Antigen-binding protein containing two Fc domains and its use

Abstract

This invention provides a fusion protein having a novel antibody structure comprising one antigen-binding site and two Fc domains. Despite having a molecular weight similar to that of human IgG, such a novel antibody structure allows up to four times more Fc domains to be present on the cell surface antigen compared to natural human antibodies. As a result, the fusion protein exhibits increased affinity for the Fcγ receptor and enhanced effector function. Therefore, this fusion protein with a novel antibody structure can be utilized as a new antibody platform.

Description

[Technical Field]

[0001] This invention relates to a novel antibody format having an antigen-binding site that specifically binds to cancer surface antigens, and two Fc domains. [Background technology]

[0002] Antibody-based therapeutics and Fc fusion proteins are a clinically important group of drugs for patients with cancer, immune disorders, infectious diseases, and inflammatory diseases. ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell phagocytosis), and CDC (complement-dependent cell-mediated cytotoxicity), induced by interactions between antibody Fc domains and the innate immune system, play a crucial role in alleviating or treating disease symptoms.

[0003] Attempts are being made to maintain the bivalent nature of antibodies and to improve effector function by increasing the number of Fc domains (Claudio Sustmann et al., MAbs. 2019; Dennis R Goulet et al., Proteins, 2020). While these platforms show improved binding affinity to the Fcγ receptor and ADCC, obtaining uniform antibodies is difficult due to the complexity of production and purification. Attempts are also underway to improve effector function by linking antibody Fc domains in series or by constructing a large number of Fc domains (US Patent Application Publication 2020 / 0040084). In this case, there is a drawback that tissue permeability may be significantly reduced due to the increase in antibody size or molecular weight. In addition, because the series-linked multimeric Fc domains have a different geometric structure from the large number of Fc domains provided by IgG antibodies bound to cell surface antigens, the series-linked multimeric Fc domains may not be able to effectively induce an increase in effector function. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Therefore, the inventors investigated ways to improve antibody function and simultaneously address the challenges of existing antibody formats designed to contain multiple Fc domains in series, as described above. [Means for solving the problem]

[0005] As a result, the inventors have developed a novel engineered antibody form that allows the Fc domain to be present up to four times more in cell surface antigens compared to natural human antibodies, even if the antibody has a molecular weight similar to that of natural human immunoglobulin G (IgG) (approximately 150 kDa).

[0006] In one embodiment of the present invention, a fusion protein is provided comprising an antigen-binding site, a first Fc domain or variant thereof linked to a first linking position of the antigen-binding site, and a second Fc domain or variant thereof linked to a second linking position of the antigen-binding site.

[0007] In another embodiment of the present invention, a fusion protein is provided comprising two antigen-binding sites linked in series (two series antigen-binding regions), a first Fc domain or variant thereof linked to a first linking position of the two series antigen-binding sites, and a second Fc domain or variant thereof linked to a second linking position of the two series antigen-binding sites. According to one embodiment, each of the two antigen-binding sites constituting the two series antigen-binding sites may be a sequence containing a CDR sequence or variable region capable of binding to different epitopes of the same antigen or to different antigens, or a sequence consisting of a variable region.

[0008] In one embodiment of the fusion protein described herein, the antigen-binding site may be a sequence comprising or consisting of a CDR sequence or variable region of an antibody. Therefore, the antigen-binding site may comprise a first peptide comprising or containing a light chain CDR sequence or light chain variable region of an antibody, and a second peptide comprising or containing a heavy chain CDR sequence or heavy chain variable region of an antibody. The first Fc domain and the second Fc domain may each be a dimer comprising two peptide sequences. The first peptide of the antigen-binding site may be bound to the first Fc domain or a variant thereof, and the second peptide of the antigen-binding site may be bound to the second Fc domain or a variant thereof.

[0009] In the fusion protein described herein, according to another embodiment, the first Fc domain and the second Fc domain may be linked to each other by covalent, non-covalent, or linker bonds, or they may not be linked to each other. In a preferred embodiment, the first Fc domain and the second Fc domain may not be linked to each other.

[0010] According to embodiments of the present disclosure, the Fc domain may be the Fc domain of wild-type (WT) immunoglobulin and may include modifications, such as amino acid substitutions, for the reactivity of the Fc domain to the Fcγ receptor (FcγR), for modulating ADCC, or for minimizing the formation of undesirable multimers of the Fc domain. The Fc domain may include CH2 and CH3 regions and may include a CH4 region and / or a hinge region, and the Fc domain should be interpreted as including an Fc domain fragment that exhibits the function of an Fc domain.

[0011] According to embodiments of the present disclosure, the antigen-binding site and the Fc domain or variant thereof may be joined directly or via a linker. For example, the antigen-binding site and the Fc domain or variant thereof may be linked between the N-terminus and the C-terminus, between N-terminus and N-terminus, or between C-terminus and C-terminus of each peptide molecule, with or without a linker.

[0012] According to an embodiment of the present disclosure, when an antigen-binding site and an Fc domain or a variant thereof are joined through a linker, the linker may be a commonly used peptide linker. For example, the linker may be a peptide consisting of about 1 to about 70 amino acid residues, about 2 to about 60 amino acid residues, about 2 to about 50 amino acid residues, about 2 to about 40 amino acid residues, about 2 to about 30 amino acid residues, about 3 to about 50 amino acid residues, about 3 to about 40 amino acid residues, about 3 to about 30 amino acid residues, about 2 to about 28 amino acid residues, about 2 to about 26 amino acid residues, about 2 to about 24 amino acid residues, about 2 to about 22 amino acid residues, about 2 to about 20 amino acid residues, about 2 to about 18 amino acid residues, about 2 to about 16 amino acid residues, about 2 to about 14 amino acid residues, about 2 to about 12 amino acid residues, or about 2 to about 10 amino acid residues. The connection between the first Fc domain and the antigen-binding site, the connection between the second Fc domain and the antigen-binding site, or both may be achieved through a linker.

[0013] In another aspect of the present invention, a pharmaceutical composition for preventing or treating cancer, comprising a fusion protein as an active ingredient, is provided.

[0014] In another aspect of the present invention, a polynucleotide encoding a fusion protein, a vector containing the polynucleotide, and a transformed cell into which the vector has been introduced are provided.

[0015] In another aspect of the present invention, a method for treating or preventing cancer, comprising the step of administering a fusion protein to a subject, is provided.

[0016] In another aspect of the present invention, the use of a fusion protein for the treatment of cancer is provided.

[0017] In another aspect of the present invention, the use of a fusion protein for use in the manufacture of a medicament for treating cancer is provided.

Advantages of the Invention

[0018] Unlike the wild-type (WT) antibody, the fusion protein having the novel antibody format of the present invention contains one or two antigen-binding sites and two Fc domains. The two Fc domains are not directly linked to each other, but each of the two Fc domains is independently linked to two different polypeptide chains constituting the antigen-binding site. Even if this novel antibody format has a size and molecular weight similar to that of human IgG, the novel antibody format may enable the Fc domain to be present on the cell surface antigen up to four times more than the native human antibody. Due to these characteristics, the fusion protein having the novel antibody format has an improved affinity (avidity) for the Fcγ receptor and can induce an improved effector function. Therefore, the fusion protein having the novel antibody format can be used for various purposes by replacing the conventional antibody.

Brief Description of the Drawings

[0019] [Figure 1a] Schematic diagram of native human immunoglobulin (IgG). [Figure 1b] Schematic diagram of a novel engineered antibody format. [Figure 1c] Schematic diagram showing cancer cells having the tumor antigen bound to antigen-specific human immunoglobulin (IgG) in a monovalent manner. [Figure 1d] Schematic diagram showing cancer cells having the tumor antigen bound to antigen-specific human immunoglobulin (IgG) in a monovalent or bivalent manner. [Figure 1e] Schematic diagram showing cancer cells having the tumor antigen bound to antigen-specific human immunoglobulin (IgG) in a bivalent manner. [Figure 1f] Schematic diagram showing cancer cells having the tumor antigen bound to a novel engineered antigen-specific antibody. [Figure 2a] Schematic diagram of a novel monovalent antibody format (WT) having two Fc domains. [Figure 2b]This is a schematic diagram of antibody (M1) in which the VH Q105C and VL A43C amino acid substitutions have been introduced into a novel monovalent antibody form having two Fc domains. [Figure 2c] This is a schematic diagram of antibody (M2) in which the CH1 F122C and CL S121C amino acid substitutions have been introduced into a novel monovalent antibody form having two Fc domains. [Figure 2d] This is a schematic diagram of antibody (M3) in which the VH G44C and VL Q100C amino acid substitutions have been introduced into a novel monovalent antibody form having two Fc domains. [Figure 3a] This is a diagram illustrating the sequence information showing the location of Cys substitutions in the VH-CH1 domain of trastuzumab. The WT sequence is sequence number 8, the sequence of mutant 1 is sequence number 10, the sequence of mutant 2 is sequence number 12, and the sequence of mutant 3 is sequence number 14. [Figure 3b] This is a diagram illustrating the sequence information showing the location of Cys substitutions in the VL-CL domain of trastuzumab. The WT sequence is sequence number 9, the sequence of mutant 1 is sequence number 11, the sequence of mutant 2 is sequence number 13, and the sequence of mutant 3 is sequence number 15. [Figure 4] This figure illustrates the results obtained by SDS-PAGE analysis of WT, M1, M2, or M3. [Figure 5a] This figure illustrates the results obtained by size exclusion chromatography analysis of WT. [Figure 5b] This figure illustrates the results obtained by size exclusion chromatography analysis of M1. [Figure 5c] This figure illustrates the results obtained by size exclusion chromatography analysis of M2. [Figure 5d] This figure illustrates the results obtained by size exclusion chromatography analysis of M3. [Figure 6a] This is a schematic diagram of an antibody (M3) in which the CL domain and hinge region are linked by a 15-mer peptide. [Figure 6b]This is a schematic diagram of antibody (V1) in which the CL domain and hinge region are linked by a 10-mer peptide. [Figure 6c] This is a schematic diagram of an antibody (V2) in which the CL domain and hinge region are linked by a 5-mer peptide. [Figure 6d] This is a schematic diagram of an antibody (V3) in which the CL domain and hinge region are directly linked without a linker. [Figure 7] This figure illustrates the results obtained by SDS-PAGE analysis of M3, V1, V2, or V3. [Figure 8a] This is a schematic diagram of the fragments generated when H01 or P01 is cleaved with papain. [Figure 8b] This is a schematic diagram of a fragment generated by papain cleavage of H01 or P01 when a disulfide bond is abnormally formed in the hinge region. [Figure 8c] This figure illustrates the results obtained by SDS-PAGE analysis of H01 papain cleavage products. [Figure 8d] This figure illustrates the results obtained by SDS-PAGE analysis of P01 papain cleavage products. [Figure 9] This figure illustrates the results obtained by SDS-PAGE analysis of H01 wt or H01. [Figure 10a] This is a schematic diagram of H01Fv1 having an Fv-(Fc)2 structure. [Figure 10b] This is a schematic diagram of H01Fv2 having an Fv-(Fc)2 structure. [Figure 10c] This is a schematic diagram of H01Fv3 having an Fv-(Fc)2 structure. [Figure 10d] This is a schematic diagram of H01Fv4 having an Fv-(Fc)2 structure. [Figure 10e] This is a schematic diagram of H01Fv5 having an Fv-(Fc)2 structure. [Figure 10f] This is a schematic diagram of H01Fv6 having an Fv-(Fc)2 structure. [Figure 10g]This is a schematic diagram of H01Fv7 having an Fv-(Fc)2 structure. [Figure 10h] This table shows the mutation locations in the Fv-(Fc)2 structure. [Figure 11] This figure illustrates the results obtained by SDS-PAGE analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 12a] This figure illustrates the results obtained by SEC analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 12b] This figure illustrates the results obtained by SEC analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 12c] This figure illustrates the results obtained by SEC analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 12d] This figure illustrates the results obtained by SEC analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 12e] This figure illustrates the results obtained by SEC analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 12f] This figure illustrates the results obtained by SEC analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 12g] This figure illustrates the results obtained by SEC analysis of the Fv-(Fc)2 structure after purification of protein A. [Figure 13a] This figure illustrates the analysis of sensorgram data regarding the binding of the purified Fv-(Fc)2 structure, H01Fv1, to human HER2. [Figure 13b] This figure illustrates the analysis of sensorgram data regarding the binding of the purified Fv-(Fc)2 structure, H01Fv2, to human HER2. [Figure 13c] This figure illustrates the analysis of sensorgram data regarding the binding of the purified Fv-(Fc)2 structure, H01Fv4, to human HER2. [Figure 13d]This figure illustrates the analysis of sensorgram data regarding the binding of the purified Fv-(Fc)2 structure, H01Fv5, to human HER2. [Figure 13e] This figure illustrates the analysis of sensorgram data regarding the binding of the purified Fv-(Fc)2 structure, H01Fv6, to human HER2. [Figure 13f] This figure illustrates the analysis of sensorgram data regarding the binding of the purified Fv-(Fc)2 structure, H01Fv7, to human HER2. [Figure 14] This figure illustrates differential scanning fluorescence spectroscopy analysis of the melting temperatures of H01, P01, trastuzumab, and pertuzumab. [Figure 15] This figure shows the competitive coupling of H01 and P01 in a biolayer interferometry (BLI) analysis of sensorgram data. [Figure 16a] This is a schematic diagram showing the binding mode of HER2 to HER2 in combination with P01 in HER2-positive cancer cells. [Figure 16b] This is a schematic diagram showing the binding mode of trastuzumab to HER2 in combination with pertuzumab in HER2-positive cancer cells. [Figure 17a] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of NCI-N87 gastric cancer cell lines after treatment with an anti-HER2 antibody (50 nM). [Figure 17b] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of BT474 breast cancer cell lines after treatment with an anti-HER2 antibody (50 nM). [Figure 17c] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of SK-OV3 ovarian cancer cell lines after treatment with an anti-HER2 antibody (50 nM). [Figure 17d] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of SNU-1 gastric cancer cell lines after treatment with an anti-HER2 antibody (50 nM). [Figure 17e]This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of SNU-5 gastric cancer cell lines after treatment with an anti-HER2 antibody (50 nM). [Figure 18a] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of NCI-N87 gastric cancer cell lines after treatment with anti-HER2 antibodies at the indicated concentrations (20, 50, and 100 nM). [Figure 18b] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of BT474 breast cancer cell lines after treatment with anti-HER2 antibodies at the indicated concentrations (20, 50, and 100 nM). [Figure 18c] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of SK-OV3 ovarian cancer cell lines after treatment with anti-HER2 antibodies at the indicated concentrations (20, 50, and 100 nM). [Figure 18d] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of SNU-1 gastric cancer cell lines after treatment with anti-HER2 antibodies at the indicated concentrations (20, 50, and 100 nM). [Figure 18e] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of SNU-5 gastric cancer cell lines after treatment with anti-HER2 antibodies at the indicated concentrations (20, 50, and 100 nM). [Figure 19a] This is a schematic diagram of H01DE4, in which the S239D and I332E mutations have been introduced into H01. [Figure 19b] This is a schematic diagram of P01DE4, in which the S239D and I332E mutations have been introduced into P01. [Figure 20a] This is a sensorogram-binding profile of H01, H01DE4, P01, or P01DE4 to human HER2. [Figure 20b] This is a sensorogram-binding profile of H01, H01DE4, P01, or P01DE4 to human HER2. [Figure 20c]This is a sensorogram-binding profile of H01, H01DE4, P01, or P01DE4 to human HER2. [Figure 20d] This is a sensorogram-binding profile of H01, H01DE4, P01, or P01DE4 to human HER2. [Figure 21a] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 21b] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 21c] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 21d] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 21e] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 21f] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 21g] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 21h]This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 1. [Figure 22a] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 22b] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 22c] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 22d] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 22e] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 22f] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 22g] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 22h]This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 2A (131R isoform). [Figure 23a] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 23b] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 23c] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 23d] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 23e] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 23f] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 23g] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 23h] This is the sensorgram binding profile of H01, P01, H01DE4, P01DE4, human IgG1, trastuzumab, pertuzumab, or margetuximab to Fcγ receptor 3A (158V isoform). [Figure 24a] This graph shows the time course of blood antibody concentrations after intravenous administration of H01, P01, trastuzumab, or pertuzumab (10 mg / kg) to Sprague-Dawley rats. [Figure 24b] This table illustrates the pharmacokinetic parameters (PK parameters) of each antibody after intravenous administration of H01, P01, trastuzumab, or pertuzumab (10 mg / kg) to Sprague-Dawley rats. [Figure 25] This is a schematic diagram of HP501, a HER2 biparatopically modified antibody. [Figure 26] This is a schematic diagram of HP501-HP516, which are HER2 biparatopically modified antibodies. [Figure 27] This figure illustrates the size exclusion chromatography analysis of HP501-HP516, which are HER2 biparatopically manipulated antibodies. [Figure 28a] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP503 to HER2. [Figure 28b] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP507 to HER2. [Figure 28c] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP511 to HER2. [Figure 28d] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP515 to HER2. [Figure 29a] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP503 to Fcγ receptor 1, Fcγ receptor 2A (131R isoform), or Fcγ receptor 3A (158V isoform). [Figure 29b] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP507 to Fcγ receptor 1, Fcγ receptor 2A (131R isoform), or Fcγ receptor 3A (158V isoform). [Figure 29c] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP511 to Fcγ receptor 1, Fcγ receptor 2A (131R isoform), or Fcγ receptor 3A (158V isoform). [Figure 29d] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP515 to Fcγ receptor 1, Fcγ receptor 2A (131R isoform), or Fcγ receptor 3A (158V isoform). [Figure 30a] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of HP503, HP507, HP511, or HP515 to neonatal Fc receptors (FcRn). [Figure 30b] This figure shows the biolayer interferometry analysis of sensorgram data regarding the binding of human IgG1, trastuzumab, pertuzumab, or margetuximab to neonatal Fc receptors (FcRn). [Figure 31a] This figure illustrates the complement-dependent cell-mediated cytotoxicity (CDC) activity of human IgG1, trastuzumab (TRA), trastuzumab + pertuzumab (TRA+PER), H01, or H01+P01 in the BT474 breast cancer cell line. [Figure 31b] This figure illustrates the CDC activity of human IgG1, trastuzumab (TRA), trastuzumab + pertuzumab (TRA+PER), H01, or H01+P01 in the NCI-N87 gastric cancer cell line. [Figure 31c] This is a schematic diagram of a monovalent (1-arm) antibody (OA_TRA) containing trastuzumab Fab. [Figure 31d] This is a schematic diagram of a Duomab antibody (Duo_TRA) containing trastuzumab Fab. [Figure 31e]This is a schematic diagram of a series of Fc antibodies containing trastuzumab Fab (series_Fc_TRA). [Figure 31f] This figure illustrates the CDC activity of human IgG1, trastuzumab (TRA), H01, 1-arm trastuzumab (OA_TRA), Duomab trastuzumab (Duo_TRA), or serial Fc trastuzumab (serial_Fc_TRA) in the NCI-N87 gastric cancer cell line, as well as the CDC activity of any of these antibodies in combination with pertuzumab. [Figure 31g] This figure illustrates the CDC activity of human IgG1, trastuzumab (TRA), H01, 1-arm trastuzumab (OA_TRA), Duomab trastuzumab (Duo_TRA), or serial Fc trastuzumab (serial_Fc_TRA) in the BT474 breast cancer cell line, as well as the CDC activity of any of these antibodies in combination with pertuzumab. [Figure 32a] This figure illustrates the ADCC activity of human IgG1, trastuzumab, or H01 in the NCI-N87 gastric cancer cell line. [Figure 32b] This figure illustrates the ADCC activity of human IgG1, trastuzumab, or H01 in the MDA-MB-453 breast cancer cell line. [Figure 32c] This figure illustrates the ADCC activity of human IgG1, trastuzumab, or H01 in the SNU-601 gastric cancer cell line. [Figure 32d] This figure illustrates the ADCC activity of human IgG1, trastuzumab, or H01 in the SNU-5 gastric cancer cell line. [Figure 33a] This figure illustrates the antitumor activity of high concentrations (5 mg / kg each) of trastuzumab, trastuzumab + pertuzumab, H01, H01 + P01, or HP507 in a CB-17 SCID mouse model of tumor xenografting of the SNU-5 gastric cancer cell line. [Figure 33b]This figure illustrates the antitumor activity of low concentrations of trastuzumab (TRA, 1 mg / kg), pertuzumab (PER, 1 mg / kg), TRA (0.5 mg / kg) + PER (0.5 mg / kg), H01 (1 mg / kg), P01 (1 mg / kg), or H01 (0.5 mg / kg) + P01 (0.5 mg / kg) in a CB-17 SCID mouse model of tumor xenografting of the SNU-5 gastric cancer cell line. [Figure 34] This figure illustrates the antitumor activity of IVIG (intravenous immunoglobulin) (50 mg / kg) alone and in combination with high concentrations (5 mg / kg each) of H01, trastuzumab, or trastuzumab plus pertuzumab in a mouse model of tumor xenografting using the SNU-601 gastric cancer cell line. [Figure 35] This figure illustrates the antitumor activity of IVIG (50 mg / kg) alone and in combination with H01 or trastuzumab at the indicated concentrations (0.2 mg / kg, 0.5 mg / kg) in a mouse model of tumor xenografting using the NCI-N87 gastric cancer cell line. [Figure 36] This is a vector map for vectors used to express the human HER2 protein in mammalian cells. [Figure 37] This figure illustrates flow cytometry quantification of HER2 expression in a CT26 mouse colorectal cancer cell line clone expressing human HER2 (CT26-HER2). [Figure 38] This figure illustrates the stability of human HER2 expression in the CT26-HER2 clone. [Figure 39] This figure illustrates the relative expression of human HER2 in the CT26-HER2 cell line (clone name: #2-60) compared to human cancer cell lines, and the amount of H1 bound to the surface of CT26-HER2 cells compared to trastuzumab. [Figure 40] This figure illustrates the antitumor activity of trastuzumab or H01 in a syngeneic CT26-HER2 mouse tumor model. [Figure 41a] This is a schematic diagram of a monovalent, manipulated mAb (monoclonal antibody) according to one embodiment of the present invention. [Figure 41b] This is a schematic diagram of a biparatopically operated mAb according to one embodiment of the present invention. [Figure 42] This figure illustrates the biolayer interferometry analysis of sensorgram data relating to the binding of a fusion protein according to one embodiment of the present invention to a target. (A) illustrates sensorgram data relating to the binding of GPM01, a monovalent engineered mAb that targets GPC-3, to human GPC-3; (B) illustrates sensorgram data relating to the binding of GPM02, a monovalent engineered mAb that targets GPC-3, to human GPC-3; (C) illustrates sensorgram data relating to the binding of GPM04, a monovalent engineered mAb that targets GPC-3, to human GPC-3; and (D) illustrates sensorgram data relating to the binding of GPB01, a biparatopic engineered mAb that targets GPC-3, to human GPC-3. (E) illustrates sensorgram data relating to the binding of GPB03, a biparatopically manipulated mAb that targets GPC-3, to human GPC-3; (F) illustrates sensorgram data relating to the binding of GPB04, a biparatopically manipulated mAb that targets GPC-3, to human GPC-3; and (G) illustrates sensorgram data relating to the binding of GPB06, a biparatopically manipulated mAb that targets GPC-3, to human GPC-3. [Figure 43] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of HepG2 liver cancer cell lines after treatment with GPC-3 antibody (100 nM). [Figure 44] This figure illustrates SDS-PAGE analysis showing inhibition of AKT phosphorylation in PC-3 prostate cancer cell lines after treatment with EphA2 antibody (100 nM). [Figure 45] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of PC-3 prostate cancer cell lines after treatment with EphA2 antibody (100 nM). [Figure 46]This figure illustrates biolayer interferometry analysis of sensorgram data regarding the binding of MEM01 or MEM06, monovalent, engineered mAbs targeting human MET, to human MET. [Figure 47] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of MKN45 gastric cancer cell lines after treatment with MET antibodies at the indicated concentrations (20, 50, and 100 nM). [Figure 48] This figure illustrates flow cytometry analysis of the amount of Fc domains present on the surface of SNU5 gastric cancer cell lines after treatment with antibodies at the indicated concentrations (50 and 100 nM). [Figure 49] This figure illustrates biolayer interferometry analysis of sensorgram data regarding the binding of monovalent, engineered mAbs targeting human EGFR to human EGFR. [Figure 50] This figure illustrates biolayer interferometry analysis of sensorgram data regarding the binding of monovalent engineered mAbs (33-1, 33-2, and 33-3) that target human CD33, or biparatopic engineered mAbs (33-4, 33-5, 33-6, and 33-7) that target CD33. [Figure 51] This figure illustrates biolayer interferometry analysis of sensorgram data regarding the binding of a monovalent, manipulated mAb targeting human CEACAM5 to human CEACAM5. [Figure 52] This figure illustrates the biolayer interferometry analysis of sensorgram data relating to the binding of a fusion protein according to one embodiment of the present invention to a target. (A) illustrates sensorgram data relating to the binding of T01, a monovalent engineered mAb that targets TROP2, to human TROP2; (B) illustrates sensorgram data relating to the binding of MSM01, a monovalent engineered mAb that targets mesothelin, to human mesothelin; and (C) illustrates sensorgram data relating to the binding of LIM01, a monovalent engineered mAb that targets LIV-1, to human LIV-1. [Figure 53a]This is a schematic diagram of a novel, manipulated antibody format. [Figure 53b] This is a schematic diagram showing a receptor bound to receptor-specific human immunoglobulin (IgG) in a monovalent form. [Figure 53c] This is a schematic diagram showing receptors bound to receptor-specific human immunoglobulin (IgG) in a monovalent or bivalent form. [Figure 53d] This is a schematic diagram showing a receptor bound to receptor-specific human immunoglobulin (IgG) in a bivalent form. [Figure 53e] This is a schematic diagram showing a receptor bound to a novel, modified antibody form. [Figure 54] This is a schematic diagram of an engineered antibody form having two Fc domains according to one embodiment of the present invention. [Figure 55a] This figure shows the sensorgram binding profiles of trastuzumab or H01 to Fcγ receptor 1. [Figure 55b] This figure shows the sensorgram binding profiles of trastuzumab or H01 to the Fcγ receptor 2A (131H isoform). [Figure 55c] This figure shows the sensorgram binding profiles of trastuzumab or H01 to Fcγ receptor 3A (158V isoform). [Figure 55d] This table illustrates the equilibrium dissociation constant (KD), association rate constant (Ka), and dissociation rate constant (Kd) values ​​for trastuzumab or H01 for Fcγ receptor 1, Fcγ receptor 2A, and Fcγ receptor 3A. [Figure 56a] This figure illustrates the binding affinity of trastuzumab or H01 to human immune cells. [Figure 56b] This figure illustrates the binding affinity of trastuzumab or H01 to human immune cells. [Figure 57] This figure illustrates the ADCC activity of trastuzumab or H01 in (A) gastric cancer cell line (SNU-5 cells) or (B) breast cancer cell line (ZR-75-1 cells). [Figure 58]This is a schematic diagram of an engineered antibody form having two Fc domains according to one embodiment of the present invention. [Figure 59a] This is a schematic diagram illustrating the binding mode of an engineered antibody form having two Fc domains, according to one embodiment of the present invention, which binds to the cell surface, or simultaneously to soluble antigens on the cell surface and around the cell. [Figure 59b] This is a schematic diagram illustrating the binding mode of an engineered antibody form having two Fc domains, according to one embodiment of the present invention, which binds to the cell surface, or simultaneously to soluble antigens on the cell surface and around the cell. [Figure 59c] This is a schematic diagram illustrating the binding mode of an engineered antibody form having two Fc domains, according to one embodiment of the present invention, which binds to the cell surface, or simultaneously to soluble antigens on the cell surface and around the cell. [Figure 59d] This is a schematic diagram illustrating the binding mode of an engineered antibody form having two Fc domains, according to one embodiment of the present invention, which binds to the cell surface, or simultaneously to soluble antigens on the cell surface and around the cell. [Figure 60] This figure illustrates the results obtained by SDS-PAGE analysis of H01(A), H04, H05, H06, H07, H08, or H09 under reducing conditions (B) or non-reducing conditions (C). [Figure 61] This figure illustrates the results obtained by SDS-PAGE analysis of (A) H01P, P01H, (B) ME13, ME14, ME15, ME16, or (C) CE05 under non-reducing conditions (NR) or reducing conditions (R). [Figure 62] This figure illustrates the results obtained by SDS-PAGE analysis of (A) EAVC-P1, EAVC-P2, EAVC-P3, EAVC-P4, EAVC-P5, EAVC-P6, EAVC-P7, EAVC-P8, or (B) EAVC-C3, EAVC-I3, EAVC-L3, EAVC-M3, or EAVC-N3 under non-reducing conditions (NR) or reducing conditions (R). [Figure 63]This figure illustrates the results obtained by SDS-PAGE analysis of (A) HAVC01, HAVC02, HAVC03, HAVC05, HAVC06, HAVC07, HAVC08, or (B) HAVC04 under non-reducing conditions (NR) or reducing conditions (R). [Figure 64a] This figure illustrates the results obtained by size exclusion chromatography analysis of (A)HAVC01, (B)HAVC02, (C)HAVC03, or (D)HAVC04. [Figure 64b] This figure illustrates the results obtained by size exclusion chromatography analysis of (A)HAVC05, (B)HAVC06, (C)HAVC07, or (D)HAVC08. [Modes for carrying out the invention]

[0020] Definition of Terms As used herein, the terms “fusion protein having two Fc domains” or “antibody having two Fc domains” refer to a fusion protein in which two Fc domains are independently conjugated to two polypeptide chains constituting an antigen-binding site. The two polypeptide chains constituting the antigen-binding site may be different from each other. For example, one of the two polypeptide chains constituting the antigen-binding site may be a sequence containing or consisting of the light chain CDR sequence or light chain variable region of the antibody, or it may be an scFv, and the other may be a sequence containing or consisting of the heavy chain CDR sequence or heavy chain variable region of the antibody, or it may be an scFv. In one embodiment, the fusion protein having two Fc domains may include a “humanized” form of a non-human antibody, which is a chimeric antibody containing a human immunoglobulin with a native CDR. In addition, the fusion protein may include a “fully human antibody” or a portion of a “human antibody.” In addition, in one embodiment, the polyspecific fusion protein or antigen-binding domain may be a “monoclonal antibody” or a portion thereof.

[0021] As used herein, the term “antibody” refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. In addition, antibodies are also called immunoglobulins. An antibody may refer to any one selected from IgG, IgE, IgM, IgD, and IgA, and may be a subclass of IgG such as IgG1, IgG2, IgG3, IgG4, or a subclass of IgA such as IgA1, IgA2, etc. In addition, an antibody may be an agonist antibody or an antagonist antibody.

[0022] As used herein, the terms “Fab” or “Fab region” refer to the region of an antibody that binds to an antigen. Conventional IgG typically contains two Fab regions. Each Fab region typically consists of one variable region and one constant region in each heavy and light chain. Specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions of the Fab region may be arranged by various means, including CrossMab Fab technology, which has a configuration in which VH and VL are substituted for each other, to give antigen-binding ability according to this disclosure.

[0023] As used herein, the term “heavy chain” refers to a polypeptide chain of approximately 50 kDa to approximately 70 kDa, where the N-terminal region includes a variable region of at least approximately 120 to 130 amino acids, and the C-terminal region includes a constant region. The constant region may be of one of five types: alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), where α, δ, and γ contain approximately 450 amino acids, and μ and ε contain approximately 550 amino acids.

[0024] As used herein, the term “light chain” refers to a polypeptide chain of approximately 25 kDa, where the N-terminal portion contains a variable region of at least approximately 100 to 110 amino acids, and the C-terminal portion contains a constant region. There are two types of light chain constant domains: kappa (κ) or lambda (λ). In addition, the constant region of the light chain is referred to as “CL”. The heavy chain C domain (CH domain) is numbered from the N-terminus to the C-terminus (e.g., CH1, CH2, CH3, etc.). Any CL and CH1 region of any of these antibody classes may be used in this disclosure. In certain embodiments, the CL and CH1 regions provided herein may be of the IgG type (e.g., IgG1).

[0025] As used herein, the terms “Fc” or “Fc region” refer to the C-terminal region of an immunoglobulin heavy chain, including the natural Fc region, recombinant Fc region, and variant Fc region. Therefore, Fc refers to the last two constant-region immunoglobulin domains of IgA, IgD, and IgG, the last three constant-region immunoglobulin domains of IgE and IgM, and the hinges located at the N-terminus of these domains. With respect to IgA and IgM, Fc may also include the J chain. With respect to IgG, Fc includes the immunoglobulin domains Cy2(CH2) and Cy3(CH3), and the hinge between Cy1 and Cy2. Although the interface of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as containing residues C226, P230, or A231 at the C-terminus, numbered according to the EU index. As used herein, “Fc polypeptide” or “Fc-derived polypeptide” refers to a polypeptide containing all or part of the Fc region. In one embodiment, the variant Fc region may be in a form in which at least one amino acid, for example about 1 to about 10 amino acids, or about 1 to about 5 amino acids, is substituted compared to the natural sequence Fc region. In addition, the variant Fc region may have at least about 80% homology, at least about 90% homology, or at least about 95% homology with the natural sequence Fc region.

[0026] As used herein, the terms "Fv," "Fv fragment," or "Fv region" refer to a polypeptide comprising the VL and VH domains of a single antibody.

[0027] As used herein, the terms "single-chain Fv" or "scFv" refer to an antibody fragment containing the VH and VL domains of an antibody within a single polypeptide chain.

[0028] As used herein, the term “variable region” refers to an antibody region comprising one or more immunoglobulin domains encoded by either the VL (including V-kappa (VK) and V-lambda (VL)) and / or VH genes that constitute the light chain (including kappa and lambda) and heavy chain immunoglobulin domain loci, respectively. The light chain or heavy chain variable region (VL or VH) consists of a “framework” or “FR” region comprising three hypervariable regions referred to as “complementarity-determining regions” or “CDR.” As used herein, the term “antigen” refers to a structure that can selectively bind to an antibody. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. Specifically, the antigen may be a polypeptide and may be a protein present on or inside a cell.

[0029] As used herein, the term “epitope” refers to an antigenic determinant, which is a portion of an antigen to which an antibody or polypeptide binds. A protein epitope may include amino acid residues directly involved in binding, as well as amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide. An epitope is the simplest form or smallest structural region of a complex antigen molecule that can bind to an antibody or receptor. Epitopes may be linear or structural / stereostructural.

[0030] As used herein, the term "Z protein" may also be referred to as "Z domain," "additional protein," or "additional domain," and "Z protein" is a protein that binds to a novel form of antibody as used herein. A "Z protein" may be any one selected from the group consisting of receptors, soluble proteins, cytokines, single-chain Fv fragments (single-chain variable fragments; scFv), antibody mimetic compounds, single-domain antibodies (sdAb), therapeutic peptides, and peptide vaccines.

[0031] As used herein, the term “vector” refers to a material for transporting or expressing nucleic acid sequences, including nucleic acid sequences encoding multispecific fusion proteins (e.g., antibodies) as described herein. Specifically, vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes.

[0032] As used herein, the term "polynucleotide," also referred to as "nucleic acid," refers to a polymer of nucleotides of any length. Specifically, the polynucleotide may be DNA or RNA.

[0033] Antibodies containing two Fc cells In one embodiment of the present invention, an antibody comprising multiple Fc domains is provided, characterized in that the ratio of antigen-binding sites to Fc domains is 1:2 or 2:2. Specifically, the antibody may be a fusion protein comprising an antigen-binding site, a first Fc domain or a variant thereof, and a second Fc domain or a variant thereof.

[0034] Here, the antigen-binding site may consist of two different polypeptide chains. In addition, each polypeptide may be ligated to a first Fc domain or a variant thereof, and to a second Fc domain or a variant thereof.

[0035] In one embodiment of the fusion protein, if the antigen-binding site includes Fab, the fusion protein may be a fusion protein in which two Fc domains are bound to the C-terminus of the CH1 region of the heavy chain and the C-terminus of the constant region of the light chain, respectively. In addition, the Fc domains and Fab may be linked via a peptide linker.

[0036] In addition, in one embodiment of the fusion protein, if the antigen-binding site is Fv, the fusion protein may be a fusion protein in which two Fc domains are bound to the C-terminus of the variable region of the heavy chain and the C-terminus of the variable region of the light chain, respectively. Furthermore, the Fc domains and Fv may be linked via a peptide linker.

[0037] This novel antibody form or structure has a molecular weight similar to that of human IgG. In addition, the fusion protein can possess antigen-binding affinity equivalent to that of human IgG-based antibodies. However, the antibody form allows the Fc domain to be present up to four times more on cell surface antigens compared to natural human antibodies. Due to these characteristics, the fusion protein can have increased affinity for the Fcγ receptor and increased effector function compared to wild-type antibodies. Each Fc domain bound to the fusion protein can have a similar level of Fc receptor (Fcγ receptor and FcRn) binding affinity to the Fc domain of an IgG-based antibody; however, due to the avidity effect, the apparent binding affinity (apparent affinity) of the fusion protein to the Fc receptor (Fcγ receptor and FcRn) may be significantly increased compared to human IgG antibodies. Furthermore, the fusion protein possesses a similar level of thermal stability to that of IgG-based antibodies.

[0038] Specifically, the fusion protein may include: (a) an antigen-binding site comprising a first polypeptide containing at least one CDR sequence and a second polypeptide containing at least one CDR sequence, wherein the first and second polypeptides form a dimer and the antigen-binding site can specifically bind to a target antigen; (b) a first Fc domain or a variant thereof which is a dimer of two polypeptide sequences in which one polypeptide sequence is conjugated to the first polypeptide of the antigen-binding site; and (c) a second Fc domain or a variant thereof which is a dimer of two polypeptide sequences in which one polypeptide sequence is conjugated to the second polypeptide of the antigen-binding site.

[0039] Here, the first polypeptide of the antigen-binding site may include CDR1, CDR2, and CDR3 of the antibody heavy chain, and the second polypeptide of the antigen-binding site may include CDR1, CDR2, and CDR3 of the antibody light chain. In addition, the first polypeptide of the antigen-binding site may further include the CH1 region of the antibody heavy chain, and / or the second polypeptide of the antigen-binding site may further include the constant region of the antibody light chain.

[0040] In addition, the Z protein may be bound to the first Fc domain or its variant, and to the second Fc domain or its variant. In particular, the Z protein may be bound to the N-terminus of the hinge domain of the first Fc domain or its variant, and to the second Fc domain or its variant.

[0041] The specific structure of the fusion protein is described in more detail below.

[0042] antigen binding site Here, the antigen-binding site can specifically bind to proteins expressed on the cell surface. Specifically, the antigen-binding site can specifically bind to cancer antigens.

[0043] In one embodiment, the antigen-binding sites are PD-L1, EGFR, EGFRvIII, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, c-Met (MET), DLL3, DR4, DR5, GD2, Nectin-4, RANKL, SLAMF7, Trop-2, LIV-1, Claudin 18.2, IL13α2, CD3, HER2, HER3, It can specifically bind to any one antigen selected from the group consisting of FGFR2, FGFR3, GPC3, ROR1, Folα, CD20, CD19, CTLA-4, VEGFR, NCAM1, ICAM-1, ICAM-2, CEACAM5, CEACAM6, carcinoembryonic antigen (CEA), CA-125, alpha-fetoprotein (AFP), MUC-1, MUC-16, PSMA, PSCA, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), immature laminin receptor, TAG-72, HPV E6 / E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA2, EphA3, mesothelin, SAP-1, survivorbin, and virus-derived antigens.

[0044] The second antigen-binding site can also specifically bind to any one antigen selected from the group of antigens described above. In one embodiment, the antigen to which the first antigen-binding site binds may be different from the antigen to which the second antigen-binding site binds. In one embodiment, the first antigen-binding site may include a sequence that specifically binds to HER2, and the second antigen-binding site may include a sequence that specifically binds to EGFR. In another embodiment, the first antigen-binding site may include a sequence that specifically binds to one epitope of the antigen, and the second antigen-binding site may include a sequence that specifically binds to different epitopes of the same antigen.

[0045] Embodiment of the antigen-binding site Here, the antigen-binding site may include a variable region that specifically binds to the antigen. Specifically, the variable region is found in cetuximab, panitumumab, nesitumumab, imugatuzumab, depatuxizumab, losatuxizumab, etevritamab, AMG-595, atezolizumab, avelumab, durvalumab, trastuzumab, pertuzumab, onarutuzumab, emibetuzumab, terisotuzumab, datopotamab, sacituzumab, lovalpituzumab, and tarrata. The heavy chain variable region and light chain variable region of any one antibody selected from the group consisting of mab, verantamab, ladiratuzumab, codrituzumab, aprutumab, bemarituzumab, bofatamab, ramucirumab, rituximab, obinutuzumab, daratumumab, and 1C1 (clone name) may be included, but are not limited to.

[0046] As an embodiment of the present invention, the embodiment may include an antigen-binding site that specifically binds to EGFR. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 175 of cetuximab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 176, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 177, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 178, L-CDR2 containing the amino acid sequence of SEQ ID NO: 179, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 180. As another example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 181 of panitumumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 182, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 183, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 184, L-CDR2 containing the amino acid sequence of SEQ ID NO: 185, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 186. As another example, the antigen-binding site may include a heavy chain variable region containing H-CDR1 represented by necitumumab's SEQ ID NO: 187, H-CDR2 represented by SEQ ID NO: 188, and H-CDR3 represented by SEQ ID NO: 189, and a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 190, L-CDR2 containing the amino acid sequence of SEQ ID NO: 191, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 192. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of imugatuzumab's SEQ ID NO: 193, H-CDR2 containing the amino acid sequence of SEQ ID NO: 194, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 195, and a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 196, L-CDR2 containing the amino acid sequence of SEQ ID NO: 197, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 198.In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 199 of depatuxizumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 200, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 201, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 202, L-CDR2 containing the amino acid sequence of SEQ ID NO: 203, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 204. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 199 of rosatuxizumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 205, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 206, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 202, L-CDR2 containing the amino acid sequence of SEQ ID NO: 203, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 204.

[0047] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to EGFRvIII. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 207 of etebritamab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 208, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 209, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 210, L-CDR2 containing the amino acid sequence of SEQ ID NO: 211, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 212. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 213 of AMG-595, H-CDR2 containing the amino acid sequence of SEQ ID NO: 214, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 215, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 210, L-CDR2 containing the amino acid sequence of SEQ ID NO: 216, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 217.

[0048] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to PD-L1. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 218 of atezolizumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 219, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 220, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 221, L-CDR2 containing the amino acid sequence of SEQ ID NO: 222, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 223. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 224 of avelumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 225, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 226, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 227, L-CDR2 containing the amino acid sequence of SEQ ID NO: 228, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 229. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 230 of durvalumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 231, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 232, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 233, L-CDR2 containing the amino acid sequence of SEQ ID NO: 234, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 235.

[0049] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to HER2. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 21 of trastuzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In addition, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 33 of pertuzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 34, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 35, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 36, L-CDR2 containing the amino acid sequence of SEQ ID NO: 37, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 38.

[0050] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to c-Met. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of onartuzumab SEQ ID NO: 236, H-CDR2 containing the amino acid sequence of SEQ ID NO: 237, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 238, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 239, L-CDR2 containing the amino acid sequence of SEQ ID NO: 240, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 241. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 242 of emibetuzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 243, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 244, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 245, L-CDR2 containing the amino acid sequence of SEQ ID NO: 246, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 247. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 248 of terisotuzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 249, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 250, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 251, L-CDR2 containing the amino acid sequence of SEQ ID NO: 252, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 253.

[0051] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to Trop-2. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of datopotamab SEQ ID NO: 254, H-CDR2 containing the amino acid sequence of SEQ ID NO: 255, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 256, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 257, L-CDR2 containing the amino acid sequence of SEQ ID NO: 258, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 259. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of SEQ ID NO: 260 of sacituzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 261, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 262, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 263, L-CDR2 containing the amino acid sequence of SEQ ID NO: 264, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 265.

[0052] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to DLL3. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 266 of lovalpituzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 267, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 268, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 269, L-CDR2 containing the amino acid sequence of SEQ ID NO: 270, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 271. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of tallatamab's SEQ ID NO: 272, H-CDR2 containing the amino acid sequence of SEQ ID NO: 273, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 274, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 275, L-CDR2 containing the amino acid sequence of SEQ ID NO: 276, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 277.

[0053] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to BCMA. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of verantamab SEQ ID NO: 278, H-CDR2 containing the amino acid sequence of SEQ ID NO: 279, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 280, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 281, L-CDR2 containing the amino acid sequence of SEQ ID NO: 282, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 283.

[0054] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to LIV-1. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of radilatuzumab SEQ ID NO: 284, H-CDR2 containing the amino acid sequence of SEQ ID NO: 285, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 286, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 287, L-CDR2 containing the amino acid sequence of SEQ ID NO: 288, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 289.

[0055] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to GPC-3. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 99 of codolituzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 100, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 101, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 102, L-CDR2 containing the amino acid sequence of SEQ ID NO: 103, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 104.

[0056] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to FGFR2. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 290 of apluzumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 291, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 292, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 293, L-CDR2 containing the amino acid sequence of SEQ ID NO: 294, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 295. In addition, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of bemarituzumab SEQ ID NO: 296, H-CDR2 containing the amino acid sequence of SEQ ID NO: 297, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 298, and may also include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 299, L-CDR2 containing the amino acid sequence of SEQ ID NO: 300, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 301.

[0057] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to FGFR3. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 302 of bofatamab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 303, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 304, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 305, L-CDR2 containing the amino acid sequence of SEQ ID NO: 306, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 307.

[0058] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to VEGFR2. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 308 of ramucirumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 309, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 310, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 311, L-CDR2 containing the amino acid sequence of SEQ ID NO: 312, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 313.

[0059] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to CD20. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of rituximab SEQ ID NO: 314, H-CDR2 containing the amino acid sequence of SEQ ID NO: 315, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 316, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 317, L-CDR2 containing the amino acid sequence of SEQ ID NO: 318, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 319. For example, the antigen-binding site may include a heavy chain variable region containing H-CDR1 containing the amino acid sequence of obinutuzumab's SEQ ID NO: 320, H-CDR2 containing the amino acid sequence of SEQ ID NO: 321, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 322, or it may include a light chain variable region containing L-CDR1 containing the amino acid sequence of SEQ ID NO: 323, L-CDR2 containing the amino acid sequence of SEQ ID NO: 324, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 325.

[0060] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to CD38. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 326 of daratumumab, H-CDR2 containing the amino acid sequence of SEQ ID NO: 327, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 328, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 329, L-CDR2 containing the amino acid sequence of SEQ ID NO: 330, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 331.

[0061] In embodiments of the present invention, the embodiment may include an antigen-binding site that specifically binds to EphA2. For example, the antigen-binding site may include a heavy chain variable region including H-CDR1 containing the amino acid sequence of SEQ ID NO: 157 of 1C1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 158, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 159, or it may include a light chain variable region including L-CDR1 containing the amino acid sequence of SEQ ID NO: 160, L-CDR2 containing the amino acid sequence of SEQ ID NO: 161, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 162.

[0062] Table 1 below shows CDR sequences of non-limiting examples of antibodies with anticancer efficacy that may be used in embodiments of the present invention.

[0063] [Table 1] JPEG2026514684000003.jpg198149 JPEG2026514684000004.jpg198149 JPEG2026514684000005.jpg214149

[0064] antigen Examples of antigens to which the antigen-binding sites described herein can specifically bind include the following non-limiting substances.

[0065] "Epidermal Growth Factor Receptor (EGFR)": EGFR is a cell membrane receptor that regulates cell growth, division, survival, and death. In various cancers, EGFR expression is increased in tumor tissue. Tumor tissue with increased EGFR is known to be invasive, metastatic, and highly resistant to anticancer drugs. In one embodiment, the substance that inhibits EGFR may be, but is not limited to, cetuximab, panitumumab, nesitumumab, imugatuzumab, depatuxizumab, or rosatuxizumab.

[0066] "Epidermal growth factor receptor variant 3 (EGFRvIII)": EGFRvIII is a mutation in which exons 2-7 of EGFR are deleted. EGFRvIII is mainly reported in glioblastoma multiforme, and most patients with EGFRvIII-positive mutations have a poor prognosis. In one embodiment, the substance inhibiting EGFRvIII may be, but is not limited to, cetuximab, panitumumab, nesitumumab, imugatuzumab, depatuxizumab, rosatuxizumab, etebritamab, or AMG-595.

[0067] "Programmed Cell Death Ligand 1 (PD-L1)": PD-L1 is a protein overexpressed on the surface of cancer cells. PD-L1 is a major cancer-specific antigen that plays a crucial role in inducing T cell exhaustion and apoptosis and achieving immune tolerance in cancer cells. In one embodiment, the PD-L1-targeted anticancer antibody may be, but is not limited to, atezolizumab, avelumab, or durvalumab.

[0068] "HER-2 / neu (Human Epidermal Growth Factor Receptor 2)": HER-2 regulates cell proliferation through the activation of PI3K / AkT. HER-2 is known to be overexpressed in metastatic breast cancer and ovarian cancer, etc., and to induce resistance to anticancer drugs. HER-2 / neu-targeted anticancer drugs may be trastuzumab or pertuzumab, but are not limited to these.

[0069] "c-Met (Mesenchymal Epithelial Transition Factor)": c-Met is a hepatocyte growth factor receptor. Amplification or mutation of c-Met is frequently reported in cancer cells and is known to promote tumor growth, metastasis, and malignancy. Specifically, inhibitors of this protein may be, but are not limited to, onartuzumab, emibetuzumab, or terisotuzumab.

[0070] "Trop-2 (tumor-associated calcium signaling transducer 2)": Trop-2 is a cellular glycoprotein involved in cancer cell growth and proliferation. It is known that Trop-2 is specifically overexpressed in non-small cell lung cancer and breast cancer. Specifically, antibodies targeting this protein may be, but are not limited to, datopotamab or sacituzumab.

[0071] "DLL3 (Delta-like ligand 3)": DLL3 is a major cancer target antigen known to be expressed in approximately 85% of small cell lung cancer patients. Specifically, antibodies targeting this protein may be, but are not limited to, lovalpituzumab or tarlatamab.

[0072] "BCMA (B-cell maturation antigen)": BCMA is an important factor in the survival and proliferation of myeloma cells and is a clinically proven target for the treatment of multiple myeloma. Specifically, the antibody targeting this protein may be, but is not limited to, velantamab.

[0073] "LIV-1 (zinc transporter ZIP6)": LIV-1 is a highly cancer-specific antigen that is overexpressed in breast cancer. Specifically, the antibody that targets this protein may be radilatuzumab, but is not limited to it.

[0074] "GPC-3 (Glypican-3)": GPC-3 is a highly cancer-specific antigen that is specifically overexpressed in liver cancer. Specifically, the antibody that targets this protein may be, but is not limited to, codolituzumab.

[0075] "FGFR (Fibroblast Growth Factor Receptor)": FGFR is a receptor for fibroblast growth factor (FGF), which regulates various biological processes including cell growth, differentiation, and migration. FGFR genes mutate readily, and these variants are commonly observed in breast cancer, uterine cancer, ovarian cancer, cervical cancer, etc. The four FGFR genes are composed of seven signaling receptors, of which FGFR2 and FGFR3 are highly cancer-specific antigens. Antibodies targeting FGFR2 or FGFR3 may be, but are not limited to, appletuzumab, bemarituzumab, or bofatamab.

[0076] "Vascular endothelial growth factor receptor (VEGFR)": VEGFR is a cell membrane receptor for vascular endothelial growth factor, which induces angiogenesis. VEGFR inhibitors inhibit tumor growth and metastasis by inhibiting angiogenesis. In one embodiment, the VEGFR inhibitor may be, but is not limited to, ramucirumab.

[0077] "CD20 (B lymphocyte antigen CD20)": CD20 is a protein expressed on the surface of B cells and is used as a target protein for the treatment of B-cell lymphoma. CD20 inhibitors may be, but are not limited to, rituximab or obinutuzumab.

[0078] "CD38 (cluster of differentiation 38)": CD38 is a protein that acts as a signal transduction receptor in immune cells and regulates cell proliferation and death. Inhibitors that target this protein may be, but are not limited to, daratumumab.

[0079] "EphA2 (EPH receptor A2)": EphA2 is overexpressed in cancer cells and has a significant impact on cancer cell growth and metastasis. Antibodies targeting this protein may be 1C1, but are not limited to it.

[0080] The antigen-binding sites that specifically bind to the antigens exemplified above may include the CDR sequences exemplified below. EGFR: 1) The VH region (SEQ ID NO: 334) containing the amino acid sequences of SEQ ID NO: 175 (VH-CDR1), SEQ ID NO: 176 (VH-CDR2), and SEQ ID NO: 177 (VH-CDR3), and the VL region (SEQ ID NO: 335) containing the amino acid sequences of SEQ ID NO: 178 (VL-CDR1), SEQ ID NO: 179 (VL-CDR2), and SEQ ID NO: 180 (VL-CDR3); 2) The VH region (SEQ ID NO: 336) containing the amino acid sequences of SEQ ID NO: 181 (VH-CDR1), SEQ ID NO: 182 (VH-CDR2), and SEQ ID NO: 183 (VH-CDR3), and the VL region (SEQ ID NO: 337) containing the amino acid sequences of SEQ ID NO: 184 (VL-CDR1), SEQ ID NO: 185 (VL-CDR2), and SEQ ID NO: 186 (VL-CDR3); 3) The VH region (SEQ ID NO: 338) containing the amino acid sequences of SEQ ID NO: 187 (VH-CDR1), SEQ ID NO: 188 (VH-CDR2), and SEQ ID NO: 189 (VH-CDR3), and the VL region (SEQ ID NO: 339) containing the amino acid sequences of SEQ ID NO: 190 (VL-CDR1), SEQ ID NO: 191 (VL-CDR2), and SEQ ID NO: 192 (VL-CDR3); 4) The VH region (SEQ ID NO: 340) containing the amino acid sequences of SEQ ID NO: 193 (VH-CDR1), SEQ ID NO: 194 (VH-CDR2), and SEQ ID NO: 195 (VH-CDR3), and the VL region (SEQ ID NO: 341) containing the amino acid sequences of SEQ ID NO: 196 (VL-CDR1), SEQ ID NO: 197 (VL-CDR2), and SEQ ID NO: 198 (VL-CDR3); 5) The VH region (SEQ ID NO: 342) containing the amino acid sequences of SEQ ID NO: 199 (VH-CDR1), SEQ ID NO: 200 (VH-CDR2), and SEQ ID NO: 201 (VH-CDR3), and the VL region (SEQ ID NO: 343) containing the amino acid sequences of SEQ ID NO: 202 (VL-CDR1), SEQ ID NO: 203 (VL-CDR2), and SEQ ID NO: 204 (VL-CDR3); 6) The VH region (SEQ ID NO: 344) containing the amino acid sequences of SEQ ID NO: 199 (VH-CDR1), SEQ ID NO: 205 (VH-CDR2), and SEQ ID NO: 206 (VH-CDR3), and the VL region (SEQ ID NO: 345) containing the amino acid sequences of SEQ ID NO: 202 (VL-CDR1), SEQ ID NO: 203 (VL-CDR2), and SEQ ID NO: 204 (VL-CDR3); EGFRvIII: 7) The VH region (SEQ ID NO: 346) containing the amino acid sequences of SEQ ID NO: 207 (VH-CDR1), SEQ ID NO: 208 (VH-CDR2), and SEQ ID NO: 209 (VH-CDR3), and the VL region (SEQ ID NO: 347) containing the amino acid sequences of SEQ ID NO: 210 (VL-CDR1), SEQ ID NO: 211 (VL-CDR2), and SEQ ID NO: 212 (VL-CDR3); 8) The VH region (SEQ ID NO: 348) containing the amino acid sequences of SEQ ID NO: 213 (VH-CDR1), SEQ ID NO: 214 (VH-CDR2), and SEQ ID NO: 215 (VH-CDR3), and the VL region (SEQ ID NO: 349) containing the amino acid sequences of SEQ ID NO: 210 (VL-CDR1), SEQ ID NO: 216 (VL-CDR2), and SEQ ID NO: 217 (VL-CDR3); PD-L1: 9) The VH region (SEQ ID NO: 350) containing the amino acid sequences of SEQ ID NO: 218 (VH-CDR1), SEQ ID NO: 219 (VH-CDR2), and SEQ ID NO: 220 (VH-CDR3), and the VL region (SEQ ID NO: 351) containing the amino acid sequences of SEQ ID NO: 221 (VL-CDR1), SEQ ID NO: 222 (VL-CDR2), and SEQ ID NO: 223 (VL-CDR3); 10) The VH region (SEQ ID NO: 352) containing the amino acid sequences of SEQ ID NO: 224 (VH-CDR1), SEQ ID NO: 225 (VH-CDR2), and SEQ ID NO: 226 (VH-CDR3), and the VL region (SEQ ID NO: 353) containing the amino acid sequences of SEQ ID NO: 227 (VL-CDR1), SEQ ID NO: 228 (VL-CDR2), and SEQ ID NO: 229 (VL-CDR3); 11) The VH region (SEQ ID NO: 354) containing the amino acid sequences of SEQ ID NO: 230 (VH-CDR1), SEQ ID NO: 231 (VH-CDR2), and SEQ ID NO: 232 (VH-CDR3), and the VL region (SEQ ID NO: 355) containing the amino acid sequences of SEQ ID NO: 233 (VL-CDR1), SEQ ID NO: 234 (VL-CDR2), and SEQ ID NO: 235 (VL-CDR3); HER2: 12) The VH region (SEQ ID NO: 356) containing the amino acid sequences of SEQ ID NO: 21 (VH-CDR1), SEQ ID NO: 22 (VH-CDR2), and SEQ ID NO: 23 (VH-CDR3), and the VL region (SEQ ID NO: 357) containing the amino acid sequences of SEQ ID NO: 24 (VL-CDR1), SEQ ID NO: 25 (VL-CDR2), and SEQ ID NO: 26 (VL-CDR3); 13) The VH region (SEQ ID NO: 27) containing the amino acid sequences of SEQ ID NO: 33 (VH-CDR1), SEQ ID NO: 34 (VH-CDR2), and SEQ ID NO: 35 (VH-CDR3), and the VL region (SEQ ID NO: 28) containing the amino acid sequences of SEQ ID NO: 36 (VL-CDR1), SEQ ID NO: 37 (VL-CDR2), and SEQ ID NO: 38 (VL-CDR3); c-Met: 14) The VH region (SEQ ID NO: 358) containing the amino acid sequences of SEQ ID NO: 236 (VH-CDR1), SEQ ID NO: 237 (VH-CDR2), and SEQ ID NO: 238 (VH-CDR3), and the VL region (SEQ ID NO: 359) containing the amino acid sequences of SEQ ID NO: 239 (VL-CDR1), SEQ ID NO: 240 (VL-CDR2), and SEQ ID NO: 241 (VL-CDR3); 15) The VH region (SEQ ID NO: 360) containing the amino acid sequences of SEQ ID NO: 242 (VH-CDR1), SEQ ID NO: 243 (VH-CDR2), and SEQ ID NO: 244 (VH-CDR3), and the VL region (SEQ ID NO: 361) containing the amino acid sequences of SEQ ID NO: 245 (VL-CDR1), SEQ ID NO: 246 (VL-CDR2), and SEQ ID NO: 247 (VL-CDR3); 16) The VH region (SEQ ID NO: 362) containing the amino acid sequences of SEQ ID NO: 248 (VH-CDR1), SEQ ID NO: 249 (VH-CDR2), and SEQ ID NO: 250 (VH-CDR3), and the VL region (SEQ ID NO: 363) containing the amino acid sequences of SEQ ID NO: 251 (VL-CDR1), SEQ ID NO: 252 (VL-CDR2), and SEQ ID NO: 253 (VL-CDR3); Trop-2: 17) The VH region (SEQ ID NO: 364) containing the amino acid sequences of SEQ ID NO: 254 (VH-CDR1), SEQ ID NO: 255 (VH-CDR2), and SEQ ID NO: 256 (VH-CDR3), and the VL region (SEQ ID NO: 365) containing the amino acid sequences of SEQ ID NO: 257 (VL-CDR1), SEQ ID NO: 258 (VL-CDR2), and SEQ ID NO: 259 (VL-CDR3); 18) The VH region (SEQ ID NO: 366) containing the amino acid sequences of SEQ ID NO: 260 (VH-CDR1), SEQ ID NO: 261 (VH-CDR2), and SEQ ID NO: 262 (VH-CDR3), and the VL region (SEQ ID NO: 367) containing the amino acid sequences of SEQ ID NO: 263 (VL-CDR1), SEQ ID NO: 264 (VL-CDR2), and SEQ ID NO: 265 (VL-CDR3); DLL3: 19) The VH region (SEQ ID NO: 368) containing the amino acid sequences of SEQ ID NO: 266 (VH-CDR1), SEQ ID NO: 267 (VH-CDR2), and SEQ ID NO: 268 (VH-CDR3), and the VL region (SEQ ID NO: 369) containing the amino acid sequences of SEQ ID NO: 269 (VL-CDR1), SEQ ID NO: 270 (VL-CDR2), and SEQ ID NO: 271 (VL-CDR3); 20) The VH region (SEQ ID NO: 370) containing the amino acid sequences of SEQ ID NO: 272 (VH-CDR1), SEQ ID NO: 273 (VH-CDR2), and SEQ ID NO: 274 (VH-CDR3), and the VL region (SEQ ID NO: 371) containing the amino acid sequences of SEQ ID NO: 275 (VL-CDR1), SEQ ID NO: 276 (VL-CDR2), and SEQ ID NO: 277 (VL-CDR3); BCMA: 21) The VH region (SEQ ID NO: 372) containing the amino acid sequences of SEQ ID NO: 278 (VH-CDR1), SEQ ID NO: 279 (VH-CDR2), and SEQ ID NO: 280 (VH-CDR3), and the VL region (SEQ ID NO: 373) containing the amino acid sequences of SEQ ID NO: 281 (VL-CDR1), SEQ ID NO: 282 (VL-CDR2), and SEQ ID NO: 283 (VL-CDR3); LIV-1: 22) The VH region (SEQ ID NO: 374) containing the amino acid sequences of SEQ ID NO: 284 (VH-CDR1), SEQ ID NO: 285 (VH-CDR2), and SEQ ID NO: 286 (VH-CDR3), and the VL region (SEQ ID NO: 375) containing the amino acid sequences of SEQ ID NO: 287 (VL-CDR1), SEQ ID NO: 288 (VL-CDR2), and SEQ ID NO: 289 (VL-CDR3); GPC-3: 23) The VH region (SEQ ID NO: 87) containing the amino acid sequences of SEQ ID NO: 99 (VH-CDR1), SEQ ID NO: 100 (VH-CDR2), and SEQ ID NO: 101 (VH-CDR3), and the VL region (SEQ ID NO: 88) containing the amino acid sequences of SEQ ID NO: 102 (VL-CDR1), SEQ ID NO: 103 (VL-CDR2), and SEQ ID NO: 104 (VL-CDR3); FGFR2: 24) The VH region (SEQ ID NO: 376) containing the amino acid sequences of SEQ ID NO: 290 (VH-CDR1), SEQ ID NO: 291 (VH-CDR2), and SEQ ID NO: 292 (VH-CDR3), and the VL region (SEQ ID NO: 377) containing the amino acid sequences of SEQ ID NO: 293 (VL-CDR1), SEQ ID NO: 294 (VL-CDR2), and SEQ ID NO: 295 (VL-CDR3); 25) The VH region (SEQ ID NO: 378) containing the amino acid sequences of SEQ ID NO: 296 (VH-CDR1), SEQ ID NO: 297 (VH-CDR2), and SEQ ID NO: 298 (VH-CDR3), and the VL region (SEQ ID NO: 379) containing the amino acid sequences of SEQ ID NO: 299 (VL-CDR1), SEQ ID NO: 300 (VL-CDR2), and SEQ ID NO: 301 (VL-CDR3); FGFR3: 26) The VH region (SEQ ID NO: 380) containing the amino acid sequences of SEQ ID NO: 302 (VH-CDR1), SEQ ID NO: 303 (VH-CDR2), and SEQ ID NO: 304 (VH-CDR3), and the VL region (SEQ ID NO: 381) containing the amino acid sequences of SEQ ID NO: 305 (VL-CDR1), SEQ ID NO: 306 (VL-CDR2), and SEQ ID NO: 307 (VL-CDR3); VEGFR2: 27) The VH region (SEQ ID NO: 382) containing the amino acid sequences of SEQ ID NO: 308 (VH-CDR1), SEQ ID NO: 309 (VH-CDR2), and SEQ ID NO: 310 (VH-CDR3), and the VL region (SEQ ID NO: 383) containing the amino acid sequences of SEQ ID NO: 311 (VL-CDR1), SEQ ID NO: 312 (VL-CDR2), and SEQ ID NO: 313 (VL-CDR3); CD20: 28) The VH region (SEQ ID NO: 384) containing the amino acid sequences of SEQ ID NO: 314 (VH-CDR1), SEQ ID NO: 315 (VH-CDR2), and SEQ ID NO: 316 (VH-CDR3), and the VL region (SEQ ID NO: 385) containing the amino acid sequences of SEQ ID NO: 317 (VL-CDR1), SEQ ID NO: 318 (VL-CDR2), and SEQ ID NO: 319 (VL-CDR3); 29) The VH region (SEQ ID NO: 386) containing the amino acid sequences of SEQ ID NO: 320 (VH-CDR1), SEQ ID NO: 321 (VH-CDR2), and SEQ ID NO: 322 (VH-CDR3), and the VL region (SEQ ID NO: 387) containing the amino acid sequences of SEQ ID NO: 323 (VL-CDR1), SEQ ID NO: 324 (VL-CDR2), and SEQ ID NO: 325 (VL-CDR3); CD38: 30) The VH region (SEQ ID NO: 388) containing the amino acid sequences of SEQ ID NO: 326 (VH-CDR1), SEQ ID NO: 327 (VH-CDR2), and SEQ ID NO: 328 (VH-CDR3), and the VL region (SEQ ID NO: 389) containing the amino acid sequences of SEQ ID NO: 329 (VL-CDR1), SEQ ID NO: 330 (VL-CDR2), and SEQ ID NO: 331 (VL-CDR3); EphA2: 31) A VH region (SEQ ID NO: 143) containing the amino acid sequences of SEQ ID NO: 157 (VH-CDR1), SEQ ID NO: 158 (VH-CDR2), and SEQ ID NO: 159 (VH-CDR3), and a VL region (SEQ ID NO: 145) containing the amino acid sequences of SEQ ID NO: 160 (VL-CDR1), SEQ ID NO: 161 (VL-CDR2), and SEQ ID NO: 162 (VL-CDR3).

[0081] Table 2 below shows exemplary polynucleotide and polypeptide sequences of signal sequences for efficient expression of fusion proteins according to various embodiments. When the above antibody is expressed in mammalian cells, the amino acid sequence of SEQ ID NO: 333 may be included as the signal sequence, but is not limited to it.

[0082] [Table 2]

[0083] Table 3 below shows the variable region polypeptide sequences of anti-cancer antibodies described as antigen-binding sites for various fusion proteins described herein. Fusion proteins according to exemplary embodiments may contain or consist of these variable region polypeptides.

[0084] [Table 3] JPEG2026514684000008.jpg208149 JPEG2026514684000009.jpg208149 JPEG2026514684000010.jpg183149

[0085] Table 4 below shows polynucleotide sequences encoding polypeptides of the variable region of anti-cancer antibodies, which are described as antigen-binding sites for various fusion proteins described herein.

[0086] [Table 4] JPEG2026514684000012.jpg206149 JPEG2026514684000013.jpg210149 JPEG2026514684000014.jpg206149 JPEG2026514684000015.jpg207149 JPEG2026514684000016.jpg206149 JPEG2026514684000017.jpg210149 JPEG2026514684000018.jpg210149 JPEG2026514684000019.jpg210149 JPEG2026514684000020.jpg43149

[0087] Fc region or its fragment Here, the first Fc domain and the second Fc domain described above may each be the Fc region of an immunoglobulin. The Fc region of an immunoglobulin may be an Fc domain variant or a wild-type Fc domain. Here, the Fc region may be the Fc region of IgG, IgA, IgE, IgD, or IgM.

[0088] As used herein, the term “Fc domain variant” may refer to a form that differs from the wild-type Fc domain in terms of its glycosylation pattern, or has a higher level of specific glycan species compared to the wild-type Fc domain, a lower level of specific glycan species compared to the wild-type Fc domain, or a deglycosylated form. In addition, aglycosylated Fc domains are included in this term. An Fc domain or its variant can be adapted to have a controlled number of sialic acid, fucosylation, or other types of glycosylation by modulating culture conditions or the host cell's gene manipulation.

[0089] In addition, the glycosylation of the Fc domain of immunoglobulins can be modified by conventional methods such as chemical methods, enzymatic methods, and gene manipulation methods using microorganisms. Furthermore, Fc domain variants may be in mixed forms of the Fc regions of immunoglobulins IgG, IgA, IgE, IgD, or IgM. Furthermore, Fc domain variants may be in which some amino acids in the Fc domain are substituted with other amino acids.

[0090] The "amino acids" 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).

[0091] In one embodiment, the variant of the Fc region may be in which amino acids 239 and / or 332 of the CH2 region are substituted with other amino acids (see Kabat numbering system). Specifically, S239 may be substituted with an amino acid other than S, specifically S239D. In addition, I332 may be substituted with an amino acid other than I, specifically I332E.

[0092] In addition, the Fc region may include a variant or structure of a knob, or a variant or structure of a hole.

[0093] As used herein, the term "knob-into-hole" refers to an Fc heterodimerization strategy for producing antibodies that specifically bind to different regions, such as bispecific antibodies, polyspecific antibodies, or heterodimer antibodies. Generally, this technique involves introducing a knob mutation at the interface of a first polypeptide (e.g., the first CH3 domain of the first antibody heavy chain) and a corresponding hole mutation at the interface of a second polypeptide (e.g., the second CH3 domain of the second antibody heavy chain) so that the knob can enter the hole to promote heterodimerization and prevent homodimerization.

[0094] A "knob" variant is constructed by replacing a small amino acid side chain at the interface of the first polypeptide (e.g., the first CH3 domain of the first antibody heavy chain) with a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A complementary "hole" variant of the same or similar size as the knob is created by replacing a large amino acid side chain at the interface of the second polypeptide (e.g., the second CH3 domain of the second antibody heavy chain) with a smaller side chain (e.g., alanine, serine, valine, or threonine). Knobs and holes may also be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0095] Examples of variants of the Fc region that promote heterodimer formation may include those described in International Publication No. 2014084607 and International Publication No. 2018059502, etc. The disclosures of International Publication No. 2014084607 and International Publication No. 2018059502 are incorporated herein by reference. International Publication No. 2014084607 may include, for example, (a-1) tryptophan (W) substituted at Lys409 of one CH3 domain, which interacts with valine (V) substituted at Asp399 of the other CH3 domain and threonine (T) substituted at Phe405 of the other CH3 domain; and (a-2) serine (S) substituted at Tyr349 of one CH3 domain, which interacts with tryptophan (W) substituted at Glu357 of the other CH3 domain, in addition Next, we describe the CH3 domain mutations, which may further include (b-1) glutamic acid (E) substituted at Lys360 in one CH3 domain that interacts with arginine (R) substituted at Gln347 in the other CH3 domain, and (b-2) glutamic acid (E) substituted at Gln347 and glutamic acid substituted at Lys360 in one CH3 domain that interact with arginine (R) substituted at Gln347 in the other CH3 domain. Here, the positions of amino acid residues follow the EU index. International Publication No. 2018059502 includes, for example, a)~e): a) L351G, L351Y, L351V, L351P, L351D, L351E, L351K, or L351W; b) T366L, T366P, T366W, or T366V; c) D399C, D399N, D399I, D399G, D399 Mutations in the Fc domain are described, including one or more mutations selected from R, D399T, or D399A; d) Y407L, Y407A, Y407P, Y407F, Y407T, or Y407H; and e) K409C, K409P, K409S, K409F, K409V, K409Q, or K409R. Here, the amino acid residue positions follow the EU index.

[0096] Structure of the fusion protein The fusion proteins have the following structural formulas: (I), (II), (III), and (IV): N'-X-(L1)nA-C' (I); N'-Y-(L2)mB-C' (II); N'-(Z1)r-(L5)sC-C' (III); and N'-(Z2)t-(L6)uD-C' (IV) It may also include a polypeptide chain represented by, In the formulas, in structural formulas (I), (II), (III), and (IV), N' is the N-terminus of each polypeptide, C' is the C-terminus of each polypeptide, - indicates connection, A, B, C, and D are monomeric polypeptide sequences of the Fc domain, each containing the CH2 and CH3 regions of immunoglobulin and optionally further containing the CH4 and / or hinge sequence. A forms a dimer with either C or D to form the first Fc domain (b), B dimerizes with the remaining one of C or D to form a second Fc domain (c); L1, L2, L5, and L6 are peptide linkers, respectively. n, m, r, s, t, and u are each independently either 0 or 1. X includes the heavy chain variable region or light chain variable region of an antibody that specifically binds to an antigen; Y comprises the light chain variable region or heavy chain variable region of an antibody that specifically binds to the antigen; X and Y pair with each other to form an antigen-binding site (a) that specifically binds to the antigen. Polypeptides (I), (II), (III), and (IV) can associate with a fusion protein containing one antigen-binding site and two Fc domains.

[0097] In addition, Z1 and Z2 may each be independently selected from the group consisting of receptors, soluble proteins, cytokines, single-chain Fv fragments (single-chain variable fragments; scFv), antibody mimetic compounds, single-domain antibodies (sdAbs), therapeutic peptides, and peptide vaccines.

[0098] Specifically, X is a first polypeptide sequence of the antigen-binding site, comprising the heavy chain CDR1, CDR2, and CDR3 sequences of an antibody that specifically binds to the first antigen, or the heavy chain variable region of an antibody that specifically binds to the first antigen; Y is a second polypeptide sequence of the antigen-binding site, comprising the light chain CDR1, CDR2, and CDR3 sequences of an antibody that specifically binds to the first antigen, or the light chain variable region of an antibody that specifically binds to the first antigen; X and Y pair with each other to form an antigen-binding site (a) that specifically binds to the antigen.

[0099] According to one embodiment, the CH3 region may be mutated to minimize interactions between A and B, and between C and D, and to promote the formation of heterodimers Fc between A and C, and between B and D. Specifically, the Fc domain monomer may include a knob variant or a hole variant that promotes the formation of an Fc heterodimer (heterodimer Fc); or the Fc domain monomer may include a variant that promotes the formation of a heterodimer by an electrostatic steering mechanism.

[0100] According to one embodiment, X in structural formula (I) may further include a heavy chain CH1 region, and / or Y in structural formula (II) may further include a light chain steady region. In addition, the fusion protein has the following structural formulas: (I'), (II'), (III), and (IV): N'-VD1-(L3)pX-(L1)nA-C' (I'); N'-VD2-(L4)qY-(L2)mB-C' (II'); N'-(Z1)r-(L5)sC-C' (III); and N'-(Z2)t-(L6)uD-C' (IV) It may also include a polypeptide chain represented by, In the formulas, in structural formulas (I'), (II'), (III), and (IV), N' is the N-terminus of the polypeptide chain, C' is the C-terminus of the polypeptide chain, - indicates connection, A, B, C, and D are monomeric polypeptide sequences of an Fc domain, each containing the CH2 and CH3 regions of an immunoglobulin and optionally further containing a CH4 and / or hinge sequence, wherein A dimers with either C or D to form a first Fc domain (b), and B dimers with the remaining C or D to form a second Fc domain (c); L1, L2, L3, L4, L5, and L6 are peptide linkers, respectively. n, m, p, q, r, s, t, and u are each either 0 or 1. VD1 consists of the heavy or light chain variable region of an antibody that specifically binds to an antigen, or the CDR1, CDR2, and CDR3 of the antibody heavy or light chain; VD2 consists of the variable regions of the antibody's light or heavy chain that specifically bind to the antigen, or the CDR1, CDR2, and CDR3 of the antibody's heavy or light chain; VD1 and VD2 pair with each other to form a second antibody variable region that specifically binds to the second antigen. X includes the heavy or light chain variable region of an antibody that specifically binds to an antigen, or the CDR1, CDR2, and CDR3 of the antibody heavy or light chain; Y comprises the variable regions of the light or heavy chain of an antibody that specifically binds to an antigen, or CDR1, CDR2, and CDR3 of the antibody heavy or light chain; X and Y pair with each other to form a first antibody variable region that specifically binds to the first antigen. VD1-(L3)pX forms the first polypeptide sequence of the antigen-binding site (a), and VD2-(L4)qY forms the second polypeptide sequence of the antigen-binding site (a).

[0101] In addition, Z1 and Z2 may each be independently selected from the group consisting of receptors, soluble proteins, cytokines, single-chain Fv fragments (single-chain variable fragments; scFv), antibody mimetic compounds, single-domain antibodies (sdAbs), therapeutic peptides, and peptide vaccines.

[0102] According to one embodiment, the CH3 region may be mutated to minimize interactions between A and B, and between C and D, and to promote the formation of heterodimers Fc between A and C, and between B and D. Specifically, the Fc domain monomer may include a knob variant or a hole variant that promotes the formation of Fc heterodimers; or the Fc domain monomer may include a variant that promotes the formation of heterodimers by an electrostatic steering mechanism.

[0103] According to one embodiment, the heavy chain variable region may further include the heavy chain CH1 region. In addition, the light chain variable region may further include the light chain steady region.

[0104] In the structures of the fusion proteins described herein, the bond between X and Y can be achieved by i) a disulfide bond formed by CH1 and Cys present in the constant region of the light chain, ii) a disulfide bond formed by Cys present in the variable region of the heavy chain and the variable region of the light chain, or iii) a disulfide bond formed by CH1 and Cys present in the constant region of the light chain, and a disulfide bond formed by Cys present in the variable region of the heavy chain and the variable region of the light chain.

[0105] Specifically, the bond between X and Y can be formed by a disulfide bond present between CH1233 and CL214 based on Kabat numbering. In addition, X and Y may further contain Cys by amino acid substitution. Examples of such variants may include mutations in the variable region, specifically mutations at 105C of VH and 43C of VL, or mutations at 44C of VH and 100C of VL, based on Kabat numbering. In one embodiment, the mutation may be Q105C of VH and A43C of VL. In addition, in one embodiment, the mutation may be G44C of VH and Q100C of VL. In addition, examples of variants in the constant region may include mutations at 122C of CH1 and 121C of CL, based on Kabat numbering. In one embodiment, the mutation may be F122C of CH1 and S121C of CL.

[0106] Linker and hinge The hinge is a hinge region derived from immunoglobulin. In one embodiment, the antibody hinge region is an IgG hinge region. The IgG hinge region provided herein may be selected, for example, from antibody hinge regions of various IgG subtypes. The table below lists exemplary IgG subtypes having a core hinge sequence that may be contained in the flexible peptide region provided herein. In addition, at least one Cys may be present within the hinge. Specifically, one, two, or three Cys may be present within the hinge.

[0107] [Table 5]

[0108] The hinge may be modified to either omit a disulfide bond or introduce an additional disulfide bond.

[0109] In addition, linkers L1 and L2 may each contain about 1 to about 70 amino acids. According to one exemplary embodiment, L1 and L2 may each contain about 5 to about 60 amino acids, about 10 to about 50 amino acids, about 15 to about 40 amino acids, or about 20 to about 30 amino acids. According to another exemplary embodiment, for example, L1 and L2 may each be peptides consisting of approximately 1 to approximately 70 amino acid residues, approximately 2 to approximately 60 amino acid residues, approximately 2 to approximately 50 amino acid residues, approximately 2 to approximately 40 amino acid residues, approximately 2 to approximately 30 amino acid residues, approximately 3 to approximately 50 amino acid residues, approximately 3 to approximately 40 amino acid residues, approximately 3 to approximately 30 amino acid residues, approximately 2 to approximately 28 amino acid residues, approximately 2 to approximately 26 amino acid residues, approximately 2 to approximately 24 amino acid residues, approximately 2 to approximately 22 amino acid residues, approximately 2 to approximately 20 amino acid residues, approximately 2 to approximately 18 amino acid residues, approximately 2 to approximately 16 amino acid residues, approximately 2 to approximately 14 amino acid residues, approximately 2 to approximately 12 amino acid residues, or approximately 2 to approximately 10 amino acid residues. Specifically, L1 and L2 may, but are not limited to, the amino acid sequence of (G4S)o (wherein o is an integer from 1 to 5) in Table 6 below. In addition, L1 and L2 may have different amino acid sequences. Furthermore, L1 and L2 may contain at least one Cys molecule. In addition, disulfide bonds may be formed through the Cys molecules present in L1 and L2.

[0110] [Table 6]

[0111] In addition, L3 and L4 may each contain about 1 to about 30 amino acids. According to one exemplary embodiment, L3 and L4 may each contain about 5 to about 25 amino acids, about 10 to about 20 amino acids, or about 15 amino acids. According to another exemplary embodiment, L3 and L4 may each be peptides consisting of about 2 to about 30 amino acid residues, about 2 to about 25 amino acid residues, about 2 to about 20 amino acid residues, about 2 to about 15 amino acid residues, about 3 to about 30 amino acid residues, about 2 to about 28 amino acid residues, about 2 to about 26 amino acid residues, about 2 to about 24 amino acid residues, about 2 to about 22 amino acid residues, about 2 to about 20 amino acid residues, about 2 to about 18 amino acid residues, about 2 to about 16 amino acid residues, about 2 to about 14 amino acid residues, about 2 to about 12 amino acid residues, or about 2 to about 10 amino acid residues. Specifically, L3 and L4 may, but are not limited to, the amino acid sequence (G4S)o (where o is an integer from 1 to 5) in Table 6 above. In addition, L3 and L4 may have different amino acid sequences.

[0112] Z Protein The Z protein may be additionally bound to the fusion protein. Here, the Z protein may be referred to as the Z1 protein or the Z2 protein. Here, the Z1 protein and the Z2 protein may be the same protein or different proteins. Here, the Z1 protein and the Z2 protein may be any one selected from the group consisting of receptors, soluble proteins, cytokines, single-chain Fv fragments (single-chain variable fragments; scFv), antibody mimetic compounds, single-domain antibodies (sdAb), therapeutic peptides, and peptide vaccines, as described above.

[0113] Here, one embodiment of the Z protein may be the extracellular domain (ECD) of a receptor or a fragment thereof. Specifically, the receptor may be a VEGF receptor, GITR (glucocorticoid-inducible TNF receptor), SIRPα, or CD80.

[0114] Here, one embodiment of the Z protein may be a soluble protein. Specifically, the soluble protein may be an IL-1R antagonist or a fragment thereof.

[0115] Here, one embodiment of the Z protein may be a cytokine. Specifically, the cytokine may be IL-2, IL-12, IL-15, IL-18, or a variant thereof.

[0116] Here, one embodiment of the Z protein may be a single-chain Fv fragment (single-chain variable fragment; scFv). Specifically, the scFv may be an scFv that specifically binds to the antigens described above. Specifically, the scFv can specifically bind to HER2, EGFR, or c-MET.

[0117] Here, one embodiment of the Z protein may be an antibody mimetic. Specifically, the antibody mimetic may be DARPin-E01.

[0118] Here, one embodiment of the Z protein may be a single-domain antibody (sdAb). Specifically, the single-domain antibody may include a CDR sequence that specifically binds to the antigen described above.

[0119] Here, one embodiment of the Z protein may be a therapeutic peptide.

[0120] Here, one embodiment of the Z protein may be a peptide vaccine. Specifically, the peptide vaccine may be any one selected from the group consisting of peptides derived from KRAS variants (G12D, G12C, G12V), p53 variants, BRAF variants, EGFRvIII, LMP1, LMP2, HPV E6 / E7, WT1, MAGE-A3, NY-ESO-1, HER2, gp100, PAP, and IDO.

[0121] Embodiments of a fusion protein A fusion protein containing one antigen-binding site and two Fc domains. i) Fusion protein with Fab as the antigen-binding site As shown in Figure 2a, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X is the heavy chain variable region and further contains CH1, and Y is the light chain variable region and contains the light chain constant region. In addition, X and Y are attached to each other by Cys in the CH1 structure and the light chain variable region to form a Fab structure, where n is 0 and CH1 is directly linked to the hinge, m is 1 and L2 contains the peptide linker. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a whole variant, and the CH3 region of C contains a knob variant. In addition, the CH3 region of B contains a whole variant, and the CH3 region of D contains a knob variant. In addition, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0122] As shown in Figure 2b, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X is the heavy chain variable region and further contains CH1, and Y is the light chain variable region and contains the light chain constant region. In addition, X and Y are attached to each other by Cys in the CH1 structure and light chain variable region to form a Fab structure, where n is 0, CH1 is directly linked to the hinge, m is 1, and L2 contains a peptide linker. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a whole variant, and the CH3 region of C contains a knob variant. In addition, the CH3 region of B contains a whole variant, and the CH3 region of D contains a knob variant. In addition, X contains a 105C mutation, and Y contains a 43C mutation, and disulfide bonds are formed between Cys. In addition, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0123] As shown in Figure 2c, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X is the heavy chain variable region and further contains CH1, and Y is the light chain variable region and contains the light chain constant region. In addition, X and Y are attached to each other by Cys in the CH1 structure and the light chain variable region to form a Fab structure, where n is 0, CH1 is directly linked to the hinge, m is 1, and L2 contains a peptide linker. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a whole variant, and the CH3 region of C contains a knob variant. In addition, the CH3 region of B contains a whole variant, and the CH3 region of D contains a knob variant. In addition, CH1 contains a 122C mutation, and the light chain constant region contains a 121C mutation, and disulfide bonds are formed between Cys. In addition, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0124] As shown in Figure 2d, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X is the heavy chain variable region and further contains CH1, and Y is the light chain variable region and contains the light chain constant region. In addition, X and Y are attached to each other by Cys in the CH1 structure and light chain variable region to form a Fab structure, where n is 0, CH1 is directly linked to the hinge, m is 1, and L2 contains a peptide linker. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a whole variant, and the CH3 region of C contains a knob variant. In addition, the CH3 region of B contains a whole variant, and the CH3 region of D contains a knob variant. In addition, X contains a 44C mutation, and Y contains a 100C mutation, and disulfide bonds are formed between Cys. In addition, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0125] As shown in Figures 6a-6d, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X is the heavy chain variable region and further contains CH1, and Y is the light chain variable region and contains the light chain constant region. In addition, X and Y are attached to each other by the CH1 structure and Cys in the light chain variable region to form a Fab structure, where n is 0 and CH1 is directly linked to the hinge. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a hole structure, and the CH3 region of C contains a knob structure. In addition, the CH3 region of B contains a hole structure, and the CH3 region of D contains a knob structure. In addition, X contains a 44C mutation, and Y contains a 100C mutation, and disulfide bonds are formed between Cys. Here, m is 0 and the light chain variable region may be directly linked to the hinge (Figure 6d). In addition, m is 1, and L2 may include a 15-mer peptide linker (Figure 6a), a 10-mer peptide linker (Figure 6b), or a 5-mer peptide linker (Figure 6c). Furthermore, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0126] As shown in Figure 19a, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X is the heavy chain variable region and further contains CH1, and Y is the light chain variable region and contains the light chain constant region. In addition, X and Y are attached to each other by Cys in the CH1 structure and light chain variable region to form a Fab structure, where n is 0, CH1 is directly linked to the hinge, m is 1, and L2 contains a peptide linker. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a whole variant, and the CH3 region of C contains a knob variant. In addition, the CH3 region of B contains a whole variant, and the CH3 region of D contains a knob variant. In addition, X contains a 44C mutation, and Y contains a 100C mutation, and disulfide bonds are formed between Cys. Here, all CH2s A, B, C, and D contain the 239D and 332E mutations. In addition, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0127] ii) Fusion protein whose antigen-binding site is Fv As shown in Figure 10, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X and Y are attached to each other by at least one Cys present in X and Y to form an Fv structure. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a whole variant, and the CH3 region of C contains a knob variant. In addition, the CH3 region of B contains a whole variant, and the CH3 region of D contains a knob variant. X may contain mutations of 44C, 105C, 122C, 44C / 105C, 44C / 122C, 105C / 126C, or 44C / 105C / 126C, and Y may contain mutations of 100C, 43C, 121C, 100C / 43C, 100C / 121C, 43C / 121C, or 100C / 43C / 121C. In addition, disulfide bonds may be formed by Cys present in L1 and L2. Furthermore, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0128] A fusion protein containing two antigen-binding sites and two Fc domains. iii) Fusion protein whose antigen-binding site is Fab As shown in FIG. 25, the fusion protein comprises polypeptides of structural formulas (I'), (II'), (III), and (IV), wherein X is a heavy chain variable region and further comprises CH1, Y is a light chain variable region and comprises a light chain constant region. In addition, X and Y are attached to each other by Cys in the CH1 structure and the light chain variable region to form a Fab structure. Here, n is 0, CH1 is directly linked to the hinge, m is 1, and L2 comprises a peptide linker. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A comprises a hole variant, and the CH3 region of C comprises a knob variant. In addition, the CH3 region of B comprises a hole variant, and the CH3 region of D comprises a knob variant. In addition, VD1 in structural formula (I') is a heavy chain variable region, VD2 in structural formula (II') is a light chain variable region, and VD1 and VD2 pair with each other to form an Fv. In addition, p and q are each 1, and L3 and L4 are peptide linkers. As a non-limiting example, X and Y can pair with each other to form the variable region of pertuzumab, and VD1 and VD2 can pair with each other to form the variable region of trastuzumab. In addition, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0129] As shown in FIG. 26, the peptide linkers L3 and L4 in structural formulas (I') and (II') may be of various lengths. In addition, the first antigen-binding site formed by the pairing between X and Y and the second antigen-binding site formed by the pairing between VD1 and VD2 may be the same or different. In addition, L1 and L2 may also comprise various peptide linkers.

[0130] A fusion protein containing one antigen-binding site including the Z protein and two Fc domains. As shown in Figure 58, the fusion protein comprises polypeptides of structural formulas (I), (II), (III), and (IV), where X is the heavy chain variable region and further contains CH1, and Y is the light chain variable region and contains the light chain constant region. In addition, X and Y are attached to each other by Cys in the CH1 structure and light chain variable region to form a Fab structure. Here, n is 0, CH1 is directly linked to the hinge, m is 1, and L2 contains a peptide linker. Here, A and C are attached to each other to form a first Fc domain, and B and D are attached to each other to form a second Fc domain. In addition, the CH3 region of A contains a hole structure, and the CH3 region of C contains a knob structure. In addition, the CH3 region of B contains a hole structure, and the CH3 region of D contains a knob structure. In addition, r, s, t, and u are 1. In addition, Z1 and Z2 are the same protein. In addition, the antigen-binding site, antigen, hinge, linker, and Fc domain are as described above.

[0131] Polynucleotides encoding fusion proteins In another embodiment of the present invention, polynucleotides encoding polypeptides of structural formulas (I), (II), (III), and / or (IV) are provided.

[0132] In another embodiment of the present invention, polynucleotides encoding polypeptides of structural formulas (I'), (II'), (III), and / or (IV) are provided.

[0133] The polynucleotide may contain nucleic acid sequences 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 with the polynucleotide encoding the polypeptide of structural formula (I), (II'), (III), and / or (IV).

[0134] The polynucleotide may further comprise nucleic acids that encode a signal sequence or a leader sequence. As used herein, the term “signal sequence” refers to a signal peptide that directs the secretion of a target protein. The signal peptide is translated and then cleaved in the host cell. Specifically, the signal sequence is an amino acid sequence that initiates the movement of a protein across the endoplasmic reticulum (ER) membrane.

[0135] Signal sequences are well known in the art in terms of their characteristics. Such signal sequences typically contain about 16 to 30 amino acid residues, although they may contain more or fewer amino acid residues. A typical signal peptide consists 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 to 12 hydrophobic residues that quiescently hold the signal sequence in place during the movement of immature polypeptides across the membrane lipid bilayer.

[0136] 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 tPa (tissue plasminogen activator), HSV gD (signal sequence of herpes simplex virus glycoprotein D), or a secretory signal sequence for growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells, including mammals, may be used. In addition, a wild-type signal sequence may be used as the signal sequence, or a signal sequence substituted with a codon that has a high expression frequency in the host cell may be used.

[0137] vectors supporting polynucleotides In another embodiment of the present invention, a vector comprising a polynucleotide is provided. The vector may comprise a polynucleotide encoding polypeptides of structural formulas (I), (II), (III), and / or (IV). In addition, the vector may comprise a polynucleotide encoding polypeptides of structural formulas (I'), (II'), (III), and / or (IV).

[0138] Vectors can be introduced into host cells to be recombined and inserted into the host cell's genome. Alternatively, vectors are understood as nucleic acid means containing polynucleotide sequences that can autonomously replicate as episomes. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and their analogues. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0139] Specifically, vectors may include plasmid DNA, phage DNA, etc., and commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis plasmids (pUB110, pTP5, etc.), yeast plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retroviruses, adenoviruses, vaccinia viruses, etc.), and insect virus vectors (baculoviruses, etc.). Since vectors exhibit different levels of protein expression and modifications depending on the host cell, it is preferable to select and use the host cell that is most appropriate for the purpose.

[0140] As used herein, the terms “gene expression” or “expression” of a target protein are understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or its fragments. Useful expression vectors may be RcCMV (Invitrogen, Carlsbad) or its variants. Expression vectors may also contain a human cytomegalovirus (CMV) promoter to promote the continuous transcription of the target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase the stability level of the post-transcriptional RNA.

[0141] Transformed cells expressing fusion proteins In another embodiment of the present invention, transformed cells that express the gene are provided. Specifically, the transformed cells may be those into which the vector has been introduced.

[0142] Host cells for transformed cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, and cells of mammalian, plant, insect, fungal, or cell origin. For example, Escherichia coli may be used as a prokaryotic cell. In addition, yeast may be used as an example of a eukaryotic cell. Furthermore, with respect to mammalian cells, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, HEK293T cells, etc., may be used. However, mammalian cells are not limited to these, and any cell known to those skilled in the art as suitable for use as a mammalian host cell may be used.

[0143] In addition, for introducing expression vectors into host cells, methods such as CaCl2 precipitation, the Hanahan method (in which efficiency is increased by using a reducing agent such as dimethyl sulfoxide (DMSO) in CaCl2 precipitation), electroporation, calcium phosphate precipitation, protoplast fusion, stirring with silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate-, lipofectamine-, and drying / inhibition-mediated transformation may be used.

[0144] As described above, the glycosylation pattern of the fusion protein (e.g., sialic acid, fucosylation, glycosylation) can be modified by manipulating glycosylation-related genes owned by the host cell in a manner known to those skilled in the art, for the purpose of optimizing the properties of the fusion protein as a therapeutic agent or for any other purpose.

[0145] Method for producing fusion proteins In another embodiment of the present invention, a method is provided for producing a fusion protein comprising an antigen-binding site, a first Fc domain or a variant thereof, and a second Fc domain or a variant thereof, the method comprising the steps of i) culturing transformed cells; and ii) recovering the produced fusion protein.

[0146] As used herein, the term "culture" refers to a method of growing microorganisms (e.g., transformed cells) under appropriately artificially controlled environmental conditions.

[0147] The method of culturing transformed cells may be carried out using methods well known in the art. Specifically, the culture may be carried out in a batch process, or may be carried out continuously in a fed-batch or repeated fed-batch process.

[0148] In addition, the step of obtaining the fusion protein from the culture product may be carried out by methods known in the art. Here, the method of obtaining is not particularly limited as long as the produced fusion protein of the present invention can be obtained. Preferably, the method of obtaining may be methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), etc.

[0149] The method of culturing transformed cells may be carried out using methods well known in the art. Specifically, the culture may be carried out in a batch process, or may be carried out continuously in a fed-batch or repeated fed-batch process.

[0150] Use of the fusion protein In another aspect of the present invention, a pharmaceutical composition for preventing or treating cancer, comprising a fusion protein as an active ingredient, is provided.

[0151] As used herein, the term "cancer" is a general term for a disease caused by cells having aggressive characteristics of dividing and growing while ignoring normal growth limits, invasive characteristics of invading surrounding tissues, and metastatic characteristics of spreading to other parts of the body, and is used in the same meaning as malignant tumor.

[0152] The pharmaceutical composition may be used for the prevention or treatment of cancer, including any one of the cancers selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, esophageal cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0153] The preferred dosage of a pharmaceutical composition varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the 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 may be contained in any amount (effective amount) depending on the application, dosage form, purpose of formulation, etc., as long as the active ingredient can exhibit therapeutic activity against tumors or, in particular, a therapeutic effect against cancer. The conventional effective amount of the active ingredient would be determined in the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, the term "effective amount" refers to the amount of the active ingredient that can induce an effect of improving or treating the condition of a disease, in particular an effect of improving or treating the condition of cancer. Such an effective amount can be experimentally determined within the scope of the common knowledge of those skilled in the art.

[0154] As used herein, the term “treatment” may be used to mean both therapeutic and preventive treatment, where prevention may be used to mean that the condition or disease in question is alleviated or reduced. In one embodiment, the term “treatment” includes both applications or any form of administration for the treatment of a disease in mammals, including humans. In addition, the term includes inhibiting or slowing the progression of a disease; restoring or repairing impaired or lost functions so that the disease is partially or completely alleviated; stimulating inefficient processes; or alleviating a serious disease.

[0155] Pharmacokinetic parameters such as bioavailability, and fundamental parameters such as clearance rate, can also affect efficacy. Therefore, "improved efficacy" (e.g., improved efficacy) may be due to improved pharmacokinetic parameters and improved efficacy, which can be measured by comparing clearance rate and other parameters such as tumor treatment or improvement in test animals or human subjects.

[0156] As used herein, the terms “therapeutic dose” or “pharmaceutical dose” refer to the amount of a compound or composition that is sufficient to treat the disease in question with a reasonable benefit-to-risk ratio applicable to medical treatment, without causing adverse effects, and that is effective in preventing or treating the disease in question. The level of the effective dose may be determined in accordance with factors including the patient’s health status, the type and severity of the disease, the activity of the drug, the patient’s sensitivity to the drug, the mode of administration, the number of doses, the route of administration and the rate of elimination, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. In one embodiment, the therapeutic dose means the amount of a drug that is effective in treating cancer.

[0157] Here, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier, insofar as it is a non-toxic substance suitable for delivery to the patient. Here, the term “pharmaceutically acceptable” means that the carrier can be adapted without having more toxicity than the target to which it is applied (prescribed) and without inhibiting the activity of the active ingredient. The carrier is used to include excipients, diluents, or adjuvants. For example, the carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, physiological saline, buffers such as PBS, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may also contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0158] Specifically, by including a pharmaceutically acceptable carrier in addition to the active ingredient, the pharmaceutical composition can be prepared into parenteral formulations according to the route of administration using conventional methods known in the art.

[0159] When a pharmaceutical composition is prepared as a parenteral formulation, it can be prepared in the form of an injection solution, transdermal patch, nasal aspirator, or suppository using a suitable carrier according to methods known in the art. When prepared as an injection solution, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as a suitable carrier; isotonic solutions such as Ringer's solution, triethanolamine, or phosphate-buffered saline (PBS) containing sterile water for injection, and 5% dextrose may be preferably used. The formulation of pharmaceutical compositions is known in the art, and specific references may be made to Remington's Pharmaceutical Sciences (Vol. 19, 1995), etc. This document is considered part of this specification.

[0160] A fusion protein or a pharmaceutical composition containing the same may be administered to a patient in a therapeutically effective or pharmaceutically effective amount.

[0161] As used herein, the term “administration” means introducing a predetermined substance into a target by an appropriate method, and the composition may be administered through any common route, insofar as the composition can reach the target tissue. The composition may be administered by intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, topical, intranasal, or rectal administration, but is not limited to these.

[0162] The preferred dosage of the pharmaceutical composition may range from 0.01 μg / kg to 10 g / kg or 0.01 mg / kg to 1 g / kg per day, depending on the patient's condition, weight, sex, age, severity of the patient's illness, and route of administration. The dosage may be administered once a day or divided into several doses per day. Specifically, administration may be once a day, 2 to 24 times a day, 1 to 2 times every 3 days, 1 to 6 times a week, 1 to 10 times every 2 weeks, 1 to 15 times every 3 weeks, 1 to 3 times every 4 weeks, or 1 to 12 times per year. However, since administration may increase or decrease depending on the route of administration, severity of the disease, sex, weight, age, etc., the scope of the present invention is not limited to such frequencies. Such dosages should not be interpreted as limiting the scope of the present invention in any embodiment.

[0163] The subjects to which the pharmaceutical composition may be applied (formulated) are mammals, including dogs, cats, and humans, and preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present invention may further contain any compound or natural extract known to have a therapeutic effect against tumors.

[0164] Therapy using fusion proteins In another aspect of the present invention, a method for preventing or treating cancer is provided, comprising the step of administering to a subject a fusion protein comprising an antigen-binding site, a first Fc domain or a variant thereof, and a second Fc domain or a variant thereof, or a pharmaceutical composition comprising the fusion protein as an active ingredient.

[0165] In another aspect of the present invention, the use of a fusion protein comprising an antigen-binding site, a first Fc domain or a variant thereof, and a second Fc domain or a variant thereof, or a pharmaceutical composition comprising the fusion protein as an active ingredient, is provided for the treatment of cancer.

[0166] The fusion proteins, pharmaceutical compositions, cancer, prevention, and treatment are as described above.

[0167] Here, the subjects may be individuals suffering from cancer. In addition, the subjects may be mammals, and preferably humans.

[0168] The route of administration, dosage, and frequency of administration of the fusion protein may vary depending on the patient's condition and the presence or absence of side effects; therefore, the fusion protein may be administered to the target by various means and in various amounts. The optimal method of administration, dosage, and frequency of administration can be appropriately selected by those skilled in the art. In addition, the fusion protein may be administered in combination with other drugs or physiologically active substances whose therapeutic effects are known for the disease being treated, or it may be formulated in the form of a combination preparation with other drugs. [Examples]

[0169] The present invention will be described in more detail thereafter by the following embodiments. However, the following embodiments are merely illustrative of the present invention, and the scope of the present invention is not limited to these embodiments.

[0170] Example 1. Design, preparation, and analysis of a novel antibody having two Fc domains. Naturally occurring human immunoglobulin G (IgG) consists of two fragment antigen-binding (Fab) domains and one fragment crystallizable (Fc) domain (Figure 1a). Human IgG binds to target antigens in a monovalent or bivalent form and, in some cases, has 0.5 to 1 Fc domain per target antigen (Figures 1c to 1e).

[0171] The objective of the present invention is to improve effector function by increasing the amount of Fc domains present per antigen, while having a molecular weight and uniform composition similar to that of antibodies. Therefore, the inventors designed a novel antibody form having two Fc domains with a molecular weight (approximately 150 kDa) similar to that of natural human IgG antibodies (Figure 1b). This form binds to cancer cell surface antigens, allowing up to four times more Fc domains to be present on the cancer cell surface compared to conventional antibodies (Figures 1b and 1f).

[0172] Trastuzumab was used as a template to implement a novel antibody form (Figure 2a). To improve the pairing between the VH-CH1 and VL-CL regions of the trastuzumab Fab domain, specific amino acids were substituted with Cys to introduce an artificial disulfide bond (Figures 2b-2d).

[0173] We designed Fabs in which glutamine (Q) at position 105 of the heavy chain and alanine (A) at position 43 of the light chain are substituted with cysteine, phenylalanine (F) at position 122 of the heavy chain and serine (S) at position 121 of the light chain are substituted with cysteine, and glycine (G) at position 44 of the heavy chain and glutamine (Q) at position 100 of the light chain are substituted with cysteine. These Fabs were designated as mutant 1, mutant 2, and mutant 3, respectively. Based on the above, the Fab-(Fc)2 structure using trastuzumab as a template was designated as the wild type (WT) (Figure 2a), and the Fab-(Fc)2 structures having Fabs corresponding to mutants 1 to 3 were designated as M1, M2, and M3, respectively (Figures 2b-2d, 3a, and 3b).

[0174] The notation of the amino acid positions constituting the antibody follows the Kabat numbering rules. To minimize unwanted Fc-related byproducts, the knob-into-hole mutation technique (Merchant et al., Nat. Biotechnol. 1998) was applied to the Fc domain of human immunoglobulin G1 (SEQ ID NO: 3) to design polypeptides with a knob mutation (S354C, T366W; SEQ ID NO: 4) and with a hole mutation (Y349C, T366S, L368A, Y407V; SEQ ID NO: 5). A (G4S)3 linker was introduced to provide additional flexibility between the CL region and the hinge (SEQ ID NO: 6). The WT consisted of polypeptides of Fc-hole (SEQ ID NO: 7), TraH-WT-knob (SEQ ID NO: 8), and TraL-WT-knob (SEQ ID NO: 9), and was prepared by co-transfection of the EXPICHO-S® (Gibco, A29127) cell line with an expression vector carrying the polynucleotides encoding these polypeptides.

[0175] M1 consists of polypeptides of Fc-hole (SEQ ID NO: 7), TraH-Q105C-knob (SEQ ID NO: 10), and TraL-A43C-knob (SEQ ID NO: 11), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotide encoding the polypeptides.

[0176] M2 consists of polypeptides of Fc-hole (SEQ ID NO: 7), TraH-F122C-knob (SEQ ID NO: 12), and TraL-S121C-knob (SEQ ID NO: 13), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotide encoding these polypeptides.

[0177] M3 consists of polypeptides of Fc-hole (SEQ ID NO: 7), TraH-G44C-knob (SEQ ID NO: 14), and TraL-Q100C-knob (SEQ ID NO: 15), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotides encoding these polypeptides.

[0178] The proteins prepared as described above were purified using an AKTA pure 25 (Cytiva) or AKTA avant 150 (Cytiva) protein isolation and purification system equipped with a CAPTURESELECT® CH1-XL pre-packed column (Thermo Scientific, 494346205). The purified products were further subjected to affinity chromatography using KappaSelect resin (Cytiva, 17545801) and then concentrated using an Amicon Ultra-15 centrifugal filter unit (Merck millipore). The absorbance at 280 nm of the final purified product was measured using a NanoDrop One trace spectrophotometer (Thermo Fisher Scientific), and the concentration was analyzed and quantified based on the sample's intrinsic decay coefficient and molecular weight.

[0179] The purified products were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) (Figures 4 and 5a-5d).

[0180] Bio-rad electrophoresis gels and systems were used for SDS-PAGE analysis, and the process was carried out under non-reducing conditions. After electrophoresis, the size of each fusion protein was determined by staining with Coomassie brilliant blue.

[0181] As a result, as shown in Figure 4, WT, M1, M2, and M3 were identified at approximately 150 kDa. Monomer bands were identified at approximately 75 kDa in WT, which lacked the additional disulfide bond introduced at the Fab interface. Similarly, trace amounts of monomer bands were identified at 75 kDa for M1 and M2. However, for M3, the monomer bands were barely identified, likely because most of the monomers readily paired due to the formation of the disulfide bond.

[0182] For size exclusion chromatography analysis, we used an Alliance® HPLC-e2695 separation module (Waters, 2695) equipped with an Agilent Bio SEC-3 HPLC column (Agilent, 5190-2511).

[0183] As shown in Figures 5a–5d, the analysis indicated that the main product was identified with a retention time of approximately 8.6 to 8.8 minutes.

[0184] Based on the M3 structure, the effect of the linker connecting the CL domain and hinge region on the structural integrity of the antibody was analyzed. M3 has a 15-mer polypeptide linker consisting of (G4S)3, V1 (SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 16) and V2 (SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 17) have polypeptide linkers of (G4S)2 and G4S, respectively, and V3 (SEQ ID NO: 7, 14, 18) is a fusion protein in which the CL domain and hinge are directly linked without a linker (Figures 6a-6d).

[0185] SDS-PAGE analysis to determine the size of the fusion protein identified the main product as approximately 150 kDa, as shown in Figure 7, while no by-products were identified. These results indicate that the presence or absence of a linker between the CL domain and the hinge does not have a significant effect on by-product formation.

[0186] Based on the above results, it was found that when the VH44 and VL100 positions of Fab are substituted with Cys, the Fab-(Fc)2 structure is stably formed.

[0187] [Table 7] JPEG2026514684000024.jpg190149 JPEG2026514684000025.jpg190149 JPEG2026514684000026.jpg184149

[0188] [Table 8] JPEG2026514684000028.jpg193149 JPEG2026514684000029.jpg190149 JPEG2026514684000030.jpg190149 JPEG2026514684000031.jpg190149 JPEG2026514684000032.jpg191149 JPEG2026514684000033.jpg191149 JPEG2026514684000034.jpg192149 JPEG2026514684000035.jpg191149 JPEG2026514684000036.jpg190149 JPEG2026514684000037.jpg213149

[0189] Table 9 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of H01. Table 10 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of H01.

[0190] [Table 9]

[0191] [Table 10]

[0192] Table 11 below shows the CDR sequences of the heavy and light chains of H01.

[0193] [Table 11]

[0194] Example 2. Preparation of a novel antibody having two Fc domains using pertuzumab as a template. M3 is characterized by a (trastuzumab Fab)-(Fc)2 structure with mutations in VH G44C and VL Q100C, hereafter referred to as "H01". Similarly, based on the VH and VL regions of pertuzumab (SEQ ID NOs. 27 and 28), a (pertuzumab Fab)-(Fc)2 structure with mutations in VH G44C and VL Q100C will hereafter be referred to as "P01".

[0195] P01 consists of polypeptides of Fc-hole (SEQ ID NO: 7), PerH-G44C-knob (SEQ ID NO: 29), and PerL-Q100C-knob (SEQ ID NO: 30), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotide encoding the polypeptides. The protein prepared as described above was purified and analyzed as a fusion protein in the same manner as in Example 1 above.

[0196] [Table 12]

[0197] [Table 13] JPEG2026514684000043.jpg43149

[0198] Table 14 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of P01. Table 15 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of P01.

[0199] [Table 14]

[0200] [Table 15]

[0201] Table 16 below shows the CDR sequences of the heavy and light chains of P01.

[0202] [Table 16]

[0203] Example 3. Analysis of H01 and P01 by-products by papain digestion. Papain recognizes a specific sequence in the hinge region and induces antibody digestion. In the case of the Fab-(Fc)2 structure, when papain digestion is performed, the Fab-(Fc)2 structure is cleaved into a Fab portion of approximately 49.3 kDa and two Fc domains of approximately 50.4 kDa (Figure 8a). However, when abnormal disulfide bonds are formed in the hinge region, unwanted interchain disulfide bond byproducts were observed. In this case, a Fab fragment of approximately 49.3 kDa and an abnormal (Fc)2 product of approximately 100.7 kDa were observed (Figure 8b).

[0204] To verify this, papain digestion of H01 and P01 was performed. Papain (Sigma, P3125) was used by diluting it to 0.1 mg / mL in digestion buffer (PBS pH 7.4 with 20 mM EDTA + 10 mM Cys-HCl). 200 μg of H01 or P01 was mixed with papain and reacted at 37°C for 2 hours, followed by SDS-PAGE.

[0205] As shown in Figures 8c and 8d, no abnormal (Fc)2 was identified in the SDS-PAGE results performed under non-reducing conditions.

[0206] Example 4. Analysis of the physical properties of H01wt In H01, a knob-into-hole mutation causes four Fc monomers to associate with two Fc dimers, resulting in the structure shown in Figure 6a.

[0207] To analyze the effect of knob-into-hole mutations on the formation of the H01 structure, the Fc hole monomer polypeptide (SEQ ID NO: 7) was replaced with a polypeptide corresponding to the wild-type IgG1 Fc monomer (SEQ ID NO: 39) (Table 17). The two knob polypeptides constituting H01 (SEQ ID NOs: 14 and 15) were also replaced with polypeptides corresponding to the wild-type IgG1 Fc monomer (SEQ ID NOs: 40 and 41) (Table 17). This novel antibody form, consisting of two wtFc polypeptides (SEQ ID NO: 39), one TraH-G44C-wtFc polypeptide (SEQ ID NO: 40), and one TraL-Q100C-wtFc polypeptide (SEQ ID NO: 41), is referred to as H01wt (Figure 9).

[0208] H01wt consists of the polypeptides wtFc (SEQ ID NO: 39), TraH-G44C-wtFc (SEQ ID NO: 40), and TraL-Q100C-wtFc (SEQ ID NO: 41), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying polynucleotides encoding these polypeptides. The protein prepared as described above was purified and analyzed in the same manner as in Example 1 above.

[0209] As shown in Figure 9, SDS-PAGE analysis under non-reducing conditions identified a small amount of H01 wt at approximately 150 kDa, with most of the H01 wt being expressed as a byproduct with an abnormal structure. On the other hand, in the case of H01 with a knob-into-hole mutation in the Fc domain, each polypeptide efficiently associated with the product identified at approximately 150 kDa.

[0210] [Table 17]

[0211] Table 18 below shows the polynucleotide sequences encoding the polypeptides HO1wt wtFc, TraH-G44C-wtFc, and TraL-Q100C-wtFc.

[0212] [Table 18] JPEG2026514684000049.jpg39149

[0213] Example 5. Characterization of the H01Fv variant Schematic diagrams of Fv-(Fc)2, in which two Fc domains are fused in parallel to the antibody Fv fragment, are shown in Figures 10a to 10g. Here, Fv consists of a VH region and a VL region. To improve the interaction of the domains at the domain interface, artificial disulfide bonds were formed by substituting amino acids with Cys at specific positions (Figures 10a to 10h, and Table 19).

[0214] [Table 19]

[0215] Table 20 shows the polypeptide sequences that make up H01Fv1 to H01Fv7.

[0216] [Table 20] JPEG2026514684000052.jpg192149 JPEG2026514684000053.jpg212149

[0217] Table 21 shows the polynucleotide sequences encoding the polypeptides that make up H01Fv1 to H01Fv7.

[0218] [Table 21] JPEG2026514684000055.jpg190149 JPEG2026514684000056.jpg191149 JPEG2026514684000057.jpg190149 JPEG2026514684000058.jpg190149 JPEG2026514684000059.jpg191149 JPEG2026514684000060.jpg190149 JPEG2026514684000061.jpg191149 JPEG2026514684000062.jpg190149 JPEG2026514684000063.jpg58149

[0219] H01Fv1 consists of the polypeptides Fc-hole-RF (SEQ ID NO: 789), H01Fv1-HC (SEQ ID NO: 790), and H01Fv1-LC (SEQ ID NO: 791), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying the polynucleotides encoding these polypeptides (Figure 10, Table 20, and Table 21). Subsequently, H01Fv1 was purified by affinity chromatography using MABSELECT® Prism A (Cytiva, 17549853). The Fc-hole polypeptide (SEQ ID NO: 7) can form an Fc-hole / Fc-hole dimer. To remove this Fc-hole / Fc-hole dimer, H435R and Y436F mutations were induced in the Fc-hole (SEQ ID NO: 7) polypeptide sequence to produce the Fc-hole-RF polypeptide (SEQ ID NO: 789). This Fc-hole-RF polypeptide can prevent Fc-hole / Fc-hole dimers from binding to the protein A resin and can remove mispaired Fc-hole / Fc-hole dimers (Figures 10 and 11).

[0220] SDS-PAGE analysis under non-reducing conditions identified the main product at approximately 130 kDa (Figure 11). In addition, monomer purity was determined by SEC analysis (Figures 12a-12f). In particular, H01Fv3 was found to exist mostly in an unpaired form at approximately 65 kDa, and the monomer purity of the fully associated form was determined to be 14.66% (Figures 11 and 12c). H01Fv1, H01Fv2, H01Fv4, H01Fv5, H01Fv6, and H01Fv7 were found to have monomer purities of 71.24%, 61.25%, 68.55%, 73.05%, 67.73%, and 79.33%, respectively.

[0221] The binding characteristics of H01Fv1, H01Fv2, H01Fv4, H01Fv5, H01Fv6, and H01Fv7 to the HER2 protein were analyzed using the biolayer interferometry (BLI) method Octet Red96e (Sartorius).

[0222] Specifically, human HER2 recombinant protein (R&D systems, 1129-ER) was loaded onto an anti-pentaHIS (HIS1K) biosensor (Sartorius, 18-5120), and then H01Fv1, H01Fv2, H01Fv4, H01Fv5, H01Fv6, and H01Fv7 were added to the binding and dissociation reactions, respectively. The binding constants were calculated, and the results are shown in Figures 13a-13f and Table 22.

[0223] [Table 22]

[0224] Example 6. Analysis of thermal stability The thermal stability of the antibody was analyzed using the Protein Thermal Shift (PROTEIN THERMAL SHIFT) dye kit (Applied biosystems, 4461146), in accordance with the manufacturer's manual.

[0225] In short, 5 μL of reaction buffer and 2.5 μL of dye included in the kit were mixed with 5 μg of trastuzumab, pertuzumab, H01, or P01, and the final volume was adjusted to 20 μL by adding PBS. The mixtures were reacted at 20°C for 30 seconds each in a C1000 thermal cycler (Bio-Rad, 1841000) equipped with a CFX96 optical reaction module (Bio-Rad, 1845096), and then the fluorescence intensity of the plates was measured while increasing the temperature from 20°C to 95°C at a rate of 1°C / min. After that, the reaction was stopped after 30 seconds at 95°C. After the reaction, the median value of relative fluorescence units (RFU) was determined, and the melting temperature (T) was measured. m An analysis of ) was conducted.

[0226] As a result, T against trastuzumab, pertuzumab, H01, and P01 m1 The values ​​were 68°C, 68°C, 66°C, and 66°C, respectively, for T against trastuzumab, pertuzumab, H01, and P01. m2 The values ​​found were 81°C, 79°C, 83°C, and 83°C, respectively. From the above results, it was found that the fusion protein according to the present invention has a Tm value similar to that of commercially available therapeutic antibodies (Figure 14 and Table 23).

[0227] [Table 23]

[0228] Example 7. Identification of competitive coupling between H01 and P01 To determine whether H01 and P01 bind to or compete for binding to different epitopes, we used Octet Red 96e, a biolayer interferometry (BLI) technique.

[0229] Human HER2 recombinant protein (R&D systems, 1129-ER) was loaded onto an anti-pentaHIS (HIS1K) biosensor, and then 100 nM human IgG1 (Bio X cell, BE0297), H01, or trastuzumab was loaded and bound to it, respectively. Subsequently, 100 nM human IgG1, P01, or pertuzumab were loaded and bound to the biosensor, respectively, and the binding of each antibody was determined.

[0230] As a result, the binding signals (nm shift from baseline) measured at equilibrium after completion of HER2 recombinant protein loading were 0.620 nm, 0.625 nm, and 0.672 nm, respectively (Figure 15 and Table 24).

[0231] The first and second analytes were sequentially bound using an association time of 900 seconds and a dissociation time of 900 seconds, and then the degree of binding was confirmed. When human IgG1 antibody was sequentially bound, it did not bind to HER2. On the other hand, sequential binding of H01 and P01, as well as sequential binding of trastuzumab and pertuzumab, was confirmed to bind to HER2, respectively.

[0232] The results indicate that the antibodies bind to different epitopes. The binding signals (nm shift from baseline) for H01+P01 and trastuzumab+pertuzumab on the HER2-supported sensor were measured to be 1.477 nm (y-axis value at 4140 s - y-axis value at 1260 s) and 0.923 nm (y-axis value at 4140 s - y-axis value at 1260 s), respectively. The binding signal (nm shift from baseline) tends to increase as more proteins bind to the biosensor surface. Therefore, this indicates that for the same amount of HER2, H01 and P01 induce more antibody binding than trastuzumab and pertuzumab (Figure 15 and Table 24).

[0233] [Table 24]

[0234] Example 8. Determination of Fc support When H01 and P01 are used in combination, a total of 16 Fc domains bind to the four HER2 antigens present on the surface of cancer cells (Figure 16a). When trastuzumab and pertuzumab are used in combination, if the binding is monovalent, 8 Fc domains bind to the four HER2 antigens present on the surface of cancer cells, and if the binding is bivalent, fewer Fc domains may bind (Figure 16b). Therefore, the combination of H01 and P01 should result in increased Fc loading on the surface of HER2-positive cancer cells compared to the combination of trastuzumab and pertuzumab, which should lead to a more potent effector function.

[0235] To confirm the above effects, antibody Fc loading on the cell surface was quantified in HER2-expressing cancer cell lines (NCI-N87 cells, BT474 cells, SK-OV-3 cells, SNU1 cells, and SNU5 cells). All cancer cell lines were cultured in RPMI-1640 medium (containing 10% FBS).

[0236] Specifically, cancer cells were inoculated into 96-well plates and then treated with human IgG1 (Bio X cell, BE0297) (50 nM), trastuzumab (TRA) (50 nM), trastuzumab (50 nM) + pertuzumab (50 nM) (TRA+PER), H01 (50 nM), or H01 (50 nM) + P01 (50 nM), respectively, and then incubated at 4°C for 30 minutes. Subsequently, the cells were treated with Alexa 488 fluorescent conjugate anti-human IgG Fcγ Fab antibody (Jackson ImmunoResearch, 109-547-008), and the antibody Fc domain bound to the cells was quantified using a flow cytometer (BD biosciences, FACSverse).

[0237] As a result, as shown in Figures 17a-17e and Table 25, the fluorescence intensity of the group treated with H01 alone was higher in all cancer cell lines compared to the group treated with TRA+PER. In addition, compared to treatment with H01 alone, an increase in Fc loading on the surface of cancer cells was observed in the group treated with H01+P01.

[0238] [Table 25]

[0239] To determine the saturation concentration for each treatment group, cells were treated with antibodies at various concentrations (final concentrations of 20 nM, 50 nM, and 100 nM) under the same conditions as described above, and the Fc domain was then quantified in the same manner as described above.

[0240] As a result, as shown in Figures 18a-18e, the saturation concentration for each treatment group was 50 nM. From the above results, it was found that treatment with 50 nM H01 alone resulted in greater Fc loading on the surface of the five cell lines compared to treatment with 50 nM trastuzumab and 50 nM pertuzumab in combination.

[0241] Example 9. Analysis of antibody binding affinity to HER2. S239D or I332E mutations in the Fc domain of antibodies improve antibody affinity to the Fcγ receptor, thereby enhancing effector function (Greg A. Lazar et al., PNAS, 2006). Therefore, H01DE4 and P01DE4 were designed by introducing S239D and I332E mutations into the Fc domains of H01 and P01, respectively (Figures 19a and 19b).

[0242] H01DE4 consists of the polypeptides Fc-hole-S239D-I332E (Table 26, SEQ ID NO: 42), TraH-G44C-knob-S239D-I332E (Table 26, SEQ ID NO: 43), and TraL-Q100C-knob-S239D-I332E (Table 26, SEQ ID NO: 44), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying the polynucleotides encoding these polypeptides.

[0243] P01DE4 consists of the polypeptides Fc-hole-S239D-I332E (Tables 26 and 27, SEQ ID NO: 42), PerH-G44C-knob-S239D-I332E (Tables 26 and 27, SEQ ID NO: 45), and PerL-Q100C-knob-S239D-I332E (Tables 26 and 27, SEQ ID NO: 46), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying the polynucleotides encoding these polypeptides.

[0244] [Table 26]

[0245] [Table 27] JPEG2026514684000070.jpg209149 JPEG2026514684000071.jpg23149

[0246] The binding characteristics of the antibodies prepared as described above were analyzed at 25°C using the biolayer interferometry (BLI) method Octet Red 96e. The buffer used for the analysis was 10× kinetic buffer (Sartorius, 18-1042) diluted in PBS (pH 7.4, Gibco, 10010), and the analysis plate was agitated at 1,000 rpm.

[0247] Human HER2 recombinant protein (R&D systems, 1129-ER) was loaded onto an anti-penta HIS (HIS1K) biosensor (Sartorius, 18-5120). Then, to measure the association rate constant (K a ), H01, H01DE4, P01, or P01DE4 at concentrations of 1 nM to 32 nM was loaded and allowed to bind to the loaded antigen for 300 seconds. Then, after dissociation for 600 seconds in kinetic buffer, the dissociation rate constant (K d ) was determined. The K a value and the K d value were analyzed using a 1:1 binding model in Octet analysis software (Sartorius) to determine the equilibrium dissociation constant (K D ) value (Figures 20a - 20d, and Table 28).

[0248]

Table 28

[0249] Example 10. Analysis of the binding affinity of antibodies to Fcγ receptors The binding constants of each antibody to Fc gamma receptors at 25°C were analyzed using Octet Red 96e, which is a biolayer interferometry (BLI).

[0250] Specifically, each of human FcγRI (R&D systems, 1257-FC), human FcγIIA (R&D systems, 1330-CD), or human FcγRIIIA (158V isoform, R&D systems, 4325-FC) containing a His tag was loaded onto an anti-penta HIS (HIS1K) biosensor, and then H01, P01, H01DE4, P01DE4, human IgG1 (Bio X cell, BE0297), trastuzumab, pertuzumab, or margetuximab was added to the binding and dissociation reactions in the same manner as above. The association rate constant (K a ), the dissociation rate constant (K d ), and the equilibrium dissociation constant (K DThe values ​​were determined (Figures 21a-21h, 22a-22h, 23a-23h, and Table 29).

[0251] [Table 29]

[0252] Example 11. Pharmacokinetic analysis in rats H01, P01, trastuzumab, or pertuzumab were administered intravenously (iv) at a concentration of 10 mg / kg per rat to 7-week-old male Sprague Dowley rats (Orientbio). Blood was collected at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, and 42 days after administration. For analysis, serum was separated from the blood, and the concentration of fusion proteins in the blood was measured by ELISA.

[0253] Specifically, 96-well ELISA plates (Corning, 3590) were coated with human HER2 recombinant protein (R&D systems, 1129-ER) and then reacted overnight at 4°C. Each well of the plate coated as described above was appropriately treated with serum and then conjugated to human HER2. After the conjugation reaction was complete, each well was treated with peroxidase-conjugated anti-human Fab goat antibody (Invitrogen, 31482), then treated with substrate to develop color, and the absorbance was measured. Standard samples of H01, P01, trastuzumab, and pertuzumab were prepared, and the concentrations of the analytes at each time point were quantified based on standard curves created from natural rat serum containing antibody standards of various concentrations.

[0254] As shown in Figures 24a, 24b, and Table 30, the half-lives of H01, P01, trastuzumab, and pertuzumab were determined to be approximately 11.8 days, 14.2 days, 7.3 days, and 11.6 days, respectively. From these results, it was found that the novel manipulated antibodies H01 and P01 have pharmacokinetic (PK) parameters at a similar level to those of the humanized antibodies trastuzumab and pertuzumab.

[0255] [Table 30]

[0256] Example 12. Design, preparation, and analysis of a novel antibody that recognizes two epitopes of HER2. To construct an antibody that recognizes two epitopes of the HER2 protein, we designed the biparatopic antibody HP501 by linking the V domains of trastuzumab and pertuzumab via a linker (Figure 25). To minimize the reduction in binding affinity due to interference between different V domains, we tested linkers with variable lengths for linking the V domains. Simultaneously, in an attempt to improve the physical integrity of the antibody, we designed 16 variants with Cys substitution mutations that can form disulfide bonds in the V domain (Figure 26 and Table 31).

[0257] Based on Kabat numbering, mutations were introduced only at position 44 of the heavy chain and position 100 of the light chain. VH linkers and VL linkers with 6 amino acid residues were designated as VH-S-linkers and VL-L-linkers, respectively, and VH linkers and VL linkers with 13 amino acid residues were designated as VH-L-linkers and VL-L-linkers, respectively (Table 31).

[0258] [Table 31]

[0259] HP501 consists of polypeptides of Fc-hole (SEQ ID NO: 7), TH-S-PH-knob (SEQ ID NO: 51), and TL-S-PL-knob (SEQ ID NO: 59), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying the polynucleotide encoding the polypeptide (Tables 31-34). The antibodies prepared as described above were purified and analyzed in the same manner as in Example 1. In addition, HP502-HP516 were expressed, purified, and analyzed in the same manner as HP501.

[0260] Tables 32-34 show the polypeptide sequences that make up the antibodies, and the polynucleotide sequences that encode them.

[0261] [Table 32] JPEG2026514684000077.jpg191149 JPEG2026514684000078.jpg191149 JPEG2026514684000079.jpg191149 JPEG2026514684000080.jpg191149 JPEG2026514684000081.jpg140149

[0262] [Table 33] JPEG2026514684000083.jpg190149 JPEG2026514684000084.jpg191149 JPEG2026514684000085.jpg191149 JPEG2026514684000086.jpg192149 JPEG2026514684000087.jpg191149 JPEG2026514684000088.jpg192149 JPEG2026514684000089.jpg191149 JPEG2026514684000090.jpg192149 JPEG2026514684000091.jpg191149 JPEG2026514684000092.jpg191149 JPEG2026514684000093.jpg191149 JPEG2026514684000094.jpg190149 JPEG2026514684000095.jpg191149 JPEG2026514684000096.jpg191149 JPEG2026514684000097.jpg192149 JPEG2026514684000098.jpg146149

[0263] [Table 34]

[0264] The purity of each antibody prepared as described above was analyzed by size exclusion chromatography in the same manner as in Example 1 above.

[0265] As shown in Figure 27 and Table 35, the analysis revealed that HP503, HP507, HP511, and HP515, in which the first V domain is wild-type and the second V domain is a Cys-substituted variant (VH 44C, VL 100C), possessed superior purity.

[0266] [Table 35]

[0267] The binding constants of HP501-HP516 to the D2 and D4 regions of the HER2 protein were determined using Octet Red 96e.

[0268] Specifically, a human HER2 recombinant protein (R&D systems, 1129-ER) was loaded onto an anti-pentaHIS (HIS1K) biosensor and then saturated with trastuzumab (100 nM) targeting the D2 region or pertuzumab targeting the D4 region.

[0269] Subsequently, each of the 16 antibodies was added at a concentration of 100 nM to the binding reaction (300 seconds) and the dissociation reaction (600 seconds), respectively, and the binding affinity of each antibody to the D2 and D4 regions was calculated.

[0270] As shown in Table 36, the binding constants of HP507, HP511, and HP515 to the D2 region were 2.285 nM, 3.267 nM, and 2.012 nM, respectively, demonstrating superior binding affinity to the D2 region compared to the other clones. HP503 had a binding constant of 8.098 nM to the D2 region, showing relatively lower binding affinity to the D2 region compared to HP507, HP511, and HP515. On the other hand, HP503 had a binding constant of 0.181 nM to the D4 region, demonstrating high binding affinity compared to HP507 (0.228 nM), HP511 (0.162 nM), and HP515 (0.227 nM).

[0271] [Table 36]

[0272] The binding constants of HP503, HP507, HP511, and HP515 to the HER2 extracellular domain (ECD) were measured using Octet Red 96e.

[0273] Specifically, human HER2 recombinant protein was loaded onto an anti-pentaHIS (HIS1K) biosensor, and then the above antibodies at various concentrations (0.25 nM, 0.5 nM, 1 nM, 2 nM, 4 nM, 8 nM) were added for binding (600 seconds) and dissociation (1800 seconds). Based on the results obtained from the above binding and dissociation reactions, the binding constant was calculated.

[0274] As shown in Figures 28a to 28d and Table 37, under the above conditions, HP503 had a coupling constant of 6.57 nM, while HP507, HP511, and HP515 had dissociation constants (<1.0E-07 1 / s) that exceeded the instrument's measurement limit.

[0275] [Table 37]

[0276] The binding constants of HP503, HP507, HP511, and HP515 to the Fc gamma receptor were analyzed using Octet Red 96e in the same manner as described above.

[0277] As shown in Figures 29a–29d and Table 38, the analysis demonstrated that HP503, HP507, HP511, and HP515 exhibit superior binding affinity to FcγRI(CD64), FcγRIIA(CD32A, 131R), or FcγRIIIA(CD16A, 158V) compared to human IgG1, trastuzumab, pertuzumab, and margetuximab (Figures 21a–21h, 22a–22h, 23a–23h, 29a–29d, and Tables 29 and 38).

[0278] [Table 38]

[0279] The binding constants of HP503, HP507, HP511, and HP515 to the neonatal Fc receptor (FcRn) were measured using an Octet Red 96e.

[0280] Specifically, HP503, HP507, HP511, HP515, human IgG1 (Bio X cell, BE0297), trastuzumab, pertuzumab, or margetuximab were loaded onto an anti-human Fab-CH1 second-generation (FAB2G) biosensor, followed by binding (120 seconds) and dissociation (120 seconds) reactions. For the analysis, kinetic buffer (Sartorius, 18-1105) was used at pH 6.0. From the above analysis results, the binding constant was calculated (Figures 30a, 30b, and Table 39).

[0281] [Table 39]

[0282] Example 13. Analysis of complement-dependent cell injury Complement-dependent cell-mediated cytotoxicity was analyzed in BT474 (HER2 3+; hyper) breast cancer cell lines or NCI-N87 (HER2 3+; hyper) gastric cancer cell lines.

[0283] Specifically, the above cell lines were inoculated into 96-well plates at a rate of 10,000 cells per well, and then treated with the cells, antibody, and human serum (Sigma, H4522) in a 1:1:1 volume ratio. Human IgG1, trastuzumab (TRA), trastuzumab + pertuzumab (TRA+PER), H01, or H01+P01 were used as antibodies, and the antibodies were sequentially diluted six times in multiples of 2 from an initial concentration of 1200 nM to be used as a 3-fold concentrate. Human serum was treated to a final concentration of 25%. The cells treated as described above were reacted for 5 hours in a humidified incubator at 37°C and 5% (v / v) CO2 conditions.

[0284] After the reaction was complete, the cells were treated with the same volume of cell titer glow reagent (Promega, G9243) as the cell culture medium, and then lysed using a plate shaker (Allsheng, MX100-4A) (500 rpm, 2 minutes). To stabilize the luminescence signal, the mixture was left at room temperature for 10 minutes, and then the luminescence was measured using a plate reader (Envision; PerkinElmer, 2105-0010), and complement-dependent cytotoxicity (CDC activity) was calculated using equation 1 below. <Equation 1> CDC activity (%) = 100 × [1 - (luminescence with experimental antibody / luminescence without antibody)]

[0285] As shown in Figures 31a and 31b, no CDC response was induced in BT474 cells treated with TRA, TRA+PER, or H01. In addition, no CDC response was induced even in NCI-N87 cells treated with human IgG1, TRA, or TRA+PER. On the other hand, a strong CDC response was confirmed to be induced in both BT474 and NCI-N87 cells when treated with the combination of H01+P01.

[0286] Similar to the present invention, techniques for increasing Fc loading and effector function of antibodies bound to the cell surface include monovalent IgG1, DuoMab, and serial Fc antibody techniques. To compare the CDC-inducing ability of antibodies of the present invention and antibodies to which these techniques are applied, monovalent IgG1 containing trastuzumab Fab (OA_TRA), DuoMab (Duo_TRA), and serial Fc antibody (serial_Fc_TRA) were constructed (Figures 31c-31e). OA_TRA consists of polypeptides OA_TRA_1, OA_TRA_2, and OA_TRA_3 (Figure 31c, Table 40, and Table 41). Duo_TRA consists of polypeptides Duo_TRA_1 and Duo_TRA_2 (Figure 31d, Table 40, and Table 41), and serial_Fc_TRA consists of polypeptides serial_Fc_TRA_1, serial_Fc_TRA_2, and serial_Fc_TRA_3 (Figure 31e, Table 40, and Table 41).

[0287] When NCI-N87 and BT474 cell lines were treated with 200 nM IgG, TRA, H01, OA_TRA, Duo_TRA, or serial_Fc_TRA, either alone or in combination with 200 nM pertuzumab, the group treated with H01 alone induced the highest CDC response (Figures 31f and 31g).

[0288] Table 40 below shows the polypeptide sequences that make up OA_TRA, Duo_TRA, and serial_Fc_TRA.

[0289] [Table 40] JPEG2026514684000106.jpg191149 JPEG2026514684000107.jpg121149

[0290] Table 41 shows the polynucleotide sequences encoding the polypeptides that make up OA_TRA, DUO_TRA, and serial_Fc_TRA.

[0291] [Table 41] JPEG2026514684000109.jpg192149 JPEG2026514684000110.jpg192149 JPEG2026514684000111.jpg191149 JPEG2026514684000112.jpg192149 JPEG2026514684000113.jpg64149

[0292] Example 14. Analysis of antibody-dependent cell-mediated cytotoxicity Antibody-dependent cell-mediated cytotoxicity of H01 was analyzed in NCI-N87 (HER2 3+; high) cells, MDA-MB-453 (HER2 2+; moderate) cells, SNU-601 (HER2 1+; low) cells, or SNU-5 (HER2 1+; low) cells.

[0293] Specifically, each of the above cells is divided into 1.0 × 10⁻⁶ cells. 4 The cells were inoculated into a 96-well plate at a concentration of individual cells / well, and then treated with H01 of the present invention. Subsequently, peripheral blood mononuclear cells (PBMCs) isolated on the same day were used as effector cells, and 1.5 × 10⁶ cells were used. 5 Cells were processed individually per well to increase the number of PBMCs to 15 times more than the number of target cells (the cancer cell line described above) (E:T ratio = 15:1). The cells were incubated for 18 hours in a humidified incubator at 37°C and 5% (v / v) CO2. After 18 hours, cytotoxicity was measured using the Cytotoxicity Detection Kit (LDH) (Roche, 11644793001), and cytotoxicity was calculated using Equation 2 below. <Equation 2> Cytotoxicity (%) = [(Test release - Spontaneous release) / (Maximum release - Spontaneous release)] × 100

[0294] In NCI-N87 (HER2 3+; high) and MDA-MB-453 (HER2 2+; moderate) cells, H01 demonstrated superior cytotoxicity at lower concentrations compared to trastuzumab (Figures 32a and 32b). In addition, cytotoxicity analysis in SNU-601 (HER2 1+; low) and SNU-5 (HER2 1+; low) cell lines showed that H01 had superior cytotoxicity compared to trastuzumab (Figures 32c and 32d).

[0295] Example 15. Evaluation of efficacy in a xenograft mouse model. The antitumor activity of the antibody according to the present invention was confirmed in a xenograft model derived from the SNU-5 (HER2 1+; low) gastric cancer cell line.

[0296] First, a 6-week-old female SCID mouse (CB-17 / NcrKoat-Prkdc scid Efficacy was evaluated using Koatech. SNU-5 cells were 1 × 10⁶ 7 The cells were suspended in PBS at a concentration of individual cells / 100 μL and then mixed in a 1:1 ratio with Matrigel® Growth Factor Reduction (GFR) Basement Membrane Matrix (Corning, 354230). The mixture (100 μL) was subcutaneously transplanted into the right flank of mice, and tumor growth was then monitored. The average tumor volume was approximately 107 mm². 3 Mice were divided into groups accordingly, and then administered intravenously once a week for a total of 6 weeks either PBS (vehicle), 5 mg / kg H01, 5 mg / kg H01 + 5 mg / kg P01, 5 mg / kg HP507, 5 mg / kg trastuzumab, or 5 mg / kg trastuzumab + 5 mg / kg pertuzumab.

[0297] As shown in Figure 33a, H01 alone demonstrated superior antitumor activity compared to trastuzumab and trastuzumab + pertuzumab. H01 + P01 induced improved antitumor activity compared to H01 alone, and HP507 was shown to induce the most potent antitumor activity.

[0298] In addition, antitumor activity was confirmed in BALB / c-nu mice (Orientbio) xenotransplanted with the SNU-5 gastric cancer cell line. In this study, 1 × 10⁶ SNU-5 cells were used. 7 The cells were suspended in PBS at a concentration of individual cells / 100 μL and then transplanted into mice in the same manner as described above. The average tumor volume was approximately 122 mm². 3 The mice were grouped accordingly, and then administered intraperitoneally (IP) twice a week for a total of 6 weeks with either PBS (vehicle), 1 mg / kg trastuzumab, 1 mg / kg pertuzumab, 0.5 mg / kg trastuzumab + 0.5 mg / kg pertuzumab, 1 mg / kg H01, 1 mg / kg P01, or 0.5 mg / kg H01 + 0.5 mg / kg P01. In addition, since antibodies were not present in the blood of SCID mice, 50 mg / kg intravenous immunoglobulin (IVIG; LIV-r, SK Plasma) was administered to all mice twice a week for 6 weeks to simulate the actual human blood environment.

[0299] As shown in Figure 33b, H01, P01, or the combination H01+P01 induced superior antitumor activity compared to trastuzumab, pertuzumab, or the combination trastuzumab+pertuzumab.

[0300] In addition, antitumor activity was confirmed in a xenograft model derived from the SNU-601 (HER2 1+; low) gastric cancer cell line. (CB-17 / NcrKoat-Prkdc) scid Koatech was used. SNU-601 cell line was divided into 1 × 10⁶ cells. 7 The cells were suspended in PBS at a concentration of individual cells / 100 μL and then transplanted into mice in the same manner as described above. The average tumor volume was approximately 142 mm². 3 Mice were divided into groups and administered intraperitoneally twice weekly for a total of 6 weeks with PBS (vehicle), 5 mg / kg H01, 5 mg / kg trastuzumab, or 5 mg / kg trastuzumab + 5 mg / kg pertuzumab. In addition, all mice were administered 50 mg / kg intravenous immunoglobulin (IVIG; LIV-r, SK Plasma) twice weekly for 6 weeks.

[0301] As shown in Figure 34, in the SNU-601 gastric cancer xenograft model, H01 alone induced the most outstanding antitumor activity.

[0302] Efficacy in a xenograft model derived from the NCI-N87 (HER2 3+; hyper) gastric cancer cell line was investigated in 6-week-old female SCID mice (CB-17 / NcrKoat-Prkdc scid NCI-N87 cells were evaluated using Koatech. 5 × 10⁶ cells were used. 6 The cells were suspended in PBS at a concentration of individual cells / 100 μL and then transplanted into mice in the same manner as described above. The average tumor volume was approximately 146 mm². 3 Mice were divided into groups accordingly, and then administered intraperitoneally twice weekly for a total of 6 weeks either PBS (vehicle), 0.2 mg / kg H01, 5 mg / kg H01, 0.2 mg / kg trastuzumab, or 5 mg / kg trastuzumab. In addition, all mice were administered 50 mg / kg intravenous immunoglobulin (IVIG; LIV-r, SK Plasma) twice weekly for 6 weeks.

[0303] As shown in Figure 35, H01 induced superior antitumor activity compared to trastuzumab at 5 mg / kg and 0.2 mg / kg. Example 16. Evaluation of antitumor activity in a CT26-HER2 syngeneic mouse model. The polynucleotide encoding the human HER2 protein (SEQ ID NO: 567, Table 42) (SEQ ID NO: 566, Table 42) was cloned into a protein expression vector containing the neomycin resistance gene (ORIGENE, PS100020) to construct the human HER2 expression vector pCMV6-AC-hHER2 (Figure 36 and Table 42).

[0304] [Table 42] JPEG2026514684000115.jpg148149

[0305] Mouse colon-derived CT26 cancer cells were transfected with the pCMV6-AC-hHER2 human HER2 expression vector using the lipofectamine 2000 transfection reagent (Invitrogen, 11668-019). Transformed cells were incubated for 14 days in culture medium containing 1 mg / mL G418 (Invivogen, ant-gn-5), and only cells transfected with the pCMV6-AC-hHER2 human HER2 expression vector were selected. The top 3% of clones in terms of HER2 expression were sorted into 96-well plates (ThermoFisher, 167008) with one cell per well using an SH800S cell sorter (SONY). Cell selection was performed by 21 days of incubation in G418-containing medium, yielding a total of eight CT26 mouse colon cancer cell line clones expressing human HER2. After staining with anti-human HER2-BV421 (BD, 744811), human HER2 expression in cell clones was monitored using a flow cytometer (Figure 37 and Table 43).

[0306] [Table 43]

[0307] Cell clones were subcultured six times for 20 days in a G418-free environment, then stained with anti-human HER2-BV421 (BD, 744811), and the level of human HER2 expression in the clones was measured. The level of human HER2 expression was not reduced in cell clones grown without G418 compared to cells grown with G418 (Figure 38 and Table 44). Hereafter, CT-26 cell clones expressing human HER2 protein will be referred to as the CT26-HER2 cell line.

[0308] [Table 44]

[0309] Cell surface Fc loading of H01 and trastuzumab was compared among parental CT26, CT26-HER2 cell lines (clones #2-60), or human cancer cell lines (SNU5, SNU601, NCI-N87). Specifically, the above cells, along with 100 nM human IgG1, trastuzumab (TRA), or H01, were mixed in a 96-well v-bottom plate (Corning, 3363) and conjugated at 4°C for 30 minutes.

[0310] Subsequently, the cells were treated with Alexa 488 fluorescent conjugate anti-human IgG Fcγ Fab antibody (Jackson ImmunoResearch, 109-547-008), and the Fc loading of the antibody in the cells was quantified using a flow cytometer.

[0311] As shown in Figure 39 and Table 45, CT26-HER2 cells (clones #2-60) were shown to express human HER2 at levels similar to those of SNU5 cells. In addition, in CT26-HER2 cells, H01 treatment resulted in increased Fc loading on the cell surface compared to trastuzumab (TRA) treatment.

[0312] [Table 45]

[0313] Next, the antitumor activity of H01 was confirmed in a syngeneic mouse model of the CT26-HER2 cell line (clone #2-60). Efficacy was evaluated using 6-week-old female Balb / c mice (Orientbio). PBS (vehicle), 5 mg / kg trastuzumab, and 5 mg / kg H01 were administered intraperitoneally twice a week for a total of 2 weeks.

[0314] As shown in Figure 40, the analysis demonstrated that H01 induced superior antitumor activity compared to trastuzumab (Figure 40).

[0315] Example 17. Design, preparation, and analysis of a novel antibody structure targeting glypican-3. Table 46 shows the polypeptide sequences of the light and heavy chains of variant antibodies that specifically recognize the glypican-3 (GPC-3) protein.

[0316] GPM01 consists of the polypeptides Fc-hole (SEQ ID NO: 7), GPM01 HC (SEQ ID NO: 67), and GPM01 LC (SEQ ID NO: 68), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotide encoding the polypeptides. The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 above. In addition, GPM02, GPM04, GPB01, GPB03, GPB04, and GPB06 were prepared, purified, and analyzed in the same manner as described above.

[0317] In this case, GPM01, GPM02, and GPM04 bind to different epitopes of the antigen in a monovalent manner and have a structure consisting of two Fc domains (Figure 41a). GPB01, GPB03, GPB04, and GPB06 have a structure in which the variable region of GPM01, GPM02, or GPM04 is linked by a polypeptide linker (SEQ ID NO: 48, SEQ ID NO: 50), bind to GPC-3 in a biparatopic manner, and have two Fc domains (Figure 41b).

[0318] [Table 46] JPEG2026514684000120.jpg191149 JPEG2026514684000121.jpg192149 JPEG2026514684000122.jpg191149 JPEG2026514684000123.jpg108149

[0319] [Table 47] JPEG2026514684000125.jpg192149 JPEG2026514684000126.jpg191149 JPEG2026514684000127.jpg191149 JPEG2026514684000128.jpg190149 JPEG2026514684000129.jpg191149 JPEG2026514684000130.jpg190149 JPEG2026514684000131.jpg190149 JPEG2026514684000132.jpg191149 JPEG2026514684000133.jpg191149 JPEG2026514684000134.jpg191149 JPEG2026514684000135.jpg191149 JPEG2026514684000136.jpg191149 JPEG2026514684000137.jpg165149

[0320] Table 48 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting GPC-3. Table 49 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting GPC-3.

[0321] [Table 48]

[0322] [Table 49] JPEG2026514684000140.jpg206149 JPEG2026514684000141.jpg149149

[0323] Table 50 below shows the CDR sequences of the heavy and light chains of engineered antibodies targeting GPC-3.

[0324] [Table 50] JPEG2026514684000143.jpg100149

[0325] The GPC-3 protein binding constants for GPM01, GPM02, GPM04, GPB01, GPB03, GPB04, and GPB06 were determined using Octet Red 96e. Specifically, human recombinant GPC-3 protein (Sino Biologicals, 10088-H08H) was loaded onto an anti-pentaHIS (HIS1K) biosensor. Each antibody was then added at various concentrations for binding (300 seconds) and dissociation (1200 seconds). The affinity of these antibodies to GPC-3 was calculated, and the results are shown in Figure 42 and Table 51.

[0326] [Table 51]

[0327] In addition, Fc loading on the surface of GPC-3-expressing cells was quantified in the HepG2 liver cancer cell line.

[0328] Specifically, HepG2 cells were treated with 100 nM human IgG1, GPM02, GPB01, GPB03, and GC3, respectively, and then reacted at 4°C for 30 minutes. Fc loading was then quantified using Alexa 488 fluorescent conjugate anti-human IgG Fcγ Fab antibody (Jackson ImmunoResearch, 109-547-008). GC33, a humanized antibody targeting GPC-3, was used as a positive control (Nakano et al., U.S. Patent No. 7,919,086).

[0329] As shown in Figure 43, higher Fc loading on the cell surface was shown to be induced by treatment with GPM02, GPB01, or GPB03 compared to GC33.

[0330] Example 18. Design, preparation, and analysis of antibody structures targeting EPH receptor A2. Table 52 shows the polypeptide sequences of the variant light chain and heavy chain of antibodies that specifically bind to the EPH receptor A2 (EphA2) protein.

[0331] EPB01 consists of the polypeptides Fc-hole (SEQ ID NO: 7), EPB01 HC (SEQ ID NO: 111), and EPB01 LC (SEQ ID NO: 112), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotide encoding the polypeptides. The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 above. In addition, EPB02, EPB03, EPB04, EPB05, EPB06, EPB07, EPB08, EPB09, EPB10, EPB11, and EPB12 were prepared, purified, and then analyzed in the same manner as described above.

[0332] The 12 antibodies have a structure in which variable regions that bind to two different epitopes of EphA2 are linked by polypeptide linkers (SEQ ID NO: 48, SEQ ID NO: 50), bind to EphA2 in a biparatopic manner, and possess two Fc domains (Figure 41b).

[0333] [Table 52] JPEG2026514684000146.jpg209149 JPEG2026514684000147.jpg209149 JPEG2026514684000148.jpg209149 JPEG2026514684000149.jpg181149

[0334] [Table 53] JPEG2026514684000151.jpg208149 JPEG2026514684000152.jpg209149 JPEG2026514684000153.jpg209149 JPEG2026514684000154.jpg209149 JPEG2026514684000155.jpg208149 JPEG2026514684000156.jpg209149 JPEG2026514684000157.jpg209149 JPEG2026514684000158.jpg208149 JPEG2026514684000159.jpg208149 JPEG2026514684000160.jpg209149 JPEG2026514684000161.jpg209149 JPEG2026514684000162.jpg209149 JPEG2026514684000163.jpg204149

[0335] Table 54 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the engineered antibody targeting EphA2. Table 55 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of the engineered antibody targeting EphA2.

[0336] [Table 54] JPEG2026514684000165.jpg208149 JPEG2026514684000166.jpg36149

[0337] [Table 55] JPEG2026514684000168.jpg206149 JPEG2026514684000169.jpg206149 JPEG2026514684000170.jpg205149 JPEG2026514684000171.jpg205149 JPEG2026514684000172.jpg205149

[0338] Table 56 below shows the CDR sequences of the heavy and light chains of engineered antibodies targeting EphA2.

[0339] [Table 56] JPEG2026514684000174.jpg208149 JPEG2026514684000175.jpg208149 JPEG2026514684000176.jpg24149

[0340] The EphA2 protein binding constants for each of EPB01, EPB02, EPB03, EPB04, EPB05, EPB06, EPB07, EPB08, EPB09, EPB10, EPB11, and EPB12 were determined using Octet Red 96e. Human recombinant EphA2 protein (Sino Biologicals, 13926-H08H) was loaded onto an anti-pentaHIS (HIS1K) biosensor, and each antibody was then added to the binding reaction (300 seconds) and dissociation reaction (1200 seconds). Based on the above results, affinity to the EphA2 protein was calculated, and the results are shown in Table 57.

[0341] [Table 57]

[0342] The ability of antibodies to inhibit the EphA2-mediated signaling pathway was analyzed using the PC-3 prostate cancer cell line.

[0343] Specifically, PC-3 cells were treated with each antibody at a concentration of 50 nM for 30 minutes, the cells were lysed, and Western blotting was performed. A 1C1 humanized antibody targeting human EphA2 was used as a positive control and was prepared based on the sequence published in the literature (Kinch et al., U.S. Patent Application Publication No. 20090304721). As primary antibodies for analysis, Akt rabbit mAb (Cell Signaling Technology, 9272), phospho-Akt(Ser473)(D9E)XP® rabbit mAb (Cell Signaling Technology, 4060), and β-actin(13E5) rabbit mAb (Cell Signaling Technology, 4970) were used. As a secondary antibody, an anti-rabbit IgG, HRP-conjugated antibody (Cell Signaling Technology, 7074) was used.

[0344] As shown in Figure 44, EPB02, EPB03, and EPB05 inhibited AKT phosphorylation at a similar level to that of the positive control (1C1).

[0345] Next, Fc loading on the cell surface was quantified in PC-3 cells. Each cell was treated with a 100 nM antibody, then incubated at 4°C for 30 minutes, and finally treated with Alexa 488 fluorescent conjugate anti-human IgG Fcγ Fab antibody. Fc loading was then quantified.

[0346] As shown in Figure 45, higher Fc loading on the cell surface was shown to be induced by treatment with EPB02, EPB03, EPB05, EPB06, EPB07, or EPB10 compared to treatment with a 1C1 humanized antibody targeting EphA2. Example 19. Design, preparation, and analysis of antibody structures targeting MET. Table 58 shows the polypeptide sequences of the variant light and heavy chains of antibodies that specifically bind to the MET protein.

[0347] MEM01 consists of polypeptides Fc-hole (SEQ ID NO: 7), MEM01 HC (SEQ ID NO: 568), and MEM01 LC (SEQ ID NO: 569), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying polynucleotides encoding these polypeptides (Figure 41a). The proteins prepared as described above were purified and analyzed in the same manner as in Example 1. In addition, MEM06 was prepared, purified, and then analyzed in the same manner as described above (Tables 58 and 59).

[0348] [Table 58]

[0349] [Table 59] JPEG2026514684000180.jpg181149

[0350] Table 60 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting MET. Table 61 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting MET.

[0351] [Table 60]

[0352] [Table 61]

[0353] Table 62 below shows the CDR sequences of the heavy and light chains of engineered antibodies targeting MET.

[0354] [Table 62]

[0355] The MET protein binding constants of MEM01 and MEM06 were determined using Octet Red 96e. Each antibody was loaded onto an anti-human Fab-CH1 second-generation (FAB2G) biosensor (Sartorius, 18-5125). Human recombinant MET protein (Sino Biologicals, 10692-H08H) was then added at various concentrations during the binding reaction (300 seconds) and dissociation reaction (600 seconds). The antibody affinity for the MET protein was calculated, and the results are shown in Figure 46 and Table 63.

[0356] [Table 63]

[0357] Fc loading on the surface of MET-expressing cells was quantified using MKN45 and SNU-5 gastric cancer cell lines. Each cell was treated with human IgG1 control, onartuzumab (produced from CHO cell lines), emibetuzumab, MEM01, or MEM06, and then Fc loading was quantified.

[0358] As a result, higher Fc loading on the surface of MET-expressing cancer cells was shown to be induced by MEM01 treatment compared to MET-targeting onartuzumab and emibetuzumab (Figures 47 and 48). Higher Fc loading on the surface of MET-expressing cancer cells was also shown to be induced by MEM06 treatment compared to emibetuzumab. MEM06 was shown to induce Fc loading at a level equivalent to that of onartuzumab (Figures 47 and 48).

[0359] Example 20. Design, preparation, and analysis of antibody structures targeting EGFR. Table 64 shows the polypeptide sequences of the variant light and heavy chains of antibodies that specifically bind to the EGFR protein.

[0360] EGM01 consists of polypeptides Fc-hole (SEQ ID NO: 7), EGM01 HC (SEQ ID NO: 590), and EGF01 LC (SEQ ID NO: 591), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying polynucleotides encoding these polypeptides. The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 (Figure 41a). In addition, EGM02, EGM03, EGM04, EGM05, and EGM06 were prepared, purified, and then analyzed in the same manner as described above.

[0361] [Table 64] JPEG2026514684000186.jpg191149 JPEG2026514684000187.jpg159149

[0362] Table 65 below shows the polynucleotide sequences encoding the heavy and light chain polypeptides of engineered antibodies targeting EGFR.

[0363] [Table 65] JPEG2026514684000189.jpg191149 JPEG2026514684000190.jpg191149 JPEG2026514684000191.jpg192149 JPEG2026514684000192.jpg190149 JPEG2026514684000193.jpg191149 JPEG2026514684000194.jpg190149 JPEG2026514684000195.jpg191149 JPEG2026514684000196.jpg39149

[0364] Table 66 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting EGFR. Table 67 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting EGFR.

[0365] [Table 66]

[0366] [Table 67] JPEG2026514684000199.jpg157149

[0367] Table 68 below shows the CDR sequences of the heavy and light chains of engineered antibodies targeting EGFR.

[0368] [Table 68]

[0369] The EGFR protein binding constants for each of EGM01-EGM05 were determined using Octet Red 96e. Each antibody was loaded onto an anti-human Fab-CH1 second-generation (FAB2G) biosensor, and human recombinant EGFR protein (Sino Biologicals, 10692-H08H) was added at various concentrations during the binding reaction (300 seconds) and dissociation reaction (600 seconds). The affinity of the antibodies to EGFR protein was calculated, and the results are shown in Figure 49 and Table 69.

[0370] [Table 69]

[0371] Example 21. Design, preparation, and analysis of a novel antibody structure targeting CD33. Table 70 shows the polypeptide sequences of the light and heavy chains of variant antibodies that specifically recognize the CD33 protein.

[0372] GPM01 consists of polypeptides Fc-hole (SEQ ID NO: 7), 33-1 HC (SEQ ID NO: 636), and 33-1 LC (SEQ ID NO: 637), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotides encoding these polypeptides (Figure 41a). The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 above. In addition, 33-2, 33-3, 33-4, 33-5, 33-6, and 33-7 were prepared, purified, and analyzed in the same manner as described above (Figures 41a, 41b, Table 70, and Table 71).

[0373] Substances 33-1, 33-2, and 33-3 bind to different epitopes of the antigen in a monovalent manner and have a structure consisting of two Fc domains (Figure 41a). Substances 33-4, 33-5, 33-6, and 33-7 have a structure in which the variable region of the CD33 antibody is linked by a polypeptide linker (SEQ ID NO: 48, SEQ ID NO: 50), bind to CD33 in a biparatopic manner, and have two Fc domains (Figure 41b).

[0374] [Table 70] JPEG2026514684000203.jpg209149 JPEG2026514684000204.jpg124149

[0375] [Table 71] JPEG2026514684000206.jpg209149 JPEG2026514684000207.jpg208149 JPEG2026514684000208.jpg209149 JPEG2026514684000209.jpg208149 JPEG2026514684000210.jpg209149 JPEG2026514684000211.jpg208149 JPEG2026514684000212.jpg51149

[0376] Table 72 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting CD33. Table 73 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting CD33.

[0377] [Table 72]

[0378] [Table 73] JPEG2026514684000215.jpg206149 JPEG2026514684000216.jpg149149

[0379] Table 74 below shows the CDR sequences of the heavy and light chains of engineered antibodies targeting CD33.

[0380] [Table 74] JPEG2026514684000218.jpg96149

[0381] The CD33 protein binding constants for each of the antibodies 33-1, 33-2, 33-3, 33-4, 33-5, 33-6, and 33-7 were determined using Octet Red 96e. Human recombinant CD33 protein (Sino Biologicals, 12238-H08H) was loaded onto an anti-pentaHIS (HIS1K) biosensor, and each antibody was then added at various concentrations for binding (600 seconds) and dissociation (1200 seconds). The affinity of these antibodies to the CD33 protein was calculated, and the results are shown in Figure 50 and Table 75.

[0382] [Table 75]

[0383] Example 22. Design, preparation, and analysis of antibody structures targeting CEACAM5. Table 76 shows the polypeptide sequences of the variant light and heavy chains of antibodies that specifically bind to the CEACAM5 protein.

[0384] CEA01 consists of the polypeptides Fc-hole (SEQ ID NO: 7), CEA01 HC (SEQ ID NO: 590), and CEA01 LC (SEQ ID NO: 591), and was prepared by co-transfection of the EXPICHO-S® cell line with an expression vector carrying the polynucleotides encoding these polypeptides (Figure 41a). The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 above. In addition, CEA02, CEA03, and CEA04 were prepared, purified, and analyzed in the same manner as described above.

[0385] [Table 76] JPEG2026514684000221.jpg76149

[0386] Table 77 below shows the polynucleotide sequences encoding the heavy and light chain polypeptides of engineered antibodies targeting CEACAM5.

[0387] [Table 77] JPEG2026514684000223.jpg209149 JPEG2026514684000224.jpg209149 JPEG2026514684000225.jpg140149

[0388] Table 78 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting CEACAM5. Table 79 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting CEACAM5.

[0389] [Table 78]

[0390] [Table 79] JPEG2026514684000228.jpg141149

[0391] Table 80 below shows the CDR sequences of the heavy and light chains of engineered antibodies targeting CEACAM5.

[0392] [Table 80]

[0393] The CEACAM5 protein binding constants for each of CEA01 to CEA04 were determined using Octet Red 96e. Human CEACAM5 recombinant protein (Sino Biologicals, 11077-H08H) was loaded onto an anti-pentaHIS (HIS1K) biosensor, and each antibody was then added at various concentrations for binding (600 seconds) and dissociation (1200 seconds). The affinity of these antibodies to human CEACAM5 protein was calculated, and the results are shown in Figure 51 and Table 81.

[0394] [Table 81]

[0395] Example 23. Design, preparation, and analysis of antibody structures targeting TROP2, mesothelin, or LIV-1. Table 82 shows the polypeptide sequences of the variant light and heavy chains of antibody T01, which specifically binds to the TROP2 protein.

[0396] T01 consists of polypeptides Fc-hole (SEQ ID NO: 7), T01 HC (SEQ ID NO: 753), and T01 LC (SEQ ID NO: 754), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying polynucleotides encoding these polypeptides (Figure 41a). The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 above.

[0397] Table 82 shows the polypeptide sequences of the variant light and heavy chains of the antibody MSM01, which specifically binds to the mesothelin protein.

[0398] MSM01 consists of polypeptides Fc-hole (SEQ ID NO: 7), MSM01 HC (SEQ ID NO: 755), and MSM01 LC (SEQ ID NO: 756), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying polynucleotides encoding these polypeptides (Figure 41a). The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 above.

[0399] Table 82 shows the polypeptide sequences of the variant light and heavy chains of the antibody LIM01, which specifically binds to the LIV-1 protein.

[0400] LIM01 consists of the polypeptides Fc-hole (SEQ ID NO: 7), LIM01 HC (SEQ ID NO: 757), and LIM01 LC (SEQ ID NO: 758), and was prepared by co-transfection of the ExpiCHO-S® cell line with an expression vector carrying the polynucleotides encoding these polypeptides (Figure 41a). The proteins prepared as described above were purified and analyzed in the same manner as in Example 1 above.

[0401] [Table 82]

[0402] Table 83 below shows the polynucleotide sequences encoding the heavy and light chain polypeptides of engineered antibodies targeting TROP2, mesothelin, or LIV-1.

[0403] [Table 83] JPEG2026514684000233.jpg202149 JPEG2026514684000234.jpg144149

[0404] Table 84 below shows the polypeptide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting TROP2, mesothelin, or LIV-1. Table 85 below shows the polynucleotide sequences encoding the polypeptides of the heavy chain variable region (VH) and light chain variable region (VL) of engineered antibodies targeting TROP2, mesothelin, or LIV-1.

[0405] [Table 84]

[0406] [Table 85]

[0407] Table 86 below shows the CDR sequences of the heavy and light chains of engineered antibodies targeting TROP2, mesothelin, or LIV-1.

[0408] [Table 86]

[0409] The binding constants of each antibody to TROP2, mesothelin, or LIV-1 were determined using Octet Red 96e. Human TROP2 recombinant protein (Sino Biologicals, 10428-H08H), human mesothelin recombinant protein (Sino Biologicals, 13128-H08H), or human LIV-1 recombinant protein (Acro biosystems, LV1-H5223) were loaded onto an anti-pentaHIS (HIS1K) biosensor. Each antibody was then added at various concentrations for binding (600 seconds) and dissociation (1200 seconds). The affinity of each antibody to TROP2, mesothelin, or LIV-1 protein was calculated, and the results are shown in Figure 52 and Table 87.

[0410] [Table 87]

[0411] Example 24. Design, preparation, and analysis of a novel antibody format having two Fc domains. Natural human immunoglobulin G1 consists of two fragment antigen-binding (Fab) domains and one fragment crystallizable (Fc) domain (Figure 1a). Human IgG1 binds to target antigens in a monovalent or bivalent form and, in some cases, has 0.5 to 1 Fc domain per target antigen (Figures 53b to 53d). The object of the present invention is to improve effector function by increasing the Fc load per antigen while having a molecular weight and uniform composition similar to that of antibodies. Therefore, the inventors designed a novel antibody form having two Fc domains with a molecular weight (approximately 150 kDa) similar to that of natural human IgG1 antibody (Figure 53a). This antibody form can bind to cancer cell surface antigens and may have up to four times the Fc load on the cancer cell surface compared to existing antibodies (Figure 53e).

[0412] Polypeptide 1 (Figure 54) of the novel antibody form according to the present invention consists of a heavy chain variable region (X), a heavy chain CH1 region (A), a linker (L1), and a hinge-CH2 region-CH3 (C) with a knob mutation; Polypeptide 2 consists of a light chain variable region (Y), a light chain CL region (B), a linker (L2), and a hinge-CH2 region-CH3 (D) with a knob mutation; Polypeptide 3 consists of a linker (L3) and a hinge-CH2 region-CH3 (E) with a hole mutation; Polypeptide 4 consists of a linker (L4) and a hinge-CH2 region-CH3 (F) with a hole mutation. In addition, the antibody form may include Cys mutations in the heavy chain variable region and light chain variable region, CH1 and light chain constant region, or in the heavy chain variable region and light chain variable region to induce additional interchain disulfide bonds, in addition to the naturally occurring disulfide bonds connecting the heavy and light chains of the Fab region. H01 is a novel antibody format consisting of two Fc regions and one trastuzumab Fab region.

[0413] Example 25. Analysis of the binding affinity of a novel antibody format having two Fc domains to the Fcγ receptor. Novel antibody forms possessing two Fc domains can exhibit improved binding affinity to the Fcγ receptor, which mediates the antibody's effector functions (ADCC, ADCP, and CDC), due to increased avidity compared to human IgG1 antibodies. To compare the binding affinity of trastuzumab and H01 to the Fcγ receptor, the binding constants of each antibody to the Fcγ receptor were determined using Octet Red 96e. Human FcγRI, human FcγIIA (131H isoform), or human FcγRIIIIA (158V isoform) were loaded onto an anti-pentaHIS (HIS1K) biosensor, and trastuzumab and H01, respectively, were added to the binding and dissociation reactions. The association rate constant (Ka) and dissociation rate constant (K) were determined. d The value was calculated using a 1:1 coupling model in octet analysis software (Sartorius), and the equilibrium dissociation constant (K) was calculated. D The value was calculated.

[0414] As shown in Figures 55a-55d and Table 88, H01 showed higher binding affinity to FcγRI(CD64), FcγRIIA(CD32A, 131H), and FcγRIIIA(CD16A, 158V) compared to trastuzumab.

[0415] [Table 88]

[0416] Example 26. Analysis of the binding affinity of a novel antibody format having two Fc domains to immune cells. Novel antibody formulations possessing two Fc domains can exhibit improved binding affinity to human immune cells expressing the Fcγ receptor, due to enhanced Fcγ receptor avidity.

[0417] To compare the binding affinity of human trastuzumab and H01 to human immune cells, each antibody was labeled with Vivotag 645 (Perkin Elmer), and the protein-to-dye ratio was measured. Peripheral blood mononuclear cells (PBMCs) (2 × 10⁶) were isolated from the blood of donor 1 (Figure 56a) and donor 2 (Figure 56b). 5 Individual cells (per well) were treated with various concentrations of the above antibodies, and then reacted at 37°C for 45 minutes. Afterward, the antibody-bound cells were washed, fixed with 2% paraformaldehyde, and permeabilized. The binding affinity of each antibody to immune cells was then determined using a flow cytometer. Binding affinity to T cells that do not express the Fcγ receptor was used as a negative control.

[0418] As shown in Figures 56a and 56b, the analysis showed that H01 bound to natural killer (NK) cells and dendritic (DC) cells more than human trastuzumab. Example 27. Analysis of antibody-dependent cell-mediated cytotoxicity in a novel antibody form having two Fc domains. The ADCC activity of trastuzumab and H01 was compared in SNU-5 gastric cancer cell lines (HER2 1+; low) and ZR-75-1 breast cancer cell lines (HER2 1+; low).

[0419] Specifically, each of the above cancer cell lines was divided into 1 × 10⁻¹⁶ cells. 4 Cells were inoculated into 96-well plates at individual cell / well concentrations and then treated with antibodies at each concentration. Subsequently, peripheral blood mononuclear cells (PBMCs) isolated on the same day were used as effector cells, and 1.5 × 10⁶ cells were used. 5 Individual cells / well were used to increase the number of PBMCs to 25 times the number of target cells (the cancer cell line described above) (E:T ratio = 15:1). The cells were incubated for 18 hours in a humidified incubator at 37°C and 5% (v / v) CO2. Cytotoxicity was measured using a cytotoxicity detection kit (LDH) and calculated using equation 2 described above.

[0420] As shown in Figure 57, cytotoxicity analysis in the SNU-5 gastric cancer cell line (HER2 1+; low) and the ZR-75-1 breast cancer cell line (HER2 1+; low) demonstrated that H01 had superior cytotoxicity compared to trastuzumab.

[0421] Example 28. Design, preparation, and analysis of a novel antibody fusion protein having two Fc domains. By fusing additional specific proteins to the N-terminus of the hinge of the novel antibody form in Example 24 above, it may acquire new therapeutic functions.

[0422] Antibody polypeptide 1 consists of a heavy chain variable region (X), a heavy chain CH1 region (A), a linker (L1), and a hinge-CH2 region-CH3 (C) with a knob mutation; polypeptide 2 consists of a light chain variable region (Y), a light chain CL region (B), a linker (L2), and a hinge-CH2 region-CH3 (D) with a knob mutation; polypeptide 3 consists of a specific additive protein (Z), a linker (L3), and a hinge-CH2 region-CH3 (E) with a hole mutation; polypeptide 4 consists of a specific additive protein (Z), a linker (L4), and a hinge-CH2 region-CH3 (F) with a hole mutation. The four polypeptides are assembled into an engineered antigen-binding protein having two Fc domains and one Fab formed by the fusion of two Z proteins. Z proteins include receptors, soluble proteins, cytokines, single-chain Fv fragments (single-chain variable fragments; scFv), antibody mimetic compounds, single-domain antibodies (sdAbs), therapeutic peptides, peptide vaccines, etc., which can be fused to novel antibody forms (Figure 58).

[0423] For example, trastuzumab and pertuzumab bind to different domains of the HER2 antigen, and therefore a novel antibody form consisting of two Fc domains, the trastuzumab Fab region, and the pertuzumab scFv(Z), could function like a biparatopic antibody that binds to two different sites on a single antigen (Figure 59a).

[0424] For example, a novel antibody configuration consisting of two Fc domains, an anti-antigen B Fab region, and a receptor (Z) can target antigen B on the cell surface and simultaneously neutralize soluble antigen C or D (Figures 59b and 59c).

[0425] For example, a novel antibody configuration consisting of two Fc domains, an anti-antigen B Fab region, and an anti-antigen E protein (Z) can simultaneously bind to antigens B and E on the cell surface to exert a therapeutic effect (Figure 59d).

[0426] H01 according to the present invention is a novel antibody format consisting of two Fc domains and one trastuzumab Fab region, and excluding the Z region (Figure 54 and Table 89).

[0427] H04, H05, H06, H07, and H08 were prepared by fusing VEGFR1 domain 2-VEGFR2 domain 3, GITR, IL-1R antagonist, IL2, antibody mimetic (E01 DARPin), and single-domain mAb (VHH domain 9G8) to the H01 antibody template (Tables 89-93).

[0428] Specifically, H01, H04, H05, H06, H07, H08, and H09 were prepared by co-transfecting the EXPICHO-S® (Gibco, A29127) cell line with polynucleotides encoding four different polypeptides. The transfected cells were incubated for 12 days, then the culture solution was filtered and purified using an AKTA Pure 25 or AKTA Avant 25 protein isolation and purification system equipped with a Capture Select® CH1-XL pre-packed column. The purified product was buffer-changed, concentrated using an Amicon Ultra-15 centrifugal filter unit, and then quantified using a NanoDrop One Trace spectrophotometer. The final purified product was identified by SDS-PAGE in the same manner as in Example 1 above.

[0429] As shown in Figure 60, four polypeptides of appropriate size were identified under reducing conditions for all of H01, H04, H05, H06, H07, H08, and H09 (Figure 60A and Figure 60B). Analysis showed that a considerable number of mispairing impurities were identified in H04 under non-reducing conditions, but H01, H05, H06, H07, H08, and H09 were successfully assembled into intact forms (Figure 60C).

[0430] Table 89 below shows information regarding the composition of the novel antibody form of the present invention.

[0431] [Table 89]

[0432] Table 90 below shows the polypeptide sequences of each region that constitute the novel antibody form.

[0433] [Table 90] JPEG2026514684000242.jpg185149 JPEG2026514684000243.jpg185149 JPEG2026514684000244.jpg186149 JPEG2026514684000245.jpg185149 JPEG2026514684000246.jpg186149 JPEG2026514684000247.jpg105149

[0434] Table 91 below shows the polynucleotide sequences encoding polypeptides in each region that constitute the novel antibody form.

[0435] [Table 91] JPEG2026514684000249.jpg184149 JPEG2026514684000250.jpg185149 JPEG2026514684000251.jpg185149 JPEG2026514684000252.jpg185149 JPEG2026514684000253.jpg185149 JPEG2026514684000254.jpg185149 JPEG2026514684000255.jpg185149 JPEG2026514684000256.jpg184149 JPEG2026514684000257.jpg184149 JPEG2026514684000258.jpg185149 JPEG2026514684000259.jpg184149 JPEG2026514684000260.jpg185149 JPEG2026514684000261.jpg185149 JPEG2026514684000262.jpg184149 JPEG2026514684000263.jpg185149 JPEG2026514684000264.jpg43149

[0436] Table 92 below shows the polypeptide sequences that make up the novel antibody form.

[0437] [Table 92] JPEG2026514684000266.jpg185149 JPEG2026514684000267.jpg185149 JPEG2026514684000268.jpg185149 JPEG2026514684000269.jpg185149 JPEG2026514684000270.jpg185149 JPEG2026514684000271.jpg185149 JPEG2026514684000272.jpg136149

[0438] Table 93 below shows the polynucleotide sequences encoding each polypeptide that constitutes the novel antibody form.

[0439] [Table 93] JPEG2026514684000274.jpg186149 JPEG2026514684000275.jpg186149 JPEG2026514684000276.jpg185149 JPEG2026514684000277.jpg186149 JPEG2026514684000278.jpg187149 JPEG2026514684000279.jpg186149 JPEG2026514684000280.jpg186149 JPEG2026514684000281.jpg186149 JPEG2026514684000282.jpg186149 JPEG2026514684000283.jpg186149 JPEG2026514684000284.jpg186149 JPEG2026514684000285.jpg186149 JPEG2026514684000286.jpg186149 JPEG2026514684000287.jpg185149 JPEG2026514684000288.jpg186149 JPEG2026514684000289.jpg186149 JPEG2026514684000290.jpg185149 JPEG2026514684000291.jpg186149 JPEG2026514684000292.jpg186149 JPEG2026514684000293.jpg186149 JPEG2026514684000294.jpg203149

[0440] Example 29. Design, preparation, and analysis of a novel scFv fusion antibody format having two Fc domains. The engineered antibody form of Example 24 described above, consisting of two Fc domains and one Fab region, can form a biparatopic engineered antibody form or a bispecific engineered antibody form in which a single-chain Fv fragment (single-chain variable fragment; scFv) is fused to the N-terminus of the hinge region, thereby possessing novel therapeutic functions (Figure 54).

[0441] Antibody polypeptide 1 consists of a heavy chain variable region (X), a heavy chain CH1 region (A), a linker (L1), and a hinge-CH2 region-CH3 (C) with a knob mutation; polypeptide 2 consists of a light chain variable region (Y), a light chain CL region (B), a linker (L2), and a hinge-CH2 region-CH3 (D) with a knob mutation; polypeptide 3 consists of a single-chain Fv fragment (Z), a linker (L3), and a hinge-CH2 region-CH3 (E) with a hole mutation; polypeptide 4 consists of a single-chain Fv fragment (Z), a linker (L4), and a hinge-CH2 region-CH3 (F) with a hole mutation (Figure 54). The four polypeptides are assembled into an engineered antigen-binding protein having two Fc domains and one Fab formed by the fusion of two single-chain Fv fragments (Figure 58).

[0442] H01P and P01H are biparatopic antigen-binding proteins having two Fc domains and one Fab region formed by the fusion of two single-chain Fv fragments. H01P and P01H specifically recognize HER2 and bind to HER2-overexpressing cancer cells in the same manner as shown in Figure 55, thereby exhibiting antitumor activity (Figures 58, 59a, and Table 94).

[0443] ME13, ME14, ME15, and ME16 are bispecific antigen-binding proteins with two Fc domains that simultaneously bind to MET and EGFR. ME13, ME14, ME15, and ME16 bind to cancer cells that simultaneously overexpress MET and EGFR in the same manner as shown in Figure 55d, thereby exhibiting antitumor activity (Figures 58, 59d, and Table 94).

[0444] CE05 is a bispecific antigen-binding protein with two Fc domains that simultaneously binds to CEACAM5 and EGFR. In cancer cells where CEACAM5 and EGFR are simultaneously overexpressed, CE05 binds in the same manner as shown in Figure 55d, thereby exhibiting antitumor activity (Figures 58, 59d, and Table 94).

[0445] H01P, P01H, ME13, ME14, ME15, ME16, and CE05 were prepared, purified, and analyzed in the same manner as in Example 28 above by co-transfecting the EXPICHO-S(trademark) cell line with polynucleotides encoding each of the four polypeptides.

[0446] As shown in Figure 61, four polypeptides of appropriate size were identified under reducing conditions for all of H01P, P01H, ME13, ME14, ME15, ME16, and CE05. Analysis showed that under non-reducing conditions, H01P, P01H, ME13, ME14, ME15, ME16, and CE05 all assembled into intact morphologies.

[0447] Table 94 below shows the composition of biparatopic or bispecific modified antibody forms.

[0448] [Table 94]

[0449] Table 95 below shows the polypeptide sequences of each domain in the biparatopic or bispecific modified antibody forms.

[0450] [Table 95] JPEG2026514684000297.jpg188149 JPEG2026514684000298.jpg188149 JPEG2026514684000299.jpg188149 JPEG2026514684000300.jpg188149 JPEG2026514684000301.jpg188149 JPEG2026514684000302.jpg201149

[0451] Table 96 below shows the polynucleotide sequences encoding polypeptides for each domain of biparatopic or bispecific manipulated antibody forms.

[0452] [Table 96] JPEG2026514684000304.jpg188149 JPEG2026514684000305.jpg187149 JPEG2026514684000306.jpg188149 JPEG2026514684000307.jpg187149 JPEG2026514684000308.jpg188149 JPEG2026514684000309.jpg188149 JPEG2026514684000310.jpg187149 JPEG2026514684000311.jpg187149 JPEG2026514684000312.jpg187149 JPEG2026514684000313.jpg187149 JPEG2026514684000314.jpg188149 JPEG2026514684000315.jpg187149 JPEG2026514684000316.jpg187149 JPEG2026514684000317.jpg187149 JPEG2026514684000318.jpg187149 JPEG2026514684000319.jpg188149 JPEG2026514684000320.jpg187149 JPEG2026514684000321.jpg37149

[0453] Table 97 below shows the polypeptide sequences that make up the novel antibody form.

[0454] [Table 97] JPEG2026514684000323.jpg187149 JPEG2026514684000324.jpg187149 JPEG2026514684000325.jpg187149 JPEG2026514684000326.jpg186149 JPEG2026514684000327.jpg187149 JPEG2026514684000328.jpg187149 JPEG2026514684000329.jpg187149 JPEG2026514684000330.jpg143149

[0455] Table 98 below shows the polynucleotide sequences encoding each polypeptide that constitutes the novel antibody form.

[0456] [Table 98] JPEG2026514684000332.jpg184149 JPEG2026514684000333.jpg184149 JPEG2026514684000334.jpg185149 JPEG2026514684000335.jpg184149 JPEG2026514684000336.jpg185149 JPEG2026514684000337.jpg185149 JPEG2026514684000338.jpg185149 JPEG2026514684000339.jpg185149 JPEG2026514684000340.jpg185149 JPEG2026514684000341.jpg184149 JPEG2026514684000342.jpg184149 JPEG2026514684000343.jpg185149 JPEG2026514684000344.jpg184149 JPEG2026514684000345.jpg185149 JPEG2026514684000346.jpg184149 JPEG2026514684000347.jpg183149 JPEG2026514684000348.jpg185149 JPEG2026514684000349.jpg184149 JPEG2026514684000350.jpg184149 JPEG2026514684000351.jpg184149 JPEG2026514684000352.jpg185149 JPEG2026514684000353.jpg185149 JPEG2026514684000354.jpg184149 JPEG2026514684000355.jpg185149 JPEG2026514684000356.jpg80149

[0457] Example 30. Design, preparation, and analysis of a novel peptide vaccine fusion antibody format having two Fc domains. The manipulated antibody format of Example 24 described above, consisting of two Fc domains and one Fab region, can fuse with a peptide vaccine at the N-terminus of its hinge, thereby providing an anti-cancer vaccine antibody format that may possess novel therapeutic functions (Figure 58).

[0458] Antibody polypeptide 1 consists of a heavy chain variable region (X), a heavy chain CH1 region (A), a linker (L1), and a hinge-CH2 region-CH3 (C) with a knob mutation; polypeptide 2 consists of a light chain variable region (Y), a light chain CL region (B), a linker (L2), and a hinge-CH2 region-CH3 (D) with a knob mutation; polypeptide 3 consists of a peptide vaccine (Z), a linker (L3), and a hinge-CH2 region-CH3 (E) with a hole mutation; polypeptide 4 consists of a peptide vaccine (Z), a linker (L4), and a hinge-CH2 region-CH3 (F) with a hole mutation (Figure 58). The four polypeptides are assembled into an engineered antigen-binding protein having two Fc domains and one Fab region fused with two or four peptide vaccines (Z) (Figure 58).

[0459] EAVC-P1, EAVC-P2, EAVC-P3, EAVC-P4, EAVC-P5, EAVC-P6, EAVC-P7, EAVC-P8, EAVC-C3, EAVC-I3, EAVC-L3, EAVC-M3, and EAVC-N3, which specifically bind to EGFR and contain the KRAS protein as the Z protein, were prepared by co-transfection of the EXPICHO-S® cell line with polynucleotides encoding four different polypeptides (Tables 99-101). The proteins prepared as described above were purified and quantified in the same manner as in Example 28, and then identified by SDS-PAGE.

[0460] As shown in Figure 62, four polypeptides of appropriate size were identified under reducing conditions for all of EAVC-P1, EAVC-P2, EAVC-P3, EAVC-P4, EAVC-P5, EAVC-P6, EAVC-P7, EAVC-P8, EAVC-C3, EAVC-I3, EAVC-L3, EAVC-M3, and EAVC-N3. Analysis showed that under non-reducing conditions, all of EAVC-P1, EAVC-P2, EAVC-P3, EAVC-P4, EAVC-P5, EAVC-P6, EAVC-P7, EAVC-P8, EAVC-C3, EAVC-I3, EAVC-L3, EAVC-M3, and EAVC-N3 were successfully assembled into intact morphologies.

[0461] In addition, HAVC01, HAVC02, HAVC03, HAVC04, HAVC05, HAVC06, HAVC07, and HAVC08, which specifically bind to HER2 and contain the KRAS protein as the Z protein, were prepared and analyzed in the same manner as described above (Tables 99-101).

[0462] As shown in Figure 63, in all of HAVC01, HAVC02, HAVC03, HAVC04, HAVC05, HAVC06, HAVC07, and HAVC08, four polypeptides of appropriate size were identified under reducing conditions. Analysis showed that under non-reducing conditions, all of HAVC01, HAVC02, HAVC03, HAVC04, HAVC05, HAVC06, HAVC07, and HAVC08 were successfully assembled into intact morphologies (Figure 63).

[0463] In addition, size exclusion chromatography (SEC) analysis was performed on HAVC01, HAVC02, HAVC03, HAVC04, HAVC05, HAVC06, HAVC07, and HAVC08. An Arc® Premier UHPLC system (Waters, 176019001, 176017033) equipped with XBridge ProBEH200 SEC guard columns (Waters, 176004334) and XBridge ProBEH200 SEC columns (Waters, 176004335) was used, with 0.15 M NaCl (pH 6.8 in PBS) as the mobile phase at a flow rate of 0.38 mL / min. A photodiode array (PDA) detector (Waters, 176019010) was used as the detector for analysis.

[0464] As shown in Figures 64a, 64b, and Table 102, the main products were identified with retention times of approximately 3.7 to 3.8 minutes.

[0465] Table 99 below shows the composition of novel engineered antibody forms conjugated with peptide vaccines.

[0466] [Table 99] JPEG2026514684000358.jpg58149

[0467] Table 100 below shows the polypeptide sequences of each domain in a novel, engineered antibody format conjugated with a peptide vaccine.

[0468] [Table 100] JPEG2026514684000360.jpg187149 JPEG2026514684000361.jpg187149 JPEG2026514684000362.jpg187149 JPEG2026514684000363.jpg187149 JPEG2026514684000364.jpg188149 JPEG2026514684000365.jpg187149 JPEG2026514684000366.jpg187149 JPEG2026514684000367.jpg187149 JPEG2026514684000368.jpg187149 JPEG2026514684000369.jpg187149 JPEG2026514684000370.jpg187149 JPEG2026514684000371.jpg188149 JPEG2026514684000372.jpg188149 JPEG2026514684000373.jpg187149 JPEG2026514684000374.jpg187149 JPEG2026514684000375.jpg187149 JPEG2026514684000376.jpg188149 JPEG2026514684000377.jpg187149 JPEG2026514684000378.jpg187149 JPEG2026514684000379.jpg87149

[0469] Table 101 below shows the polypeptide sequences that make up the novel antibody structure.

[0470] [Table 101] JPEG2026514684000381.jpg187149 JPEG2026514684000382.jpg188149 JPEG2026514684000383.jpg187149 JPEG2026514684000384.jpg187149 JPEG2026514684000385.jpg187149 JPEG2026514684000386.jpg187149 JPEG2026514684000387.jpg188149 JPEG2026514684000388.jpg187149 JPEG2026514684000389.jpg188149 JPEG2026514684000390.jpg187149 JPEG2026514684000391.jpg187149 JPEG2026514684000392.jpg187149 JPEG2026514684000393.jpg188149 JPEG2026514684000394.jpg187149 JPEG2026514684000395.jpg187149 JPEG2026514684000396.jpg187149 JPEG2026514684000397.jpg187149 JPEG2026514684000398.jpg187149 JPEG2026514684000399.jpg188149 JPEG2026514684000400.jpg32149

[0471] Table 102 below shows the results obtained by purity analysis of novel engineered antibody forms conjugated with peptide vaccines: HAVC01, HAVC02, HAVC03, HAVC04, HAVC05, HAVC06, HAVC07, and HAVC08.

[0472] [Table 102]

Claims

1. (a) an antigen-binding site comprising a first polypeptide containing at least one complementarity-determining region (CDR) sequence and a second polypeptide containing at least one complementarity-determining region (CDR) sequence, wherein the first polypeptide and the second polypeptide form a dimer, and the antigen-binding site is capable of specifically binding to a target antigen, (b) A first Fc domain or a variant thereof which is a dimer of two polypeptide sequences, in which one polypeptide sequence is conjugated to the first polypeptide of the antigen-binding site, and (c) A second Fc domain or variant thereof, which is a dimer consisting of two polypeptide sequences, in which one polypeptide sequence is conjugated to the second polypeptide of the antigen-binding site. A fusion protein containing [the specified ingredient].

2. The fusion protein according to claim 1, wherein the first polypeptide of the antigen-binding site comprises antibody heavy chains CDR1, CDR2, and CDR3, and the second polypeptide of the antigen-binding site comprises antibody light chains CDR1, CDR2, and CDR3.

3. The fusion protein according to claim 2, wherein the first polypeptide of the antigen-binding site further comprises the CH1 region of the antibody heavy chain, and / or the second polypeptide of the antigen-binding site further comprises the constant region of the antibody light chain.

4. The fusion protein according to claim 1, wherein the antigen-binding site specifically binds to a protein expressed on the cell surface.

5. The antigen-binding sites are PD-L1, EGFR, EGFRvIII, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, c-Met, DLL3, DR4, DR5, GD2, Nectin-4, RANKL, SLAMF7, Trop-2, LIV-1, Claudin-18.2, IL-13α2, CD3, HER2, HER3, FGFR2, FGFR3, The fusion protein according to claim 1, which specifically binds to any one selected from the group consisting of GPC3, ROR1, Folα, CD20, CD19, CTLA-4, VEGFR, NCAM1, ICAM-1, ICAM-2, CEACAM5, CEACAM6, carcinoembryonic antigen (CEA), CA-125, alpha-fetoprotein (AFP), MUC-1, MUC-16, PSMA, PSCA, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), immature laminin receptor, TAG-72, HPV E6 / E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA2, EphA3, mesothelin, SAP-1, survivorbin, and virus-derived antigens.

6. The fusion protein according to claim 1, further comprising a Z protein.

7. The fusion protein according to claim 6, wherein the Z protein is bound to the N-terminuses of the first Fc domain and the second Fc domain.

8. The fusion protein according to claim 6, wherein the Z protein is bound to the first Fc domain and the second Fc domain via a peptide linker.

9. The fusion protein according to claim 8, wherein the peptide linker includes a hinge.

10. The fusion protein according to claim 6, wherein the Z protein is one selected from the group consisting of a receptor, a soluble protein, a cytokine, a single-chain Fv fragment (single-chain variable fragment; scFv), an antibody mimetic, a single-domain antibody (sdAb), a therapeutic peptide, and a peptide vaccine.

11. The following structural formulas (I), (II), (III), and (IV): N'-X-(L1)n-A-C' (I); N'-Y-(L2)m-B-C' (II); N'-(Z1)r-(L5)s-C-C' (III); and N'-(Z2)t-(L6)u-D-C' (IV) Contains polypeptides, In the formulas, in the structural formulas (I), (II), (III), and (IV), N' is the N-terminus of each polypeptide, C' is the C-terminus of each polypeptide, - indicates connection, A, B, C, and D are monomeric polypeptide sequences of an Fc domain, each comprising the CH2 and CH3 regions of an immunoglobulin and optionally further comprising a CH4 and / or hinge sequence, wherein A dimers with either C or D to form the first Fc domain (b), and B dimers with the remaining C or D to form the second Fc domain (c); L1, L2, L5, and L6 are peptide linkers, n, m, r, s, t, and u are each independently 0 or 1. X is a first polypeptide sequence of the antigen-binding site, which includes the heavy chain CDR1, CDR2, CDR3 sequences of an antibody that specifically binds to the first antigen, or the heavy chain variable region of an antibody that specifically binds to the first antigen; Y is a second polypeptide sequence of the antigen-binding site, which includes the light chain CDR1, CDR2, CDR3 sequences of an antibody that specifically binds to the first antigen, or the light chain variable region of an antibody that specifically binds to the first antigen; X and Y pair with each other to form the antigen-binding site (a) which specifically binds to the antigen. Z1 and Z2 are each independently selected from the group consisting of receptors, soluble proteins, cytokines, single-chain Fv fragments (single-chain variable fragments; scFv), antibody mimetic compounds, single-domain antibodies (sdAb), therapeutic peptides, and peptide vaccines. The fusion protein according to claim 1.

12. The fusion protein according to claim 11, wherein X in the structural formula (I) further comprises a heavy chain CH1 region, and / or Y in the structural formula (II) further comprises a light chain constant region.

13. The following structural formulas: (I'), (II'), (III), and (IV): N'-VD1-(L3)p-X-(L1)n-A-C' (I'); N'-VD2-(L4)q-Y-(L2)m-B-C' (II'); N'-(Z1)r-(L5)s-C-C' (III); and N'-(Z2)t-(L6)u-D-C' (IV) Contains polypeptides, In the formula, in the structural formulas (I'), (II'), (III), and (IV), N' is the N-terminus of each polypeptide, C' is the C-terminus of each polypeptide, - indicates connection, A, B, C, and D are monomeric polypeptide sequences of an Fc domain, each comprising the CH2 and CH3 regions of an immunoglobulin and optionally further comprising a CH4 and / or hinge sequence, wherein A dimers with either C or D to form the first Fc domain (b), and B dimers with the remaining C or D to form the second Fc domain (c); L1, L2, L3, L4, L5, and L6 are peptide linkers, n, m, p, q, r, s, t, and u are each 0 or 1. VD1 consists of the variable region of the heavy or light chain of the antibody that specifically binds to the antigen, or CDR1, CDR2, and CDR3 of the antibody heavy or light chain; VD2 consists of the variable region of the antibody's light or heavy chain that specifically binds to the antigen, or CDR1, CDR2, and CDR3 of the antibody's heavy or light chain; VD1 and VD2 pair up with each other to form a second antibody variable region that specifically binds to the second antigen. X comprises a variable region of the heavy or light chain of an antibody that specifically binds to an antigen, or CDR1, CDR2, and CDR3 of the antibody heavy or light chain; Y comprises a variable region of the light or heavy chain of an antibody that specifically binds to an antigen, or CDR1, CDR2, and CDR3 of the antibody heavy or light chain; X and Y pair with each other to form a first antibody variable region that specifically binds to the first antigen. VD1-(L3)p-X forms the first polypeptide sequence of the antigen-binding site (a), and VD2-(L4)q-Y forms the second polypeptide sequence of the antigen-binding site (a). Z1 and Z2 are each independently selected from the group consisting of receptors, soluble proteins, cytokines, single-chain Fv fragments (single-chain variable fragments; scFv), antibody mimetic compounds, single-domain antibodies (sdAb), therapeutic peptides, and peptide vaccines. The fusion protein according to claim 1.

14. The fusion protein according to claim 13, wherein the heavy chain variable region further comprises a heavy chain CH1 region, and the light chain variable region further comprises a light chain constant region.

15. The Fc domain monomer includes a knob variant or a whole variant that promotes the formation of an Fc heterodimer (heterodimer Fc); or The Fc domain monomer includes a variant that promotes heterodimer formation by an electrostatic steering mechanism, The fusion protein according to claim 11 or 13.

16. The bond between X and Y is i) Through the disulfide bond formed by CH1 and Cys present in the light chain constant region, ii) Through disulfide bonds formed by Cys present in the heavy chain variable region and the light chain variable region, or iii) Disulfide bonds formed by CH1 and Cys present in the light chain constant region, and disulfide bonds formed by Cys present in the heavy chain variable region and the light chain variable region The fusion protein according to claim 12 or 14 is achieved.

17. The bond between X and Y is a CH based on Kabat numbering. １ In addition to the disulfide bond between 233 and CL214, i) The disulfide bond between VH105 and VL43; ii) Disulfide bond between VH44 and VL100; or iii)CH １ Disulfide bond between 122 and CL121 The fusion protein according to claim 14, further comprising:

18. A pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a fusion protein described in any one of claims 1 to 17.

19. The pharmaceutical composition according to claim 18, wherein the cancer is one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, esophageal cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

20. Transformed cells expressing the fusion protein described in any one of claims 1 to 17.

21. A method for preventing or treating cancer, comprising the step of administering the fusion protein described in claim 1 to a target.

22. Use of the fusion protein described in claim 1.

23. Use of the fusion protein according to claim 1 for use in the manufacture of a pharmaceutical product for treating cancer.

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