A dual antigen-targeting protein complex containing an antibody or its antigen-binding fragment, and an affibody.

Abstract

The present invention relates to a dual antigen-targeting protein complex comprising an antibody or its antigen-binding fragment and an affibody. More specifically, the dual antigen-targeting protein complex comprises an EGFR (Epidermal Growth Factor Receptor) or PSMA (Prostate Specific Membrane Antigen) target site containing an antibody or its antigen-binding fragment, and a CD137 target site containing an affibody bound to the antibody or its antigen-binding fragment. When the dual antigen-targeting protein complex or a composition containing it is used, cancer can be effectively prevented or treated.

Description

Detailed description of the invention

[0001] [Technical field] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0062007, filed with the Korean Intellectual Property Office on 12 May 2023, and the disclosures of said patent application are incorporated herein by reference.

[0002] The present invention relates to a biantigen-targeting protein complex comprising an antibody or its antigen-binding fragment, and an affibody.

[0003] [Background technology] Monoclonal antibodies (mAbs) have proven to be a useful tool for treating various types of cancer. However, the increasing clinical use of mAbs has highlighted the limitations of monotherapy for cancer. These limitations are particularly evident in cancers with complex mechanisms or those that rapidly develop resistance or tolerance after monotherapy.

[0004] Bispecific antibodies, or biantigen-targeting protein complexes, are artificial proteins capable of simultaneously binding to two different types of antigens or two different epitopes. Such bispecific protein complexes are opening up a wide range of applications, including T cell redirection to tumor cells, dual targeting of different disease mediators, and delivery of payloads to target sites. For example, the approval of catumaxomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) represents significant milestones in the research and development of bispecific antibodies.

[0005] In relation to the limitations of monotherapy mentioned above, therapeutic approaches that utilize two drugs acting on different targets to achieve synergistic effects are attracting attention. Drugs acting on dual targets have been shown to exhibit higher therapeutic efficacy compared to single drugs, and there is an urgent need to develop dual antigen-targeting protein complexes that are structurally stable yet possess excellent anticancer effects.

[0006] [Overview of the prefecture] [Problems the invention aims to solve] The inventors of this invention have diligently conducted research to develop a dual antigen-targeting protein complex with excellent anticancer effects. As a result, they have demonstrated that by binding antibodies or their antigen-binding fragments and aphibodies that target each antigen, it is possible to produce a dual antigen-targeting protein complex with excellent binding affinity to each antigen and excellent anticancer effects, thus completing the present invention.

[0007] Therefore, an object of the present invention is to provide a biantigen-targeting protein complex comprising a first antigen target site containing an antibody or its antigen-binding fragment, and a second antigen target site containing an affibody bound to the antibody or its antigen-binding fragment.

[0008] Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the biantigen target protein complex.

[0009] A further object of the present invention is to provide a recombinant vector containing the nucleic acid molecule.

[0010] A further object of the present invention is to provide a host cell containing the recombinant vector.

[0011] A further object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising a biantigen-targeting protein complex comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site containing an antibody or an antigen-binding fragment thereof, and a CD137 target site containing an affibody bound to the antibody or the antigen-binding fragment thereof.

[0012] Still another object of the present invention is to provide a method for preventing or treating cancer using a bispecific antigen-targeting protein complex comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site containing an antibody or an antigen-binding fragment thereof, and a CD137 target site containing an affibody bound to the antibody or an antigen-binding fragment thereof.

[0013] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0014] [Means for Solving the Problems] In one aspect of the present invention, the present invention provides a bispecific antigen-targeting protein complex comprising a first antigen target site containing an antibody or an antigen-binding fragment thereof, and a second antigen target site containing an affibody bound to the antibody or an antigen-binding fragment thereof.

[0015] The present inventors have made intensive research efforts to develop a bispecific antigen-targeting protein complex having an excellent anti-cancer effect. As a result, it has been clarified that when an antibody or an antigen-binding fragment thereof and an affibody that target each antigen are bound, a bispecific antigen-targeting protein complex having excellent binding force to each antigen and an excellent anti-cancer effect can be produced.

[0016] As used herein, the term "bispecific antigen-targeting protein complex" may be, for example, but not limited to, a bispecific antibody to which an antibody having two different antigen-binding sites is bound, a fusion protein or a chimeric protein to which an antibody and an antigen-binding peptide are bound. Preferably, in the present invention, the bispecific antigen-targeting protein complex means a form in which an antibody or an antigen-binding fragment thereof and an affibody are bound.

[0017] As used herein, the term "bispecific antigen-targeting protein complex" means a protein structure capable of simultaneously recognizing and binding two different antigens.

[0018] One embodiment of the present invention is a dual antigen-targeting protein complex in which an antibody or antigen-binding fragment and an affibody are linked. The antibody or antigen-binding fragment and the affibody of the present invention are covalently linked to each other, and according to one embodiment of the present invention, the dual antigen-targeting protein complex may be embodied in the form of a fused protein or a conjugate.

[0019] Therefore, the antibody or its antigen-binding fragment and the affibody may be linked by chemical conjugation (known as an organic chemical method) or by other means (for example, by expressing the complex as a fusion protein, directly or indirectly via a linker (e.g., an amino acid linker)).

[0020] In a specific embodiment of the present invention, the antibody or antigen-binding fragment and the affibody forming the protein complex are linked by at least one linker. In this case, the linker may consist of an amino acid sequence represented by the general formula (GnSm)p or (SmGn)p: Here, n, m, and p are independent of each other. n is an integer between 1 and 7; m is an integer between 0 and 7; The sum of n and m is an integer less than or equal to 8; and p is an integer between 1 and 7.

[0021] In another specific embodiment of the present invention, the linker is n=1 to 5 and m=0 to 5. In a more specific embodiment, n=4 and m=1.

[0022] In a more specific example, the linker is GGGGS.

[0023] In this specification, the term "antibody" includes not only the complete antibody form but also the antigen-binding fragment of the antibody molecule.

[0024] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond.

[0025] The constant regions of the heavy chain have gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant regions of the light chain have kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WBSaunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, MA (1984)).

[0026] In this specification, the term "antigen-binding fragment" refers to a fragment that possesses antigen-binding function, and includes Fab, F(ab'), F(ab')2, chemically linked F(ab')2, and Fv. Among the antibody fragments, Fab has a structure that includes variable regions of the light chain and heavy chain, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment having only heavy chain variable regions and light chain variable regions. Recombination techniques for producing Fv fragments are disclosed in PCT international publication patent applications WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In two-chain Fv, the heavy chain variable region and the light chain variable region are linked by non-covalent bonds, while in single-chain Fv, the heavy chain variable region and the single-chain variable region are generally linked by a peptide linker via covalent bonds or directly linked at the C-terminus, thus forming a dimer-like structure similar to two-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restrictive cleavage of the entire antibody with papain, and F(ab')2 fragments can be obtained by cleavage with pepsin), or they can be produced using genetic recombination techniques.

[0027] In this invention, the antibody is specifically in scFv form or in complete antibody form. The constant region of the heavy chain may be selected from one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). The constant region of the light chain may be kappa-type or lambda-type.

[0028] In this specification, the term "heavy chain" refers to either a full-length heavy chain or a fragment thereof, comprising a variable region domain VH containing an amino acid sequence having sufficient variable region elements to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. In this specification, the term "light chain" refers to either a full-length light chain or a fragment thereof, comprising a variable region domain VL containing an amino acid sequence having sufficient variable region elements to confer specificity to an antigen, and a constant region domain CL.

[0029] In this specification, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USD Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (CDR-H1, CDR-H2, and CDR-H3) and the light chain (CDR-L1, CDR-L2, and CDR-L3) each contain three CDRs. The CDRs provide key contact residues when the antibody binds to an antigen or epitope.

[0030] In this specification, the antibody or its antigen-binding fragment includes full-length or original polyclonal or monoclonal antibodies, as well as its antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fab3, Fv, and their variants), fusion proteins containing one or more antibody moieties, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies (scFv), scFv-Fc, bispecific antibodies, multispecific antibodies, glycosylated variants of antibodies as other modified sequence forms of immunoglobulin molecules containing antigen recognition sites of the required specificity, amino acid sequence variants of antibodies, and antibodies modified by covalent bonding. Specific examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFs (dual acting Fabs), bispecific antibodies (two-in-one antibodies), SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs. The catalog of antibodies and their antigen-binding fragments is not limited to these.

[0031] In this specification, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4.

[0032] Therefore, the HVR and FR sequences generally appear in the following order in VH (or VL / Vk): (a) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and (b)FRL1(Framework region 1 of Light chain)-CDRL1(complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

[0033] In this specification, the terms "variable region" or "variable domain" refer to the domain of the antibody heavy or light chain involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). (Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Alternatively, antibodies that bind to a specific antigen may be separated by their VH or VL domains from antibodies that bind to the antigen and screen for a library of complementary VL or VH domains, respectively.

[0034] In this specification, terms such as “specifically bind” mean that an antibody or its antigen-binding fragment, or other component such as scFv, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10⁻¹⁶. -6 M or less (for example, 9x10) -7 M, 8x10 -7 M, 7x10 -7 M, 6x10 -7 M, 5x10-7 M, 4 x 10 -7 M, 3 x 10 -7 M, 2 x 10 -7 M, or 1 x 10 -7 M), preferably 1 x 10 -7 M or less (e.g., 9 x 10 -8 M, 8 x 10 -8 M, 7 x 10 -8 M, 6 x 10 -8 M, 5 x 10 -8 M, 4 x 10 -8 M, 3 x 10 -8 M, 2 x 10 -8 M, or 1 x 10 -8 M), more preferably 1 x 10 -8 M or less (e.g., 9 x 10 -9 M, 8 x 10 -9 M, 7 x 10 -9 M, 6 x 10 -9 M, 5 x 10 -9 M, 4 x 10 -9 M, 3 x 10 -9 M, 2 x 10 -9 M, or 1 x 10 -9 M) and characterized by an equilibrium dissociation constant (e.g., a smaller KD indicates a stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0035] As used herein, the term "Affinity" means the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used in this application, "binding affinity" represents the intrinsic binding affinity that reflects the 1:1 interaction between the components of a binding pair (e.g., an antibody and an antigen). The affinity of molecule Y and its partner Y can generally be represented by the dissociation constant (Kd). Affinity may be measured by conventional methods known in the art, including those described in this application.

[0036] Furthermore, in this specification, the term "human antibody" refers to an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences, possessing an amino acid sequence corresponding to such an antibody. This definition of a human antibody excludes humanized antibodies that contain non-human antigen-binding residues.

[0037] In this specification, the term "chimeric" antibody means an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, and the rest of the heavy chain and / or light chain originates from another source or species.

[0038] In this specification, the term "humanized antibody" refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing the minimal sequence derived from a non-human (e.g., mouse) antibody non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues in the recipient complementarity-determining region (CDR) are replaced with CDR residues from a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in the recipient antibody or in the incorporated CDR or framework sequence. Such modifications are made to further improve and optimize antibody performance. Generally, the humanized antibody will contain at least one and typically two substantially all variable domains, wherein all or substantially all of the CDR region in the domain corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region has the sequence of the FR region of a human immunoglobulin. The humanized antibody contains at least a portion or substantial of the constant region (Fc region) of the immunoglobulin.

[0039] The aforementioned variants have "substantial similarity," meaning that when the two peptide sequences are optimally aligned, such as by the program GAP or BESTFIT using default gap weighting values, they share at least about 90% sequence identity, more preferably at least about 95%, 98%, or 99% sequence identity. Preferably, non-identical residue positions are distinguished from one another by conservative amino acid substitutions. A "conservative amino acid substitution" is when an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of the protein. When two or more amino acid sequences are distinguished from one another by conservative substitutions, the percentage or degree of similarity may be upregulated to compensate for the conservative nature of the substitutions.

[0040] Such amino acid mutations are based on the relative similarities of amino acid side-chain substitutions, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, morphology, and type of amino acid side-chain substitutions reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar morphologies. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0041] When introducing mutations, the hydropathic index of amino acids may be taken into consideration. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0042] The hydrophobic amino acid index is extremely important in conferring interactive biological functions to proteins. It is a well-known fact that similar biological activity can only be achieved by substituting amino acids with similar hydrophobic indices. When introducing mutations by referring to the hydrophobic index, substitutions are preferably made between amino acids that show a difference in hydrophobic index of ±2, more preferably within ±1, and even more preferably within ±0.5.

[0043] On the other hand, it is well known that substitutions between amino acids with similar hydrophilicity values ​​result in proteins with equal biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When introducing mutations by referring to hydrophilicity values, substitutions are preferably performed between amino acids that exhibit a hydrophilicity difference of ±2, more preferably within ±1, and even more preferably within ±0.5.

[0044] Amino acid exchanges in proteins that do not alter the overall molecular activity are well known in this field (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0045] The antibody or antigen-binding fragment of the present invention includes an antibody or antigen-binding fragment that includes a small change from the above-described amino acid sequence, i.e., a modification that has little effect on the tertiary structure and the function of the antibody. Therefore, in a given embodiment, even if it does not match the above-described sequence, it may have at least 100%, 93%, 95%, 96%, 97%, or 98% or more similarity.

[0046] In this specification, "affibody" is also referred to as "Z body" or "Zb". An affibody is a small protein consisting of 58 amino acid residues. In the protein sequence of such an affibody molecule, the 13 amino acids that form a binding to the target protein can bind to various target antigens depending on the amino acid sequence, and random sequences are possible, allowing for the construction of a library. Similar to antibodies, affibody molecules capable of binding to various target antigens can be selected from a library using screening methods such as phage display and yeast two-hybrid (Y2H). Furthermore, because affibody molecules have a very small molecular weight of 6.5 kDa, compared to IgG-type antibodies which generally have a molecular weight of 150 kDa, they diffuse systemically upon administration to the human body and are rapidly removed by renal filtration. Therefore, affibody molecules are mainly applied in the research and development of diagnostic samples (Goldstein R et al., 2013, Expert Rev Anticancer Ther.).

[0047] In one embodiment of the present invention, the biantigen-targeting protein complex has an aphibody dimer bound to the C-terminus of the heavy chain of an antibody or its antigen-binding fragment.

[0048] In one specific example of the present invention, the biantigen-targeting protein complex has an aphibody dimer bound to the C-terminus of the heavy chain of the antibody or its antigen-binding fragment: The heavy chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 33, or SEQ ID NO: 57; The affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0049] In the present invention, a dimer may be formed by the bonding of two aphibody monomers having the same amino acid sequence. The dimer may be formed by hydrogen bonds, covalent bonds, or other intermolecular forces between the individual aphibody units. Preferably, each aphibody constituting the aphibody dimer is linked by at least one linker. In this case, the linker may consist of an amino acid sequence represented by the general formula (GnSm)p or (SmGn)p: Here, n, m, and p are independent of each other. n is an integer between 1 and 7; m is an integer between 0 and 7; The sum of n and m is an integer less than or equal to 8; and p is an integer between 1 and 7.

[0050] In another specific embodiment of the present invention, the linker is n=1 to 5 and m=0 to 5. In a more specific embodiment, n=4 and m=1.

[0051] In a more specific example, the linker is GGGGS.

[0052] In one embodiment of the present invention, the biantigen-targeting protein complex has aphibody monomers bound to the C-terminuses of the heavy chain and light chain of the antibody or its antigen-binding fragment, respectively.

[0053] In one specific example of the present invention, the biantigen-targeting protein complex has aphibody monomers bound to the C-terminuses of the heavy chain and light chain of the antibody or its antigen-binding fragment, respectively: The heavy chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 33, or SEQ ID NO: 57; The light chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 34, or SEQ ID NO: 58; and The affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0054] In one embodiment of the present invention, the biantigen-targeting protein complex has an aphibody dimer bound to the C-terminus of the light chain of an antibody or its antigen-binding fragment.

[0055] In one specific example of the present invention, the biantigen-targeting protein complex has an aphibody dimer bound to the C-terminus of the light chain of the antibody or its antigen-binding fragment: The light chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 34, or SEQ ID NO: 58; and The affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; In one embodiment of the present invention, the first antigen target site targets EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen); and the second antigen target site targets CD137.

[0056] In this specification, the term "EFGR (Epidermal Growth Factor Receptor)" refers to a receptor tyrosine kinase present on the surface of cells. EFGRs are primarily involved in regulating important cellular processes such as cell growth, differentiation, and survival. When ligands such as epidermal growth factor bind to EGFR, intracellular signaling pathways are activated, promoting cell division and growth. EGFR is often overexpressed or mutated in various cancers, particularly non-small cell lung cancer, colorectal cancer, and head and neck cancer, and this plays a crucial role in cancer growth and progression.

[0057] In this specification, the term "PSMA (Prostate-Specific Membrane Antigen)" refers to a cell membrane protein primarily expressed in prostate cells. PSMA expression tends to increase in prostate cancer, meaning that PSMA can be used as a target for the diagnosis and treatment of prostate cancer. PSMA also possesses enzymatic activity and can be involved in nutrient absorption and signal transduction in the extracellular environment.

[0058] In this specification, the term "CD137" refers to a co-stimulatory molecule expressed on the surface of T cells in the immune system. CD137 plays a crucial role in increasing T cell activation and survival. Binding of CD137 to its ligand promotes T cell activation and enhances the immune response. In cancer treatment, CD137 is being studied as a strategy to increase the anti-cancer activity of T cells when used in combination with immune checkpoint inhibitors.

[0059] In one embodiment of the present invention, the affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0060] The affibody consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 represents an affibody that targets CD137.

[0061] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 3 to 8, respectively; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 27 to 32, respectively; or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 51 to 56, respectively.

[0062] In the present invention, Sequence IDs 3 to 8 each represent the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody or antigen-binding fragment that targets EFGR. Sequence IDs 27 to 32 each represent the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody or antigen-binding fragment that targets EFGR. Sequence IDs 51 to 56 each represent the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody or antigen-binding fragment that targets PSMA.

[0063] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain consisting of amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; a heavy chain and a light chain consisting of amino acid sequences shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively; or a heavy chain and a light chain consisting of amino acid sequences shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively. In the present invention, SEQ ID NO: 9 and SEQ ID NO: 10 each represent the heavy chain and light chain sequences of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NO: 33 and SEQ ID NO: 34 each represent the heavy chain and light chain sequences of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NO: 57 and SEQ ID NO: 58 each represent the heavy chain and light chain sequences of an antibody or antigen-binding fragment that targets PSMA.

[0064] In one embodiment of the present invention, the dual antigen target protein complex includes a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 11 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 12; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 19 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 20; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 35 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 36; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 43 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 44; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 59 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 60; or a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 67 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 68. In the present invention, SEQ ID NOs: 11, 19, 35, and 43 refer to sequences in which an aphibody monomer targeting CD137 is bound to the heavy chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NOs: 59 and 67 refer to sequences in which an aphibody monomer targeting CD137 is bound to the heavy chain of an antibody or antigen-binding fragment that targets PSMA.

[0065] In one embodiment of the present invention, the dual antigen target protein complex includes a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 13 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 14; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 21 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 22; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 37 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 38; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 45 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 46; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 61 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 62; or a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 69 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 70. In the present invention, SEQ ID NOs: 13, 21, 37, and 45 refer to sequences in which an aphibody dimer targeting CD137 is bound to the heavy chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NOs: 61 and 69 refer to sequences in which an aphibody dimer targeting CD137 is bound to the heavy chain of an antibody or antigen-binding fragment that targets PSMA.

[0066] In one embodiment of the present invention, the biantigen target protein complex comprises a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 15 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 16 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 23 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 24 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 39 is bound, and an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 40 is bound The present invention includes a light chain to which a fibrous monomer consisting of the amino acid sequence shown in SEQ ID NO: 47 is bound, and a light chain to which a fibrous monomer consisting of the amino acid sequence shown in SEQ ID NO: 48 is bound, and a heavy chain to which a fibrous monomer consisting of the amino acid sequence shown in SEQ ID NO: 63 is bound, and a light chain to which a fibrous monomer consisting of the amino acid sequence shown in SEQ ID NO: 64 is bound, or a heavy chain to which a fibrous monomer consisting of the amino acid sequence shown in SEQ ID NO: 71 is bound, and a light chain to which a fibrous monomer consisting of the amino acid sequence shown in SEQ ID NO: 72 is bound. In the present invention, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 39, and SEQ ID NO: 47 mean sequences in which an a fibrous monomer targeting CD137 is bound to the heavy chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NO: 63 and SEQ ID NO: 71 mean sequences in which an a fibrous monomer targeting CD137 is bound to the heavy chain of an antibody or antigen-binding fragment that targets PSMA.

[0067] In the present invention, Sequence IDs 16, 24, 40, and 48 refer to sequences in which an aphibody monomer targeting CD137 is bound to the light chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, Sequence IDs 64 and 72 refer to sequences in which an aphibody monomer targeting CD137 is bound to the light chain of an antibody or antigen-binding fragment that targets PSMA.

[0068] In one embodiment of the present invention, the biantigen target protein complex includes a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 17 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 18 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 25 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 26 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 41 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 42 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 49 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 50 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 65 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 66 is bound; or a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 73 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 74 is bound. In the present invention, Sequence IDs 18, 26, 42, and 50 refer to sequences in which an aphibody dimer targeting CD137 is bound to the light chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, Sequence IDs 66 and 74 refer to sequences in which an aphibody dimer targeting CD137 is bound to the light chain of an antibody or antigen-binding fragment that targets PSMA.

[0069] In one embodiment of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the biantigen target protein complex.

[0070] In this specification, the term "nucleic acid molecule" comprehensively includes DNA (gDNA and cDNA) and RNA molecules. In nucleic acid molecules, the basic building blocks, nucleotides, include not only natural nucleotides but also analogues in which the sugar or base site has been altered (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman & Peyman, Chemical Reviews, 90:543-584 (1990)).

[0071] It will be obvious to those skilled in the art that the nucleotide sequence encoding the antibody or its antigen-binding fragment of the present invention may be any nucleotide sequence encoding the amino acid sequence constituting the antibody or its antigen-binding fragment, and is not limited to any specific nucleotide sequence.

[0072] This is because even if a mutation occurs in the nucleotide sequence, expressing the mutated nucleotide sequence as a protein may not result in any change in the protein sequence. This is called codon degeneracy. Therefore, the nucleotide sequence includes a nucleotide sequence containing functionally homogeneous codons or codons encoding the same amino acid (for example, due to codon degeneracy, there are six codons for arginine or serine), or codons encoding biologically homogeneous amino acids.

[0073] Considering the mutations having the same biological activity as described above, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody or its antigen-binding fragment is interpreted to also include sequences that exhibit substantial identity thereto. This substantial identity is defined as at least 60% homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), and more specifically, 70% homology (e.g., 71%, 72%, 73%, 74%, or 75%), when the sequences are aligned to correspond as closely as possible with any other sequence and analyzed using algorithms commonly used in the industry. This means sequences exhibiting 76%, 77%, 78%, or 79% homology, more specifically 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), particularly specifically 90% or more homology (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and most specifically 95% or more homology (e.g., 95%, 96%, 97%, 98%, or 99%). All integers in the range of 60% to 100% and decimals between them are included within the scope of the present invention in relation to % homology.

[0074] Alignment methods for sequence comparison are publicly known in this industry. Various methods and algorithms for alignment are disclosed in Smith and Waterman, Adv.Appl.Math.2:482 (1981); Needleman and Wunsch, J.Mol.Bio.48:443 (1970); Pearson and Lipman, Methods in Mol.Biol.24:307-31 (1988); Higgins and Sharp, Gene73:237-44 (1988); Higgins and Sharp, CABIOS5:151-3 (1989); Corpet et al., Nuc.Acids Res.16:10881-90 (1988); Huang et al., Comp.Appl.BioSci.8:155-65 (1992); and Pearson et al., Meth.Mol.Biol.24:307-31 (1994). BLAST (NCBI Basic Local Alignment Search Tool) (Altschul et al., J.Mol.Biol.215:403-10(1990)) is accessible from the NBCI (National Center for Biological Information) and other sources, and can be used online in conjunction with sequence analysis programs such as blastp, blastn, blastx, tblastn, and tblastx. BLAST can be accessed from the BLAST page on the NCBI website. Methods for comparing sequence homology using this program can be found on the BLAST help page on the NCBI website.

[0075] In one embodiment of the present invention, the nucleic acid molecule may contain one or more of the amino acid sequences of SEQ ID NOs. 75 to 148.

[0076] Sequence ID 75 or 76 indicates a nucleotide sequence encoding an affibody that targets CD137.

[0077] In the present invention, Sequence IDs 77 to 82 each represent nucleotide sequences encoding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody or antigen-binding fragment that targets EFGR. Sequence IDs 101 to 106 each represent nucleotide sequences encoding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody or antigen-binding fragment that targets EFGR. Sequence IDs 125 to 130 each represent nucleotide sequences encoding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody or antigen-binding fragment that targets PSMA.

[0078] In the present invention, Sequence ID No. 83 and Sequence ID No. 84 each represent nucleotide sequences encoding the heavy and light chains of an antibody or antigen-binding fragment that targets EFGR. In the present invention, Sequence ID No. 107 and Sequence ID No. 108 each represent nucleotide sequences encoding the heavy and light chains of an antibody or antigen-binding fragment that targets EFGR. In the present invention, Sequence ID No. 131 and Sequence ID No. 132 each represent nucleotide sequences encoding the heavy and light chains of an antibody or antigen-binding fragment that targets PSMA.

[0079] In the present invention, SEQ ID NOs. 85, 93, 109, and 117 represent nucleotide sequences encoding an antibody or antigen-binding fragment that targets EFGR, to which an aphibody monomer targeting CD137 is bound. SEQ ID NOs. 86, 94, 110, and 118 represent nucleotide sequences encoding the light chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NOs. 133 and 141 represent nucleotide sequences encoding an antibody or antigen-binding fragment that targets PSMA, to which an aphibody monomer targeting CD137 is bound. SEQ ID NOs. 134 and 142 represent nucleotide sequences encoding the light chain of an antibody or antigen-binding fragment that targets PSMA.

[0080] In the present invention, SEQ ID NOs. 87, 95, 111, and 119 represent nucleotide sequences encoding an antibody or antigen-binding fragment that targets EFGR, with an aphibody dimer targeting CD137 bound to its heavy chain. SEQ ID NOs. 88, 96, 112, and 120 represent nucleotide sequences encoding the light chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NOs. 135 and 143 represent nucleotide sequences encoding an antibody or antigen-binding fragment that targets PSMA, with an aphibody dimer targeting CD137 bound to its heavy chain. SEQ ID NOs. 136 and 144 represent nucleotide sequences encoding the light chain of an antibody or antigen-binding fragment that targets PSMA.

[0081] In the present invention, SEQ ID NOs. 89, 97, 113, and 121 represent nucleotide sequences encoding a heavy chain of an antibody or antigen-binding fragment targeting EFGR to which an aphibody monomer targeting CD137 is bound. In the present invention, SEQ ID NOs. 137 and 145 represent nucleotide sequences encoding a heavy chain of an antibody or antigen-binding fragment targeting PSMA to which an aphibody monomer targeting CD137 is bound.

[0082] In the present invention, SEQ ID NOs. 90, 98, 114, and 122 represent nucleotide sequences encoding a system in which an aphibody monomer targeting CD137 is bound to the light chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NOs. 138 and 146 represent nucleotide sequences encoding a system in which an aphibody monomer targeting CD137 is bound to the light chain of an antibody or antigen-binding fragment that targets PSMA.

[0083] In the present invention, SEQ ID NOs. 91, 99, 115, and 123 represent nucleotide sequences encoding the heavy chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NOs. 92, 100, 116, and 124 represent nucleotide sequences encoding an aphibody dimer that targets CD137 bound to the light chain of an antibody or antigen-binding fragment that targets EFGR. In the present invention, SEQ ID NOs. 139 and 147 represent nucleotide sequences encoding the light chain of an antibody or antigen-binding fragment that targets PSMA. In the present invention, SEQ ID NOs. 140 and 148 represent nucleotide sequences encoding an aphibody dimer that targets CD137 bound to the light chain of an antibody or antigen-binding fragment that targets PSMA.

[0084] In one embodiment of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0085] In this specification, the term "vector" refers to a means for expressing a target gene in a host cell, and includes, but is not limited to, plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0086] According to one embodiment of the present invention, in the vector of the present invention, the nucleic acid molecule is operatively linked to the promoter.

[0087] In this specification, the term “operatively linked” means a functional link between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, thereby the regulatory sequence modulates the transcription and / or decoding of the other nucleic acid sequence.

[0088] The recombinant vector system of the present invention may be constructed by various methods known to the art, the specific methods of which are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0089] The vectors of the present invention may typically be constructed as vectors for cloning or vectors for expression. Furthermore, the vectors of the present invention may be constructed using prokaryotic or eukaryotic cells as hosts.

[0090] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, promoters derived from the genome of mammalian cells (e.g., metallothione promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Roussarcoma virus (RSV) promoter) may be used, and generally have a polyadenylated sequence as a transcription termination sequence.

[0091] The vector of the present invention may be fused with other sequences to facilitate the purification of the antibody expressed therefrom. Examples of sequences to be fused include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0092] On the other hand, the expression vector of the present invention includes, as a selective label, antibiotic resistance genes commonly used in the industry, such as resistance genes to ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0093] Selectively, the vector can further deliver genes encoding reporter molecules (e.g., luciferase and glucuronidase).

[0094] According to one embodiment of the present invention, the expression vector is a recombinant vector for host cell expression in which a nucleic acid molecule encoding a protein complex to which an antibody or its antigen-binding fragment and an affibody are bound is inserted, and which is operatively linked to the nucleotide sequence of the nucleic acid molecule and includes a promoter that causes the formation of an RNA molecule in the host cell, and a poly(A) signal sequence that acts in the host cell to cause polyadenylation of the 3' end of the RNA molecule.

[0095] In one embodiment of the present invention, the present invention provides a host cell containing the recombinant vector.

[0096] Any host cell known in the art may be used to stably and continuously clone and express the vector of the present invention. Suitable eukaryotic host cells for the vector include, but are not limited to, yeast (Saccharomyce cerevisiae), insect cells, monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK-293 cells.

[0097] In this specification, the terms “transformed,” “transduced,” or “transfected” refer to the process by which an exogenous nucleic acid is transmitted or introduced into a host cell. A “transformed,” “transduced,” or “transfected” cell is a cell that has been transformed, transduced, or transfected with an exogenous nucleic acid, and such cell includes the cell itself and its progeny cells obtained by subculturing.

[0098] The vector of the present invention can be delivered into host cells by microinjection (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, F. Let al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated phenotypic infection (Wong, T. Ket al., Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene banbarment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) when the host cell is a eukaryotic cell.

[0099] In the present invention, a recombinant vector injected into a host cell can express the above-mentioned protein complex that has been recombinantly introduced into the host cell, and in such cases, a large amount of the protein complex can be obtained. For example, if the expression vector contains a lac promoter, gene expression can be induced by treating the host cell with IPTG.

[0100] The culture is usually carried out under aerobic conditions, such as shaking culture or rotation in a rotating incubator. The culture temperature is preferably in the range of 10 to 40°C, and the culture time is generally 5 hours to 7 days. The pH of the culture medium is preferably maintained in the range of 3.0 to 9.0 during culture. The pH of the culture medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. During culture, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be added as needed to maintain and express the recombinant vector. When culturing host cells transformed with a recombinant expression vector having an induceable promoter, an inducer suitable for the medium may be added as needed. For example, if the expression vector contains a lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) may be added, and if it contains a trp promoter, indoleacrylic acid may be added to the medium.

[0101] In one aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising a biantigen-targeting protein complex comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site containing an antibody or an antigen-binding fragment thereof, and a CD137 target site containing an affibody bound to the antibody or the antigen-binding fragment thereof.

[0102] In one embodiment of the present invention, the cancer is selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, lung cancer, non-small cell lung cancer, liver cancer, hepatocellular carcinoma, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, rectal cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, squamous cell carcinoma, peritoneal cancer, kidney cancer, testicular cancer, esophageal cancer, and ureteral cancer. However, the cancer may be, for example, carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, Schwann cell tumor, pyelonephryoma, adenocarcinoma, or melanoma any cancer that overexpresses EGFR or PSMA on the surface of cancer cells.

[0103] Since the pharmaceutical compositions of the present invention utilize the antibody or its antigen-binding fragment and a protein complex containing the affibody as described above as active ingredients, the description of the common elements of both is omitted to avoid excessive complexity in this specification.

[0104] In one embodiment of the present invention, the cancer may be selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, lung cancer, non-small cell lung cancer, liver cancer, hepatocellular carcinoma, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, rectal cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, squamous cell carcinoma, peritoneal cancer, kidney cancer, testicular cancer, esophageal cancer, and ureteral cancer. However, the cancer may be, for example, carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, Schwann cell tumor, pyelonephryoma, adenocarcinoma, or melanoma any cancer that overexpresses EGFR or PSMA on the surface of cancer cells.

[0105] The pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to these components, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0106] The pharmaceutical composition of the present invention can be administered orally or parenterally, for example, by intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intrasternal injection, local administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0107] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity, and a skilled, ordinary physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. In this specification, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat the diseases described above.

[0108] In this specification, the term "prevention" means the treatment of a disease or disease condition that prevents or protects against it. In this specification, the term "treatment" means the reduction, suppression, sedation, or eradication of a disease condition.

[0109] The pharmaceutical compositions of the present invention may be manufactured in unit dose form by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains, or by being contained in multi-dose containers. The dosage form may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may further contain a dispersant or stabilizer.

[0110] In one embodiment of the present invention, the biantigen-targeting protein complex has an aphibody dimer bound to the C-terminus of the heavy chain of an antibody or its antigen-binding fragment.

[0111] In one specific example of the present invention, the biantigen-targeting protein complex has an aphibody dimer bound to the C-terminus of the heavy chain of the antibody or its antigen-binding fragment: The heavy chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 33, or SEQ ID NO: 57; and The affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0112] In one embodiment of the present invention, the biantigen-targeting protein complex has aphibody monomers bound to the C-terminuses of the heavy chain and light chain of the antibody or its antigen-binding fragment, respectively.

[0113] In one specific example of the present invention, the biantigen-targeting protein complex has aphibody monomers bound to the C-terminuses of the heavy chain and light chain of the antibody or its antigen-binding fragment, respectively: The heavy chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 33, or SEQ ID NO: 57; The light chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 34, or SEQ ID NO: 58; and The affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0114] In one embodiment of the present invention, the biantigen-targeting protein complex has an aphibody dimer bound to the C-terminus of the light chain of an antibody or its antigen-binding fragment.

[0115] In one specific example of the present invention, an aphibody dimer is bound to the C-terminus of the light chain of an antibody or its antigen-binding fragment: The light chain of the antibody or its antigen-binding fragment consists of the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 34, or SEQ ID NO: 58; and The affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0116] In one embodiment of the present invention, the dual antigen target protein complex includes a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 11 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 12; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 19 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 20; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 35 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 36; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 43 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 44; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 59 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 60; or a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 67 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 68.

[0117] In one embodiment of the present invention, the dual antigen target protein complex includes a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 13 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 14; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 21 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 22; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 37 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 38; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 45 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 46; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 61 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 62; or a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 69 is bound, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 70.

[0118] In one embodiment of the present invention, the biantigen target protein complex comprises a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 15 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 16 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 23 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 24 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 39 is bound, and an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 40 is bound This includes products containing a light chain, a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 47 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 48 is bound, a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 63 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 64 is bound, or a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 71 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 72 is bound.

[0119] In one embodiment of the present invention, the dual antigen target protein complex includes a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 17 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 18 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 25 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 26 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 41 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 42 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 49 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 50 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 65 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 66 is bound; or a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 73 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 74 is bound.

[0120] In one aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising the step of administering a biantigen-targeting protein complex or a pharmaceutical composition containing the same, comprising a target site comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site comprising an affibody bound to the antibody or the antigen-binding fragment thereof, and a CD137 target site comprising an affibody bound to the antibody or the antigen-binding fragment thereof.

[0121] Since the cancer prevention or treatment method of the present invention involves administering a biantigen-targeting protein complex or a pharmaceutical composition containing the same to the target, common information related to the biantigen-targeting protein complex or a pharmaceutical composition containing the same is omitted from this specification to avoid excessive complexity.

[0122] [Effects of the invention] The features and advantages of the present invention can be summarized as follows:

[0123] (a) The present invention provides a biantigen-targeting protein complex comprising a first antigen target site containing an antibody or an antigen-binding fragment thereof, and a second antigen target site containing an affibody bound to the antibody or the antigen-binding fragment thereof.

[0124] (b) The present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the biantigen target protein complex.

[0125] (c) The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0126] (d) The present invention provides a host cell containing the recombinant vector.

[0127] (e) The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a biantigen-targeting protein complex comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site containing an antibody or an antigen-binding fragment thereof, and a CD137 target site containing an affibody bound to the antibody or the antigen-binding fragment thereof.

[0128] (f) The present invention provides a method for the prevention or treatment of cancer, comprising the step of administering a biantigen-targeting protein complex or a pharmaceutical composition containing the same, comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site containing an antibody or an antigen-binding fragment thereof, and a CD137 target site containing an affibody bound to the antibody or the antigen-binding fragment thereof.

[0129] (g) When using the biantigen target protein complex of the present invention or a composition containing the same, cancer can be effectively prevented or treated.

[0130] [Brief description of the drawing] [Figure 1] This is a schematic diagram illustrating the optimized form of a biantigen-targeting protein complex.

[0131] [Figure 2] This shows the results of producing various forms of cetuximab-based bi-antigen target protein complexes using animal cells and confirming whether or not their production was successful using SDS-PAGE.

[0132] [Figure 3A] This shows the results of comparing the binding affinity of CET_ZAAD01D and CET_ZAAD01M4 to CD137 and EGFR proteins.

[0133] [Figure 3B] This shows the results of comparing the binding affinity of CET_ZAAD05D and CET_ZAAD05M4 to CD137 and EGFR proteins.

[0134] [Figure 3C] This shows the results of comparing the binding affinity of CET_ZAAD01D and CET_ZAAD01D_LC to CD137 and EGFR proteins.

[0135] [Figure 3D] This shows the results of comparing the binding affinity of CET_ZAAD05D and CET_ZAAD05D_LC to CD137 and EGFR proteins.

[0136] [Figure 4] This shows the results of comparing the binding affinity of various forms of cetuximab-based dual antigen target protein complexes to activated CEMT cells expressing CD137.

[0137] [Figure 5A] This shows the results of a comparative study of the anticancer effects of various forms of cetuximab-based dual antigen target protein complexes CET_ZAAD01D, CET_ZAAD01M4, CET_ZAAD05D, and CET_ZAAD05M4 using cytotoxicity tests.

[0138] [Figure 5B] This shows the results of a comparative study of the anticancer effects of various forms of cetuximab-based dual antigen target protein complexes CET_ZAAD01D, CET_ZAAD0d_LC, ZAAD05D, and CET_ZAAD05D_LC using cytotoxicity tests.

[0139] [Figures 6A and 6B] Comparison of the EGFR protein-dependent CD8 T cell activity of various forms of cetuximab-based biantigen target protein complexes.

[0140] [Figure 7] Comparison of the HT29 cell-dependent CD8 T cell activation capacity of various forms of cetuximab-based biantigen target protein complexes.

[0141] [Figures 8A-8D] Results of comparing the purity of various forms of cetuximab-based bi-antigen target protein complexes using SEC-HPLC.

[0142] [Figure 9] This shows the results of cytotoxicity tests confirming the anticancer effects of various forms of biantigen-targeting protein complexes based on 15E3.

[0143] [Figure 10] This shows the results of producing a dual antigen-targeting protein complex based on an antibody that targets PSMA and confirming it by SDS-PAGE.

[0144] [Figure 11] Results of analysis of the LNCap cell-dependent CD8 T cell activation ability of a dual antigen-targeting protein complex based on an antibody targeting PSMA. [Modes for carrying out the invention] The present invention will be described in more detail below using examples. These examples are merely for the purpose of illustrating the present invention more concretely, and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited to these examples, given the gist of the invention.

[0145] [Examples] Example 1: Preparation of various forms of bi-antigen target protein complexes To create a protein complex targeting a biantigen, an antibody and an affibody were conjugated to produce the complex. To confirm the optimal morphology of the produced protein complex, the morphology shown in Figure 1 was secured by gene cloning. After producing the protein complex with the above morphology using animal cells, its size and purity were confirmed by SDS-PAGE.

[0146] 1 x 10⁶ HEK293F cells 6 100 mL of culture medium at a concentration of cells / mL was prepared and cultured with shaking at 37°C, 8% CO2, and RPM 125. 5 mL of culture medium was placed in a sterile 15 mL tube, and the expression vector DNA for expressing the heavy and light chain DNA of the antibody was added and mixed to prepare the DNA. PEI (Polysciences, cat.#.23966) was added to the 15 mL tube containing the culture medium and DNA, mixed, and then added to HEK293F cells, which were cultured for 7 days. Recombinant proteins in the culture medium were purified using Protein A resin (GE healthcare, cat.#.17-5438-03), and the buffer was replaced using diafiltration (Satorius, cat.#.VS2002). The purified recombinant proteins were quantified by measuring absorbance at a wavelength of 280 nm. The purified recombinant proteins were analyzed by 4-20% gradient SDS-PAGE.

[0147] Using a basic configuration in which an affibody is attached to the C-terminus of the antibody's heavy chain, various optimized configurations of dual antigen-targeting protein complexes were constructed. The configuration in which an affibody dimer is added to the C-terminus of the antibody's heavy chain was called the D form, the configuration in which affibody is added to both the C-terminus of the antibody's heavy and light chains was called the M4 form, and the configuration in which an affibody dimer is added to the C-terminus of the antibody's light chain was called the D_LC form. The structures of the constructed dual antigen-targeting protein complexes are shown in Figure 1.

[0148] Example 2: Production of various forms of biantigen-targeting protein complexes based on an antibody (cetuximab) that targets EFGR. The biantigen-targeting protein complexes described above were constructed using cetuximab antibody and CD137-binding affibodies (ZAAD01 / ZAAD05). The names of the D, M4, and D_LC forms of the biantigen-targeting complex proteins constructed using cetuximab and affibodies were designated as CET_ZAAD01D / CET_ZAAD05D, CET_ZAAD01M4 / CET_ZAAD05M4, and CET_ZAAD01D_LC / CET_ZAAD05D_LC, respectively.

[0149] The SDS-PAGE results were used to confirm whether each biantibody was successfully produced to the intended size.

[0150] The results are shown in Figure 2.

[0151] As shown in Figure 2, increased molecular weight was observed in the heavy chain bands of CET_ZAAD01 and CET_ZAAD05 compared to CET, and increased molecular weight was observed in the heavy chain bands of CET_ZAAD01D and CET_ZAAD05D compared to CET_ZAAD01 and CET_ZAAD05. Increased molecular weight was observed in the light and heavy chain bands of CET_ZAAD01M4 and CET_ZAAD05M4 compared to CET. Decreased molecular weight and increased molecular weight were observed in the heavy chain bands and light chain bands compared to CET_ZAAD01D_LC and CET_ZAAD05D_LC compared to CET_ZAAD01D and CET_ZAAD05D. Therefore, it was confirmed that biantibodies of each form were successfully produced (reducing conditions in Figure 2). Some of the optimized biantibody candidates (CET_ZAAD01D / CET_ZAAD05D / CET_ZAAD05_LC) showed bands larger than normal size (non-reducing condition, Figure 2).

[0152] Example 3: Comparison of target binding ability of antigen-targeted protein complexes based on an antibody (cetuximab) that targets EFGR. The binding affinity of purified biantigen-targeting protein complexes to target proteins or cells expressing them was analyzed. The binding of CD137 to EGFR protein was confirmed using ELISA with eight produced biantigen-targeting protein complexes. For ELISA, plates coated with hCD137-ECD-Fc and hEGFR-ECD-Fc proteins at a concentration of 1 ug / mL were treated with the eight purified biantibodies at 7 points, starting at 60 nM and diluting to 1 / 5. After treatment with a secondary antibody (anti-hIgG-Fab-HRP (Jackson, JAC-109-035-097)), a color reaction was generated using TMB (biofx, TMBC-1000-01). OD450 values ​​were measured using an ELISA reader (Victor X3 Perkinelmer), and EC50 values ​​were determined using a graph prism.

[0153] The results are shown in Figures 3A, 3B, 3C, 4D, Table 1, and Table 2.

[0154] As shown in Figure 3A, no difference was observed in the degree of binding to the CD137 protein between CET_ZAAD01, CET_ZAAD01D, and CET_ZAAD01M4. As shown in Table 1, no difference was observed in the actual EC50 values. As shown in Figure 3B, the degree of binding to the CD137 protein increased in CET_ZAAD05D and CET_ZAAD05M4 compared to CET_ZAAD05, and as shown in Table 1, the actual EC50 values ​​improved by more than 10 times.

[0155] [Table 1]

[0156] As shown in Figures 3C, 3D, and Table 2, the graphs confirm that the degree of bonding in CET_ZAAD01D_LC and CDT_ZAAD05D_LC is reduced or similar to that of CET_ZAAD01 and CET_ZAAD05.

[0157] [Table 2]

[0158] To confirm the binding ability of various forms of dual antigen-targeting protein complexes to CD137-expressing cells, CEMT cells (ATCC, CCL-119) were subjected to 1X10⁻¹⁰⁻¹⁰⁻⁴ 7 Prepare 10 mL of water, treat with PMA (Sigma, P1585) at a concentration of 50 ng / mL and ionomycin (Sigma, I9657) at a concentration of 1 ug / mL, then culture for 16 hours to activate. Activated CEMT cells (ATCC, CCL-119) were then placed in 5 x 10⁻¹⁵ 5 Cells were prepared at the concentration of / tube, centrifuged at 1200 rpm for 3 minutes to collect, and washed with PBS containing 5% FBS. Subsequently, the cells were treated with a 0.48 nM biantigen target protein complex and cultured on ice for 1 hour. Then, the cells were washed three times with 200 μL of PBS containing 5% FBS by centrifuging at 1200 rpm for 3 minutes. Next, the cells were treated with anti-human-Fc-FITC (LT,A11013) at a concentration of 1 ug / mL and cultured on ice for 45 minutes while blocking light. After washing the cells three times with 200 μL of PBS containing 5% FBS by centrifuging at 1200 rpm for 3 minutes, the fluorescence intensity was measured using a Beckman Coulter FACS instrument.

[0159] The results are shown in Figure 4.

[0160] As shown in Figure 4, when the degree of binding to activated CEMT cells was examined, the MFI values ​​were similar for CET_ZAAD01D, increased for CET_ZAAD01M4, and decreased for CET_ZAAD01D_LC compared to CET_ZAAD01. Compared to CET_ZAAD05, the MFI values ​​were similar for CET_ZAAD05D, increased for CET_ZAAD01M4, and similar for CET_ZAAD05D_LC.

[0161] Example 4: Comparison of cytotoxicity of a dual antigen-targeting protein complex based on an antibody (cetuximab) that targets EFGR. The potential anticancer effect was confirmed by comparing the degree of cytotoxicity of the produced biantigen-targeting protein complex under co-culture conditions of PBMCs and EGFR-expressing cells. One day prior, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) at a concentration of 5 μg / mL was coated onto each 96-well round plate. Three washes were performed with 100 μL of culture medium (RPMI1640, 10% FBS), followed by treatment with 100 μL of culture medium (RPMI1640, 10% FBS) and blocking at 37°C for 1 hour. The culture medium (RPMI1640, 10% FBS) on the plate was completely removed, and DLD-1, which expresses luciferase, was added to the culture medium in a 1.0 x 10⁶ dose. 5 50 μL of a concentration of / mL was used for treatment. Subsequently, eight types of biantigen target protein complexes were treated at 6 points, starting from 1 nM and diluting to 1 / 10, with 50 μL of each. Meanwhile, blood from healthy donors was diluted 1 / 2 with PBS, placed in a Leucosep tube (greiner bio-one, 227290) with Picoll (GE healthcare, 17-1440-12) floating on it, and centrifuged at 1000 g for 30 minutes. Only the layer of PBMCs located on Picoll (GE healthcare, 17-1440-12) was collected. The PBMCs were washed twice with 300 g of PBS for 10 minutes, and then the number of PBMCs was measured. 1.0 x 10⁻⁶ in culture medium. 550 μL of a solution at a concentration of / mL was used for treatment and cultured for 3 days. Cell down was performed on the 96-well plates that had been cultured for 3 days using a centrifuge at 2000 RPM for 2 minutes. The culture solution was transferred in 50 μL / well portions to 96-well V-bottom plates and stored for subsequent experiments. 3X Lysis Buffer (75 mm Tris (pH 8.0), 30% Glycerol, 3% Triton X-100) was dispensed into the remaining 96-well round plates in 50 μL / well portions and mixed thoroughly. The mixture was allowed to react at room temperature for 15 minutes. After the reaction was complete, the samples were dispensed into 96-well white plates in 50 μL / well portions, and then luciferase assay reagent (Promega, G7940) was dispensed into 50 μL / well portions and mixed thoroughly. The mixture was allowed to react at room temperature for 15 minutes while blocking out light. After the reaction was complete, measurements were taken using a VITOR™ X3.

[0162] The results are shown in Figures 5A and 5B.

[0163] As shown in Figures 5A and 5B, cytotoxicity tests confirmed that the D, M4, and D_LC protein complexes exhibited increased cytotoxicity compared to the basic form of the dual antigen target protein complex. Compared to CET_ZAAD01, CET_ZAAD01D and CET_ZAAD01M4 showed increased cytotoxicity, with the cell death rate improving from approximately 50% to approximately 70% at its maximum. Compared to CET_ZAAD05, CET_ZAAD05D and CET_ZAAD05M4 showed an improvement in cell death rate from approximately 40% to 65% at its maximum. CET_ZAAD01D_LC exhibited similar cytotoxicity to CET_ZAAD01D, and CET_ZAAD05D_LC also exhibited similar cytotoxicity to CET_ZAAD05D.

[0164] Example 5: Comparison of CD8 T cell activation capabilities of various forms of biantigen-targeting protein complexes based on an antibody (cetuximab) that targets EFGR. The EGFR protein-dependent CD8 T cell activation ability was compared using various forms of the produced biantigen-targeting protein complexes. One day prior, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) at a concentration of 3 μg / mL was coated onto each 96-well round plate. The following day, the plates were washed three times with 100 μL of sterile PBS. Each plate was again coated with 50 μL of hEGFR-ECD-Fc at a concentration of 5 μg / mL and incubated at 37°C for 3 hours. Three washes were performed with 100 μL of R10 medium (media) (RPMI1640, 10% FBS, 10 mM Hepes), followed by treatment with 100 μL of R10 medium (media) (RPMI1640, 10% FBS, 10 mM Hepes) and blocking at 37°C for 1 hour. The R10 medium (RPMI1640, 10% FBS, 10 mM Hepes) on the plate was completely removed, and six types of biantibodies were treated at 4 points with 50 μL for 30 minutes, starting at 5 nM and diluted to 1 / 5. Meanwhile, blood from a healthy donor was diluted 1 / 2 with PBS, placed in a Leucosep tube (greiner bio-one, 227290) with Picoll (GE healthcare, 17-1440-12) floating on it, and centrifuged at 1000 g for 30 minutes. Only the layer of PBMCs located on Picoll (GE healthcare, 17-1440-12) was collected. After repeating the washing process twice with 300 g of PBS containing 2% PBMCs for 10 minutes, the number of PBMCs was measured. CD8-positive T cells were prepared using PBMCs and the CD8 isolation kit (Myltenybiotec / 130-096-495). The number of CD8+ T cells was measured to 1.4 x 10⁻⁶. 6After soaking the cells in R10 medium (RPMI1640, 10% FBS, 10 mM Hepes) to a concentration of / mL, 50 μL of the solution was applied to plates treated with the biantigen target protein complex. Three days after treatment, the plates were centrifuged at 2000 RPM for 10 minutes, and the supernatant was collected and used to proceed with the IFN-γ measurement kit (BD, 555142). The degree of CD8 T cell activity was compared by measuring CD8 T cell proliferation (WST-8) and IFN-γ.

[0165] The results are shown in Figures 6A and 6B.

[0166] As shown in Figures 6A and 6B, the activity capacity of CD8 T cells was superior in the D and M4 morphologies compared to the basic morphology of the biantigen-targeting protein complex. As shown in Figure 6A, CD8 T cell proliferation increased in CET_ZAAD01D and CET_ZAAD01M4 compared to CET_ZAAD01, and increased in CET_ZAAD05D and CET_ZAAD05M4 compared to CET_ZAAD05. As shown in Figure 6B, similar results were observed for secreted IFN-γ as for cell proliferation.

[0167] Furthermore, a comparison was made using HT29 cells, which express EGFR, in a CD8 T cell activation test.

[0168] One day prior, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) at a concentration of 3 μg / mL was coated onto each 96-well round plate. The following day, three washes were performed using 100 μL of sterile PBS. Three washes were then performed using 100 μL of R10 medium (RPMI1640, 10% FBS, 10 mM Hepes), followed by 100 μL of R10 medium (RPMI1640, 10% FBS, 10 mM Hepes) treatment and blocking at 37°C for 1 hour. The R10 medium (RPMI1640, 10% FBS, 10 mM Hepes) on the plate was completely removed, and the number of HT29 cells was measured. 5After soaking R10 medium to a concentration of / mL, the samples were plated and treated with 50 μL. Six types of biantigen target protein complexes were treated at 4 points with 50 μL for 30 minutes, starting at 5 nM and diluting to 1 / 5. Meanwhile, blood from healthy donors was diluted 1 / 2 with PBS, placed in a Leucosep tube (greiner bio-one, 227290) with Picoll (GE healthcare, 17-1440-12) floating on it, and centrifuged at 1000 g for 30 minutes. Only the layer of PBMCs located on Picoll (GE healthcare, 17-1440-12) was collected. After repeating the washing process twice with PBS containing 2% PBMCs and centrifuging at 300 g for 10 minutes, the number of PBMCs was measured. CD8-positive T cells were prepared using PBMCs and the CD8 isolation kit (Myltenybiotec / 130-096-495). The number of CD8+ T cells was measured to 1.4 x 10⁻⁶. 6 After soaking the cells in R10 medium (media) (RPMI1640, 10% FBS, 10 mM Hepes) to a concentration of / mL, 50 μL of the solution was applied to plates treated with the biantigen target protein complex. Three days after treatment, the plates were centrifuged at 2000 RPM for 10 minutes, and the supernatant was collected and used to proceed with the IFN-γ measurement kit (BD, 555142) process. The degree of CD8 T cell activity was compared by measuring IFN-γ expression levels.

[0169] The results are shown in Figure 7.

[0170] As shown in Figure 7, it was confirmed that the amount of secreted IFN-γ was significantly increased in CET_ZAAD01D and CET_ZAAD01M4 compared to CET_ZAAD01, and that the expression level of secreted IFN-γ was also significantly increased in CET_ZAAD05D and CET_ZAAD05M4 compared to CET_ZAAD05.

[0171] Example 6: SEC-HPLC analysis of various forms of biantigen-targeting protein complexes based on an antibody (cetuximab) targeting EFGR. The purity of the produced biantigen-targeted protein complexes was compared by SEC-HPLC analysis. To confirm purity, the biantigen-targeted protein complexes were prepared at a concentration of 1 mg / mL and filtered through a 0.2 μm PES filter. The filtered biantigen-targeted protein complexes were dispensed into HPLC analysis screw vials in 20 μL portions. A mobile phase buffer (0.1 M potassium phosphate + 0.2 M arginine, pH 6.8) was connected to the HPLC instrument, and the SEC_HPLC (TOSOH, SWXL3000-7.8 mm x 30 cm) column used for analysis was also connected. The method was set to a flow rate of 0.8 mL / min, detection at 280 nm, injection volume of 10 μL, and temperature of 25 °C. The analysis order and sample names were entered into the instrument's program according to the number of samples to be analyzed, and the instrument was run to analyze each biantigen-targeted protein complex. The detection time, peak area, etc., of each sample were analyzed using the HPLC analysis program.

[0172] The results are shown in Figures 8A, 8B, 8C, 8D, and Table 3.

[0173] As shown in Figures 8A, 8B, 8C, 8D, and Table 3, purity analysis was performed on six optimized bivalent antibodies using SEC_HPLC. The main peak time was compared with that of CET, and the purity of each antibody was analyzed accordingly. As shown in Figures 8A, 8B, 8C, 8D, and Table 3, CET_ZAAD01 showed a purity of 98%, CET_ZAAD01D approximately 83%, and CET_ZAAD01M4 approximately 97%. CET_ZAAD05 showed a purity of approximately 97%, CET_ZAAD05D approximately 64%, and CET_ZAAD05M4 approximately 89%.

[0174] [Table 3]

[0175] The overall evaluation results of various forms of biantigen-targeting protein complexes based on cetuximab are shown in Table 4.

[0176] [Table 4]

[0177] Example 7: Comparison of cytotoxicity of various forms of biantigen-targeting protein complexes based on an antibody (15E3) that targets EFGR. A dual antigen-targeting protein complex based on the EGFR-specific antibody 15E3 was prepared using the method described in Example 1, and the potential anticancer effect was confirmed by comparing the degree of cytotoxicity under co-culture conditions of PBMCs and EGFR-expressing cells. One day prior, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) at a concentration of 5 μg / mL was coated onto each 96-well round plate. Three washes were performed using 100 μL of culture medium (RPMI1640, 10% FBS), followed by treatment with 100 μL of culture medium (RPMI1640, 10% FBS) and blocking at 37°C for 1 hour. The culture medium (RPMI1640, 10% FBS) on the plate was completely removed, and DLD-1 expressing luciferase was added to the culture medium in a 1.0 x 10⁶ dose. 5 After dispensing at a concentration of 1 / mL, the plate was treated with 50 μL. Then, eight types of biantibodies were treated at 6 points, starting from 1 nM and diluting to 1 / 10, with 50 μL each. Meanwhile, blood from a healthy donor was diluted 1 / 2 with PBS, placed in a Leucosep tube (greiner bio-one, 227290) with Picoll (GE healthcare, 17-1440-12) suspended in it, and centrifuged at 1000 g for 30 minutes. Only the layer of PBMCs located on the Picoll (GE healthcare, 17-1440-12) was collected. After repeating the washing process twice using PBS containing 2% PBMCs and centrifuging at 300 g for 10 minutes, the number of PBMCs was measured. 1.0 x 10⁻⁶ cells were placed in the culture medium. 5After dispensing at 1 / mL, 50 μL was added to the plate and incubated for 3 days. Cell down was performed on the 96-well plates incubated for 3 days using a centrifuge at 2000 RPM for 2 minutes. The culture solution was transferred to 96-well V-bottom plates in 50 μL / well portions and stored for subsequent experiments. 3X lysis buffer (75 mm Tris (pH 8.0), 30% glycerol, 3% Triton X-100) was dispensed into the remaining 96-well round plates in 50 μL / well portions and mixed thoroughly. The mixture was allowed to react at room temperature for 15 minutes. After the reaction was complete, the samples were dispensed into 96-well white plates in 50 μL / well portions, and luciferase analysis reagent (Promega, G7940) was dispensed into 50 μL / well portions and mixed thoroughly. The mixture was allowed to react at room temperature for 15 minutes while blocking out light. After the reaction was complete, measurements were taken using a VITOR™ X3 device. Confirmation was performed on two donors.

[0178] The results are shown in Figures 9A and 9B.

[0179] As shown in Figures 9A and 9B, analysis of cytotoxicity revealed that the M4 morphology protein complex exhibited superior cytotoxicity compared to the basic morphology of the biantigen-targeting protein complex.

[0180] As shown in Figure 9A, the graph confirms that the cytotoxicity of 15E3_ZAAD01M4 is increased compared to 15E3_ZAAD01. In the first donor, the cell viability decreased from approximately 50% to approximately 30% at its maximum value, confirming improved cytotoxicity. In the second donor, the cell viability decreased from approximately 70% to approximately 35% at its maximum value, confirming improved cytotoxicity.

[0181] As shown in Figure 9B, compared to 15E3_ZAAD05, 15E3_ZAAD05M4 showed improved cytotoxicity in the first donor, with cell viability decreasing from approximately 60% to 35% at its maximum value, and in the second donor, cell viability decreasing from approximately 85% to approximately 35% at its maximum value, indicating improved cytotoxicity.

[0182] Example 8: Production of various forms of biantigen-targeting protein complexes based on antibodies targeting PSMA Various forms of dual antigen-targeting protein complexes based on antibodies targeting PSMA were prepared using the J591 antibody and affibo (ZAAD01 / ZAAD05) as described in Example 1.

[0183] Based on the PSMA target antibody J591, we created dual antigen-targeting protein complexes in two forms: the D form, in which an affibody dimer is added to the C-terminus of the antibody's heavy chain, and the M4 form, in which affibodies are added to both the C-terminuses of the antibody's heavy and light chains. These complexes were named J591_ZAAD01D / J591_ZAAD05D and J591_ZAAD01M4 / J591_ZAAD05M4, respectively.

[0184] As shown in Figure 10, SDS-PAGE results confirmed the successful construction of each form of the biantigen-targeting protein complex (reduced conditions in Figure 10). Among the optimized biantibody candidates, J591_ZAAD01D / J591_ZAAD05D clearly showed bands larger than normal size (non-reduced conditions in Figure 10). This is similar to the results for the cetuximab-based antibody.

[0185] Example 9: Comparison of CD8 T cell activation capabilities of various forms of biantigen-targeting protein complexes based on PSMA-targeting antibodies. We compared the PSMA-dependent CD8 T cell activation ability using a dual antigen target protein complex based on the J591 antibody, which is specific to PSMA. CD8 T cell activation tests were performed using LNCap cells, which express PSMA, and MKN45 cells, which do not express PSMA. One day prior, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) at a concentration of 3 μg / mL was coated onto each 96-well round plate. The following day, the plates were washed three times with 100 μL of sterile PBS. They were then washed three times with 100 μL of R10 medium (RPMI1640, 10% FBS, 10 mM Hepes), treated with 100 μL of R10 medium (RPMI1640, 10% FBS, 10 mM Hepes), and blocked at 37°C for 1 hour. Completely remove the R10 medium (RPMI1640, 10% FBS, 10 mM Hepes) from the plate and count the number of LNCap cells and MKN45 cells in a 7x10 grid. 5 After soaking R10 medium to a concentration of / mL, the plate was treated with 50 μL. Six types of biantigen target protein complexes were treated with 50 μL at 25 nM for 30 minutes. Meanwhile, blood from a healthy donor was diluted 1 / 2 with PBS, placed in a Leucosep tube (greiner bio-one, 227290) with Picoll (GE healthcare, 17-1440-12) floating on it, and centrifuged at 1000 g for 30 minutes. Only the layer of PBMCs located on Picoll (GE healthcare, 17-1440-12) was collected. The PBMCs were centrifuged at 300 g for 10 minutes using PBS containing 2% PBMCs, washed twice, and then the number of PBMCs was measured. CD8-positive T cells were prepared from the PBMCs using the CD8 isolation kit (Myltenybiotec / 130-096-495). The number of CD8+ T cells was measured and 1.4 x 10⁻⁶ 6After soaking the cells in R10 medium (RPMI1640, 10% FBS, 10 mM Hepes) to a concentration of / mL, 50 μL was applied to plates treated with the biantigen target protein complex. Three days after treatment, the plates were centrifuged at 2000 RPM for 10 minutes, and the supernatant was collected. This supernatant was then used to perform the IFN-γ assay kit (BD, 555142) and the granzyme B assay kit (R&D systems, DY2906-05) according to their respective processes. The degree of CD8 T cell activity was compared by measuring IFN-γ and granzyme B.

[0186] The results are shown in Figure 11.

[0187] As shown in Figure 11, the CD8 T cell activation ability of the D and M4 forms of the biantigen target protein complex was superior to that of the basic form. Increased secretion of IFN-γ and granzyme B was confirmed in J591_ZAAD01D and J591_ZAAD01M4 compared to J591_ZAAD01, and increased secretion of IFN-γ and granzyme B was also confirmed in J591_ZAAD05D and J591_ZAAD05M4 compared to J591_ZAAD05. [Brief explanation of the drawing]

[0188] [Figure 1] This diagram schematically illustrates the optimized form of a biantigen-targeting protein complex. [Figure 2] This report details the results of producing various forms of cetuximab-based bi-antigen target protein complexes using animal cells, and confirming the success of their production using SDS-PAGE. [Figure 3A] This is a comparison of the binding affinity of CET_ZAAD01D and CET_ZAAD01M4 to CD137 and EGFR proteins. [Figure 3B] This is a comparison of the binding affinity of CET_ZAAD05D and CET_ZAAD05M4 to CD137 and EGFR proteins. [Figure 3C]This is a comparison of the binding affinity of CET_ZAAD01D and CET_ZAAD01D_LC to CD137 and EGFR proteins. [Figure 3D] This is a comparison of the binding affinity of CET_ZAAD05D and CET_ZAAD05D_LC to CD137 and EGFR proteins. [Figure 4] This study compares the binding affinity of various cetuximab-based dual antigen target protein complexes to activated CEMT cells expressing CD137. [Figure 5A] This is a comparative study of the anticancer effects of various forms of cetuximab-based biantigen target protein complexes (CET_ZAAD01D, CET_ZAAD01M4, CET_ZAAD05D, and CET_ZAAD05M4) using cytotoxicity tests. [Figure 5B] This is a comparative study of the anticancer effects of various forms of cetuximab-based biantigen target protein complexes (CET_ZAAD01D, CET_ZAAD0d_LC, ZAAD05D, and CET_ZAAD05D_LC) using cytotoxicity tests. [Figure 6A] This is a comparison of the EGFR protein-dependent CD8 T cell activation capabilities of various forms of cetuximab-based biantigen target protein complexes. [Figure 6B] This is a comparison of the EGFR protein-dependent CD8 T cell activation capabilities of various forms of cetuximab-based biantigen target protein complexes. [Figure 7] This is a comparison of the HT29 cell-dependent CD8 T cell activation capacity of various forms of cetuximab-based biantigen target protein complexes. [Figure 8A] This is a comparison of the purity of various forms of cetuximab-based bi-antigen target protein complexes using SEC-HPLC. [Figure 8B] This is a comparison of the purity of various forms of cetuximab-based bi-antigen target protein complexes using SEC-HPLC. [Figure 8C]This is a comparison of the purity of various forms of cetuximab-based bi-antigen target protein complexes using SEC-HPLC. [Figure 8D] This is a comparison of the purity of various forms of cetuximab-based bi-antigen target protein complexes using SEC-HPLC. [Figure 9A] This is the result of cytotoxicity tests confirming the anticancer effects of various forms of biantigen-targeting protein complexes based on 15E3. [Figure 9B] This is the result of cytotoxicity tests confirming the anticancer effects of various forms of biantigen-targeting protein complexes based on 15E3. [Figure 10] This is the result of producing a dual antigen-targeting protein complex based on an antibody that targets PSMA and confirming it using SDS-PAGE. [Figure 11] This is the result of an analysis of the LNCap cell-dependent CD8 T cell activation ability of a dual antigen-targeting protein complex based on an antibody that targets PSMA.

Claims

1. A bi-antigen target protein complex comprising a first antigen target site containing an antibody or its antigen-binding fragment, and a second antigen target site containing an affibody bound to the antibody or its antigen-binding fragment.

2. The bi-antigen target protein complex according to claim 1, wherein an aphibody dimer is bound to the C-terminus of the heavy chain of an antibody or its antigen-binding fragment.

3. The bi-antigen target protein complex according to claim 1, wherein an aphibody monomer is bound to the C-terminus of the heavy chain and light chain of the antibody or its antigen-binding fragment, respectively.

4. The bi-antigen target protein complex according to claim 1, wherein an aphibody dimer is bound to the C-terminus of the light chain of an antibody or its antigen-binding fragment.

5. The dual antigen-targeting protein complex according to claim 1, wherein the first antigen target site targets EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen), and the second antigen target site targets CD137.

6. The bi-antigen target protein complex according to claim 1, wherein the affibody consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

7. The dual antigen-targeting protein complex according to claim 1, wherein the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 3 to 8, respectively; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 27 to 32, respectively; or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences shown in SEQ ID NOs. 51 to 56, respectively.

8. The dual antigen-targeting protein complex according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain and a light chain consisting of the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively, a heavy chain and a light chain consisting of the amino acid sequences shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively, or a heavy chain and a light chain consisting of the amino acid sequences shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively.

9. The dual antigen-targeting protein complex according to claim 1, comprising a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 11 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 12; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 19 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 20; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 35 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 36; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 43 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 44; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 59 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 60; or a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 67 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO:

68.

10. The dual antigen-targeting protein complex according to claim 1, comprising a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 13 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 14; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 21 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 22; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 37 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 38; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 45 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 46; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 61 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 62; or a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 69 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO:

70.

11. The aforementioned biantigen-targeting protein complex includes a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 15 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 16 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 23 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 24 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 39 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 40 is bound; SEQ ID NO: 4 The biantigen target protein complex according to claim 1, comprising a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in 7 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 48 is bound, comprising a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 63 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 64 is bound, or comprising a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 71 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 72 is bound.

12. The dual antigen-targeting protein complex according to claim 1, comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 17 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 18 is bound; comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 25 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 26 is bound; comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 41 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 42 is bound; comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 49 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 50 is bound; comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 65 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 66 is bound; or comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 73 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 74 is bound.

13. A nucleic acid molecule comprising a nucleotide sequence encoding a biantigen target protein complex according to any one of claims 1 to 12.

14. A recombinant vector comprising the nucleic acid molecule described in claim 13.

15. A host cell comprising the recombinant vector according to claim 14.

16. A pharmaceutical composition for the prevention or treatment of cancer comprising a biantigen-targeting protein complex comprising a biantigen-targeting protein complex comprising an EFGR (Epidermal Growth Factor Receptor) or PSMA (Prostate-Specific Membrane Antigen) target site containing an antibody or its antigen-binding fragment, and a CD137 target site containing an affibody bound to the antibody or its antigen-binding fragment.

17. The pharmaceutical composition for the prevention or treatment of cancer according to claim 16, wherein the double antigen target protein complex has an aphibody dimer bound to the C-terminus of the heavy chain of an antibody or its antigen-binding fragment.

18. The pharmaceutical composition for the prevention or treatment of cancer according to claim 16, wherein the biantigen-targeting protein complex has aphibody monomers bound to the C-terminuses of the heavy chain and light chain of an antibody or its antigen-binding fragment, respectively.

19. The pharmaceutical composition for the prevention or treatment of cancer according to claim 16, wherein the double antigen target protein complex has an aphibody dimer bound to the C-terminus of the light chain of an antibody or an antigen-binding fragment thereof.

20. The pharmaceutically active ingredient for the prevention or treatment of cancer according to claim 16, wherein the dual antigen target protein complex comprises a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 11 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 12; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 19 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 20; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 35 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 36; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 43 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 44; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 59 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 60; or a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 67 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO:

68.

21. The pharmaceutically active ingredient for the prevention or treatment of cancer according to claim 16, wherein the dual antigen target protein complex comprises a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 13 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 14; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 21 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 22; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 37 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 38; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 45 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 46; a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 61 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 62; or a heavy chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 69 is bound and a light chain consisting of the amino acid sequence shown in SEQ ID NO:

70.

22. The aforementioned biantigen target protein complex includes a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 15 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 16 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 23 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 24 is bound; a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 39 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 40 is bound; and SEQ ID NO: 47 A pharmaceutical composition for the prevention or treatment of cancer according to claim 16, comprising a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 48 is bound, comprising a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 63 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 64 is bound, or comprising a heavy chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 71 is bound, and a light chain to which an aphibody monomer consisting of the amino acid sequence shown in SEQ ID NO: 72 is bound.

23. The pharmaceutically active ingredient for the prevention or treatment of cancer according to claim 16, wherein the dual antigen target protein complex comprises a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 17 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 18 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 25 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 26 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 41 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 42 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 49 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 50 is bound; a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 65 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 66 is bound; or a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 73 and a light chain to which an aphibody dimer consisting of the amino acid sequence shown in SEQ ID NO: 74 is bound.

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