized by a single antibody, it can specifically target FAP highly expressed in tumors and specifically localize IL-12 at the tumor site, thereby efficiently treating cancer. When the fusion protein is administered in combination with a known anti-cancer agent, a synergistic effect is observed with the anti-cancer agent treatment.

Description

[Technical field]

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

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

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

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

[0005] Therefore, the present inventors have conducted research to develop a pharmaceutical composition having an effective anti-cancer effect. As a result, the present inventors have found a synergistic anti-cancer effect when a fusion protein containing IL-12 and an anti-FAP antibody and an anti-cancer drug are administered together, thereby completing the present invention. [Means for solving the problem]

[0006] In one aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, comprising 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 (fibroblast activation protein alpha), and an anticancer agent.

[0007] In another aspect of the present invention, there is provided a kit for preventing or treating cancer, comprising as active ingredients a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen-binding site that specifically binds to a FAP, and an anti-cancer agent. Effect of the Invention

[0008] A fusion protein comprising IL-12 and an antigen binding site that specifically binds to FAP can show anti-cancer effects by IL-12. In addition, it has been found that when an anti-FAP antibody is realized by one antibody, IL-12 specifically accumulates in tumors with high FAP expression, thereby reducing systemic toxicity and showing tumor-specific anti-cancer effects. In other words, cancer can be effectively treated by specifically targeting FAP that is highly expressed in tumors and specifically localizing IL-12 at the tumor site. In particular, when the fusion protein and anti-cancer drug are administered together, a synergistic anti-cancer effect is seen. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1 shows the results obtained by identifying T1.01, T1.02, T1.03, T1.04, T1.05, T1.06 and T1.07 according to one embodiment using SDS-PAGE. [Diagram 2] FIG. 1 shows the results obtained by identifying T1.08, T1.09, T1.10, T1.11, T1.12, T1.13, T1.14 and T1.15 according to one embodiment using SDS-PAGE. [Diagram 3] FIG. 1 shows the results obtained by identifying T1.16, T1.17, T1.18, T1.19 and T1.21 according to one embodiment using SDS-PAGE. [Figure 4] FIG. 1 shows the results obtained by identifying T1.01m, T1.02m, T1.03m, T1.04m, T1.05m, T1.06m and T1.07m according to one embodiment using SDS-PAGE. [Diagram 5] FIG. 1 shows the results obtained by identifying T1.08m, T1.09m, T1.10m, T1.11m, T1.12m, T1.13m and T1.15m according to one embodiment using SDS-PAGE. [Figure 6] FIG. 1 shows the results obtained by identifying T1.16m, T1.17m, T1.18m, T1.19m, T1.20m, T1.21m and T1.22m according to one embodiment using SDS-PAGE. [Figure 7] 1 is a graph showing the binding affinity of T1.01 and T1.02 to recombinant human FAP (rhFAP) by surface plasmon resonance analysis. [Figure 8] 1 is a graph showing the binding affinity of T1.03 to recombinant human FAP by surface plasmon resonance analysis. [Figure 9] FIG. 1 shows the degree of binding of T1.04 and T1.05 to recombinant human FAP by surface plasmon resonance analysis. [Figure 10] FIG. 1 shows the degree of binding of T1.06 and T1.07 to recombinant human FAP by surface plasmon resonance analysis. [Figure 11] FIG. 1 shows the degree of binding of T1.08 and T1.09 to recombinant human FAP by surface plasmon resonance analysis. [Figure 12] FIG. 1 shows the degree of binding of T1.10 and T1.11 to recombinant human FAP by surface plasmon resonance analysis. [Figure 13] 1 is a graph showing the binding affinity of T1.01m and T1.02m to recombinant mouse FAP by surface plasmon resonance analysis. [Figure 14] 1 is a graph showing the binding affinity of T1.03m to recombinant mouse FAP by surface plasmon resonance analysis. [Figure 15] 1 is a graph showing the binding affinity of T1.12, T1.01, T1.02 and T1.03 to HEK293-hFAP cells and HEK293 cells by flow cytometry. [Figure 16] 1 is a graph showing the binding affinity of T1.12m, T1.01m, T1.02m and T1.03m to B16F10-mFAP cells and B16F10 cells by flow cytometry. [Figure 17] 1 is a graph showing the binding affinity of T1.01, T1.02, T1.03 and T1.12 to IL-12 receptor β1 by enzyme-linked immunosorbent assay. [Figure 18] Graph showing results obtained by identifying T1.01m, T1.02m, T1.03m and T1.12m and IL-12 activity via absorbance in HEK Blue reporter cells. [Figure 19] 1 is a graph showing T1.01, T1.02, T1.03 and T1.12 and IL-12 activity mediated signaling capacity of human T cells. [Figure 20] FIG. 1 is a graph showing IL-12 activity of T1.01, T1.02 and T1.03 in the presence of low molecular weight heparin (LMWH) through the signaling capacity of human T cells. [Figure 21]Graph showing results obtained by measuring the ability of human T cells to secrete the cytokine IFN-γ by T1.12, T1.01, T1.02 and T1.03. [Figure 22] Graph showing results obtained by measuring the ability of human NK cells to secrete the cytokine IFN-γ by T1.12, T1.01, T1.02 and T1.03. [Diagram 23] This is a graph showing the tumor growth inhibition ability of T1.01m and T1.02m by measuring the change in tumor size in a mouse tumor model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26. [Figure 24] This graph shows the results obtained by measuring the tumor size for each subject to identify the tumor growth inhibitory ability of T1.01m and T1.02m in a mouse tumor model in which mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26 were co-injected. [Diagram 25] This is a graph showing the tumor growth inhibition ability of T1.01m and T1.02m administered at each concentration in a mouse tumor model transplanted with the mouse FAP-overexpressing colon cancer cell line CT26, by measuring the change in tumor size for each subject. [Figure 26] FIG. 1 is a graph showing the change in CT26 tumor size after establishing a tumor re-induction model by transplanting CT26 cells and 4T1 cells into mice, in which a complete response was achieved in a mouse tumor model in which mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26 were co-injected. [Figure 27] FIG. 1 is a graph showing the change in 4T1 tumor size after establishing a tumor re-induction model by transplanting CT26 cells and 4T1 cells into mice, in which a complete response was achieved in a mouse tumor model in which mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line CT26 were co-injected. [Figure 28]This is a graph showing the tumor growth inhibition ability of T1.01m, T1.02m and T1.03m administered at various concentrations to each subject in a mouse model implanted with mouse FAP-overexpressing CT26 cells, by measuring the tumor size for each subject. [Figure 29] This is a graph showing the tumor growth inhibition ability for each subject by administering T1.03m at each concentration, by measuring the change in tumor size for each subject in a mouse model implanted with mouse FAP-overexpressing CT26 cells. [Diagram 30] This is a graph showing the tumor growth inhibition ability of T1.03m administered at each concentration by measuring the change in tumor size for each subject in a mouse model implanted with mouse FAP-overexpressing B16F10 cells. [Diagram 31] This is a graph showing the tumor growth inhibition ability of T1.03m administered at each concentration by measuring the change in tumor size for each subject in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and lung cancer cell line LLC1. [Diagram 32] This is a graph showing the results obtained by measuring tumor size 10 days after treatment with T1.16m, T1.17m, T1.03m, or simultaneous administration of T1.16m and T1.17m in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and lung cancer cell line LLC1. [Figure 33a] Figure 13 shows the results obtained by measuring tumor size 9 days after treatment with T1.03m and aPD-L1, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and lung cancer cell line LLC1. [Figure 33b] This is a graph showing the results obtained by measuring tumor size 9 days after treatment with T1.03m and aCTLA-4, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and lung cancer cell line LLC1. [Figure 33c]This graph shows the results obtained by measuring tumor size 9 days after treatment with T1.03m and aPD-1, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and lung cancer cell line LLC1. [Figure 34a] Figure 13 shows the results obtained by measuring tumor size 16 days after treatment with T1.03m and aPD-L1, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 34b] Figure 1 shows the results over time obtained by measuring tumor size up to 16 days after treatment with T1.03m and aPD-L1, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 35a] Figure 13 shows the results obtained by measuring tumor size 11 days after treatment with T1.03m and aPD-L1, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 35b] Figure 13 shows the results over time obtained by measuring tumor size up to 11 days after treatment with T1.03m and aPD-L1, respectively, or co-administration in a mouse model co-injected with murine FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 36a] This is a graph showing the results obtained by measuring the tumor size 10 days after treatment with T1.03m and aTIGIT, respectively, or co-administration in a mouse model in which mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38 were co-injected. [Figure 36b]1 is a graph showing the results over time obtained by measuring tumor size up to 10 days after treatment with T1.03m and aTIGIT, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 37a] This is a graph showing the results obtained by measuring tumor size 10 days after treatment with T1.03m and aLAG-3, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 37b] This is a graph showing the results over time obtained by measuring tumor size up to 10 days after treatment with T1.03m and aLAG-3, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 38a] 13 is a graph showing the results obtained by measuring tumor size 10 days after treatment with T1.03m and aTIM-3, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 38b] FIG. 13 is a graph showing results over time obtained by measuring tumor size up to 10 days after treatment with T1.03m and aTIM-3, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 39a] 1 is a graph showing the results obtained by measuring tumor size 11 days after treatment with T1.03m and cyclophosphamide, respectively, or co-administration, in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 39b]FIG. 13 is a graph showing results over time obtained by measuring tumor size up to 11 days after treatment with T1.03m and cyclophosphamide, respectively, or co-administration, in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 40a] Graph showing results obtained by measuring tumor size 11 days after treatment with T1.03m and fluorouracil, respectively, or co-administration, in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 40b] FIG. 13 is a graph showing results over time obtained by measuring tumor size up to 11 days after treatment with T1.03m and fluorouracil, respectively, or co-administration, in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 41a] This is a graph showing the results obtained by measuring tumor size 11 days after treatment with T1.03m and paclitaxel, respectively, or co-administration, in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 41b] This is a graph showing the results over time obtained by measuring tumor size up to 11 days after treatment with T1.03m and paclitaxel, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 42a] FIG. 13 is a graph showing the results obtained by measuring tumor size 11 days after treatment with T1.03m and Camptosar, respectively, or co-administration, in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Figure 42b]This is a graph showing the results over time obtained by measuring tumor size up to 11 days after treatment with T1.03m and Camptosar, respectively, or co-administration in a mouse model co-injected with mouse FAP-overexpressing fibroblasts NIH-3T3 and colon cancer cell line MC38. [Diagram 43] Schematic diagrams of an anti-FAP / IL-12 fusion protein (first from the left), an anti-FAP / IL-12 fusion protein with dual variable domains (second), an anti-FAP / IL-12 dual antibody with a FAP-specific single-chain variable fragment (scFv) attached (third and fourth), and an anti-FAP / IL-12 fusion protein with IL-12 attached to the C-terminus of the Fc (fifth) are shown. [Diagram 44] Schematic diagrams of a FAP-specific Fab / FAP-specific single-chain variable fragment (scFv) and IL-12 fusion protein (left), a fusion protein dimer comprising a FAP-specific Fab and IL-12 (center), and a FAP-specific single-chain variable fragment (scFv) and IL-12 fusion protein (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Combination treatment comprising a fusion protein comprising IL-12 and an anti-FAP antibody, and an anti-cancer agent In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising 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 (fibroblast activation protein alpha), and an anticancer agent.

[0011] The fusion protein not only exhibits anti-cancer effects by IL-12 and specifically accumulates IL-2 in tumors with high FAP expression, but also exhibits tumor-specific anti-cancer effects and reduces systemic toxicity, and the fusion protein can be used in combination with various conventionally used anti-cancer treatments. Specifically, the conventional treatments that can be used in combination can be selected from the group consisting of chemotherapeutic anti-cancer agents, oncolytic viruses, immune checkpoint inhibitors, and combinations thereof.

[0012] Anticancer drugs As used herein, the term "chemical anticancer agent" is also referred to as an antitumor agent or a cytotoxic agent. The term "chemical anticancer agent" is a general term for drugs that exhibit anticancer activity by directly affecting DNA, blocking the DNA replication, transcription, and translation processes, or by interfering with the synthesis of nucleic acid precursors in metabolic pathways and inhibiting cell division. Antitumor agents act not only on tumor cells but also on normal cells, and exhibit cytotoxicity. Chemical anticancer agents can be used for maintenance therapy. Furthermore, as used herein, the term "maintenance therapy" refers to treating cancer with drugs after the first anticancer treatment, and refers to a treatment method carried out to prevent or delay the recurrence of cancer.

[0013] The chemical anti-cancer agent can be any one selected from the group consisting of alkylating agents, microtubule inhibitors, antimetabolites, and topoisomerase inhibitors.

[0014] In one embodiment, the alkylating agent can be cyclophosphamide or cisplatin. The microtubule inhibitor can be paclitaxel. The antimetabolite can be fluorouracil or gemcitabine. The topoisomerase inhibitor can be camptosar or adriamycin.

[0015] As used herein, the term "oncolytic virotherapy" refers to a treatment that kills cancer by inserting specific genes into a replicative, infectious virus that targets cancer cells. The oncolytic virotherapy can be Talimogene Laherparepvec (T-VEC).

[0016] As used herein, the term "immune checkpoint inhibitors" refers to substances that inhibit the activity of immune checkpoint proteins that inhibit the differentiation, proliferation, and activity of immune cells; immune checkpoint inhibitors are known to eliminate cancer cells by preventing them from performing functions that evade the immune system.

[0017] In one embodiment, the immune checkpoint inhibitor may be any one selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, and an anti-LAG3 antibody.

[0018] In one embodiment, the immune checkpoint inhibitor may be any one selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and durvalumab.

[0019] The fusion protein can be used in combination with an anti-cancer vaccine, etc. Furthermore, the anti-cancer drug can be used not only with the above-mentioned anti-cancer drugs, but also with an anti-cancer vaccine, etc.

[0020] In addition, the anti-cancer drug may include one or more anti-cancer drugs. Specifically, the fusion protein can be used with two anti-cancer drugs. For example, a chemical anti-cancer drug and an anti-cancer virus; an anti-cancer virus and an immune checkpoint inhibitor; and a chemical anti-cancer drug and an immune checkpoint inhibitor.

[0021] In addition, the fusion protein can be used with three anti-cancer drugs. Anti-cancer drugs different from the above two anti-cancer drugs may be further included and used. In one embodiment, the anti-cancer drugs may be chemical anti-cancer drugs, anti-cancer viruses and immune checkpoint inhibitors.

[0022] Anti-cancer Use of Fusion Proteins Another aspect of the present invention provides a composition for maintaining anti-cancer activity, comprising, as an active ingredient, a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen-binding site that specifically binds to a FAP.

[0023] The fusion proteins are as described below.

[0024] As mentioned above, "maintaining anticancer properties" refers to treating cancer after an initial anticancer treatment. In particular, "maintaining anticancer properties" refers to a treatment method that improves the efficacy of cancer treatment by preventing or delaying the recurrence of cancer.

[0025] At this point, at least one additional anti-cancer agent may be included for maintenance therapy. At this point, the anti-cancer agents are as described above. Kit containing fusion protein and anticancer drug Another aspect of the present invention provides a kit for preventing or treating cancer, comprising, as active ingredients, a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen-binding site that specifically binds to a FAP, and an anti-cancer agent.

[0026] Fusion protein comprising IL-12 and FAP antibody The fusion protein comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to a FAP may comprise an immunoglobulin Fc region. At this point, the IL-12 or a variant thereof may be a variant having low heparin-binding ability.

[0027] In one embodiment, the fusion protein may be a fusion protein comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen binding site that specifically binds to a FAP.

[0028] In another embodiment, the fusion protein may be a dimer of a fusion protein comprising IL-12 or a variant thereof and an antigen binding site that specifically binds to a FAP.

[0029] IL-12 or its variants As used herein, the term "IL-12" is a heterodimeric cytokine composed of p35 and p40 subunits encoded by two separate genes, IL-12A and IL-12B, respectively. IL-12 is produced by antigen-presenting cells such as macrophages and binds to receptors on the cell surface of activated T cells and NK cells. IL-12 promotes the proliferation of T cells and NK cells and enhances the cytotoxic effect of T cells, NK cells and macrophages. In addition, IL-12 induces the production of IFN-γ, TNF-α and GM-CSF, and acts to induce the activation of Th1 cells. On the other hand, IL-12 binds to the IL-12 receptor, a heterodimeric receptor formed by IL-12Rβ1 and IL-12Rβ2.

[0030] As used herein, the term "variant" of IL-12 includes an amino acid sequence having a similar or identical function to that of the wild-type IL-12 protein. In particular, the amino acid sequences of the p35 and p40 subunits constituting IL-12 may have substitutions, insertions or deletions compared to the wild-type.

[0031] IL-12 or a variant thereof may include IL-12A(p35) or a variant thereof, and IL-12B(p40) or a variant thereof.

[0032] 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. In one embodiment, the IL-12 or variant thereof has the following structural formula (I) or (II): N'-Y-[linker (1)]oZ-C' (I) N'-Z-[Linker (1)]oY-C' (II) [In structural formulas (I) and (II), N' may be the N-terminus of the fusion protein; C' may be the C-terminus of the fusion protein; Y may be IL-12A or a variant thereof; Z may be IL-12B or a variant thereof; The linker (1) may be a peptide linker, o may be 0 or 1. It may also be a fusion protein comprising:

[0033] One embodiment of IL-12 or a variant thereof may have a structure in which IL-12B or a variant thereof, a peptide linker, and IL-12A or a variant thereof are bound in this order from the N-terminus. Additionally, one embodiment of IL-12 or a variant thereof may have a structure in which IL-12A or a variant thereof, a peptide linker, and IL-12B or a variant thereof are bound in this order from the N-terminus.

[0034] As used herein, the term "IL-12A" refers to the 35 kDa subunit (p35) of IL-12, which binds via a disulfide bond to the 40 kDa subunit (p40) of IL-12 to produce the active cytokine.

[0035] The amino acid sequence of IL-12A may be that set forth in GenBank Accession No. AAK84425 (for the amino acid sequence of mouse p35, see GenBank Accession No. AAA39292). Additionally, the IL-12A(p35) gene is a sequence encoding the IL-12A subunit, and may be a nucleotide sequence corresponding to the coding sequence (CDS) in the sequence set forth in GenBank Accession No. AF404773 (for the mouse sequence, see GenBank Accession No. M86672).

[0036] As used herein, the term "variant" of IL-12A includes an amino acid sequence having a similar or identical function to that of the wild-type IL-12A protein. In particular, a "variant" of IL-12A means that the amino acid sequence of IL-12A has a substitution, insertion or deletion compared to the wild-type. In particular, an IL-12A variant may be a fragment of IL-12A, and may have an amino acid sequence in which amino acids 1 to 22 of SEQ ID NO: 122 are deleted. More particularly, an IL-12A variant may have the amino acid sequence of SEQ ID NO: 75.

[0037] Additionally, the IL-12A used in one embodiment of the present invention may be the amino acid sequence of SEQ ID NO: 75 as human IL-12A. In one embodiment, mouse IL-12A may be the amino acid sequence of SEQ ID NO: 87.

[0038] As used herein, the term "IL-12B" refers to the 40 kDa subunit (p40) of IL-12, which binds to the 35 kDa subunit (p35) of IL-12 via a disulfide bond to form IL-12, and to the subunit p19 of IL-23 to form IL-23. On the other hand, IL-12B is also called natural killer cell stimulating factor 2.

[0039] The amino acid sequence of "IL-12B" may be that set forth in GenBank Accession No. AAD56386 (for the amino acid sequence of mouse p40, see GenBank Accession No. AAA39296). Additionally, the IL-12B(p40) gene is a sequence encoding the IL-12B subunit, and may be a nucleotide sequence corresponding to the coding sequence (CDS) in the sequence set forth in GenBank Accession No. AF180563 (for the mouse sequence, see GenBank Accession No. M86671).

[0040] Additionally, the IL-12B used in one embodiment of the invention may be human IL-12B, in particular, the IL-12B may have the amino acid sequence of SEQ ID NO: 74. In one embodiment, mouse IL-12B may have the amino acid sequence of SEQ ID NO: 86.

[0041] As used herein, the term "variant" of IL-12B includes an amino acid sequence that has a similar or identical function to that of the wild-type IL-12B protein. In particular, a "variant" of IL-12B means that the amino acid sequence of IL-12B has substitutions, insertions, or deletions compared to the wild-type.

[0042] In particular, the IL-12B variant may be one in which the 1st to 22nd amino acids of SEQ ID NO: 121 have been deleted. More particularly, the IL-12B variant may be one in which 1 to 8 amino acids have been substituted in the sequence of SEQ ID NO: 74. In addition, the IL-12B variant may be one in which the 1st to 22nd amino acids of SEQ ID NO: 123 have been deleted. More particularly, the IL-12B variant may be one in which up to 1 to 8 amino acids have been substituted in the sequence of SEQ ID NO: 86.

[0043] The variant of IL-12B(p40) may have a substitution in an amino acid involved in heparin binding of IL-12.

[0044] In one embodiment, the variant of IL-12B(p40) may be a form in which the lysine (K) binding to heparin in IL-12B is replaced by another amino acid. In particular, the variant of IL-12B(p40) may be a form in which the lysines at positions 258, 260, 263 and 264 of SEQ ID NO: 74 are replaced by other amino acids. In particular, the variant of IL-12B(p40) may comprise an amino acid sequence obtained by at least one substitution selected from the group consisting of K258A, K260A, K263A and K264A in the amino acid sequence of SEQ ID NO: 74. In particular, the variant of IL-12B(p40) may comprise an amino acid sequence obtained by substitution of K258A and K263A in the amino acid sequence of SEQ ID NO: 74 or may further comprise the mutations K260A and / or K264A in the amino acid sequence of SEQ ID NO: 74.

[0045] In one embodiment, the variant of IL-12B(p40) may be a form in which the arginine (R) or lysine (K) binding to heparin in IL-12B is replaced by another amino acid. In particular, the variant of IL-12B(p40) may be a form in which the arginine at position 254 and / or the lysine at positions 255, 256 and 260 of SEQ ID NO: 86 are replaced by other amino acids. In particular, the variant of IL-12B(p40) may comprise an amino acid sequence obtained by at least one substitution selected from the group consisting of R254A, K255A, K256A and K260A in the amino acid sequence of SEQ ID NO: 86. Specifically, it may comprise the mutations R254A, K255A, K256A and K260A in the amino acid sequence of SEQ ID NO: 86.

[0046] In one embodiment, a variant of IL-12B(p40) may comprise the amino acid sequence of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:89 or SEQ ID NO:91. In one embodiment, the human IL-12B variant has the following structural formula A: [Structural formula A] X 1 -LX 2 [In the formula, X 1 is the amino acid sequence of SEQ ID NO: 125, X 2 is the amino acid sequence of SEQ ID NO: 126, L is VA 1 -A 2 -QA 3 -K * -A 4 -A 5 -A 6 -A 7 -K * -A 8 is an amino acid sequence consisting of It may consist of.

[0047] A 1 is R or Q, and A 2 is V, A or I, and A 3 is G or R * And A 4 is S, N or K * And A 5 is K * , N or E, and A 6 is R or K, and A 7 is E, M or T, and A 8 is K * Or E.

[0048] " * At least one amino acid residue marked with " may be substituted with a nonpolar amino acid residue selected from the group consisting of, but not limited to, A, G, I, L, M, F, P, and V.

[0049] In one embodiment, variants of IL-12B(p40) may contain substitutions of amino acids involved in heparin binding to reduce the cytokine-associated toxicity of IL-12 while maintaining its killing effect on cancer cells. FAP and antigen-binding sites that specifically bind to FAP As used herein, the term "FAP (fibroblast activation protein)" refers to fibroblast activation protein, a protein that is expressed on the cell surface and is presented in the environment of tumor cells of various tumor types or in tumor cells of various tumor types. FAP is known to be selectively overexpressed on the surface of cancer-associated fibroblasts (CAFs) in the tumor microenvironment.

[0050] In addition, FAPs are homodimers containing two N-glycosylated subunits with a C-terminal extracellular domain in which the enzyme catalytic domain is located, and the glycosylated forms of FAPs have both post-prolyl dipeptidyl peptidase and gelatinase activities.

[0051] On the other hand, FAP is a prolyl endopeptidase, a membrane-bound gelatinase of about 170 kDa. Prolyl endopeptidase FAP can recognize and cleave a specific amino acid sequence. Therefore, FAP is also called FAPα, separase, or circulating antiplasmin cleavage enzyme.

[0052] The amino acid sequence of FAP may be that set forth in GenBank Accession No. AAC51668, and human FAP was first identified in cultured fibroblasts by use of monoclonal antibody (mAb) F19 (see International Patent Application Publication No. WO93 / 05804). FAP is expressed in many cancers, and due to its restricted expression in normal tissues, it serves as a promising antigenic target for imaging, diagnosis and treatment of various carcinomas.

[0053] FAP includes any variant, isoform, and species homolog of human FAP expressed by cells naturally or in cells transfected with FAP genes. The antibodies of the present invention can bind to FAPs present on the membrane or cell surface of cancer cells (tumor cells or cells in the tumor stroma) and can trigger ADCC or other effector-mediated killing of cancer cells. In addition, the antibodies of the present invention can be used to block FAP enzymatic activity (e.g., serine peptidase, gelatinase, collagenase activity), FAP-mediated ECM degradation, and FAP-mediated cell invasion or migration.

[0054] As used herein, the term "specifically binds" refers to binding that is distinct from non-specific interactions. Specific binding can be determined by competition between the target and a similar control molecule that does not have binding activity.

[0055] As used herein, the term "antigen-binding site (epitope)" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody. An antigen-binding site is a group of molecules that usually have specific structural and charge characteristics, such as amino acids or sugar side chains. An antigen-binding site that specifically binds to a FAP can be a collective term for molecules capable of antigen-antibody binding specifically to a FAP.

[0056] In addition, the antibody or fragment thereof can be used in any form as long as it contains an antigen-binding domain capable of specifically binding to a FAP.

[0057] The antigen-binding site may contain other amino acids that are not directly involved in binding or whose effects are blocked by amino acid residues in the antigen-binding site.

[0058] In particular, the antigen-binding site may be an antibody or a fragment thereof that specifically binds to the FAP.

[0059] As used herein, the term "antibody" refers to a molecule that contains an antigen-binding site and an immunologically active fragment of an immunoglobulin molecule that contains an antigen-binding site. The immunoglobulin molecule may be an immunoglobulin molecule of IgG, IgE, IgM, IgD, IgA, IgY or a subclass thereof. Antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFv (including, e.g., monospecific and bispecific antibodies), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of these antibodies.

[0060] As used herein, the term "antibody fragment" refers to a portion of an antibody, such as an F(ab') 2 , F(ab) 2 Regardless of structure, an antibody fragment can be any of a variety of fragments, including Fab', Fab, Fv, scFv. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.

[0061] In one embodiment, an antigen-binding site that specifically binds to a FAP may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 96, an HCDR2 of SEQ ID NO: 97, and an HCDR3 of SEQ ID NO: 98; and a light chain variable region comprising an LCDR1 of SEQ ID NO: 99, an LCDR2 of SEQ ID NO: 100, and an LCDR3 of SEQ ID NO: 101. Here, in one embodiment, an antigen-binding site that specifically binds to a FAP may have a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 103.

[0062] Additionally, in one embodiment, an antigen-binding site that specifically binds to a FAP may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 104, HCDR2 of SEQ ID NO: 105, and HCDR3 of SEQ ID NO: 106; and a light chain variable region comprising LCDR1 of SEQ ID NO: 107, LCDR2 of SEQ ID NO: 108, and LCDR3 of SEQ ID NO: 109. Here, in one embodiment, an antigen-binding site that specifically binds to a FAP may have a heavy chain variable region of SEQ ID NO: 110 and a light chain variable region of SEQ ID NO: 111.

[0063] In one embodiment, the antigen-binding site that specifically binds to the FAP may be an scFv, where the scFv may comprise the heavy and light chain CDRs described above. In one embodiment, the scFv that specifically binds to the FAP may have the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 84.

[0064] In addition, an antigen-binding site that specifically binds to a FAP can include a known anti-FAP antibody or a fragment thereof. An anti-FAP antibody or a fragment thereof can refer to, without limitation, an antibody known to those skilled in the art.

[0065] The anti-FAP antibody or fragment thereof may comprise at least one variable region selected from the group consisting of NG-641, AMG-506 / MP-0310, 28H1 of RG-7827, 4B9 of RG-7827, OMTX-705, 3F2, 4G8, 3D9, 4B3, 19G1, 20G8, 5B8, 5F1, 14B3, 16F1, 16F8, O3C9, 29B11, O2D7, and 23C10.

[0066] In another example, the antibody may be an anti-FAP antibody or a fragment thereof disclosed in Korean Patent Application Publication No. KR20200037826A, U.S. Patent Application Publication No. US2020-0385488A1, U.S. Patent No. US3,924,445B2, U.S. Patent No. US9,011,847B2, or U.S. Patent Application Publication No. US2017-007716A1.

[0067] In one embodiment, the anti-FAP antibody may comprise the variable region of NG-641. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 127, HCDR2 of SEQ ID NO: 128, and HCDR3 of SEQ ID NO: 129, and a light chain variable region comprising LCDR1 of SEQ ID NO: 130, LCDR2 of SEQ ID NO: 131, and LCDR3 of SEQ ID NO: 132. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 248, and a light chain variable region of SEQ ID NO: 249.

[0068] In one embodiment, the anti-FAP antibody may comprise the variable region of 28H1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 133, an HCDR2 of SEQ ID NO: 134, and an HCDR3 of SEQ ID NO: 135, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 136, an LCDR2 of SEQ ID NO: 137, and an LCDR3 of SEQ ID NO: 138. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 250, and a light chain variable region of SEQ ID NO: 251.

[0069] In one embodiment, the anti-FAP antibody may comprise the variable region of 4B9. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 139, an HCDR2 of SEQ ID NO: 140, and an HCDR3 of SEQ ID NO: 141, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 142, an LCDR2 of SEQ ID NO: 143, and an LCDR3 of SEQ ID NO: 144. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 252, and a light chain variable region of SEQ ID NO: 253.

[0070] In one embodiment, the anti-FAP antibody may comprise the variable region of OMTX-705. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 145, an HCDR2 of SEQ ID NO: 146, and an HCDR3 of SEQ ID NO: 147, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 148, an LCDR2 of SEQ ID NO: 149, and an LCDR3 of SEQ ID NO: 150. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 254, and a light chain variable region of SEQ ID NO: 255.

[0071] In one embodiment, the anti-FAP antibody may comprise the variable region of 3F2. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 151, HCDR2 of SEQ ID NO: 152, and HCDR3 of SEQ ID NO: 153, and a light chain variable region comprising LCDR1 of SEQ ID NO: 154, LCDR2 of SEQ ID NO: 155, and LCDR3 of SEQ ID NO: 156. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 256, and a light chain variable region of SEQ ID NO: 257.

[0072] In one embodiment, the anti-FAP antibody may comprise the variable region of 4G8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 157, HCDR2 of SEQ ID NO: 158, and HCDR3 of SEQ ID NO: 159, and a light chain variable region comprising LCDR1 of SEQ ID NO: 160, LCDR2 of SEQ ID NO: 161, and LCDR3 of SEQ ID NO: 162. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 258, and a light chain variable region of SEQ ID NO: 259.

[0073] In one embodiment, the anti-FAP antibody may comprise the variable region of 3D9. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 163, HCDR2 of SEQ ID NO: 164, and HCDR3 of SEQ ID NO: 165, and a light chain variable region comprising LCDR1 of SEQ ID NO: 166, LCDR2 of SEQ ID NO: 167, and LCDR3 of SEQ ID NO: 168. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 260, and a light chain variable region of SEQ ID NO: 261.

[0074] In one embodiment, the anti-FAP antibody may comprise the variable region of 4B3. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 169, HCDR2 of SEQ ID NO: 170, and HCDR3 of SEQ ID NO: 171, and a light chain variable region comprising LCDR1 of SEQ ID NO: 172, LCDR2 of SEQ ID NO: 173, and LCDR3 of SEQ ID NO: 174. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 262, and a light chain variable region of SEQ ID NO: 263.

[0075] In one embodiment, the anti-FAP antibody may comprise the variable region of 19G1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 175, an HCDR2 of SEQ ID NO: 176, and an HCDR3 of SEQ ID NO: 177, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 178, an LCDR2 of SEQ ID NO: 179, and an LCDR3 of SEQ ID NO: 180. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 264, and a light chain variable region of SEQ ID NO: 265.

[0076] In one embodiment, the anti-FAP antibody may comprise the variable region of 20G8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 181, HCDR2 of SEQ ID NO: 182, and HCDR3 of SEQ ID NO: 183, and a light chain variable region comprising LCDR1 of SEQ ID NO: 184, LCDR2 of SEQ ID NO: 185, and LCDR3 of SEQ ID NO: 186. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 266, and a light chain variable region of SEQ ID NO: 267.

[0077] In one embodiment, the anti-FAP antibody may comprise the variable region of 5B8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 187, HCDR2 of SEQ ID NO: 188, and HCDR3 of SEQ ID NO: 189, and a light chain variable region comprising LCDR1 of SEQ ID NO: 190, LCDR2 of SEQ ID NO: 191, and LCDR3 of SEQ ID NO: 192. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 268, and a light chain variable region of SEQ ID NO: 269.

[0078] In one embodiment, the anti-FAP antibody may comprise the variable region of 5F1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 193, an HCDR2 of SEQ ID NO: 194, and an HCDR3 of SEQ ID NO: 195, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 196, an LCDR2 of SEQ ID NO: 197, and an LCDR3 of SEQ ID NO: 198. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 270, and a light chain variable region of SEQ ID NO: 271.

[0079] In one embodiment, the anti-FAP antibody may comprise the variable region of 14B3. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 199, an HCDR2 of SEQ ID NO: 200, and an HCDR3 of SEQ ID NO: 201, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 202, an LCDR2 of SEQ ID NO: 203, and an LCDR3 of SEQ ID NO: 204. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 272, and a light chain variable region of SEQ ID NO: 273.

[0080] In one embodiment, the anti-FAP antibody may comprise the variable region of 16F1. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:205, an HCDR2 of SEQ ID NO:206, and an HCDR3 of SEQ ID NO:207, and a light chain variable region comprising an LCDR1 of SEQ ID NO:208, an LCDR2 of SEQ ID NO:209, and an LCDR3 of SEQ ID NO:210. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:274, and a light chain variable region of SEQ ID NO:275.

[0081] In one embodiment, the anti-FAP antibody may comprise the variable region of 16F8. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO:211, HCDR2 of SEQ ID NO:212, and HCDR3 of SEQ ID NO:213, and a light chain variable region comprising LCDR1 of SEQ ID NO:214, LCDR2 of SEQ ID NO:215, and LCDR3 of SEQ ID NO:216. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:276, and a light chain variable region of SEQ ID NO:277.

[0082] In one embodiment, the anti-FAP antibody may comprise the variable region of O3C9. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO:217, HCDR2 of SEQ ID NO:218, and HCDR3 of SEQ ID NO:219, and a light chain variable region comprising LCDR1 of SEQ ID NO:220, LCDR2 of SEQ ID NO:221, and LCDR3 of SEQ ID NO:222. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:278, and a light chain variable region of SEQ ID NO:279.

[0083] In one embodiment, the anti-FAP antibody may comprise the variable region of 22A3. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 223, HCDR2 of SEQ ID NO: 224, and HCDR3 of SEQ ID NO: 225, and a light chain variable region comprising LCDR1 of SEQ ID NO: 226, LCDR2 of SEQ ID NO: 227, and LCDR3 of SEQ ID NO: 228. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 280, and a light chain variable region of SEQ ID NO: 281.

[0084] In one embodiment, the anti-FAP antibody may comprise the variable region of 29B11. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:229, an HCDR2 of SEQ ID NO:230, and an HCDR3 of SEQ ID NO:231, and a light chain variable region comprising an LCDR1 of SEQ ID NO:232, an LCDR2 of SEQ ID NO:233, and an LCDR3 of SEQ ID NO:234. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:282, and a light chain variable region of SEQ ID NO:283.

[0085] In one embodiment, the anti-FAP antibody may comprise the variable region of O2D7. In particular, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 235, HCDR2 of SEQ ID NO: 236, and HCDR3 of SEQ ID NO: 237, and a light chain variable region comprising LCDR1 of SEQ ID NO: 238, LCDR2 of SEQ ID NO: 239, and LCDR3 of SEQ ID NO: 240. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO: 284, and a light chain variable region of SEQ ID NO: 285.

[0086] In one embodiment, the anti-FAP antibody may comprise the variable region of 23C10. In particular, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:241, an HCDR2 of SEQ ID NO:242, and an HCDR3 of SEQ ID NO:243, and a light chain variable region comprising an LCDR1 of SEQ ID NO:244, an LCDR2 of SEQ ID NO:245, and an LCDR3 of SEQ ID NO:246. In addition, the anti-FAP antibody may comprise a heavy chain variable region of SEQ ID NO:286, and a light chain variable region of SEQ ID NO:287.

[0087] In one embodiment, the anti-FAP antibody may be AMG-506 / MP-0310. In particular, the antibody may comprise the amino acid sequence of SEQ ID NO:247.

[0088] Any anti-FAP antibody can be used as long as it can specifically bind to FAP present in cancer cells and induce the death of the cancer cells. Structure of the first monomer The first monomer comprises IL-12 or a variant thereof.

[0089] The first monomer may further comprise an antigen binding site that specifically binds to the FAP.

[0090] In one embodiment, the first monomer may comprise IL-12 or a variant thereof and an Fc region fragment or a variant thereof.

[0091] In one embodiment, the first monomer may comprise IL-12 or a variant thereof, an Fc region fragment or a variant thereof, and an antigen binding site that specifically binds to a FAP. The first monomer has the following structural formula (III) or (IV): N'-X-[linker (2)]p-Fc region fragment or variant thereof-[linker (3)]q-(T)r-C' (III) N'-(T)r-[linker (2)]q-Fc region fragment or variant thereof-[linker (3)]pX-C' (IV) [In structural formulas (III) and (IV), N' may be the N-terminus of the fusion protein; C' may be the C-terminus of the fusion protein; X may be of structural formula (I) or (II), T can be an antigen-binding site that specifically binds to a FAP, The linkers (2) and (3) may be peptide linkers; p, q and r may each independently be 0 or 1. may include.

[0092] 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.

[0093] Here, the first monomer may comprise the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:32.

[0094] Here, when r is 1 in structural formula (III), the first monomer may be in a form in which an anti-FAP antibody monomer is bound to IL-12 or a variant thereof.

[0095] Here, in structural formula (III), when r is 1, the first monomer may include the amino acid sequence of SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:302, or SEQ ID NO:303.

[0096] Here, in structural formula (IV), when r is 1, the first monomer may comprise the amino acid sequence of SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300, or SEQ ID NO:301.

[0097] In one embodiment, when r is 1, structural formula (III) is represented by the following structural formulas (III') and (III"): N'-X-[linker (2)]p-Fc region fragment or variant thereof-[linker (3)]q-(T')-C' (III') N'-(T")-C' (III") [In structural formulas (III') and (III"), T' is a heavy chain region of an antibody that specifically binds to a FAP, or a light chain region of an antibody, comprising a variable region and a CH1 region; T" is a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including the variable region and the CH1 region; wherein T' and T" bind to each other to form a variable region of an antibody, the variable region specifically binds to a FAP; The linkers (2) and (3) are peptide linkers, p and q are each independently 0 or 1; N', X and C' are as defined above. may include.

[0098] In one embodiment, when r is 1, structural formula (IV) has the following structural formulas (IV') and (IV"): N'-(T')-[linker (2)]q-Fc region fragment or variant thereof-[linker (3)]pX-C' (IV'); and N'-(T")-C' (IV") [In structural formulas (IV') and (IV"), T' is a heavy chain region of an antibody that specifically binds to a FAP, or a light chain region of an antibody, comprising a variable region and a CH1 region; T" is a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including the variable region and the CH1 region; wherein T' and T" bind to each other to form a variable region of an antibody, the variable region specifically binds to a FAP; The linkers (2) and (3) are peptide linkers, p and q are each independently 0 or 1; N', X and C' are as defined above. may include.

[0099] Here, the first monomer may comprise the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:2; SEQ ID NO:11 and SEQ ID NO:2; SEQ ID NO:27 and SEQ ID NO:19; SEQ ID NO:28 and SEQ ID NO:19; SEQ ID NO:294 and SEQ ID NO:2; SEQ ID NO:295 and SEQ ID NO:2; SEQ ID NO:300 and SEQ ID NO:19; or SEQ ID NO:301 and SEQ ID NO:19. Structure of the second monomer The second monomer may further comprise an antigen binding site that specifically binds to the FAP.

[0100] In one embodiment, the second monomer may comprise a first FAP binding site and a second FAP binding site.

[0101] The antigen-binding site that specifically binds to a FAP may be a Fab, scFv, Fv, or fragments thereof, where a first FAP-binding site may be a Fab and a second FAP-binding site may be an Fv or scFv.

[0102] The second FAP binding site may be attached to the N-terminus or C-terminus of the second monomer. Specifically, the second monomer may additionally be attached to the C-terminus of the heavy chain, the C-terminus of the light chain, or the C-terminus of the variable region.

[0103] The second monomer has the following structural formula (V): N'-(R)s-[linker (4)]tQ-[linker (5)]u-Fc region fragment or variant thereof-[linker (6)]v-(W)a-C' (V) [In structural formula (V), N' may be the N-terminus of the fusion protein; C' may be the C-terminus of the fusion protein; R and Q may be antigen binding sites that specifically bind to the FAP; W may be an scFv that specifically binds to a FAP; or IL-12 of structural formula (I) or (II) or a variant thereof, each of the linkers (4), (5) and (6) may be a peptide linker; s, t, u, v and a may each independently be 0 or 1. may include.

[0104] In one embodiment, structural formula (V) may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300, and SEQ ID NO:301. In one embodiment, structural formula (V) has the following structural formulas (V') and (V"): N'-(R')s-[linker (4)]t-Q'-[linker (5)]u-Fc domain fragment or variant thereof-[linker (6)]p-(W)a-C' (V') N'-(R")s-[linker (4)]tQ"-[linker (7)]x-(W)b-C' (V") [In structural formulas (V') and (V"), R' may be a heavy chain region of an antibody that specifically binds to a FAP, including a variable region and a CH1 region, or a light chain region of an antibody, R″ can be a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including the variable region and the CH1 region; wherein R′ and R″ may be combined together to form a variable region of an antibody, the variable region specifically binding to a FAP; Q' can be a heavy chain region of an antibody that specifically binds to a FAP, including a variable region and a CH1 region, or a light chain region of an antibody, Q″ can be a light chain region of an antibody that specifically binds to a FAP, or a heavy chain region of an antibody, including a variable region and a CH1 region; wherein Q′ and Q″ may be linked together to form a variable region of an antibody, the variable region specifically binding to a FAP; W may be an scFv that specifically binds to a FAP; or IL-12 of structural formula (I) or (II) or a variant thereof, each of the linkers (4), (5), (6) and (7) may be a peptide linker; s, t, u, x, a, and b may each independently be 0 or 1. may include.

[0105] 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.

[0106] 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.

[0107] In one embodiment, the second monomer may comprise a heavy chain consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain consisting of the amino acid sequence of SEQ ID NO: 103; or a heavy chain consisting of the amino acid sequence of SEQ ID NO: 110 and a light chain consisting of the amino acid sequence of SEQ ID NO: 111.

[0108] In addition, when W is an scFv, W may have the amino acid sequence of SEQ ID NO:72 or SEQ ID NO:84.

[0109] Peptide Linker As used herein, the term "peptide linker" refers to a peptide used to provide physicochemical distance or connect domains in a fusion protein. The linker may include an immunoglobulin hinge region.

[0110] The peptide linkers (1) to (7) refer to peptide linkers composed of amino acids. In particular, the peptide linkers (2) and (5) may be composed of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, 10 to 60 consecutive amino acids, or 12 to 50 amino acids. The peptide linkers are represented by the following general formula (G 4 S)n, where n is an integer from 1 to 10. 4 In S), n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0111] In one embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO:93, SEQ ID NO:94 or SEQ ID NO:95.

[0112] The peptide linker (2) or (5) may consist of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, or 10 to 60 consecutive amino acids, or 12 to 50 amino acids. In one embodiment, the peptide linker (2) may consist of 30 amino acids. In addition, the peptide linker (2) may contain at least one cysteine. In particular, the peptide linker (2) may contain one, two or three cysteines. In addition, the peptide linker (2) may be derived from an immunoglobulin hinge and may be a cysteine ​​residue having a cysteine ​​residue (G). 4 S)n (wherein n is an integer from 1 to 10). In particular, the peptide linker (2) may include 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, and SEQ ID NO:119.

[0113] In addition, the peptide linkers (3), (4), (6) and (7) may consist of 1 to 30 consecutive amino acids, 5 to 20 consecutive amino acids, or 10 to 15 consecutive amino acids. 4 S)n, where n is an integer from 1 to 10. 4 In S), n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0114] In addition, in particular, the peptide linker (2), (3) or (5) may comprise SEQ ID NO:93 or SEQ ID NO:94.

[0115] In one embodiment of the invention, the peptide linker (2) or (5) may be a peptide linker comprising the amino acid sequence of SEQ ID NO: 118 or 120. Fc region or a fragment thereof Here, the immunoglobulin fragment may be an Fc region of an immunoglobulin. The Fc region of an immunoglobulin may be a wild-type Fc domain or an Fc domain variant. Here, the Fc region may be an Fc region of IgG, IgA, IgE, IgD or IgM. In particular, the Fc region may be derived from IgG1 or IgG2a.

[0116] As used herein, the term "Fc domain variant" may refer to a form that differs from a wild-type Fc domain in terms of glycosylation pattern, has higher glycosylation compared to the wild-type Fc domain, has lower glycosylation compared to the wild-type Fc domain, or has a deglycosylated form. In addition, "Fc domain variant" includes a non-glycosylated Fc domain. The Fc domain or its variants may be configured to have a regulated number of sialic acids, fucosylation, or glycosylation by host culture conditions or genetic engineering.

[0117] In addition, the glycosylation of the Fc domain of an immunoglobulin can be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a mixed form of the Fc region of each of the immunoglobulins IgG, IgA, IgE, IgD, or IgM. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are replaced with other amino acids.

[0118] 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).

[0119] In addition, the Fc region may comprise a knob or hole structure.

[0120] As used herein, the term "knobs-into-holes" is a design strategy for producing antibodies that specifically bind to different regions, such as bispecific, multispecific, or heterodimeric antibodies. Knobs-into-holes are also called knobs-in-holes. In general, this technique involves introducing a knob at the interface of a first polypeptide (e.g., the first CH3 domain of a first antibody heavy chain) and a corresponding hole at the interface of a second polypeptide (e.g., the second CH3 domain of a second antibody heavy chain) such that the knob can be positioned within the hole to promote heterodimer formation and prevent homodimer formation.

[0121] A "knob" is constructed by replacing a small amino acid side chain from the interface of a first polypeptide (e.g., the first CH3 domain of a first antibody heavy chain) with a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A complementary "hole" of the same or similar size in the knob is created by replacing a large amino acid side chain from the interface of a second polypeptide (e.g., the second CH3 domain of a second antibody heavy chain) with a smaller side chain (e.g., alanine, serine, valine, or threonine). Knobs and holes can be created by altering the nucleic acid encoding the polypeptide, for example by site-directed mutagenesis or peptide synthesis.

[0122] In one embodiment, the knob structure of IgG1 may be SEQ ID NO: 13 or 288, and the hole structure may be SEQ ID NO: 12 or 289. The knob structure or hole structure may be in a form in which the amino acids at positions 146, 148, and 187 are substituted with other amino acids. Specifically, in one embodiment of the present invention, the knob structure or hole structure may be in a form in which one or more selected from the group consisting of T146W, T146S, L148A, and Y187V are substituted. In particular, the knob structure may be in a form in which one or more selected from the group consisting of T146W, T146S, L148A, and Y187V are substituted with T146W, and the hole structure may be in a form in which one or more selected from the group consisting of T146W, T146S, L148A, and Y187V are substituted with T146S, L148A, and Y187V.

[0123] On the other hand, in one embodiment of the present invention, the knob structure of IgG2a may be SEQ ID NO: 30 or 290, and the hole structure may be SEQ ID NO: 29 or 291. The knob structure or the hole structure may be in a form in which the amino acids at positions 152, 154, and 193 of the amino acid sequence of SEQ ID NO: 305 are substituted with other amino acids. In particular, in one embodiment of the present invention, the knob structure or the hole structure may be in a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152W or T152S, M154A, and Y193V. In particular, the knob structure may be in a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152W, and the hole structure may be in a form in which the amino acid sequence of SEQ ID NO: 305 is substituted with T152S, M154A, and Y193V.

[0124] In one embodiment, the Fc domain variant may comprise a DANG mutation or an NG mutation, where "DANG mutation" refers to the D265A / N297G mutation to eliminate effector function in human IgG1 or mouse IgG2a.

[0125] Effector functions mediated by the Fc region in IgG molecules include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), etc. Generally, these effector functions require association of the Fc region with a binding domain (e.g., an antibody variable domain).

[0126] Effector functions can be altered by substitutions in the amino acid sequence of the non-mutated Fc region, and Fc regions with altered effector functions can be designed, for example, by modifying C1q binding and / or FcR binding, thereby altering CDC and / or ADCC activity. Thus, "DANG mutation" means that the effector functions mediated by the Fc region are removed from the IgG molecule, such that undesired effector functions do not arise during antibody production.

[0127] In one embodiment, the DANG mutation may be in the form of a D45A / N77G substitution of amino acids in human IgG1 of SEQ ID NO: 289. Additionally, the DANG mutation may be in the form of a D51A / N83G substitution of amino acids in mouse IgG2a of SEQ ID NO: 291. Fusion protein structure The fusion protein may be an antibody. In particular, the antibody may be a heterodimeric antibody comprising a knob-in-hole structure.

[0128] The fusion protein can comprise structural formula (III) and structural formula (V); or structural formula (IV) and structural formula (V).

[0129] More specifically, one embodiment of the fusion protein may comprise a first monomer of (i) or (ii) below, and a second monomer of (iii), (iv), (v) or (vi) below: Example of the first monomer: (i) When r is 0, the structural formula (III) (ii) When r is 1, the structural formula (IV) Examples of second monomers: (iii) When s, a, and b are 0, the structural formula (V') and the structural formula (V") are (iv) When s is 1 and a and b are 0, the structural formula (V') and the structural formula (V") (v) When s and b are 0 and a is 1, the structural formula (V') and the structural formula (V") (vi) When b is 1, and s and a are 0, Structural Formula (V') and Structural Formula (V").

[0130] In one embodiment, the fusion protein may include a first monomer including the above (i) and a second monomer including the above (iii) (first from the left in FIG. 35). Here, the fusion protein may include at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22. In detail, the fusion protein may include SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5; SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:21; or SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:22.

[0131] In one embodiment, the fusion protein may comprise a first monomer comprising the above (i) and a second monomer comprising the above (iv) (second from the left in FIG. 35). Here, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24. In particular, the fusion protein may comprise SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:20, SEQ ID NO:23 and SEQ ID NO:24; or SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0132] In one embodiment, the fusion protein may comprise a first monomer comprising the above (i) and a second monomer comprising the above (v) (third from the left in FIG. 35). Here, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:25. In particular, the fusion protein may comprise SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:8; SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8; SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:25; or SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:25.

[0133] In one embodiment, the fusion protein may comprise a first monomer comprising the above (i) and a second monomer comprising the above (vi) (fourth from the left in FIG. 35). Here, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:26. In particular, the fusion protein may comprise SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:9; SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:9; SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:26; or SEQ ID NO:18, SEQ ID NO:22 and SEQ ID NO:26.

[0134] In one embodiment, the fusion protein may include a first monomer including the above (ii) and a second monomer including the above (iii) (fifth from the left in FIG. 35). Here, the fusion protein may include at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:27, and SEQ ID NO:28. In detail, the fusion protein may include SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:10; SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:11; SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:27; or SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:28.

[0135] In addition, in one embodiment, the fusion protein dimer comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to a FAP may be a fusion protein dimer comprising a monomer of structural formula (III) and a monomer of structural formula (V) (left side of FIG. 36 ), where r in structural formula (III) is 1, and s, a, and t in structural formula (V) are 0. Here, structural formula (V) may comprise (V′) and (V″), where s, a, t, and b in (V′) and (V″) are 0. In addition, the Fc region of the dimer has a knob-in-hole structure that allows different fusion proteins to bind to each other.

[0136] In particular, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:298 and SEQ ID NO:299. In particular, the fusion protein may comprise SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:292; SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:293; SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:298; or SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:299.

[0137] In addition, the fusion protein dimer comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to FAP may be a fusion protein dimer comprising the structure of structure (V) (center of FIG. 36 ). Here, the fusion protein dimer may comprise (V') and (V"), where in structural formula (V), s and t are 0, and a is 1. Additionally, in (V') and (V"), s, t and b are 0, and a is 1. Additionally, the fusion protein dimer may be a fusion protein dimer comprising the structure of structural formula (IV). Here, in structural formula (IV), r is 1.

[0138] In particular, the fusion protein may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:19, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300 and SEQ ID NO:301. In particular, the fusion protein may comprise SEQ ID NO:2 and SEQ ID NO:294; SEQ ID NO:2 and SEQ ID NO:295; SEQ ID NO:19 and SEQ ID NO:300; or SEQ ID NO:19 and SEQ ID NO:301.

[0139] In one embodiment, the fusion protein dimer comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to FAP may be a fusion protein dimer comprising the structure of structural formula (III) (right side of FIG. 36), where r in structural formula (III) is 1.

[0140] In particular, the fusion protein may comprise SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:302 or SEQ ID NO:303.

[0141] The multispecific fusion protein herein may be in a chemically modified form. In one embodiment, the fusion protein can be chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage and / or binding to cellular ligands or other proteins. Many of these chemical modifications can be performed by known techniques.

[0142] Polynucleotides encoding fusion proteins In another aspect of the invention, there is provided a polynucleotide encoding the first monomer or a polynucleotide encoding the second monomer.

[0143] In one embodiment, the first monomeric polypeptide may comprise an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, or SEQ ID NO:303.

[0144] In one embodiment, the polynucleotide encoding the first monomer may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:67.

[0145] In one embodiment, the second monomeric polypeptide may comprise an amino acid sequence having about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:300, or SEQ ID NO:301.

[0146] In one embodiment, the polynucleotide encoding the second monomer may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:66, SEQ ID NO:306.

[0147] The polynucleotide may further comprise a nucleic acid encoding a signal sequence or leader sequence. As used herein, the term "signal sequence" refers to a signal peptide that directs the secretion of a target protein. The signal peptide is translated and then cleaved in the host cell. In particular, a signal sequence is an amino acid sequence that initiates the transport of a protein across the endoplasmic reticulum (ER) membrane.

[0148] Signal sequences are well known in the art for their characteristics. Such signal sequences typically contain 16-30 amino acid residues, but may contain more or fewer than these amino acid residues. A typical signal peptide is composed of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4-12 hydrophobic residues that anchor the signal sequence during transport of the immature polypeptide through the membrane lipid bilayer.

[0149] After initiation, the signal sequence is cleaved in the lumen of the ER by a cellular enzyme commonly known as a signal peptidase. Here, the signal sequence may be a secretory signal sequence of tPa (tissue plasminogen activator), HSV gDs (signal sequence of herpes simplex virus glycoprotein D), or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells, including mammals, can be used. In addition, a wild-type signal sequence can be used, or a signal sequence substituted with a codon having a high expression frequency in a host cell can be used.

[0150] Use of a pharmaceutical composition comprising a fusion protein and an anticancer agent A composition comprising as an active ingredient of the present invention a fusion protein comprising IL-12 or a variant thereof and an antigen-binding site that specifically binds to a FAP, and an anti-cancer agent, may be used to treat or prevent cancer, and / or may increase the efficacy of treatment.

[0151] Therefore, the present invention can provide the use of the fusion protein for treating cancer.Furthermore, the use can be for treating cancer in combination with an anti-cancer drug.Furthermore, the present invention provides the use of the fusion protein for preparing a medicament for treating cancer in combination with an anti-cancer drug.

[0152] The fusion protein and the fusion protein dimer are as described above.

[0153] Here, the cancer may be any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0154] The preferred dose of the pharmaceutical composition varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing tumors of the present invention, the active ingredient can be contained in any amount (effective amount) depending on the use, dosage form, purpose of formulation, etc., as long as the active ingredient can exhibit activity to treat tumors or, in particular, therapeutic effect against cancer. The usual effective amount of the active ingredient is determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, the term "effective amount" refers to the amount of active ingredient capable of inducing an effect of improving or treating the condition of a disease, in particular, an effect of improving or treating the condition of a cancer. Such an effective amount can be determined experimentally within the scope of common sense of a person skilled in the art.

[0155] As used herein, the term "treatment" can be used to mean both therapeutic and prophylactic treatment. Here, prevention can be used to mean that a condition or disease in a subject is alleviated or ameliorated. In one embodiment, the term "treatment" includes both application or any form of administration to treat a disease in a mammal, including a human. In addition, the term "treatment" includes inhibiting or slowing the progression of a disease, restoring or repairing a decreased or lost function such that the disease is partially or completely alleviated; stimulating an inefficient process; or reducing the severity of the disease.

[0156] As used herein, the term "efficacy" can be determined by one or more parameters, such as survival over a period of time, such as 1 year, 5 years, or 10 years, or disease-free survival. Additionally, the parameters may include inhibition of the size of at least one tumor in an individual.

[0157] Pharmacokinetic parameters such as bioavailability and fundamental parameters such as clearance rate can also affect efficacy. Thus, "enhanced efficacy" (e.g., improved efficacy) can result from enhanced pharmacokinetic parameters and enhanced efficacy, which can be measured by comparing parameters such as clearance rate and tumor treatment or improvement in test animals or human subjects.

[0158] As used herein, the term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount of a compound or composition effective to prevent or treat the disease in question, sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatments and without causing side effects. The level of the effective amount can be determined by factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the mode of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, co-administered or co-used drugs, and other factors well known in the medical art. In one embodiment, a therapeutically effective amount refers to an amount of drug effective to treat cancer.

[0159] Here, the pharmaceutical composition may further include a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fat, wax, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0160] In particular, by including a pharma- ceutical composition in addition to the active ingredient, a pharma- ceutical composition can be prepared into a parenteral formulation by using a conventional method known in the art depending on the administration route. Here, the term "pharma- ceutical acceptable" means that the carrier does not inhibit the activity of the active ingredient and at the same time does not have a toxicity greater than that which can be accommodated by the subject to which it is applied (prescribed).

[0161] When the pharmaceutical composition is prepared into a parenteral preparation, the parenteral preparation can be prepared in the form of injection, transdermal patch, nasal inhalant, or suppository using a suitable carrier according to a method known in the art.When it is made into an injection, sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof can be used as a suitable carrier, and isotonic liquid such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, and 5% dextrose can be preferably used.The formulation of pharmaceutical compositions is known in the art, and can be referred to Remington's Pharmaceutical Sciences (19th edition, 1995) for details.The same document is considered to be a part of this specification.

[0162] The preferred dose of the pharmaceutical composition may range from 0.01 μg / kg to 10 g / kg, or from 0.01 mg / kg to 1 g / kg per day, depending on the patient's condition, body weight, sex, age, severity of the patient, and route of administration. The dose can be administered once a day or divided into several doses per day. Such doses should not be considered as limiting the scope of the present invention in any way.

[0163] 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.

[0164] Combination treatment methods including fusion proteins and anti-cancer agents In yet another aspect of the present invention, there is provided a method for treating or preventing cancer, comprising the step of administering to a subject a fusion protein or a dimer thereof comprising a first monomer comprising IL-12 or a variant thereof and a second monomer comprising an antigen-binding site that specifically binds to a FAP, and an anti-cancer agent.

[0165] Here, the subject may be a subject suffering from cancer. In addition, the subject may be a mammal, preferably a human. The fusion protein or its dimer, and the anticancer agent are as described above.

[0166] The administration route, dosage, and administration frequency of the fusion protein or fusion protein dimer may vary depending on the patient's condition and the presence or absence of side effects, so the fusion protein or fusion protein dimer can be administered to the subject in various ways and amounts.The optimal administration method, dosage, and administration frequency can be selected within an appropriate range by those skilled in the art.In addition, the fusion protein or fusion protein dimer can be administered in combination with other drugs or bioactive substances (e.g., anticancer drugs) that are known to have therapeutic effects on the disease to be treated, or can be formulated in the form of a combination preparation with other drugs. EXAMPLES

[0167] 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. Preparation Example 1. Overview of anti-FAP / IL-12 IgG1 DANG, human fusion protein

[0168] [Table 1]

[0169] [seq1] consists of the human anti-FAP heavy chain variable region sequence and human IgG1 Fc, with the effector function eliminated by DANG mutations (D265A, N297G) and the knob structure formed by the T366W mutation.

[0170] [seq2] consists of the human anti-FAP light chain sequence.

[0171] [seq3] consists of human IL-12 p40 (beta) region sequence and linker GGGGSGGGGSGGGGS, human interleukin-12 p35 (alpha) region sequence and linker GGGGSGGGGS, and human IgG1 Fc, with effector function eliminated by DANG mutations (D265A, N297G) and hole structure formed by T366S, L368A and Y407V mutations.

[0172] [seq4] consists of a sequence in which the lysine (K) at positions 280 and 285, which are involved in heparin binding in the human IL-12 p40 (beta) region, is mutated to alanine (A), the linker GGGGSGGGSGGGGGS, the human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and human IgG1 Fc hole DANG.

[0173] [seq5] consists of a sequence in which the lysine (K) at amino acids 280, 282, 285, and 286 involved in heparin binding in the human IL-12 p40 (beta) region is mutated to alanine (A), the linker GGGGSGGGGSGGGGS, the human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and human IgG1 Fc hole DANG.

[0174] [seq6] consists of the human anti-FAP heavy chain variable region sequence, linker GGGGSGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, and human IgG1 Fc knob DANG.

[0175] [seq7] consists of the human anti-FAP light chain variable region sequence, linker GGGGSGGGSGGGGGS, and the human anti-FAP light chain sequence.

[0176] [seq8] consists of the human anti-FAP heavy chain variable region sequence, human IgG1 Fc knob DANG, linker GGGGSGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0177] [seq9] consists of a human anti-FAP light chain sequence, a linker GGGGSGGGSGGGGGS, a human anti-FAP heavy chain variable region sequence, a linker GGGGSGGGGSGGGGSGGGGS, and a human anti-FAP light chain variable region sequence.

[0178] [seq10] is composed of a human anti-FAP heavy chain sequence, a human IgG1 Fc hole DANG, a linker GGGSGGGGSGGGGGS, a p40 (beta) region sequence of human IL-12, a linker GGGGSGGGGSGGGGS, and a human IL-12 p35 (alpha) region sequence.

[0179] [seq11] consists of the human anti-FAP heavy chain sequence, human IgG1 Fc hole DANG, linker GGGSGGGGSGGGGGS, human IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, and human IL-12 p35 (alpha) domain sequence.

[0180] [seq12] is composed only of human IgG1 Fc hole DANG.

[0181] [seq13] is composed only of human IgG1 Fc knob DANG.

[0182] [seq14] consists of the human anti-FAP heavy chain variable region sequence and human IgG1 Fc DANG.

[0183] [seq15] consists of the p40 (beta) domain sequence of human IL-12 containing four amino acid mutations involved in heparin binding, the linker GGGGSGGGSGGGGGS, the human IL-12 p35 (alpha) domain sequence, the linker GGGGSGGGGS, and human IgG1 Fc DANG.

[0184] [seq16] is composed of the heavy chain sequence of a human anti-CD20 antibody and a human IgG1 Fc knob DANG.

[0185] [seq17] is the light chain sequence of a human anti-CD20 antibody.

[0186] [seq292] is composed of human IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and human IgG1 Fc hole DANG, linker GGGGSGGGGSGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0187] [seq293] is composed of human IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and human IgG1 Fc hole DANG, linker GGGSGGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0188] [seq294] is composed of human anti-FAP heavy chain sequence, human IgG1 Fc DANG, linker GGGSGGGGSGGGGGS, human IL-12 p40 (beta) domain sequence, linker GGGGSGGGGSGGGGS, and human IL-12 p35 (alpha) domain sequence.

[0189] [seq295] consists of human anti-FAP heavy chain sequence, human IgG1 Fc DANG, linker GGGGSGGGSGGGGS, human IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGGSGGGSGGGGGS, and human IL-12 p35 (alpha) domain sequence.

[0190] [seq296] is composed of human IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS and human IgG1 Fc DANG, linker GGGSGGGGSGGGGGS, human anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and human anti-FAP light chain variable region sequence.

[0191] [seq297] is composed of human IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, the linker GGGGSGGGSGGGGGS, human IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and human IgG1 Fc DANG, the linker GGGSGGGGSGGGGGS, the human anti-FAP heavy chain variable region sequence, the linker GGGGSGGGGSGGGGSGGGGS, and the human anti-FAP light chain variable region sequence.

[0192] 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-in-hole structure and the effector function has been removed.

[0193] T1.02 (seq1, seq2, seq4) is a bispecific antibody that consists of a human anti-FAP sequence targeting human FAP and a human IL-12 sequence containing two amino acid mutations involved in heparin binding, forming a knob-in-hole structure and eliminating the effector function.

[0194] T1.03 (seq1, seq2, seq5) is a bispecific antibody that consists of a human anti-FAP sequence targeting human FAP and a human IL-12 sequence containing four amino acid mutations involved in heparin binding, forming a knob-in-hole structure and eliminating the effector function.

[0195] T1.04 (seq3, seq6, seq7) is a bispecific antibody in which a dual variable domain immunoglobulin (DVD-Ig) type human anti-FAP sequence and a human IL-12 sequence form a knob-in-hole structure and the effector function has been removed.

[0196] T1.05 (seq5, seq6, seq7) is a bispecific antibody in which a knob-in-hole structure is formed between a DVD-Ig type human anti-FAP sequence and a human IL-12 sequence containing four amino acid mutations involved in heparin binding, and the effector function is eliminated.

[0197] T1.06 (seq2, seq3, seq8) is a bispecific antibody in which a single-chain variable fragment (scFv) of a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the heavy chain and a human IL-12 sequence form a knob-in-hole structure, and the effector function has been removed.

[0198] T1.07 (seq2, seq5, seq8) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the heavy chain and a human IL-12 sequence containing four amino acid mutations involved in heparin binding form a knob-in-hole structure, thereby eliminating the effector function.

[0199] T1.08 (seq1, seq3, seq9) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the light chain and a human IL-12 sequence form a knob-in-hole structure, and the effector function has been removed.

[0200] T1.09 (seq1, seq5, seq9) is a bispecific antibody in which a human anti-FAP sequence targeting human FAP is linked to the C-terminus of the light chain and a human IL-12 sequence containing four amino acid mutations involved in heparin binding form a knob-in-hole structure, thereby eliminating the effector function.

[0201] T1.10 (seq1, seq2, seq10) is an antibody in which a human anti-FAP sequence targeting human FAP and a human anti-FAP sequence in which a human IL-12 sequence is linked to the C-terminus of the heavy chain form a knob-in-hole structure.

[0202] T1.11 (seq1, seq2, seq11) is an antibody that forms a knob-in-hole structure with a human anti-FAP sequence that targets human FAP and a human IL-12 sequence containing four amino acid mutations involved in heparin binding linked to the C-terminus of the heavy chain.

[0203] T1.12 (seq1, seq2, seq12) is a knobs-in-hole effector function-deleted antibody that has only the human anti-FAP sequence targeting human FAP.

[0204] T1.13 (seq3, seq13) is a knobs-in-hole effector function deleted antibody with only human IL-12 sequence.

[0205] T1.14 (seq4, seq13) is a knobs-in-hole effector function ablated antibody that contains only human IL-12 sequence containing two amino acid mutations involved in heparin binding.

[0206] T1.15 (seq5, seq13) is a knob-in-hole effector function-deleted antibody that contains only human IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0207] T1.16 (seq2, seq14) is a human anti-FAP antibody that targets human FAP.

[0208] T1.17(seq15) is an Fc fusion protein that contains only the human IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0209] 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-in-hole structure and the effector function has been removed.

[0210] T1.19 (seq4, seq16, seq17) is a bispecific antibody that consists of a human anti-CD20 sequence that targets human CD20 and a human IL-12 sequence that contains two amino acid mutations involved in heparin binding, forming a knobs-in-hole structure and eliminating effector functions.

[0211] T1.20 (seq5, seq16, seq17) is a bispecific antibody that consists of a human anti-CD20 sequence that targets human CD20 and a human IL-12 sequence that contains four amino acid mutations involved in heparin binding, forming a knob-in-hole structure and eliminating the effector function.

[0212] T1.21 (seq12, seq16, seq17) is a knobs-in-hole effector function-deleted antibody that has only the human anti-CD20 sequence to target human CD20.

[0213] T1.23 (seq1, seq2, seq292) is a bispecific antibody in which a human anti-FAP sequence and a human IL-12 sequence to which the scFv of the human anti-FAP sequence is linked at the C-terminus form a knob-in-hole structure and the effector function has been removed.

[0214] T1.24 (seq1, seq2, seq293) is a bispecific antibody in which a human anti-FAP heavy chain sequence and a human IL-12 sequence containing four amino acid mutations involved in heparin binding are linked to the C-terminus of the scFv of the human anti-FAP sequence, forming a knobs-in-hole structure and removing the effector function.

[0215] T1.25 (seq2, seq294) is an Fc fusion protein dimer containing a human anti-FAP sequence linked to the C-terminus of the heavy chain with a human IL-12 sequence.

[0216] T1.26 (seq2, seq295) is an Fc fusion protein dimer containing a human anti-FAP sequence linked to the C-terminus of the heavy chain with a human IL-12 sequence containing amino acid mutations (four) involved in heparin binding.

[0217] T1.27(seq296) ​​is an Fc fusion protein dimer consisting of a human IL-12 sequence linked to the C-terminus of a single chain variable fragment (scFv) of a human anti-FAP sequence.

[0218] T1.28 (seq297) is an Fc fusion protein dimer composed of a human IL-12 sequence containing four amino acid mutations involved in heparin binding linked to the C-terminus of a scFv of a human anti-FAP sequence.

[0219] [seq1] Anti-hu FAP HC hu IgG1 Fc knob DANG QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0220] [seq2] anti - hu FAP LC DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0221] [seq3] hu scIL - 12 - hu IgG1 Fc whole DANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEIDITKDK TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVLFPPKPKDTLMISRTPE VTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK

[0222] [seq4]hu scIL-12 mut1-hu IgG1 FcホールDANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASKREAKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEIDKDK TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVLFPPKPKDTLMISRTPE VTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK

[0223] [seq5]hu scIL-12 mut2-hu IgG1 FcホールDANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0224] [seq6](Anti - hu FAP VH)2 - hu IgG1 Fc Nob DANG QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0225] [seq7](Anti - hu FAP VL)2 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0226] [seq8] anti-hu FAP HC hu IgG1 Fc knob DANG - anti-hu FAP scFv QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK

[0227] [seq9] anti-hu FAP LC - anti-hu FAP scFv DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK

[0228] [seq10] Anti-hu FAP HCP hu IgG1 Fc whole DANG-hu scIL-12 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGKSKREKDRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0229] [seq11]Anti-hu FAP HC hu IgG1 Fc whole DANG-hu scIL-12 mut2 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0230] [seq12]hu IgG1 Fc whole DANG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0231] [seq13]hu IgG1 Fc knob DANG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0232] [seq14]Anti - hu FAP HC hu IgG1 Fc DANG QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0233] [seq15]hu scIL-12 mut2-hu IgG1 Fc DANG IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0234] [seq16] Anti-hu CD20 HC hu IgG1 Fc Nob DANG QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0235] [seq17] Anti - hu CD20 LC QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0236] [seq292] hu scIL - 12 - hu IgG1 Fc whole DANG - anti - hu FAP scFv

[0237] [seq293]hu scIL-12 mut2-hu IgG1 Fc Other-chain hu FAP scFv

[0238] [seq294]Anti-hu FAP HCP hu IgG1 Fc DANG-hu scIL-12 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGKSKREKDRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0239] [seq295]Anti-hu FAP HC hu IgG1 Fc DANG-hu scIL-12 mut2 QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSGGGSGIWELKKDVYVVELDWYPDAPGEMVV LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVPDTWSTPHSYFSLTFCVQVQGASAREAADRVFTDK TSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIVTIDRVMSYLNAS

[0240] [seq296]hu scIL-12-hu IgG1 Fc DANG-anti-hu FAP scFv

[0241] [seq297]hu scIL-12 mut2-hu IgG1 Fc DANG-anti-hu FAP scFv

[0242] Preparation Example 2. Overview of anti-FAP / IL-12 IgG2a DANG, mouse fusion protein

[0243] [Table 2]

[0244] [seq18] is composed of a mouse anti-FAP heavy chain variable region sequence and a mouse IgG2a Fc, with effector function eliminated by DANG mutations (D265A, N297G) and the knob structure formed by a T321W mutation.

[0245] [seq19] consists of the mouse anti-FAP light chain sequence.

[0246] [seq20] consists of mouse IL-12 p40 (beta) region sequence, linker GGGGSGGGGSGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc, with effector function eliminated by DANG mutations and hole structure formed by T321S, M323A, and Y362V mutations.

[0247] [seq21] consists of a sequence in which the lysine (K) at amino acids 277 and 282 involved in heparin binding in the mouse IL-12 p40 (beta) region is mutated to alanine (A), the linker GGGGSGGGSGGGGGS, the mouse IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and mouse IgG2a Fc hole DANG.

[0248] [seq22] consists of a sequence in which the arginine (R) at amino acid 276, which is involved in heparin binding in the mouse IL-12 p40 (beta) region, and the lysines (K) at amino acids 277, 278, and 282 are mutated to alanine (A), the linker GGGGSGGGSGGGGGS, the mouse IL-12 p35 (alpha) region sequence, the linker GGGGSGGGGS, and mouse IgG2a Fc hole DANG.

[0249] [seq23] consists of the mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, and mouse IgG2a Fc knob DANG.

[0250] [seq24] consists of the mouse anti-FAP light chain variable region sequence, the linker GGGGSGGGSGGGGGS, and the mouse anti-FAP light chain sequence.

[0251] [seq25] consists of the mouse anti-FAP heavy chain variable region sequence, mouse IgG2a Fc knob DANG, linker GGGGSGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0252] [seq26] consists of the mouse anti-FAP light chain sequence, linker GGGGSGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0253] [seq27] is composed of mouse anti-FAP heavy chain sequence, mouse IgG2a Fc hole DANG, linker GGGGSGGGSGGGGGS, mouse IL-12 p40 (beta) domain sequence, linker GGGGSGGGSGGGGGS, and mouse IL-12 p35 (alpha) domain sequence.

[0254] [seq28] consists of mouse anti-FAP heavy chain sequence, mouse IgG2a Fc hole DANG, linker GGGGSGGGGSGGGGS, mouse IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, and mouse IL-12 p35 (alpha) domain sequence.

[0255] [seq29] is composed only of mouse IgG2a Fc hole DANG.

[0256] [seq30] is composed only of mouse IgG2a Fc knob DANG.

[0257] [seq31] is composed of mouse anti-FAP heavy chain variable region and mouse IgG2a Fc DANG.

[0258] [seq32] consists of the p40 (beta) domain sequence of mouse IL-12 containing four amino acid mutations involved in heparin binding, the linker GGGGSGGGSGGGGGS, the mouse IL-12 p35 (alpha) domain sequence, the linker GGGGSGGGGS, and mouse IgG2a Fc DANG.

[0259] [seq33] consists of the heavy chain variable region sequence of the mouse anti-CD20 antibody 18B12 and a mouse IgG2a knob DANG.

[0260] [seq34] consists of the light chain sequence of the mouse anti-CD20 antibody 18B12.

[0261] [seq35] is composed only of mouse IgG2a Fc DANG.

[0262] [seq298] is composed of mouse IL-12 p40 (beta) region sequence, linker GGGSGGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS and mouse IgG2a Fc hole DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0263] [seq299] is composed of mouse IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc hole DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0264] [seq300] consists of mouse anti-FAP heavy chain sequence, mouse IgG2a Fc DANG, linker GGGGSGGGSGGGGGS, mouse IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, and mouse IL-12 p35 (alpha).

[0265] [seq301] consists of mouse anti-FAP heavy chain sequence, mouse IgG1 Fc DANG, linker GGGGSGGGSGGGGGS, mouse IL-12 p40 (beta) domain sequence containing four amino acid mutations involved in heparin binding, linker GGGGSGGGSGGGGGS, and mouse IL-12 p35 (alpha) domain sequence.

[0266] [seq302] is composed of mouse IL-12 p40 (beta) region sequence, linker GGGGSGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0267] [seq303] is composed of mouse IL-12 p40 (beta) region sequence containing four amino acid mutations involved in heparin binding, linker GGGSGGGGSGGGGGS, mouse IL-12 p35 (alpha) region sequence, linker GGGGSGGGGS, and mouse IgG2a Fc DANG, linker GGGSGGGGSGGGGGS, mouse anti-FAP heavy chain variable region sequence, linker GGGGSGGGGSGGGGSGGGGS, and mouse anti-FAP light chain variable region sequence.

[0268] 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-in-hole structure and the effector function has been removed.

[0269] T1.02m (seq18, seq19, seq21) is a bispecific antibody that consists of a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing two amino acid mutations involved in heparin binding, forming a knob-in-hole structure and eliminating the effector function.

[0270] T1.03m (seq18, seq19, seq22) is a bispecific antibody consisting of a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, forming a knob-in-hole structure and eliminating the effector function.

[0271] T1.04m (seq20, seq23, seq24) is a bispecific antibody that consists of a dual variable domain immunoglobulin (DVD-Ig) type mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence forming a knob-in-hole structure and has removed effector functions.

[0272] T1.05m (seq22, seq23, seq24) is a bispecific antibody that combines a mouse anti-FAP sequence targeting mouse FAP of the DVD-Ig type with a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding to form a knob-in-hole structure, thereby eliminating the effector function.

[0273] T1.06m (seq19, seq20, seq25) is a bispecific antibody in which a mouse anti-FAP sequence containing a single-chain variable fragment (scFv) of a mouse anti-FAP sequence targeting mouse FAP at the C-terminus of the heavy chain forms a knob-in-hole structure with a mouse IL-12 sequence, and the effector function has been removed.

[0274] T1.07m (seq19, seq22, seq25) is a bispecific antibody that has a knob-in-hole structure formed by a mouse anti-FAP sequence containing an scFv of the mouse anti-FAP sequence targeting mouse FAP at the C-terminus of the heavy chain and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, thereby eliminating the effector function.

[0275] T1.08m (seq18, seq20, seq26) is a bispecific antibody in which the mouse anti-FAP sequence, which targets mouse FAP and contains an scFv of the mouse anti-FAP sequence at the C-terminus of the light chain, forms a knob-in-hole structure with the mouse IL-12 sequence, and the effector function has been removed.

[0276] T1.09m (seq18, seq22, seq26) is a bispecific antibody that has a knob-in-hole structure formed by a mouse anti-FAP sequence containing a mouse anti-FAP sequence scFv targeting mouse FAP at the C-terminus of the light chain and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, thereby eliminating the effector function.

[0277] T1.10m (seq18, seq19, seq27) is an antibody that forms a knob-in-hole structure with a mouse anti-FAP sequence that targets mouse FAP and a mouse anti-FAP sequence in which a mouse IL-12 sequence is linked to the C-terminus of the heavy chain.

[0278] T1.11m (seq18, seq19, seq28) is an antibody in which a mouse anti-FAP sequence targeting mouse FAP and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding are linked to the C-terminus of the heavy chain, forming a knob-in-hole structure.

[0279] T1.12m (seq18, seq19, seq29) is a knobs-in-hole effector function-deleted antibody that has only the mouse anti-FAP sequence targeting mouse FAP.

[0280] T1.13m (seq20, seq30) is a knobs-in-hole effector function-deleted antibody that contains only the mouse IL-12 sequence.

[0281] T1.14m (seq21, seq30) is a knobs-in-hole effector function ablated antibody that contains only the mouse IL-12 sequence containing two amino acid mutations involved in heparin binding.

[0282] T1.15m (seq22, seq30) is a knobs-in-hole effector function ablated antibody that contains only the mouse IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0283] T1.16m (seq19, seq31) is a mouse anti-FAP antibody that targets mouse FAP.

[0284] T1.17m (seq32) is an Fc fusion protein dimer that contains only the mouse IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0285] T1.18m (seq20, seq33, seq34) is a bispecific antibody that consists of a mouse anti-CD20 (18B12) sequence targeting mouse CD20 and a mouse IL-12 sequence forming a knob-in-hole structure and has deleted effector functions.

[0286] T1.19m (seq21, seq33, seq34) is a bispecific antibody that consists of a mouse anti-CD20 (18B12) sequence targeting mouse CD20 and a mouse IL-12 sequence containing two amino acid mutations involved in heparin binding, forming a knob-in-hole structure and eliminating effector function.

[0287] T1.20m (seq22, seq33, seq34) is a bispecific antibody that consists of a mouse anti-CD20 sequence targeting mouse CD20 and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, forming a knobs-in-hole structure and eliminating effector function.

[0288] T1.21m (seq29, seq33, seq34) is a knobs-in-hole effector-function-deleted antibody that has only the mouse anti-CD20 sequence to target mouse CD20.

[0289] T1.22m(seq35) is a protein that contains only the mouse Fc sequence with effector functions removed.

[0290] T1.23m (seq18, seq19, seq298) is a bispecific antibody in which a mouse anti-FAP sequence and a mouse IL-12 sequence to which the scFv of the mouse anti-FAP sequence is linked at the C-terminus form a knob-in-hole structure, and the effector function has been removed.

[0291] T1.24m (seq18, seq19, seq299) is a bispecific antibody consisting of a mouse anti-FAP heavy chain sequence and a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding, linked to the C-terminus of an scFv of the mouse anti-FAP sequence, forming a knob-in-hole and abolishing effector functions.

[0292] T1.25m (seq19, seq300) is an Fc fusion protein dimer containing a mouse anti-FAP sequence linked to the C-terminus of the heavy chain with a mouse IL-12 sequence.

[0293] T1.26m (seq19, seq301) is an Fc fusion protein dimer containing a mouse anti-FAP sequence linked to the C-terminus of the heavy chain with a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding.

[0294] T1.27m (seq302) is an Fc fusion protein dimer consisting of a mouse IL-12 sequence linked to the C-terminus of a scFv of a mouse anti-FAP sequence.

[0295] T1.28m (seq303) is an Fc fusion protein dimer composed of a mouse IL-12 sequence containing four amino acid mutations involved in heparin binding linked to the C-terminus of a scFv of a mouse anti-FAP sequence.

[0296] [seq18]Anti-mu FAP HC mu IgG2a Fc knob DANG QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0297] [seq19]Anti-mu FAP LC QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWK IDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0298] [seq20]mu scIL-12-mu IgG2a Fc whole DANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSPDSRAVTCGMALSLAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELEAR QQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGGSGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPKIKDVLMISLS PIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFCKKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKFSRTPGK

[0299] [seq21]mu scIL-12 mut1-mu IgG2a FcホールDANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSPDSRAVTCGMALSLAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELEAR QQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRAKEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGGSGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPKIKDVLMISLS PIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFCKKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKFSRTPGK

[0300] [seq22]mu scIL-12 mut2-mu IgG2a FcホールDANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0301] [seq23](Anti-mu FAP VH)2, mu IgG2a Fc nob DANG QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0302] [seq24](Anti-mu FAP VL)2 QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0303] [seq25]Anti-mu FAP HC mu IgG2a Fc knob DANG - Anti-mu FAP scFv QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0304] [seq26]Anti-mu FAP LC - Anti-mu FAP scFv QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0305] [seq27] Anti-mu FAP HC mu IgG2a Fc whole DANG-mu scIL-12 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0306] [seq28]mut FAP HC mu IgG2a Fc-mut DANG-mu scIL-12 mut2 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0307] [seq29]mu IgG2a Fc whole DANG EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0308] [seq30]mu IgG2a Fc knob DANG EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0309] [seq31]Anti-mu FAP HC mu IgG2a Fc DANG QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0310] [seq32]mu scIL-12 mut2-mu IgG2a Fc DANG MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0311] [seq33] Anti-mu CD20 HC mu IgG2a Fc Nobe DANG QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPGQGLEWIGVIDPSDNYTKYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYFCAREGYYGSSPWFAYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0312] [seq34] Anti-mu CD20 LC QIVMSQSPAILSASPGEKVTMTCRARSSVSYIHWYQQKPGSSPKPWIYATSNLASGVPGRFSGSGSGTSYSLTITRVEAEDAATYYCQQWSSKPPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0313] [seq35] mu IgG2a Fc DANG EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0314] [seq298] mu scIL-12-mu IgG2a Fc whole DANG-anti-mu FAP scFv

[0315] [seq299]mu scIL-12 mut2-mu IgG2a Fc hole DANG-anti-mu FAP scFv

[0316] [seq300]Anti-mu FAP HC mu IgG2a Fc DANG-mu scIL-12 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0317] [seq301]Anti-mu FAP HC mu IgG2a Fc DANG-mu scIL-12 mut2 QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYGSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQAAAEKMAETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0318] [seq302]mu scIL-12-mu IgG2a Fc DANG-anti-mu FAP scFv

[0319] [seq303] mu scIL-12 mut2-mu IgG2a Fc DANG-anti-mu FAP scFv

[0320] Production Example 1. Production of fusion protein The reagents and equipment are described in Tables 3 and 4 below.

[0321] [Table 3]

[0322] [Table 4]

[0323] The synthesized DNA fragments were amplified by PCR, and the PCR products were gel purified. The pTT5 vector was cleaved by restriction enzymes EcoRI and BamHI, and then gel purified. Each PCR product and the linearized vector were ligated using the In-Fusion kit. The resulting vector was transformed into ECOS101 DH5α competent cells, and the cells were cultured on 2xYT agar plates containing 100 μg / ml ampicillin. All the operation processes were performed according to the standard transformation protocol. The positive recombinants were identified by colony PCR, and sequence verification sequencing was performed on the recombinant plasmids. A single colony was selected, and the seed culture was inoculated into 5 ml of 2xYT medium containing 100 μg / ml ampicillin. The culture was shaken at 37°C for 8 hours.

[0324] The seed culture was then diluted in 200 ml of selective 2xYT medium at a ratio of 1:1,000. The culture was incubated at 37°C for 16 hours with shaking. The bacterial cells were harvested by centrifugation at 4,700 rpm for 10 minutes at 4°C. The bacterial pellet was resuspended in 12 ml of RES-EF buffer. Then, 12 ml of LYS-EF buffer was added and the sealed tube was mixed thoroughly by vigorously inverting, and then incubated at room temperature for 5 minutes. 12 ml of NEU-EF buffer was added to the lysate and mixed thoroughly and quickly by vigorously inverting.

[0325] Prior to pouring the lysate into the NucleoBond™ Xtra column filter, the lysate tube was inverted three times to prepare a uniform suspension of the precipitate and to prevent clogging of the filter. The NucleoBond™ Xtra column filter and NucleoBond™ Xtra column were then washed with 10 ml of filter wash buffer FIL-EF. The NucleoBond™ Xtra column filter was removed by pulling and inverting the column. The NucleoBond™ Xtra column was washed with 90 ml of wash buffer ENDO.

[0326] The NuCryobond™ Xtra column was washed with 45 ml of wash buffer WASH-EF. Plasmid DNA was eluted with 15 ml of elution buffer ELU. The eluate was collected in a 50 ml centrifuge tube. The eluted plasmid DNA was precipitated by adding 10.5 ml of isopropanol at room temperature. After vortexing, the mixture was left for 2 minutes.

[0327] Then, 5 ml of 70% ethanol was added to the pellet. The ethanol was carefully and completely removed from the tube using a pipette tip. The pellet was dried at room temperature (20°C). The DNA pellet was then resuspended in 1,000 μl of HO. 2 Dissolved in O.

[0328] Preparation Example 2. Cell transfection and protein expression Preparation example 2.1. Cell transfection The materials and reagents used are shown in Table 5 below.

[0329] [Table 5]

[0330] 293F seed stock containing complete medium was incubated at 130 rpm, 37 °C, and 8% CO 2 The cells were maintained in an incubator shaker at 0.3–0.4 × 10 6The cells were cultured at a concentration of 2.6 × 10 cells / ml and the medium was changed every 2–3 days. 24 h before transfection, a new passage number of 293F cells was added to each well. 6 The cells were incubated at 130 rpm, 37°C, and 8% CO 2 On the day of transfection, the cell density was increased to 5.0 × 10 using fresh medium. 6 The transfection reagent was adjusted to 1 L cells / ml. The transfection was performed in a 3 L shaker flask with a total volume of 1 L. 0.4 mg of HC and 0.6 mg of LC plasmids were diluted in 50 ml of OPTI MEM I and filtered through a 0.22 μm filter. Then, 2 mg of PEI was diluted in 50 ml of OPTI MEM I to prepare the transfection reagent.

[0331] The diluted PEI was added to the DNA mixture and mixed immediately, then incubated at room temperature for 15 min. The DNA-PEI mixture was diluted to 2.6 × 10 6 The cells were then incubated at 130 rpm, 37°C, and 8% CO 2 The cells were continuously cultured for 24 hours in an incubator shaker at 130 rpm. 24 hours after transfection, 10% peptone was added to 1 / 20 of the culture solution to a final concentration of 0.5%. The cells were then incubated at 130 rpm, 37°C, and 8% CO 2 The cells were continuously cultured in an incubator shaker at 4 °C for 2 h. Cell density / viability was measured and recorded daily for 2–5 days after transfection. Cells were harvested for purification 7 days after transfection or when cell viability was less than 70%.

[0332] Example 2.2. Protein purification The reagents, buffer compositions, and equipment used in protein purification are shown in Tables 6 to 8 below.

[0333] [Table 6]

[0334] [Table 7]

[0335] [Table 8]

[0336] The protein was purified using a Mabselect Sure column. Specifically, the supernatant was collected by centrifugation at 2,000×g at 4°C for 20 min. The supernatant was then filtered through a Sartopore2 filter. The clarified supernatant was loaded onto a 5 ml MabSelect Sure column equilibrated with Buffer A. The column was then washed with Buffer A until the A280 absorbance reached baseline. The column was washed with 10 CV Buffer B. The column was washed with 10 CV Buffer A. The bound protein was eluted with 6 CV Buffer C, and 1 / 6 volume of Buffer D was added to neutralize the eluted material. SDS-PAGE and SEC-HPLC analysis were performed. The protein was then purified using a HIC column. The protein was then dialyzed against Buffer E overnight at 4°C. The supernatant was loaded onto a HIC column equilibrated with Buffer E. The column was then washed with Buffer E until the A280 absorbance reached baseline. Bound proteins were eluted by gradient elution (10 CV Buffer F 0% to 40%). Bound proteins were eluted with 2 CV 100% Buffer F and subjected to SDS-PAGE analysis.

[0337] After the proteins were purified, they were collected in one place and then the proteins were dialyzed overnight against the final buffer at 4° C. Then, SDS-PAGE and SEC-HPLC analysis were performed.

[0338] As a result, as shown in FIG. 1 to FIG. 6, it was found that the human protein and mouse protein of one embodiment were purified.

[0339] Preparation Example 3. Improved bispecific antibody production Preparation 3.1 Identification of improvements in the production of fusion proteins containing human IL-12 The change in the production of human anti-FAP / IL-12 bispecific antibodies due to human IL-12 mutations is shown in Table 9 below. The brackets indicate the production scale of the protein in units of L. The number before the brackets is the production amount per liter, calculated by dividing the amount of protein by the production scale.

[0340] [Table 9]

[0341] As shown in Table 9 above, increasing numbers of mutations in the amino acids involved in heparin binding in the human IL-12 p40(beta) region improved yields after protein purification via a Protein A column (MabSelect Sure column). Bispecific antibody T1.02, in which the lysines (K) at amino acids 280 and 285 were mutated to alanines (A), was found to have improved yield compared to T1.01. Bispecific antibody T1.03, in which the lysines (K) at amino acids 280, 282, 285, and 286 were mutated to alanines (A), was found to have approximately 2.5-fold improved yield compared to T1.01.

[0342] Preparation Example 3.2. Identification of improvements in the production of fusion proteins containing mouse IL-12 Table 10 below shows the change in mouse anti-FAP / IL-12 bispecific antibody production due to mouse IL-12 mutations.

[0343] [Table 10]

[0344] As shown in Table 10 above, as the number of mutations in the amino acids involved in heparin binding in the murine IL-12 p40(beta) region increased, the amount of protein increased after purification of the protein through a Protein A column (MabSelect Sure column).

[0345] In each table, the amount of protein after passing through the Protein A column was compared by plotting the total protein production at production scale. Bispecific antibody T1.02m, in which the lysine (K) at amino acid positions 277 and 282 was mutated to alanine (A), and bispecific antibody T1.03m, in which the arginine (R) at amino acid position 276 was mutated to alanine (A) and the lysine (K) at amino acid positions 277, 278, and 282 was mutated to alanine (A), were found to have gradually improved protein production compared to T1.01m and T1.02m. Furthermore, production of T1.03m using a CHO cell line was found to be approximately 13-fold improved compared to production using a HEK293 cell line.

[0346] Production Example 4. Cell lines and cell line culture HEK-Blue IL-12 cell line, which was transfected with IL-12 receptor gene and STAT4-inducible SEAP reporter gene into human embryonic kidney fibroblast HEK293 cells, was obtained from Invivogen (San Diego, USA). HEK-Blue IL-12 cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO), 100 μg / ml normocin, and 1×HEK-Blue selection.

[0347] Human embryonic kidney fibroblast cell line HEK293, mouse colon cancer cell line CT26-WT, mouse melanoma cell line B16F10 and mouse fibroblast cell line NIH-3T3 were obtained from ATCC (American Type Culture Collection, Manasas, VA, USA). HEK293 cells, B16F10 cells and NIH-3T3 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.

[0348] Cell lines (CT26-mFAP, B16F10-mFAP, and NIH-3T3-mFAP) in which mouse FAP (fibroblast activation protein alpha) was overexpressed in CT26-WT, B16F10, and NIH-3T3 cells were prepared using lentivirus capable of delivering mouse FAP gene. CT26-mFAP cells were maintained in RPMI-1640 (GIBCO) containing 10% FBS (GIBCO) and 10 μg / ml puromycin. B16F10-mFAP cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO) and 10 μg / ml puromycin. NIH-3T3-mFAP cells were maintained in DMEM (GIBCO) containing 10% FBS (GIBCO) and 5 μg / ml puromycin.

[0349] Example 5. Isolation and activation of human immune cells Blood packs were obtained from the Korean Red Cross with Institutional Review Board (IRB) approval, and peripheral blood mononuclear cells (PBMCs) were isolated and frozen. Thawed PBMCs were subjected to positive selection, and human NK cells were isolated using the Easy sep (Stem cell, Vancouver, BC, CA) kit. The isolated NK cells were activated and maintained in RPMI-1640 (GIBCO) and 10% FBS containing 50 IU / ml recombinant human IL-2 (rhIL-2, R&D systems, Minnesota, USA).

[0350] Thawed PBMCs were subjected to negative selection and human T cells were isolated using the Easy sep (Stem cell) kit. Human T cells were cultured on plates coated with 1 μg / ml anti-CD3 (OKT3, Invitrogen) and activated for 72 hours. Human T cells were maintained in RPMI-1640 (GIBCO) containing 10% FBS (GIBCO).

[0351] Example 1. Identification of binding affinity of fusion proteins Example 1.1. Identification of Binding of Fusion Proteins to Recombinant Human FAP The binding of T1.01, T1.02 and T1.03 to recombinant human FAP was identified by surface plasmon resonance (SPR).

[0352] Specifically, the surface of a CM5 chip was activated with a 1:1 mixture of 50 nM NHS (N-hydroxysuccinimide) and 200 nM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 25 μg / ml of anti-human IgG (Fc) antibody was immobilized at a rate of 10 μl / min for 400 seconds. The remaining active ester groups were blocked with 1 M ethanolamine. T1.01, T1.02 and T1.03 were diluted to 2 μg / ml and reacted with anti-human IgG (Fc) antibody on the immobilized CM5 chip. Recombinant human FAP was diluted to 200 nM in 1×HBS-EP+ buffer and diluted by serial dilution. The diluted recombinant human FAP was reacted at a rate of 30 μl / min. The association and dissociation times were 180 and 400 seconds, respectively. After dissociation, stabilization was performed for 60 seconds, followed by regeneration with 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min for 30 seconds.

[0353] As a result, as shown in Figures 7 and 8, it was found that T1.01, T1.02 and T1.03 could specifically bind to recombinant human FAP.

[0354] The binding of T1.04, T1.05, T1.06, T1.07, T1.08, T1.09, T1.10 and T1.11 to recombinant human CD20 was identified by surface plasmon resonance (SPR). The surface of a CM5 chip was activated with a 1:1 mixture of 50 nM NHS (N-hydroxysuccinimide) and 200 nM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 25 μg / ml of anti-human IgG (Fc) antibody was immobilized at a rate of 10 μl / min for 400 s. The remaining active ester groups were blocked with 1 M ethanolamine.

[0355] Specifically, human proteins T1.04, T1.05, T1.06, T1.07, T1.08, T1.09, T1.10, and T1.11 were diluted to 1 μg / ml, 1 μg / ml, 1 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.5 μg / ml, 0.5 μg / ml, and 1 μg / ml, respectively, and reacted on an anti-human IgG (Fc) antibody-immobilized CM5 chip. Recombinant human FAP was diluted to 25 nM or 50 nM in 1×HBS-EP+ buffer and diluted by serial dilution. The diluted recombinant human FAP was reacted at a rate of 30 μl / min. Association and dissociation times were 180 s and 400 s, respectively. After dissociation, stabilization was performed for 60 s, and then regeneration was performed for 30 s with 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min.

[0356] As a result, as shown in Table 11 and Figures 9 and 10, it was found that T1.04, T1.05, T1.06 and T1.07 can bind to recombinant human FAP and specifically target FAP. Furthermore, as shown in Table 12 and Figures 11 and 12, it was found that T1.08, T1.09, T1.10 and T1.11 can bind to recombinant human FAP and specifically target FAP.

[0357] [Table 11]

[0358] [Table 12] Example 1.2. Identification of binding of mouse proteins to recombinant mouse FAP The binding of T1.01m, T1.02m and T1.03m to recombinant mouse FAP was identified by surface plasmon resonance (SPR).

[0359] The surface of a CM5 chip was activated with a 1:1 mixture of 50 nM NHS (N-hydroxysuccinimide) and 200 nM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 25 μg / ml of anti-mouse IgG (Fc) antibody was immobilized at a rate of 10 μl / min for 400 seconds. The remaining active ester groups were blocked with 1 M ethanolamine. T1.01m, T1.02m, and T1.03m were diluted to 2 μg / ml and reacted with anti-mouse IgG (Fc) antibody on the immobilized CM5 chip. Recombinant mouse FAP was diluted to 200 nM in 1×HBS-EP+ buffer and serially diluted. The diluted recombinant mouse FAP was reacted at a rate of 30 μl / min. Association and dissociation times were 180 and 400 seconds, respectively. After dissociation, stabilization was performed for 60 seconds, followed by regeneration with 10 mM glycine (pH 1.5) solution at a rate of 30 μl / min for 30 seconds.

[0360] As a result, as shown in Figures 13 and 14, it was found that T1.01m, T1.02m and T1.03m could bind to recombinant mouse FAP and specifically target FAP.

[0361] Example 1.3. Identification of binding to FAP-expressing cells The extent of binding of T1.12, T1.01, T1.02 and T1.03 to HEK293 and HEK293-hFAP cells was determined.

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

[0363] As a result, as shown in Figure 15, T1.12, T1.01, T1.02 and T1.03 were found to bind to the human FAP-expressing cell line by 99% or more. These results indicate that the fusion protein of one embodiment can bind to the human FAP-expressing cell line and specifically target the FAP.

[0364] Furthermore, the extent of binding of T1.12m, T1.01m, T1.02m and T1.03m to B16F10 and B16F10-mFAP cells was determined.

[0365] Specifically, the cell line was cultured at 1 × 10 5 Cells were prepared by suspending in FACS buffer at 100 μl per cell and treated with 1 μg each of T1.12m, T1.01m, T1.02m and T1.03m. Cells were washed twice with FACS buffer. Cells were stained with anti-mouse IgG2a antibody (Biolegend). Negative controls were stained with anti-mouse IgG2a antibody (Biolegend) alone. The expression rate of stained cells was measured using a BD LSR and analyzed using FlowJo software.

[0366] As a result, as shown in Figure 16, T1.12m, T1.01m, T1.02m and T1.03m were found to bind to the FAP-expressing cell line by 99% or more. These results indicate that the fusion protein of one embodiment can bind to the mouse FAP-expressing cell line and specifically target FAP.

[0367] Example 1.4. Identification of fusion protein binding to recombinant human IL-12 receptor Considering that the fusion protein IL-12 must bind to the IL-12 receptor for action, T1.01, T1.02, T1.03 and T1.12 were found to bind to the recombinant human IL-12 receptor.

[0368] T1.01, T1.02 and T1.03 were immobilized on a plate and recombinant human IL-12 receptor protein conjugated with horseradish peroxidase (HRP) was bound to them. Specifically, the following steps were performed: (i) T1.01, T1.02, T1.03 and T1.12 were suspended at 5 μg / ml and serially diluted. The diluted T1.01, T1.02, T1.03 and T1.12 were aliquoted into 96-well immunoplates and treated for 24 hours.

[0369] (ii) The wells were washed with a washing solution and blocked with a 1% BSA (bovine serum albumin, Sigma) solution for 1 hour.

[0370] (iii) The wells were washed with washing solution and then treated with 1 nM IL-12 receptor beta 1-biotin (IL-12Rβ1-biotin; Acrobiosystems, Newark, USA) for 2 h.

[0371] (iv) The wells were washed with washing solution and then treated with streptavidin-HRP for 20 minutes.

[0372] (v) The wells were washed with wash solution and then treated with substrate solution for 20 minutes.

[0373] (vi) The wells were treated with stop solution and the absorbance was measured at 450 nm.

[0374] As a result, it was found that T1.01, T1.02 and T1.03 bind to the recombinant human IL-12 receptor protein in a concentration-dependent manner, as shown in Figure 17. T1.12 was used as a control.

[0375] Example 2. Identification of the ability of fusion proteins to induce signal transduction Example 2.1. Identification of signal transduction capabilities in IL-12-recognizing cells Considering that IL-12 is known to initiate signal transduction by inducing phosphorylation of the signal transduction mediator STAT4 (signal transducer and activator of transcription 4), the signal transduction ability of an embodiment fusion protein in IL-12 recognizing cells expressing the IL-12 receptor when IL-12 binds to the IL-12 receptor was evaluated.

[0376] Specifically, 2.8 × 10 HEK-Blue IL-12 cell line, which is an IL-12-recognizing cell line, was used. 5 The cells were diluted to 1000 cells / ml and dispensed at 180 μl / well. Recombinant human IL-12, T1.01m, T1.02m, T1.03m and T1.12m were suspended at 4 nM and diluted by serial dilution. HEK-Blue IL-12 cells were treated with diluted recombinant human IL-12, T1.01m, T1.02m, T1.03m and T1.12m. After 24 h of treatment, cell supernatants were collected and mixed with QUANTI-Blue Solution (Invivogen) in a 96-well plate. Reporter expression levels were measured at 620 nm using a spectrophotometer (Thermo Fisher).

[0377] As a result, as shown in Figure 18, in the case of IL-12, T1.01m, T1.02m and T1.03m treated groups, IL-12 was recognized and STAT4 was induced to phosphorylate and express the reporter gene, as identified by absorbance at 620 nm. In contrast, it was identified that the control T1.12m did not recognize IL-12 when IL-12-recognizing cells were treated with T1.12m. These results indicate that the fusion protein of one embodiment specifically binds to the IL-12 receptor to induce signal transduction.

[0378] Example 2.2. Identification of signaling capabilities in human T cells To identify whether IL-12 initiates signal transduction by inducing phosphorylation of the signal transduction mediator STAT4 when it binds to the IL-12 receptor on human T cells, human T cells activated with anti-CD3 (OKT3, Invitrogen) were analyzed using the AlphaLISA SureFire Ultra p-STAT4 (Tyr693) Assay Kit (PerkinElmer, Massachusetts, USA).

[0379] Specifically, human proteins IL-12, T1.01, T1.02, T1.03 and T1.12 were suspended at 100ng / ml and diluted by serial dilution. Human T cells activated with anti-CD3 (OKT3, Invitrogen) were treated with diluted recombinant human IL-12, T1.01, T1.02, T1.03 and T1.12. After 2 hours of treatment, cells were lysed and treated with a reagent (PerkinElmer) to detect p-STAT4 (phosphorylated STAT4). pSTAT4 was detected using a spectrophotometer at an excitation wavelength of 680nm and an emission wavelength of 615nm.

[0380] As a result, the half maximal effective concentration (EC50) of the group treated with recombinant human IL-12 was 8.945 pM, the EC50 of the group treated with T1.01 was 14.48 pM, the EC50 of the group treated with T1.02 was 56.51 pM, and the EC50 of the group treated with T1.03 was 147.2 pM, as shown in Figure 19. In contrast, no phosphorylated STAT4 was detected in the control group treated with T1.12.

[0381] These results indicate that IL-12 mutations attenuated T cell signaling capacity.

[0382] Example 2.3. Identification of changes in signaling capacity of fusion proteins by low molecular weight heparin in human T cells Next, we identified the enhancement of the signaling capacity of IL-12 mutant (IL-12 mut1) fusion protein by low molecular weight heparin using human T cells activated with anti-CD3 (OKT3, Invitrogen) and the AlphaLISA SureFire Ultra p-STAT4 (Tyr693) assay kit.

[0383] In particular, the signaling capacity in human T cells in the presence or absence of low molecular weight heparin (LMWH) was identified by observing whether induction of phosphorylation of the signaling mediator STAT4 was attenuated when attenuated IL-12 mut2 bound to the IL-12 receptor in human T cells.

[0384] Specifically, recombinant human IL-12, T1.01, T1.02 and T1.03 were suspended at 100ng / ml and diluted by serial dilution. Activated human T cells were treated with diluted recombinant human IL-12, T1.01, T1.02 and T1.03 together with low molecular weight heparin. After 2 hours of treatment, cells were lysed and treated with a reagent (PerkinElmer) for detecting p-STAT4. pSTAT4 was detected using a spectrophotometer at an excitation wavelength of 680nm and an emission wavelength of 615nm.

[0385] As a result, as shown in FIG. 20, the half maximal effective concentration (EC50) of the group treated with T1.01 and low molecular weight heparin was increased by 9.4-fold compared to the group treated with T1.01, the half maximal effective concentration (EC50) of the group treated with T1.02 and low molecular weight heparin was increased by 5.5-fold compared to the group treated with T1.02, and the half maximal effective concentration (EC50) of the group treated with T1.03 and low molecular weight heparin was increased by 4-fold compared to the group treated with T1.03.

[0386] These results indicate that when the IL-12 mutation (IL-12 mut2) was added to a pre-existing IL-12 mutation (IL-12 mut1), the degree of enhancement of signaling capacity by the addition of low molecular weight heparin was reduced and IL-12 was effectively attenuated.

[0387] Example 3. Identification of cytokine secretion ability of fusion proteins Example 3.1. Enzyme Immunoassays for Measuring Cytokines For cytokine measurements, supernatants stored at -20°C were subjected to enzyme-linked immunosorbent assay (ELISA, R&D systems) using the following steps: (i) Capture antibodies were diluted according to the Certificate of Analysis (CoA) and coated onto 96-well plates for 24 hours. (ii) The wells were washed with a washing solution and blocked with a 1% BSA (bovine serum albumin, Sigma) solution for 1 hour. (iii) The wells were washed with a washing solution and then treated with the samples for 2 hours. (iv) The wells were washed with a washing solution, then the detection antibody was diluted according to the certificate of analysis and aliquoted into the wells, and then the wells were treated with it for 2 hours. (v) The wells were washed with washing solution and then treated with streptavidin-HRP for 20 minutes. (vi) The wells were washed with washing solution and then treated with substrate solution for 20 minutes. (vii) The wells were treated with stop solution and the absorbance was measured at 450 nm.

[0388] Example 3.2. Identification of cytokine secretion ability in human T cells Considering that IFN-γ (interferon-gamma), a major immune response, is secreted when human T cells are treated with IL-12, we evaluated the IFN-γ secretion ability of human T cells when treated with recombinant human IL-12, T1.12, T1.01, T1.02, and T1.03.

[0389] Specifically, 5 × 10 human T cells activated with anti-CD3 (OKT3, Invitrogen) were 5 Cells / ml were diluted and dispensed at 200 μl / well into 96-well plates. Recombinant human IL-12, T1.12, T1.01, T1.02 and T1.03 were diluted to 10 nM and dispensed at 20 μl / well. After 48 hours of treatment, cell supernatants were collected and samples were stored at -80°C.

[0390] As a result, it was found that the samples treated with recombinant human IL-12 had an average of 1,640 pg / ml IFN-γ, the samples treated with T1.01 had an average of 1,115 pg / ml IFN-γ, the samples treated with T1.02 had an average of 845 pg / ml IFN-γ, and the samples treated with T1.03 had an average of 607 pg / ml IFN-γ, as shown in Figure 21. In contrast, the samples treated with T1.12 as a control were found to have no IFN-γ.

[0391] These results indicate that T1.01, T1.02 and T1.03 act on human T cells to induce IFN-γ secretion.

[0392] Example 3.3. Identification of cytokine secretion capacity in human NK cells Considering that IFN-γ (interferon-gamma), a major immune response, is secreted when human NK cells are treated with IL-12, the inventors evaluated the IFN-γ secretion ability of human NK cells activated with recombinant human IL-2 (R&D systems) when treated with recombinant human IL-12, T1.12, T1.01, T1.02 and T1.03.

[0393] Specifically, 5 × 10 human NK cells activated with recombinant human IL-2 (R&D Systems) were 5 Cells / ml were diluted and dispensed at 200 μl / well into 96-well plates. Recombinant human IL-12, T1.12, T1.01, T1.02 and T1.03 were diluted to 10 nM and dispensed at 20 μl / well. After 24 hours of treatment, cell supernatants were harvested and samples were stored at -80°C.

[0394] As a result, as shown in Figure 22, it was found that the samples treated with recombinant human IL-12 had an average of 180 pg / ml of IFN-γ, the samples treated with T1.01 had an average of 205 pg / ml of IFN-γ, the samples treated with T1.02 had an average of 133 pg / ml of IFN-γ, and the samples treated with T1.03 had an average of 80 pg / ml of IFN-γ. In contrast, the samples treated with T1.12 as a control were found to have no IFN-γ. These results indicate that T1.01, T1.02 and T1.03 act on human NK cells to induce IFN-γ secretion.

[0395] Example 4. Evaluation of anticancer effect of fusion protein in colon cancer animal model Example 4.1. Evaluation of anti-cancer effects in a colon cancer animal model by co-injection of FAP-expressing fibroblasts The fusion protein is in the form of a double antibody having an anti-FAP sequence structure and an IL-12 sequence structure. Considering that FAP is overexpressed in fibroblasts present in many tumor microenvironments, mouse FAP was overexpressed in mouse fibroblast cell line NIH3T3 cells, and then the tumor growth inhibitory ability was evaluated in a tumor animal model in which the cells were co-injected 1:1 with the mouse colon cancer cell line CT26.

[0396] We prepared FAP-expressing tumor animal models using mouse colon cancer cell line CT26 and mouse fibroblast cell line NIH-3T3. Next, we prepared NIH-3T3 as a FAP-expressing cell line.

[0397] Specifically, NIH-3T3 cells and CT26 cells overexpressing mouse FAP in culture were incubated at 1 × 10 6 The cells were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) at a concentration of cells / 50 μl, mixed at a ratio of 1:1, and then implanted with 100 μl per mouse into the subcutaneous region of the right flank of 6-week-old BALB / c mice using a 1 cc syringe (25G) to obtain FAP-expressing fibroblast co-injected animal models. Tumor size was measured using a digital Vernier caliper by measuring the short and long axes of the tumor, and tumor size was expressed as (short axis, mm). 2 Measurements were taken twice weekly using the calculation formula: × (long axis, mm) × 0.5.

[0398] Next, the efficacy of the drug was evaluated in patients with tumors 100 mm 3 When the tumor growth rate reached 100 μl, the mice were randomly divided into groups. Each experimental group consisted of 10 mice, and as a control, 100 μl of 50 μg T1.24m was intraperitoneally administered three times at 3-day intervals. In the test groups, 10 μg and 50 μg T1.02m and 50 μg T1.01m were intraperitoneally administered three times at 3-day intervals to evaluate the tumor growth inhibition ability.

[0399] As a result, as shown in Figure 23, when comparing the tumor size on day 14, the groups administered 10μg and 50μg of T1.02m were found to have superior tumor growth inhibition abilities of 93.2% and 92.7%, respectively, compared to the group administered 50μg of T1.24m as a control. Furthermore, it was observed that the group administered 50μg of T1.01m had 91.9% tumor growth inhibition compared to the group administered 50μg of T1.24m as a control.

[0400] Furthermore, as shown in FIG. 24, 24 days after treatment, complete response subjects were observed in the groups receiving T1.01m and T1.02m compared to the control.

[0401] These results demonstrate that the fusion protein of one embodiment has excellent anti-cancer effects in an animal model of colon cancer.

[0402] Example 4.2. Evaluation of anticancer effect in FAP-expressing colon cancer animal model In the case of colorectal cancer, considering the high expression of FAP in the tumor cells themselves, to evaluate the anticancer effect, the tumor growth inhibition ability was evaluated in a tumor model in which overexpression of mouse FAP was induced in the mouse colon cancer cell line CT26 cells.

[0403] To prepare a mouse FAP-expressing tumor animal model, FAP overexpression was induced in the mouse colon cancer cell line CT26. Specifically, FAP-overexpressing CT26 cells in culture were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) and then injected into the left dorsal subcutaneous region of 6-week-old BALB / c mice using 1 cc syringe (25G) at 100 μl of 1 × 10 cells / mouse. 6 Tumor cells were transplanted to obtain a mouse FAP-expressing colon cancer animal model. Tumor size was measured using a digital Vernier caliper by measuring the short and long axes of the tumor, and tumor size was expressed as (short axis, mm). 2 Measurements were taken twice weekly using the calculation formula: × (long axis, mm) × 0.5.

[0404] Next, the efficacy of the drug was evaluated in patients with tumors 100 mm 3 When the tumor growth rate reached 100 μl, the mice were randomly divided into groups. Each experimental group consisted of 8 animals, and 100 μl of 50 μg T1.24m was administered intraperitoneally 4 times at 3-day intervals as a control. In the test groups, 100 μl of 10 μg and 100 μg T1.01m, and 2 μg, 10 μg and 100 μg T1.02m were administered intraperitoneally 4 times at 3-day intervals to evaluate the tumor growth inhibition ability.

[0405] As a result, as shown in Figure 25, after 18 days of administration, the groups administered 10μg and 100μg of T1.01m were found to have 84.9% and 85.6% higher tumor growth inhibition abilities, respectively, compared to the group administered 50μg of T1.24m as a control. Furthermore, it was observed that the groups administered T1.02m had 80.1%, 90.5% and 89.8% higher tumor growth inhibition abilities per dose, compared to the group administered T1.24m as a control.

[0406] Furthermore, 30 days after administration, each administration group showed complete response (CR) subjects along with tumor growth inhibition, and no change in body weight was observed in response to administration. These results indicate that the fusion protein of one embodiment has excellent anti-cancer effects in a colon cancer animal model.

[0407] Example 4.3. Evaluation of fusion protein inhibition of recurrent solid tumor growth in complete response mice To evaluate whether tumor recurrence could be inhibited by inducing a long-term immune response after fusion protein treatment, subjects who achieved complete response were rechallenged with tumors by administering T1.01m to a tumor model implanted with FAP-overexpressing CT26 cells. Untreated BALB / c mice were used as control mice.

[0408] Control mice and mice that achieved a complete response by administration of T1.01m were subcutaneously injected with 4T1 cells in the left flank and with CT26 cells in the right flank to obtain tumor recurrence models.

[0409] Specifically, in a model transplanted with CT26 cells, a tumor recurrence model was induced in mice that showed a complete response to administration of T1.01m. CT26 cells and 4T1 cells were administered at a dose of 1 × 10 6 Suspended in Hank's Balanced Salt Solution (HBSS, Gibco) at a concentration of cells / 100 μl, CT26 cells were implanted into the subcutaneous region of the right flank of mice using a 1 cc syringe (25G). Tumor formation and growth were assessed by subcutaneous implantation of 4T1 cells into the left flank of the same mice. Nine animals were used in the control group and seven animals in the test group.

[0410] As a result, it was found that both the transplanted 4T1 tumor and the CT26 tumor grew in the control mice without any prior treatment, whereas in the mice that achieved a complete response after administration of T1.01m, the 4T1 tumor grew but the growth of the CT26 tumor was inhibited, as shown in Figures 26 and 27. These results indicate that the fusion protein of one embodiment can inhibit tumor recurrence.

[0411] Example 4.4. Evaluation of reduction in activity due to IL-12 amino acid mutations in fusion proteins To reduce in vivo toxicity due to excessive activation of the immune system by IL-12, the present inventors used an IL-12 sequence structure containing a mutation in the amino acid sequence that binds to heparin to induce a decrease in activity by blocking binding to the IL-12 receptor, which was identified by a tumor animal model.

[0412] Among the anti-FAP antibodies, T1.02m, T1.03m, and T1.01m, in which the amino acids involved in heparin binding are mutated, were used as controls. Next, T1.02m has a mouse IL-12 sequence structure containing two amino acid mutations involved in heparin binding together with the anti-FAP sequence structure. T1.03m is a fusion protein having an IL-12 sequence structure containing four amino acid mutations involved in heparin binding together with the anti-FAP sequence structure.

[0413] To evaluate the change in IL-12 activity depending on the presence and degree of amino acid mutation, 5 × 10 5 FAP-overexpressing mouse colon cancer CT26 was prepared by resuspending in PBS at a concentration of cells / 100 μl, and the tumor growth inhibition ability was evaluated in a tumor animal model prepared by transplanting the cells into 5-week-old female BALB / c mice.

[0414] Specifically, the tumor size in the model was 100 mm 3 When the mice reached 100 μl of phosphate buffered saline (PBS) was administered once via tail vein injection in the control group, and T1.01m, T1.02m, and T1.03m were each administered intravenously once at a dose of 10 μg.

[0415] As a result, as shown in FIG. 28, it was found that all of the T1.01m, T1.02m and T1.03m administration groups had superior tumor growth inhibition abilities compared to the control 15 days after administration.

[0416] Furthermore, the group administered T1.03m, which has four IL-12-containing mutations of amino acids involved in heparin binding together with the mouse anti-FAP sequence, was found to have reduced tumor growth inhibition ability compared to the group administered T1.01m. These results indicate that the group administered T1.03m, which has a mouse IL-12 sequence structure containing four amino acid mutations involved in heparin binding, had reduced IL-12 activity compared to the other experimental groups and controls.

[0417] Example 4.5. Evaluation of anti-cancer effect of single administration of IL-12 amino acid mutant fusion protein To evaluate the anticancer effect of the fusion protein, which has reduced IL-12 activity due to a sequence structure containing four amino acid mutations involved in heparin binding, we evaluated its tumor growth inhibition ability in a tumor model implanted with FAP-overexpressing mouse colon cancer cell line CT26 cells.

[0418] Specifically, tumor cells were transplanted into the right dorsal flank of 6-week-old BALB / c mice, and the tumor size of the animal model was 70 to 100 mm. 3 When the tumor size reached 10 μg, the mice were randomly divided into groups and administered. PBS was administered as a control, and 100 μl of T1.03m was administered intravenously once at doses of 2 μg, 10 μg, and 100 μg as a test group. The tumor size was measured in the same manner as in Example 4.1.

[0419] As a result, as shown in Figure 29, it was observed that the groups administered 2μg, 10μg and 100μg of T1.03m had a higher tumor growth inhibition ability compared to the control. No change in body weight was observed during the test period. These results indicate that T1.03m, which has reduced IL-12 activity due to its sequence structure containing four amino acid mutations involved in heparin binding, has an excellent anti-cancer effect above a certain concentration.

[0420] Example 5. Evaluation of the anti-cancer effect of the fusion protein in a melanoma animal model To identify the anti-cancer effect of the fusion protein through immune activation, we overexpressed mouse FAP in the mouse melanoma cell line B16F10, which is known to have low immune cell infiltration, and then implanted it into C57BL / 6J mice to evaluate its ability to inhibit tumor growth.

[0421] Specifically, the cultured cells were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) and then injected into the subcutaneous region of the left flank of 6-week-old C57BL / 6J mice at 100 μl of 1 × 10 cells / mouse. 6 Tumor cells were implanted.

[0422] Next, to evaluate the efficacy of the drug, the tumor size was 70-100 mm 3 When the mice reached the threshold of 100 μg / kg, they were randomly divided into groups: PBS was administered as a control group, and T1.03m was administered intravenously once at doses of 10 μg and 100 μg as a test group.

[0423] As a result, as shown in Figure 30, it was observed that both groups administered with 10 μg and 100 μg of T1.01m had high tumor growth inhibition ability compared to the control. No change in body weight was observed during the test period. These results indicate that T1.03m, which has reduced IL-12 activity due to its sequence structure containing four amino acid mutations involved in heparin binding, has excellent anti-cancer effect even in melanoma animal models.

[0424] Example 6. Evaluation of the anticancer effect of the fusion protein in an animal model of lung cancer Example 6.1. Evaluation of the anti-cancer effects of IL-12 amino acid mutant fusion proteins in lung cancer animal models Considering that FAP is overexpressed in fibroblasts present in the tumor microenvironment, we overexpressed FAP in mouse fibroblast cell line NIH-3T3 cells and then co-injected them with mouse lung cancer cell line LLC1 cells at a 1:1 ratio to prepare a tumor animal model and evaluate its tumor growth inhibition ability.

[0425] Specifically, FAP-overexpressing NIH-3T3 cell line and LLC1 cells were cultured at 1 × 10 6 The cells were resuspended in Hank's Balanced Salt Solution (HBSS, Gibco) at a concentration of cells / 50 μl, mixed at a 1:1 ratio, and then 100 μl per mouse was implanted into the subcutaneous region of the right flank of 6-week-old C57BL / 6J mice using a 1 cc syringe (25G).

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

[0427] As a result, as shown in Figure 31, it was observed that both groups administered with 10μg or 100μg of T1.03m had superior tumor growth inhibition ability compared to the control. No change in body weight was observed during the study period. These results indicate that T1.03m, which has reduced IL-12 activity due to its sequence structure containing four amino acid mutations involved in heparin binding, recognizes target factors in the tumor microenvironment, thereby having superior anti-cancer effect even in lung cancer animal models.

[0428] Example 6.2. Evaluation of cancer targeting effects in lung cancer animal models by co-injection of fusion proteins To compare the anti-cancer effects of the dual antibody fusion protein, in which the anti-FAP sequence structure and the IL-12 sequence structure form a knob-in-hole structure, the tumor growth inhibition ability was identified in an animal model in which NIH-3T3 cells overexpressing FAP, a mouse fibroblast cell line, and LLC1, a mouse lung cancer cell line, were co-injected at a 1:1 ratio.

[0429] Specifically, the efficacy of the drug was evaluated in patients with tumors between 70 and 100 mm 3 When the tumor growth rate reached 100%, the mice were randomly divided into groups. As a control, PBS was administered intraperitoneally twice a week. The groups administered T1.16m or T1.17m alone, the group administered T1.16m and T1.17m in combination (T1.16m+T1.17m), and the group administered T1.03m were used as test groups to evaluate the tumor growth inhibition ability. T1.16m and T1.17m, which are dimers of anti-FAP and IL-12, were administered intraperitoneally twice a week at a dose of 0.01 μg, respectively, and T1.03m, a monomeric fusion protein, was administered intraperitoneally twice a week at a dose of 0.02 μg to observe the tumor growth inhibition ability.

[0430] As a result, as shown in Figure 32, it was observed that the group administered with T1.03m had superior tumor growth inhibition ability at 10 days after administration compared to the control. Comparing the group administered with T1.16m or T1.17m alone and each combination administration group, it was found that the group administered with T1.03m alone had extremely good tumor growth inhibition ability. These results indicate that the synergistic effect of the fusion protein of one embodiment is significant when only FAP is targeted or when compared with IL-12 without a cancer target.

[0431] Example 7. Confirmation of anticancer effect when fusion protein and anticancer drug are administered simultaneously Example 7.1. Confirmation of anticancer effect by simultaneous administration of immune checkpoint inhibitors in a lung cancer mouse model To compare the anti-cancer efficacy of the fusion protein in which the anti-FAP sequence structure and the IL-12 sequence structure form a knob-in-hole structure and an immune checkpoint inhibitor when administered simultaneously, the tumor growth inhibition ability was confirmed in a mouse animal model. Specifically, a mouse animal model was used in which NIH-3T3 cells, a mouse fibroblast cell line that overexpresses FAP, and LLC1, a mouse lung cancer cell line, were co-injected at a 1:1 ratio.

[0432] Drug efficacy evaluation was performed on tumors with an average size of 110-120 mm 3When the tumor growth inhibition ability was evaluated, the tumor growth inhibition ability was evaluated using the following experimental groups: T1.03m single administration group, immune checkpoint inhibitor aPD-L1 (clone 10F.9G2, BioXCell), aCTLA-4 (clone 9D9, BioXCell), or aPD-1 (clone RMP1-14, BioXCell) single administration group, T1.03m and aPD-L1 simultaneous administration group (Fig. 33a), T1.03m and aCTLA-4 simultaneous administration group (Fig. 33b), and T1.03m and aPD-1 simultaneous administration group (Fig. 33c). T1.03m was intravenously injected once at a dose of 10 μg, and immune checkpoint inhibitor was administered once every 3 days at a dose of 200 μg, and the tumor growth inhibition ability was observed.

[0433] As a result, as shown in Figures 33a to 33c, superior tumor growth inhibition ability was observed in the immune checkpoint inhibitor co-administration group compared to the control group and the T1.03m administration group 9 days after administration. These results indicate that T1.03m, an anti-FAP and IL-12 monomer fusion protein, has a significant synergistic effect in a lung cancer mouse model when co-administered with an immune checkpoint inhibitor.

[0434] Example 7.2. Confirmation of anti-cancer effect by simultaneous administration of immune checkpoint inhibitors in a mouse model of colon cancer In order to compare the anti-cancer effects of the bispecific antibody fusion protein in which the anti-FAP sequence structure and the IL-12 sequence structure form a knob-in-hole structure and are co-administered with an immune checkpoint inhibitor, the tumor growth inhibition ability was confirmed in a mouse animal model. Specifically, a mouse animal model was used in which NIH-3T3 cells, a mouse fibroblast cell line that overexpresses FAP, and MC38, a mouse colon cancer cell line, were co-injected at a 1:1 ratio.

[0435] Specifically, the efficacy of the drug was evaluated in patients with tumors with an average size of 90 to 160 mm 3When the tumor growth inhibition capacity reached 100%, they were randomly divided into groups, and PBS was intraperitoneally administered once every 3 days as the control group. The T1.03m single-administration group, aPD-L1 immune checkpoint inhibitor (clone 10F.9G2, BioXCell) single-administration group, and T1.03m and aPD-L1 co-administration group were used as experimental groups to evaluate the tumor growth inhibition capacity. T1.03m was intravenously injected once at a dose of 2 μg, and the immune checkpoint inhibitor was administered once every 3 days at a dose of 200 μg to observe the tumor growth inhibition capacity.

[0436] As a result, as shown in Figures 34a and 34b, 16 days after administration, superior tumor growth inhibition ability was observed in the immune checkpoint inhibitor co-administration group compared to the control group and the T1.03m administration group. These results indicate that the anti-FAP and IL-12 monomer fusion protein, T1.03m, has a significant synergistic effect in a colon cancer mouse model when co-administered with an immune checkpoint inhibitor.

[0437] In addition, to further evaluate the tumor growth inhibition ability, the T1.03m single-administration group, the aPD-L1 immune checkpoint inhibitor (clone 10F.9G2, BioXCell) single-administration group, and the T1.03m and aPD-L1 co-administration group were used as experimental groups. T1.03m was intravenously injected once at a dose of 10 μg, and the immune checkpoint inhibitor was administered once every 3 days at a dose of 200 μg, and the tumor growth inhibition ability was observed.

[0438] As a result, as shown in Figures 35a and 35b, 11 days after administration, superior tumor growth inhibition ability was observed in the aPD-L1 immune checkpoint inhibitor co-administration group compared to the control group and T1.03m administration group.

[0439] In addition, the patients were randomly assigned to receive either a single dose of T1.03m, or a single dose of the immune checkpoint inhibitors aTIGIT (clone 1G9, BioXCell), aLAG-3 (clone C9B7W, BioXCell), or aTIM-3 (clone RMT3-23, BioXCell). The co-administration group of T1.03m and aTIGIT (FIG. 36), the co-administration group of T1.03m and aLAG-3 (FIG. 37), and the co-administration group of T1.03m and aTIM-3 (FIG. 38) were used as experimental groups to evaluate the tumor growth inhibition ability. T1.03m was intravenously injected once at a dose of 10 μg, and the immune checkpoint inhibitor was administered once every 3 days at a dose of 200 μg, and the tumor growth inhibition ability was observed.

[0440] As a result, as shown in Figures 36a and 36b, 10 days after administration, superior tumor growth inhibition ability was observed in the T1.03m and aTIGIT co-administration group compared with the control group and the T1.03m single administration group.

[0441] Furthermore, as shown in Figures 37a and 37b, 10 days after administration, superior tumor growth inhibition ability was observed in the T1.03m and aLAG-3 co-administration group compared with the control group and the T1.03m single administration group.

[0442] Furthermore, as shown in Figures 38a and 38b, 10 days after administration, superior tumor growth inhibition ability was observed in the T1.03m and aTIM-3 co-administration group compared with the control group and the T1.03m single administration group.

[0443] These results demonstrate that the anti-FAP and IL-12 monomeric fusion protein, T1.03m, has significant synergistic effects in a mouse model of colon cancer when co-administered with immune checkpoint inhibitors.

[0444] Example 7.3. Confirmation of anticancer effect by simultaneous administration of chemical anticancer drugs in a mouse model of colon cancer To compare the anti-cancer efficacy of the bispecific antibody fusion protein in which the anti-FAP sequence structure and the IL-12 sequence structure form a knob-in-hole structure and a chemical anti-cancer drug, the tumor growth inhibition ability was confirmed in a mouse animal model. Specifically, a mouse animal model was used in which NIH-3T3 cells, a mouse fibroblast cell line that overexpresses FAP, and MC38, a mouse colon cancer cell line, were co-injected at a 1:1 ratio.

[0445] Specifically, the efficacy of the drug was evaluated in patients with tumors with an average size of 110 to 120 mm 3 When the tumors reached the threshold, they were randomly divided into groups and PBS was administered intraperitoneally once every 4 days as a control group. The T1.03m single administration group, single administration of the chemical anticancer drugs cyclophosphamide (Endoxan Injection, Bukwang Pharm.Co.,Ltd.), fluorouracil (5-FU Injection, JW Pharmaceutical), paclitaxel (Neotax Injection, JW Pharmaceutical), or camptosar (Irinotecan Injection, Pfizer), co-administration group of T1.03m and cyclophosphamide (Figure 39), co-administration group of T1.03m and fluorouracil (Figure 40), co-administration group of T1.03m and paclitaxel (Figure 41), and co-administration group of T1.03m and camptosar (Figure 42) were used as experimental groups to evaluate the tumor growth inhibition ability.

[0446] As a result, as shown in Figures 39a and 39b, 11 days after administration, superior tumor growth inhibition ability was observed in the T1.03m and cyclophosphamide co-administration group compared to the control group and the T1.03m single administration group.

[0447] Furthermore, as shown in Figures 40a and 40b, 11 days after administration, superior tumor growth inhibition ability was observed in the T1.03m and fluorouracil co-administration group compared with the control group and the T1.03m single administration group.

[0448] Furthermore, as shown in Figures 41a and 41b, 11 days after administration, superior tumor growth inhibition ability was observed in the T1.03m and paclitaxel co-administration group compared with the control group and the T1.03m single administration group.

[0449] Furthermore, as shown in Figures 42a and 42b, 11 days after administration, superior tumor growth inhibition ability was observed in the T1.03m and Camptosar co-administration group compared with the control group and the T1.03m administration group.

[0450] These results imply that the anti-FAP and IL-12 monomeric fusion protein, T1.03m, has significant synergistic effects in a mouse model of colon cancer when co-administered with a chemical anticancer agent.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising: 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 (fibroblast activation protein alpha); and Anticancer drugs 23. A pharmaceutical composition comprising:

2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the IL-12 or the variant thereof comprises IL-12A (p35) or a variant thereof, and IL-12B (p40) or a variant thereof.

3. 3. The pharmaceutical composition for preventing or treating cancer according to claim 2, wherein the variant of IL-12B(p40) comprises an amino acid sequence obtained by one or more substitutions selected from the group consisting of K258A, K260A, K263A and K264A in the amino acid sequence of SEQ ID NO:

74.

4. 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the first monomer and the second monomer form a knob-into-hole structure.

5. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the anticancer agent is any one selected from the group consisting of a chemical anticancer agent, an anticancer virus, and an immune checkpoint inhibitor.

6. 6. The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein the chemical anticancer agent is any one selected from the group consisting of alkylating agents, microtubule inhibitors, metabolic antagonists, and topoisomerase inhibitors.

7. The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein the chemical anticancer agent is any one selected from the group consisting of cyclophosphamide, cisplatin, fluorouracil, gemcitabine, camptosar, paclitaxel and adriamycin.

8. The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein said anti-cancer virus is Talimogene Laherparepvec (T-VEC).

9. The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein the immune checkpoint inhibitor is any one selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, and an anti-LAG3 antibody.

10. The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein the immune checkpoint inhibitor is any one selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab and durvalumab.

11. A pharmaceutical composition for preventing or treating cancer, comprising: The pharmaceutical composition of claim 1, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, melanoma, bladder cancer, esophageal cancer, head and neck cancer, skin cancer, colorectal cancer and lymphoma.

12. A kit for preventing or treating cancer, comprising: As an active ingredient, 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; and Anticancer drugs Kit including:

13. A method for preventing or treating cancer, comprising the step of administering to a subject a fusion protein or a dimer thereof, the 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, and an anti-cancer agent.

Images