Novel Anti-ceacam5 antibody and therapeutic agent use thereof

Abstract

The present invention relates to a novel antibody specifically binding to CEACAM5, and a therapeutic use thereof. The antibody according to the present invention can be in the form of a monoclonal antibody or a bispecific antibody, and the bispecific antibody can be a biparatopic antibody. The antibody according to the present invention is useful for the treatment of cancer. The antibody according to the present invention can have various antibody-based modalities that can be used for cancer treatment, and the antibody-based modalities include antibody-drug conjugates (ADCs).

Description

Novel anti-CEACAM5 antibodies and their therapeutic uses

[0001] The present invention relates to a novel antibody that specifically binds to CEACAM5 and its therapeutic use. The antibody according to the present invention may be in the form of a monoclonal antibody or a bispecific antibody, and the bispecific antibody may be a biparatopic antibody. The antibody according to the present invention is useful for cancer treatment. The antibody according to the present invention may take the form of various antibody-based modalities that can be used for cancer treatment, including antibody-drug conjugates (ADCs). The present invention relates to a composition for cancer treatment comprising the antibody or an antibody-based modality comprising the antibody. The present invention also relates to a method for cancer treatment comprising administering the antibody or an antibody-based modality comprising the antibody.

[0002] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. The CEA family belongs to the immunoglobulin superfamily. The 18-gene CEA family is subdivided into two subgroups: the carcinoembryonic antigen-related cell adhesion protein (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup (Kammerer & Zimmermann, BMC Biology 2010, 8:12).

[0003] The CEACAM subfamily includes CEACAM1, CEACAM3, CEACAM5, CEACAM6, CEACAM7, and CEACAM8, and these proteins are expressed in various cell types and perform a wide range of biological functions, including cell adhesion, immune response regulation, and pathogen recognition.

[0004] CEACAM5 (designated CEA and also known as CD66e) is a glycoprotein with a molecular weight of approximately 180 kDa. CEACAM5, containing seven domains, is anchored to the cell membrane via glycosyl-phosphatidyl-inositol (GPI). The seven domains include a single N-terminal Ig variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to Ig constant domains.

[0005] CEACAM5 is known to be expressed primarily in epithelial cells in normal adult tissues, but its expression levels are known to be significantly increased in embryonic tissues and many cancerous tissues. CEACAM5 is particularly overexpressed in solid tumors such as colon, gastric, pancreatic, non-small cell lung, and breast cancers, and has been reported to be closely associated with disease progression and prognosis. Given its high tumor specificity and limited expression in normal tissues, CEACAM5 is emerging as a tumor-associated antigen useful for targeted therapy.

[0006]

[0007] The contents described in the background art of this specification are merely reference materials to facilitate understanding of the present invention, and should not be construed as being recognized as prior art.

[0008] Cancer is a leading cause of death worldwide and remains a challenging disease for many patients. Conventional cancer treatments primarily rely on surgery, radiation therapy, chemotherapy, and targeted therapies. While these treatments have shown some therapeutic efficacy in certain cancer types, they often fail to achieve complete cure due to issues such as genetic and phenotypic heterogeneity of cancer cells, the development of resistance, non-specific toxicity to normal cells, and high recurrence rates.

[0009] Immunotherapy, including immune checkpoint inhibitors, has recently emerged as a promising new treatment strategy. However, these immune-based treatments also suffer from limitations, such as limited targeting, unpredictable immune responses, and disparities in efficacy. Furthermore, some patients respond only to these treatments, or immune-related adverse reactions are reported. Consequently, there is a growing demand for novel, precise anticancer treatments that can more selectively target tumors and overcome the limitations of existing treatments.

[0010] Antibody therapeutics, particularly those targeting antigens specifically expressed in tumor cells but with limited expression in normal cells, offer the potential to overcome the limitations of existing treatments in terms of therapeutic selectivity and safety. This approach is being explored for application to various solid tumors and can be utilized as antibodies themselves or in antibody-drug conjugates (ADCs). Accordingly, there is a growing need for novel antibodies that recognize tumor-specific antigens and therapeutic strategies based on them.

[0011] Meanwhile, the inventors of the present invention noted that antibodies recognizing tumor-specific antigens can be more effective when they are biparatopic. Biparatopic antibodies are designed so that a single antibody molecule can simultaneously recognize two different epitopes within the same target protein. This is distinct from typical monoclonal or bispecific antibodies and can provide stronger binding affinity for a single target protein or enhanced intracellular delivery efficiency. This structure can offer advantages in antigen clustering and promoting target internalization.

[0012] Biparatopic antibodies have the advantage of simultaneously improving tumor selectivity and therapeutic efficacy by recognizing multiple epitopes on a single cancer target protein. Specifically, when targeting proteins with complex domain structures and multifunctionality, such as CEACAM5, biparatopic antibodies can enhance the efficacy of ADC therapeutics by more effectively inducing antibody internalization. Furthermore, by securing multiple binding sites within a single antibody, the possibility of evading drug resistance can be reduced.

[0013] Just because monoclonal antibodies recognize different epitopes doesn't mean that biparatopic antibodies, created by combining them, will always exhibit beneficial effects. Simply recognizing different epitopes isn't enough; for example, the two epitopes must be spatially aligned on the target protein to induce effective clustering, although this isn't necessarily theoretical. Furthermore, one of the key advantages of biparatopic antibodies is their ability to induce cellular internalization, effectively releasing the ADC drug and enhancing therapeutic effects, such as tumor cell death. However, this internalization ability can also vary depending on the monoclonal antibodies used in the combination.

[0014] Considering the technical needs described above and the efficacy of biparatopic antibodies, the present invention seeks to provide a novel antibody capable of specifically binding to CEACAM5. More specifically, the present invention aims to provide an antibody that targets CEACAM5, which is overexpressed in solid tumors, and selectively recognizes and binds to cancer cells while also enabling superior drug delivery, thereby providing a novel therapeutic agent with enhanced tumor specificity and therapeutic efficacy.

[0015]

[0016] The tasks of the present invention are not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those skilled in the art from the description below.

[0017] The present invention provides a novel antibody or antigen-binding fragment thereof that specifically binds to CEACAM5. The novel antibody or antigen-binding fragment thereof according to the present invention is implemented as a variable region sequence including specific CDR sequences, and specifically includes a set of six specific CDR sequences (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3) selected from SEQ ID NOs: 1 to 36. The antibody may be in the form of a monoclonal antibody or a bispecific antibody, and the bispecific antibody may be a biparatopic antibody.

[0018] One aspect of the present invention is a novel biparatopic antibody that specifically binds to CEACAM5. The biparatopic antibody according to the present invention can recognize and bind to two or more different epitopes present on the CEACAM5 protein. Specifically, the biparatopic antibody according to the present invention comprises six specific sets of CDR sequences (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3) selected from SEQ ID NOs: 1 to 36. Prior to the present invention, no biparatopic antibody designed to recognize and bind to two or more different epitopes present on the CEACAM5 protein was known.

[0019] Another aspect of the present invention is a novel monoclonal antibody or antigen-binding fragment thereof that specifically binds to CEACAM5. These monoclonal antibodies or antigen-binding fragments thereof can selectively recognize and bind to cancer cells by effectively targeting CEACAM5, which is specifically overexpressed in solid tumors. Furthermore, these monoclonal antibodies or antigen-binding fragments thereof can be used as part of a biparatopic antibody according to the present invention, thereby demonstrating enhanced tumor specificity and therapeutic efficacy.

[0020] Another aspect of the present invention provides an ADC comprising a novel anti-CEACAM5 antibody according to the present invention. The present invention also provides a pharmaceutical composition for treating cancer comprising, as an active ingredient, a biparatopic antibody according to the present invention or an ADC comprising the same. The present invention also provides a method for treating cancer, comprising administering to a patient in need of such treatment a biparatopic antibody according to the present invention or an ADC comprising the same.

[0021] Hereinafter, the present invention will be described in more detail through examples. However, these examples are provided merely to illustrate the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

[0022] The anti-CEACAM5 antibody of the present invention exhibits excellent specificity and binding affinity for CEACAM5, which is specifically overexpressed in solid tumors, and can enhance tumor selectivity and therapeutic efficacy. In particular, the biparatopic antibody of the present invention simultaneously recognizes two or more different epitopes on CEACAM5, thereby inducing enhanced binding affinity and intracellular internalization compared to existing antibodies that bind to a single epitope. These characteristics not only enhance the therapeutic efficacy of the antibody itself, but also significantly improve the efficiency of drug delivery when applied in the form of an antibody-drug conjugate (ADC).

[0023] Furthermore, the antibody according to the present invention can minimize nonspecific binding to normal tissues and reduce toxic reactions associated with treatment by leveraging the characteristic of CEACAM5 being selectively expressed in solid tumors. In particular, the biparatopic structure offers advantages in cancer cell-specific targeting and drug resistance avoidance, making it useful for overcoming the limitations of existing treatments.

[0024] Therefore, the present invention provides a novel antibody capable of simultaneously achieving selective targeting of cancer cells and improved therapeutic efficiency, and a therapeutic composition comprising the same, thereby being used as an effective treatment method for various cancers, including solid cancers.

[0025] The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0026] Figure 1 shows the results of SDS-PAGE analysis of purified human CEACAM5 protein.

[0027] Figure 2 is a schematic diagram showing the immunization process of CEACAM5 protein.

[0028] Figure 3 shows the results of ELISA measurement of CEACAM5 antigen in mouse anti-CEACAM5 serum obtained by immunization with CEACAM5 protein.

[0029] Figure 4a shows the results of ELISA measurements for screening of parental antibodies that bind to CEACAM5.

[0030] Figure 4b shows the results of ELISA measurements for the selection of subclone antibodies derived from selected parental clone antibodies.

[0031] Figures 4c to 4h show the results of measuring the binding affinity of selected and purified antibodies to CEACAM5-expressing cell lines.

[0032] Figure 5 shows the results of measuring the binding affinity of selected antibodies to the CEACAM5 protein.

[0033] Figures 6a to 6d show the results of measuring the binding affinity of selected antibodies to CEACAM5-expressing cells.

[0034] Figure 7 shows the results of epitope identification of the h81E3-06 antibody that binds to human CEACAM5 protein.

[0035] Figures 8a and 8b show the results of measuring the cell internalization effect when the biparatopic antibody according to the present invention and the corresponding bivalent monoclonal antibodies were administered in the form of ADC, measured in several cell lines (HPAC, HPAF-II, MKN-45, H3122, and LS174T).

[0036] Figures 9a and 9b are results showing cell viability according to the concentration of ADC when a biparatopic antibody according to the present invention is administered in the form of ADC, and represent the results measured in CEACAM5 positive and negative cell lines.

[0037] Figures 10a and 10b show changes in tumor volume and body weight of mice over time when the biparatopic antibody and tusamitamab according to the present invention were administered in the form of ADC, respectively.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the terms used herein are those well known and commonly used in the art.

[0039] The present invention provides a novel antibody or antigen-binding fragment thereof that specifically binds to CEACAM5. The novel antibody or antigen-binding fragment thereof according to the present invention is implemented as a variable region sequence including specific CDR sequences, and specifically includes a set of six specific CDR sequences (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3) selected from SEQ ID NOs: 1 to 36. The antibody may be in the form of a monoclonal antibody or a bispecific antibody, and the bispecific antibody may be a biparatopic antibody.

[0040] The term “specifically binds” as used herein means the ability of an antibody or antigen-binding fragment to selectively recognize and bind to a target antigen, such as CEACAM5, and not substantially bind to a non-target antigen or structurally similar related antigen (e.g., CEACAM1, CEACAM6, CEACAM8, etc.) under the same conditions.

[0041] The term “substantially does not bind” as used herein includes cases where the binding signal that the antibody exhibits for a non-target antigen is statistically significantly lower than the binding signal for the target antigen, and generally the relative binding signal may be 10% or less, or 20% or less. Such specific binding can be confirmed through known experimental methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), cell-based competitive binding assay, epitope binning analysis, etc. The results of binding affinity, dissociation rate, or binding selectivity measured through the above methods can be used as a technical basis for determining the expression that the antibody “specifically binds” to the target antigen as used herein.

[0042] The term “antibody” as used herein refers to conventional antibodies and fragments thereof, as well as single-domain antibodies and fragments thereof, particularly variable heavy chains of single-domain antibodies and chimeric, humanized, bispecific or multispecific antibodies. There are five types of antibodies, IgM, IgD, IgG, IgA and IgE, each of which includes a heavy chain produced from heavy chain constant region genes μ, δ, γ, α, and ε. In antibody technology, IgG is mainly used, and there are four types of isotypes, IgG1, IgG2, IgG3, and IgG4, and the structural and functional properties of each may be different. In addition, the IgG forms a very stable Y-shaped structure (molecular weight, approximately 150 kDa) composed of only two heavy chain (approximately 50 kDa) proteins and two light chain (approximately 25 kDa) proteins. The light and heavy chains of antibodies are divided into variable regions, whose amino acid sequences differ from antibody to antibody, and constant regions, whose amino acid sequences are the same. The heavy chain constant region contains CH1, H (hinge), CH2, and CH3 domains. Each domain consists of two β-sheets, which are connected by intramolecular disulfide bonds. The two variable regions of the heavy and light chains combine to form an antigen binding site. This site exists in each of the two Y-shaped arms. The part that can bind to the antigen is called the Fab (antibody binding fragment), and the part that cannot bind to the antigen is called the Fc (crystalizable fragment). The Fab and Fc are connected by a flexible hinge region.

[0043] The term "heavy chain" as used herein, when used in reference to an antibody, may refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domains, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0044] The term "light chain," as used herein, when used in connection with an antibody, may refer to any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are known in the art. In a specific embodiment, the light chain is a human light chain.

[0045] The term "immunoglobulin" as used herein refers to an immune molecule from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, and include but are not limited to human IgG1, IgG2, IgG3, and IgG4. Many of the molecules described herein are immunoglobulins. As used herein, "isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. Furthermore, immunoglobulins are typically tetrameric molecules composed of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa).

[0046] The term "antigen-binding fragment" as used herein refers to a protein comprising a portion of an antibody that is capable of specifically binding to an antigen but lacks some amino acids compared to the full-length chain of the antibody, wherein the antigen-binding portion comprises a portion that binds to an antigen or a target protein and, optionally, a scaffold or framework portion, such that the antigen-binding portion adopts a conformation that facilitates binding of the antigen-binding molecule to the antigen. Representative examples of types of antigen-binding molecules include, but are not limited to, scFv, human, mouse or rabbit antibodies; humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; diabodies; triabodies; tetrabodies; Fab, Fab', F(ab')2, Fv fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof. Such antigen-binding molecules may comprise alternative protein scaffolds or artificial scaffolds, for example, having grafted complementarity determining regions (CDRs) or CDR derivatives. The scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to stabilize the three-dimensional structure of the antigen-binding molecule, as well as completely synthetic scaffolds, for example, comprising biocompatible polymers. Furthermore, the antigen-binding molecule may have one or more binding sites. When more than one binding site is present, the binding sites may be identical or different. That is, the antibodies within the present disclosure may be single-chain composite polypeptides having antigen-binding capacity and comprising amino acid sequences that are homologous or similar to the variable regions of immunoglobulin light and heavy chains, i.e., linked VH-VL or single-chain Fv (scFv).

[0047] The term "CDR" as used herein refers to the hypervariable region, which has a different amino acid sequence for each antibody within the heavy and light chain variable regions of an antibody, and which binds to an antigen. Looking at the three-dimensional structure of an antibody, the CDR has a loop shape on the surface of the antibody, and below the loop is a framework region (FR) that structurally supports the CDR. There are three loop structures in each heavy chain and light chain, and these six loop regions are combined to directly contact the antigen. More specifically, the framework region can help maintain the appropriate conformation of the CDR to promote binding between the antigen-binding molecule and the antigen. There are three CDRs in each variable region of the heavy chain and light chain, and these are designated as CDR1, CDR2, and CDR3 for each variable region. The exact boundaries of the CDRs are defined differently in different systems.

[0048] The terms "variable region" or "variable domain" as used herein are used interchangeably and refer to a portion of an antibody, typically a portion of the light or heavy chain, typically the amino-terminal end of the antibody, that differs extensively in sequence among antibodies and is used in the binding and specificity of a particular antibody to a particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while more highly conserved regions of the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the antibody's antigen interaction and specificity.

[0049] The term "avidity" as used herein refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antigen binding molecule such as an antibody) and its binding partner (e.g., an antigen). "Avidity" is distinguished from "affinity" which refers to the strength of a single antigen-antibody interaction. The binding or affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Avidity or affinity can be measured and / or expressed in a number of ways known in the art, including but not limited to the equilibrium dissociation constant (KD) and the equilibrium association constant (KA). KD is calculated from the equation koff / kon, while KA is calculated from the equation kon / koff. Kon refers to, for example, the association rate constant of an antibody for an antigen, and koff refers to, for example, the dissociation rate constant of an antibody for an antigen. kon and koff can be determined by standard techniques known to those skilled in the art, such as BIAcore® or KinExA or surface plasmon resonance.

[0050] The term “sequence identity” as used herein refers to the number of residues present at the same position when two amino acid sequences or nucleic acid sequences are aligned, expressed as a percentage of the total length of the sequences. When it is said herein that a specific sequence “has at least X% sequence identity,” X can be, for example, 85%, 90%, 95%, 98%, or 99%.

[0051] Sequence identity is typically calculated using BLAST (Basic Local Alignment Search Tool), ClustalW, EMBOSS, or other well-known sequence alignment algorithms, using default parameters. For example, when aligning amino acid sequences using BLASTP, identity values ​​calculated using the BLOSUM62 matrix and gap penalty as default values ​​can be used as the basis.

[0052] In addition, the term “sequence identity” used herein may include a value calculated according to an optimized global alignment or local alignment that takes into account insertions, deletions, substitutions, etc. during sequence alignment, and is also used as a standard for describing the range of functional equivalents that can maintain the technical effect of the invention.

[0053] The term “conservative amino acid substitution” as used herein refers to a substitution between amino acids having similar physicochemical properties (e.g., charge, size, hydrophobicity, polarity, etc.), and includes substitutions that can substantially maintain the structural stability or biological function of the protein.

[0054] For example, substitutions within the following amino acid groups may be conservative substitutions:

[0055] Hydrophobic amino acid group: Ala, Val, Leu, Ile, Met

[0056] Polar uncharged amino acid group: Ser, Thr, Gln, Asn

[0057] Acidic amino acid group: Asp, Glu

[0058] Basic amino acid group: Lys, Arg, His

[0059] Aromatic amino acid group: Phe, Tyr, Trp

[0060] The term “having one or more conservative amino acid substitutions” as used herein includes that the function of the protein (e.g., antigen binding ability) is substantially maintained even when the original amino acid is replaced with a physicochemically similar amino acid as described above.

[0061] To determine whether conservative amino acid substitutions maintain protein function, antigen-antibody binding affinity assays, cell-based functional assays, etc. can generally be used. For example, if a variant antibody containing a substituted amino acid substantially retains binding ability to CEACAM5 compared to an antibody containing the original sequence (e.g., retains binding ability of at least 80% of the original) or substantially retains biological activities such as cellular internalization and target cancer cell killing ability, the substitution may be considered a conservative substitution. Such conservative substitutions are examples of variants that can be derived by those skilled in the art through routine experiments based on the core CDR sequences or variable region sequences specified in the embodiments of the invention, and are recognized as substantial modifications of the technical concepts of the embodiments described in the specification. Therefore, when the specification states that a “conservative amino acid substitution” includes an amino acid, it is interpreted to include substitutions with amino acids that are structurally similar and functionally equivalent.

[0062] One aspect of the present invention is a novel biparatopic antibody that specifically binds to CEACAM5. The biparatopic antibody according to the present invention can recognize and bind to two or more different epitopes present on the CEACAM5 protein. Specifically, the biparatopic antibody according to the present invention comprises six specific CDR sequence sets (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3) selected from SEQ ID NOs: 1 to 36.

[0063] As used herein, the term “biparatopic antibody” refers to an antibody that binds to two or more different epitopes of the same antigen (in the present invention, CEACAM5). Typically, a biparatopic antibody comprises different combinations of variable regions within the antibody.

[0064] The term "epitope" as used herein refers to a portion of an antigen molecule to which an antibody or antigen-binding fragment can recognize and specifically bind. An epitope may generally be a continuous linear amino acid sequence (linear epitope) or a discontinuous / conformational sequence formed by the three-dimensional structure of the antigen protein (conformational epitope).

[0065] The term “binds to different epitopes” as used herein means that an antibody or antigen-binding fragment can independently bind to two or more spatially distinct sites on CEACAM5. Such binding properties can be confirmed by a variety of experimental methods, including competitive binding assays, epitope binning, X-ray crystallography, cryo-EM, or mutagenesis mapping. Two epitopes can be considered different as long as the amino acid sequences constituting the epitopes are not completely identical.

[0066] A biparatopic antibody or antigen-binding fragment thereof according to the present invention may comprise the following six specific sets of CDR sequences (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3).

[0067] (1) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 1;

[0068] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2,

[0069] A heavy chain CDR3 comprising an amino acid sequence represented by sequence number 3,

[0070] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4,

[0071] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and

[0072] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6;

[0073] (2) Heavy chain CDR1 comprising the amino acid sequence represented by sequence number 7;

[0074] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8,

[0075] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9,

[0076] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10,

[0077] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and

[0078] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12;

[0079] (3) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 13;

[0080] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14,

[0081] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15,

[0082] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16,

[0083] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17, and

[0084] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 18;

[0085] (4) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 19;

[0086] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20,

[0087] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21,

[0088] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 22,

[0089] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and

[0090] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 24,

[0091] (5) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 25;

[0092] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26,

[0093] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 27,

[0094] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 28,

[0095] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and

[0096] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 30; or

[0097] (6) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 31;

[0098] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32,

[0099] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 33,

[0100] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 34,

[0101] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and

[0102] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 36;

[0103]

[0104] A biparatopic antibody or antigen-binding fragment thereof according to the present invention may comprise the following heavy chain variable region sequence and light chain variable region sequence.

[0105] (1) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0106] (2) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0107] (3) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0108] (4) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 43, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 44, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0109] (5) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 46, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; or

[0110] (6) A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 47, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 48, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto.

[0111]

[0112] The biparatopic antibody or antigen-binding fragment thereof according to the present invention may comprise two different sets of CDR sequences among (1) to (6) below. Each set of CDR sequences is composed of a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3.

[0113] (1) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 1;

[0114] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2,

[0115] A heavy chain CDR3 comprising an amino acid sequence represented by sequence number 3,

[0116] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4,

[0117] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and

[0118] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6;

[0119] (2) Heavy chain CDR1 comprising the amino acid sequence represented by sequence number 7;

[0120] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8,

[0121] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9,

[0122] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10,

[0123] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and

[0124] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12;

[0125] (3) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 13;

[0126] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14,

[0127] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15,

[0128] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16,

[0129] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17, and

[0130] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 18;

[0131] (4) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 19;

[0132] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20,

[0133] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21,

[0134] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 22,

[0135] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and

[0136] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 24,

[0137] (5) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 25;

[0138] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26,

[0139] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 27,

[0140] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 28,

[0141] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and

[0142] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 30;

[0143] (6) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 31;

[0144] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32,

[0145] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 33,

[0146] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 34,

[0147] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and

[0148] A light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.

[0149] The biparatopic antibody or antigen-binding fragment thereof according to the present invention may comprise two different sets of CDR sequences among (1) to (6) below. Each set of CDR sequences is composed of a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3.

[0150] (1) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 1;

[0151] Heavy chain CDR2 consisting of an amino acid sequence represented by sequence number 2,

[0152] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 3,

[0153] Light chain CDR1 consisting of the amino acid sequence represented by sequence number 4,

[0154] A light chain CDR2 consisting of an amino acid sequence represented by sequence number 5, and

[0155] A light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6;

[0156] (2) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 7;

[0157] Heavy chain CDR2 consisting of the amino acid sequence represented by sequence number 8,

[0158] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 9,

[0159] A light chain CDR1 consisting of an amino acid sequence represented by sequence number 10,

[0160] A light chain CDR2 consisting of an amino acid sequence represented by sequence number 11, and

[0161] A light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 12;

[0162] (3) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 13;

[0163] Heavy chain CDR2 consisting of the amino acid sequence represented by sequence number 14,

[0164] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 15,

[0165] Light chain CDR1 consisting of the amino acid sequence represented by sequence number 16,

[0166] A light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 17, and

[0167] A light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 18;

[0168] (4) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 19;

[0169] Heavy chain CDR2 consisting of the amino acid sequence represented by sequence number 20,

[0170] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 21,

[0171] A light chain CDR1 consisting of an amino acid sequence represented by sequence number 22,

[0172] A light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 23, and

[0173] A light chain CDR3 consisting of an amino acid sequence represented by sequence number 24,

[0174] (5) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 25;

[0175] Heavy chain CDR2 consisting of the amino acid sequence represented by sequence number 26,

[0176] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 27,

[0177] A light chain CDR1 consisting of an amino acid sequence represented by sequence number 28,

[0178] A light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 29, and

[0179] A light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 30;

[0180] (6) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 31;

[0181] Heavy chain CDR2 consisting of the amino acid sequence represented by sequence number 32,

[0182] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 33,

[0183] A light chain CDR1 consisting of an amino acid sequence represented by sequence number 34,

[0184] A light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 35, and

[0185] Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 36.

[0186] The biparatopic antibody or antigen-binding fragment thereof according to the present invention may comprise two different sets of variable region sequences among (1) to (6) below. Each set of variable region sequences is composed of a heavy chain variable region sequence and a light chain variable region sequence.

[0187] (1) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0188] (2) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0189] (3) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0190] (4) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 43, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 44, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0191] (5) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 46, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions;

[0192] (6) A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 47, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 48, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto.

[0193] A biparatopic antibody or antigen-binding fragment thereof according to the present invention comprises, for example, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6; and a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12.

[0194] A biparatopic antibody or antigen-binding fragment thereof according to the present invention comprises, for example, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6; and a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 9, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 11, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 12.

[0195] A biparatopic antibody or antigen-binding fragment thereof according to the present invention comprises, for example, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6; and a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 18.

[0196] A biparatopic antibody or antigen-binding fragment thereof according to the present invention comprises, for example, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6; and a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 15, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 16, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 17, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 18.

[0197] In certain embodiments, the biparatopic antibody or antigen-binding fragment thereof according to the present invention does not substantially bind to soluble CEACAM5. “Soluble CEACAM5” refers to CEACAM5 that is not present on the cell surface but is present in serum or body fluids. A significant amount of CEACAM5 expressed on the cell surface is shed from the cell surface and exists in the form of soluble CEACAM5, and this soluble CEACAM5 is believed to be a factor that neutralizes antibodies administered for therapeutic purposes, reducing the amount of antibody delivered to solid tumor tissues, and consequently hinders the clinical efficacy of anti-CEACAM5 antibody therapeutics.

[0198] The term "substantially does not bind" as used herein may mean that the binding signal for soluble CEACAM5 is, for example, 10% or less, or 20% or less, lower than the binding signal of the antibody to cell surface-expressed CEACAM5 in a comparative experiment under the same conditions. This binding specificity can be assessed using techniques such as flow cytometry, ELISA, or SPR.

[0199] Another aspect of the present invention is a novel monoclonal antibody or antigen-binding fragment thereof that specifically binds to CEACAM5.

[0200] The term “monoclonal antibody” as used herein means an antibody molecule generated based on a single B cell clone or its equivalent genetic sequence, having a single specificity for a particular antigen (epitope), and comprising uniform heavy and light chain variable region sequences.

[0201] Monoclonal antibodies are typically designed to bind to a single epitope, meaning that both arms (antigen-binding arms) within the antibody molecule have identical binding properties. The monoclonal antibodies of the present invention can be produced using hybridoma technology, recombinant DNA technology, human antibody expression libraries, and the like, and include fully human antibodies, humanized antibodies, chimeric antibodies, and drug conjugates thereof.

[0202] A monoclonal antibody or antigen-binding fragment thereof according to the present invention may comprise the following six specific CDR sequence sets (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3).

[0203] (1) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 7,

[0204] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8,

[0205] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9,

[0206] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10,

[0207] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and

[0208] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12; or

[0209] (2) Heavy chain CDR1 comprising the amino acid sequence represented by sequence number 25;

[0210] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26,

[0211] A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 27,

[0212] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 28,

[0213] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and

[0214] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 30.

[0215] An antibody that specifically binds to CEACAM5, or an antigen-binding fragment thereof.

[0216] A monoclonal antibody according to the present invention may comprise the following six specific CDR sequence sets (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3).

[0217] (1) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 7;

[0218] Heavy chain CDR2 consisting of the amino acid sequence represented by sequence number 8,

[0219] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 9,

[0220] A light chain CDR1 consisting of an amino acid sequence represented by sequence number 10,

[0221] A light chain CDR2 consisting of an amino acid sequence represented by sequence number 11, and

[0222] A light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 12; or

[0223] (2) Heavy chain CDR1 consisting of an amino acid sequence represented by sequence number 25;

[0224] Heavy chain CDR2 consisting of the amino acid sequence represented by sequence number 26,

[0225] Heavy chain CDR3 consisting of the amino acid sequence represented by sequence number 27,

[0226] A light chain CDR1 consisting of an amino acid sequence represented by sequence number 28,

[0227] A light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 29, and

[0228] Light chain CDR3 consisting of the amino acid sequence represented by sequence number 30.

[0229] A monoclonal antibody according to the present invention may include the following heavy chain variable region sequence and light chain variable region sequence.

[0230] (1) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; or

[0231] (2) A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 46, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto.

[0232] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof according to the invention does not substantially bind to soluble CEACAM5.

[0233] ADC

[0234] Another aspect of the present invention provides an ADC comprising a novel anti-CEACAM5 antibody (biparatopic antibody or monoclonal antibody) according to the present invention.

[0235] The term "antibody-drug conjugate (ADC)" as used herein refers to an antibody molecule coupled to a small molecule drug, such as a therapeutic agent or marker, for example, a growth inhibitory agent (e.g., a cytotoxic agent). Specifically, an ADC refers to a drug structure in which a small molecule drug (payload, e.g., a cytotoxic anticancer agent) is covalently conjugated to an antibody that binds to a specific target antigen on the surface of cancer cells via a linker. The antibody molecule may be coupled directly or indirectly to a non-antibody moiety, such as a drug, for example, via a linker.

[0236] The antibody included in the ADC according to the present invention may include any antibody disclosed herein (e.g., an antibody comprising a set of six specific CDR sequences (heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3) selected from SEQ ID NOs: 1 to 36), and the antibody may be a biparatopic antibody or a monoclonal antibody.

[0237] As used herein, the terms “growth inhibitory agent” or “antiproliferative agent” refer to a molecule or compound that inhibits the growth of cells, such as tumor cells, in vitro and / or in vivo. In some embodiments, the growth inhibitory agent is a cytotoxic drug (also referred to as a “cytotoxic agent”).

[0238] The term “cytotoxic drug” as used herein refers to a substance that directly or indirectly inhibits or prevents the function of a cell and / or causes cell destruction. The term “cytotoxic drug” includes, for example, chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzymatically active toxins, toxoids, vincas, taxanes, maytansinoids or maytansinoid analogs, tomomycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065 and CC-1065 analogs.

[0239] Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I, which is involved in altering the topology of DNA by catalyzing the transient cleavage and rejoining of single strands of DNA. Topoisomerase I inhibitors are highly toxic, for example, to dividing mammalian cells. Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and its analogs, such as topotecan, irinotecan, silatecan, cositecan, exatecan, lurtodecane, gimatecan, belotecan, rubitecan, and deruxtecan.

[0240] In the ADC according to the present invention, the antibody (or antigen-binding fragment thereof) is covalently linked to at least one growth inhibitory agent via a linker. The term "linker" as used herein refers to a chemical moiety comprising a covalent bond and / or any atomic chain that covalently attaches the growth inhibitory agent to the antibody. Linkers that can be used in ADCs are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photostabilized groups, peptidase-labile groups, and esterase-labile groups. Conjugation of an antibody (or antigen-binding fragment thereof) according to the present invention with a cytotoxic drug or other growth inhibitory agent may be accomplished, for example, by N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g. dimethyl adipimidate HCL), active esters (e.g. disuccinimidyl suberate), aldehydes (e.g. Various bifunctional protein coupling agents can be used, including but not limited to, glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in [Vitetta et al (1987)].Carbon-labeled 1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies (WO 94 / 11026).

[0241] In embodiments of the present invention, the linker may be a "cleavable linker," which may facilitate the release of a cytotoxic drug or other growth inhibitory agent within or near a cell, such as a tumor cell. In some embodiments, the linker is a linker that is cleavable in the endosome of a mammalian cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker (see, e.g., U.S. Patent No. 5,208,020) may be used.

[0242] In various embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the drug from the antibody in the intracellular environment. In another embodiment, the linker unit is non-cleavable and the drug is released, for example, by antibody degradation.

[0243] In various embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., a lysosome, an endosome, or a cyst). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease, including but not limited to a lysosomal or endosomal protease. The peptide linker can be comprised of two or more amino acids or three or more amino acids. The cleavage agent can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug into the target cell. For example, a peptide linker that can be cleaved by the thiol-dependent protease cathepsin-B, which is abundantly expressed in cancer tissue, can be used (e.g., a Phe-Leu or Gly-Phe-Leu-Gly linker). However, this is only an example and is not limiting, and various linkers consisting of 5 to 9 amino acids suitable for drug conjugation may also be used.

[0244] In various embodiments, the cleavable linker may be pH sensitive, i.e., susceptible to hydrolysis at a certain pH value. A pH-sensitive linker may be hydrolyzed under acidic conditions. For example, an acid-labile linker (e.g., a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitamide, an orthoester, an acetal, a ketal, etc.) that can be hydrolyzed in lysosomes may be used. Exemplary linkers are relatively stable under neutral pH conditions, such as blood, but are unstable below pH 5.5 or below pH 5.0, which is the approximate pH of the lysosome. In various embodiments, the hydrolyzable linker may be a thioether linker (e.g., a thioether linked to a therapeutic agent via an acylhydrazone linkage).

[0245] In various embodiments, the linker may be cleavable under reducing conditions (i.e., a disulfide linker). Various disulfide linkers may be used, including SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), which can be formed using SPDB and SMPT.

[0246] Suitable methods for preparing the ADCs of the present invention are well known in the art (see, e.g., Hermanson GT, Bioconjugate Techniques, Third Edition, 2013, Academic Press). For example, methods for conjugating cytotoxic drugs to antibodies via linkers covalently attached to cysteine ​​residues of interchain disulfide bridges of the antibody are well known.

[0247] In general, the ADC according to the present invention can be obtained by a process including, but not limited to, the following steps.

[0248] (i) preparing a compound comprising a linker and a growth inhibitor (e.g., a cytotoxic drug);

[0249] (ii) a step of contacting an optionally buffered aqueous solution of the antibody according to the present invention with a solution of the drug-linker compound obtained in (i);

[0250] (iii) optionally separating the conjugate formed in (ii) from the untreated antibody and / or drug-linker compound.

[0251] Aqueous solutions of antibodies can be buffered with buffers, such as histidine, potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer can be selected depending on the properties of the antibody. The drug-linker compound can be dissolved in, for example, an organic polar solvent, such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

[0252] For conjugation to cysteine ​​residues of the antibody, the antibody is subjected to reduction (e.g., using TCEP) prior to step (ii). Reduction conditions suitable for reducing only interchain disulfide bonds are known in the art.

[0253] The reaction temperature for conjugation is typically 20 to 40°C. The reaction time may vary, typically 1 to 24 hours. The reaction between the antibody and the drug-linker compound can be monitored by size exclusion chromatography (SEC) using a refractometer and / or UV detector. If the conjugate yield is too low, the reaction time may be extended.

[0254] To carry out the separation in step (iii), a number of different chromatographic methods can be employed by those skilled in the art: the conjugate can be purified, for example, by SEC, adsorption chromatography (e.g., ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed-support chromatography, such as hydroxyapatite chromatography, or high-performance liquid chromatography (HPLC), such as reverse-phase HPLC. Purification by dialysis or filtration or diafiltration can also be employed.

[0255] After steps (ii) and / or (iii), the conjugate-containing solution may be subjected to an additional purification step (iv), for example by chromatography, ultrafiltration and / or diafiltration. If reduction is performed prior to conjugation, such an additional purification step, for example by chromatography, ultrafiltration and / or diafiltration, may also be performed on the antibody-containing solution after the reduction reaction.

[0256] The conjugate is recovered at the end of this process in an aqueous solution. The drug-to-antibody ratio (DAR) is a number that can vary depending on the properties of the antibody and drug-linker compound used, along with the experimental conditions used for conjugation (e.g., the ratio of (drug-linker compound) to (antibody), reaction time, and the nature of the solvent and cosolvent (if any). Therefore, contact of an antibody with a drug-linker compound can produce a mixture containing several different conjugates due to different drug-to-antibody ratios. Therefore, the determined DAR is an average value.

[0257] The antibodies or antigen-binding fragments provided herein may be conjugated to a "bioactive agent." As used herein, the term "bioactive agent" encompasses any synthetic or natural compound that binds to an antigen and / or enhances or mediates a desired biological effect, thereby enhancing a cell death toxin. Non-limiting examples of direct effects include modulation, stimulation, and / or inhibition of growth signals, modulation, stimulation, and / or inhibition of anti-apoptotic signals, modulation, stimulation, and / or inhibition of apoptotic or necrotic signals, modulation, stimulation, and / or inhibition of the ADCC cascade, and modulation, stimulation, and / or inhibition of the CDC cascade.

[0258] When an ADC according to the present invention comprises a biparatopic antibody, such an ADC can significantly increase the level of internalization into cells expressing CEACAM5 compared to a corresponding ADC composition comprising a bivalent monoclonal (single-specific) antibody, thereby enhancing the intracellular delivery efficiency of the drug and its tumor cell killing effect. These effects have been confirmed in the examples herein.

[0259] The term “corresponding ADC composition comprising two monoclonal (monospecific) antibodies” as used herein refers to an ADC structure comprising an antibody in which both arms (antigen-binding arms) within the antibody molecule are composed of antibodies with the same sequence (i.e., antibodies that specifically bind to the same epitope), and other components (drugs, linkers, etc.) are the same as the biparatopic antibody-based ADC of the present invention. In other words, it refers to a conventional ADC that is set as a reference for comparing and evaluating the structural and functional advantages of biparatopic antibodies by maintaining the same remaining elements while changing only the specificity structure of the antibodies compared to the biparatopic antibody-based ADC of the present invention. In general, a bivalent monospecific antibody-based ADC may exhibit limited characteristics compared to a biparatopic antibody-based ADC in terms of cell internalization ability or target cell selectivity, and the present invention can provide improved therapeutic efficacy based on such structural differences.

[0260]

[0261] Methods for preparing a pharmaceutical composition for the prevention or treatment of cancer comprising an antibody or an antigen-binding fragment thereof, a drug conjugate comprising the antibody or an antigen-binding fragment thereof, an engineered cell comprising the antibody or an antigen-binding fragment thereof, or an antibody or an antigen-binding fragment thereof, a drug conjugate, or an engineered cell as an active ingredient according to one embodiment of the present invention, and administering the same to a subject in need thereof are well known to or can be easily determined by those skilled in the art. The route of administration thereof may be, for example, oral, parenteral, inhalation, or topical. The term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all of these forms of administration are expressly recognized as being within the scope of the present invention, examples of the form of administration may be solutions for injection, particularly intravenous or intraarterial injection, or drip solutions.

[0262] Typically, pharmaceutical compositions for injection may include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), optionally stabilizers (e.g., human albumin), etc. However, in another method compatible with the teachings herein, the immune cells of the present invention, cell therapeutic agents comprising the same, or pharmaceutical compositions for the prevention or treatment of solid tumors may be delivered directly to the site of a disadvantaged cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0263] The pharmaceutical composition used in the present invention comprises a pharmaceutically acceptable carrier, including, for example, an ion exchanger, alumina, aluminum stearate, lecithin, serum proteins, such as human serum proteins, buffering agents, such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic agents, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0264] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the present invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, preferably 0.05 M, phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements, and those based on Ringer's dextrose, for example. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present.

[0265] More specifically, pharmaceutical compositions suitable for injectability include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the compositions must be sterile and fluid to the extent that they are readily syringable. They must be stable under the conditions of manufacture and storage, and preferably protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Formulations suitable for use in the therapeutic methods disclosed herein are described below.

[0266] Prevention of microbial activity can be achieved with various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it may be desirable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0267] In various embodiments, sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield the active ingredient plus any additional desired ingredients from a sterile-filtered solution thereof as described above. Injectable formulations are processed and filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. In addition, the formulations can be packaged and sold in the form of kits such as those described below.

[0268] Parenteral formulations may be administered as a single bolus dose, as an infusion, or as a maintenance dose following a loading bolus dose. These compositions may be administered at specific fixed or variable intervals, such as once daily or on an "as needed" basis.

[0269] Certain pharmaceutical compositions used in the present invention may be administered orally in any acceptable dosage form, including, for example, capsules, tablets, aqueous suspensions, or solutions. Certain pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions may be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers, and / or other conventional solubilizers or dispersants to enhance bioavailability.

[0270] The amount of antibody or fragment, variant, or derivative thereof that can be combined with a carrier material to form a single dosage form will vary depending on the host being treated and the specific route of administration. The composition may be administered as a single dose, multiple doses, or over a set period of time by infusion. Furthermore, the dosage regimen may be adjusted to provide the optimal desired response (therapeutic or prophylactic).

[0271] The antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, can be administered to such humans or animals in conventional dosage forms prepared by combining the antibodies of the present invention with conventional pharmaceutically acceptable carriers or diluents according to known techniques. Those skilled in the art will recognize that the form and properties of the pharmaceutically acceptable carrier or diluent will be dictated by the amount of active ingredient to be combined, the route of administration, and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of CEACAM5 antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, may prove particularly effective.

[0272] As used herein, the term “therapeutically effective dose or amount” or “effective amount” means an amount of a CEACAM5 antibody or antigen-binding fragment thereof that, when administered, elicits a positive therapeutic response in the treatment of a patient having a disease to be treated.

[0273] The therapeutically effective amount of the composition of the present invention for treating certain types of cancer, such as head and neck cancer, prostate cancer, colon cancer, breast cancer, and lung cancer; inflammatory diseases, including autoimmune diseases, such as arthritis, multiple sclerosis, and inflammatory diseases of the central nervous system (CNS) and peripheral nervous system (PNS) will vary depending on the means of administration, the target site, the physiological condition of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human animals, including transgenic animals, may also be treated. The therapeutic dose can be titrated to optimize safety and efficacy using conventional methods known to those skilled in the art.

[0274] The amount of at least one CEACAM5 antibody, or binding fragment thereof, can be readily determined by one skilled in the art without undue experimentation in light of the present disclosure. Factors affecting the route of administration and the respective amount of at least one antibody, antigen-binding fragment, variant, or derivative thereof include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the subject receiving treatment. Similarly, the amount of the CEACAM5 antibody, or fragment, variant, or derivative thereof, to be administered will depend on the route of administration and whether the subject will receive a single dose or multiple doses of the agent.

[0275] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided only to illustrate the present invention and the present invention is not limited thereto.

[0276]

[0277] Example 1: Immunization for production of CEACAM5 antibodies

[0278] The process of producing CEACAM5 antibodies is described below with reference to Figures 1 to 3.

[0279] First, the CHO-S cell line used for protein production was cultured in an 8% CO2 orbital shaker incubator, and one day before genetic transformation, 100 mL of 3' CHO-S cells were added to a 500 mL Erlenmeyer flask (Corning) and expanded. Afterwards, each gene produced using a transfection kit (Gibco) was introduced into the CHO-S cells for transformation, and the recombinant CHO-S cells were cultured in an 8% CO2 orbital shaker incubator for 10 days. After the culture was completed, the culture supernatant was collected by centrifugation and injected into a Ni Sepharose affinity column. Wash buffer was flowed through the column to remove proteins nonspecifically bound to the resin, and finally, Elution buffer was flowed through the column to elute the proteins to be purified. After replacing the buffer of each protein elution collection with PBS, SDS-PAGE analysis was performed on each final prepared protein sample to evaluate its purity.

[0280] Figure 1 shows the results of SDS-PAGE analysis of purified human CEACAM5 protein, including the results of SDS-PAGE analysis for the entire sequence of CEACAM5 protein (CEACAM5 (35-685)) and the results of SDS-PAGE analysis for the A3-B3 domain of CEACAM5 protein (CEACAM5 A3-B3 (496-685)).

[0281]

[0282] Below, the in vivo CEACAM5 antibody production process in a mouse model is described through Fig. 2.

[0283] First, the prepared CEACAM5 protein (100 mg) and Complete Freund's adjuvant (Sigma) were mixed in a 1:1 ratio and injected intraperitoneally into 5 BALB / c mice at a dose of 100 mL / mouse.

[0284] Two weeks after the first immunization, CEACAM5 protein (100 mg) and Incomplete Freund's adjuvant (Sigma) were mixed in a 1:1 ratio and injected intraperitoneally into mice at a dose of 100 mL / mouse. Two weeks after the second immunization, CEACAM5 protein (100 mg) and Incomplete Freund's adjuvant (Sigma) were mixed in a 1:1 ratio and injected intraperitoneally into mice at a dose of 100 mL / mouse.

[0285] One week after the final immunization, 100 mL of serum was collected from the submandibular vein of the mice, and the antibody titer against human CEACAM5 was determined using ELISA.

[0286]

[0287] Referring to Figure 3, CEACAM5 full, CEACAM1, and CEACAM6 recombinant proteins were prepared at a concentration of 0.5 μg / ml each.

[0288] The high binding 96-well microplate prepared as above was coated with 100 ml / well of CEACAM5 full-length recombinant protein at a concentration of 0.5 ml / ml, and incubated at 4 °C for 16 h. After that, the coated microplate was washed four times with PBS-T (1x PBS, 0.5% Tween-20), and then PBS-T containing 3% BSA was added and incubated at room temperature for 1 h. After washing four times with PBS-T (1x PBS, 0.05% Tween-20), the serum collected from the mouse was serially diluted twofold (1:1000 to 512,000) with 1X DPBS, starting from 1:1000, and 100 μL / well of each dilution solution was added to a designated microplate well. After reacting for 2 hours at room temperature, 250 μL / well of PBS-T (1x PBS, 0.05% Tween-20) was added to the microplate and washed, and the washing process was repeated four times.

[0289] After washing four times with PBS-T, 100 μL / well of HRP-anti-mouse IgG, FcR (Jackson Immuno Research), which binds to mouse antibodies, was added and reacted at room temperature for 1 hour. Then, the cells were washed four times with PBS-T, 100 μL / ml of TMB substrate solution was added, and reacted at room temperature for 10 minutes. Finally, 100 μL / ml of stop solution solution was added, and the absorbance was measured at 450 nm.

[0290]

[0291] As a result, referring to Figure 3, it was confirmed that CEACAM5-specific antibodies were produced in all five mice immunized with CEACAM5 full protein, and one mouse with a high titer value among the five was selected to proceed to the next step, hybridoma cell production.

[0292]

[0293] Example 2: Hybridoma production and parental antibody screening for CEACAM5 antibody production

[0294] Below, the selection of a parent clone for selecting an antibody that specifically binds to CEACAM5 is described in detail with reference to Fig. 4a.

[0295]

[0296] Figure 4a is a diagram showing the results of screening for a parental clone that binds to the CEACAM5 full-length protein.

[0297] SP2 / 0 cells, which are mouse myeloma cells, and B cells obtained from immunized mouse spleen were mixed in a 1:1 ratio and 2X10 6 / mL to prepare hybridoma cells and culture them in 96-well plates (10 plates). Using the culture medium obtained from the divided cultured parental clones (1 parental clone / well), clones that bind to full-length CEACAM5 were identified by ELISA, and also whether they nonspecifically bind to CEACAM1 and CEACAM6 with similar homology was confirmed. After selecting parental clones that bind to full-length CEACAM5 protein but not to other CEACAM proteins, subclones were selected.

[0298] Specifically, 100 μL / well of CEACAM5, CEACAM1, and CEACAM6 full-length recombinant proteins were coated at a concentration of 0.5 μL / mL onto a high binding 96-well microplate, and incubated at 4°C for 16 hours. The coated microplate was then washed four times with PBS-T (1x PBS, 0.5% Tween-20), and then PBS-T containing 3% BSA was added and incubated at room temperature for 1 hour (blocking). After washing four times with 250 mL / well of PBS-T, 100 mL / well of culture medium from the parental clone was added to the wells of the designated microplate, and then reacted for 2 hours at room temperature, and then washed four times with 250 mL / well of PBS-T (1x PBS, 0.05% Tween-20).

[0299] Afterwards, 100 μL / well of HRP-anti-mouse IgG, FcR (Jackson Immuno Research), which binds to mouse antibodies, was added and reacted at room temperature for 1 hour. Afterwards, the plate was washed four times with 250 mL / well of PBS-T, and 100 μL / ml of TMB substrate solution was added and reacted at room temperature for 10 minutes. Finally, 100 μL / ml of stop solution solution was added and the absorbance was measured at 450 nm.

[0300] As a result, referring to Fig. 4a, when using a hybridoma cell (parental clone) culture medium, clones showing high binding affinity to CEACAM5 full length and low binding affinity to CEACAM1 and CEACAM6 with similar homology were selected, and it was confirmed that parental clones 36-B5, 94-E12, 83-G8, and 81-E3 showed high binding affinity specific to CEACAM5 full length.

[0301]

[0302] Example 3: Subclone antibody screening for production of CEACAM5 antibodies.

[0303] Below, subclone selection for selecting antibodies that specifically bind to CEACAM5 is described in detail with reference to FIG. 4b.

[0304]

[0305] First, CEACAM5 full, CEACAM1, and CEACAM6 recombinant proteins were prepared at a concentration of 0.5 μg / ml each. The prepared protein solutions were dispensed into designated high-binding 96-well microplates at 100 μL / well and incubated at 4 °C for 16 h to coat. The coated microplates were washed with 250 μL / well of PBS-T (1x PBS, 0.05% Tween-20), and the washing process was repeated four times.

[0306] Afterwards, 200 μL / well of PBS-T containing 3% BSA was added to the microplate and incubated at room temperature for 1 hour. The microplate was washed by adding 250 μL / well of PBS-T (1x PBS, 0.05% Tween-20). The washing process was repeated four times.

[0307] The culture medium collected from the parental clone was added to the wells of the designated microplate at 100 μL / well and incubated at room temperature for 2 hours. The microplate was washed with 250 μL / well of PBS-T (1x PBS, 0.05% Tween-20), and the washing process was repeated 4 times.

[0308] HRP-anti-mouse IgG, Fcγ (Jackson Immuno Research), which binds to mouse antibodies, was added to a microplate at 100 mL / well and reacted at room temperature for 1 hour. The microplate was washed with 250 mL / well of PBS-T (1x PBS, 0.05% Tween-20), and the washing process was repeated four times.

[0309] TMB substrate solution was added to a microplate at 100 mL / well and reacted at room temperature for 10 minutes. Stop solution was added to a microplate at 100 mL / well and the absorbance was measured at 450 nm.

[0310]

[0311] CHO-K1-CEACAM5 full cells were added at 2.5X10^5 / 100 μL to a 96-well plate, centrifuged, and the supernatant was removed. 100 μL / well of the culture medium obtained from the hybridoma cells was added, and incubated at 4 °C for 50 minutes. After the culture reaction, the cells were washed twice with 150 μL of 1X PBS, and then 1 μL of Alexa Fluor647-anti-mouse IgG, Fcγ was added per sample, and 80 μL of 1X PBS was added, and the reaction was carried out at 4 °C for 40 minutes in a light-protected state. After the secondary antibody reaction, the cells were washed twice with 150 μL of 1X PBS, and 250 μL / well of 1X PBS was added, and the cells were analyzed using a flow cytometer.

[0312] As a result, referring to Fig. 4b, it was confirmed that all 36B5H8, 81E3A8, 83G8D11, and 94E12E2 subclones derived from the selected parent clone specifically bind to CEACAM5, but not to CEACAM1 or CEACAM6.

[0313]

[0314] Hereinafter, the results of measuring the binding affinity of antibodies selected and purified according to one embodiment of the present invention to a CEACAM5 expressing cell line will be described in detail with reference to FIGS. 4c to 4h.

[0315]

[0316] To measure the binding affinity of the antibodies purified as above to the CEACAM5-expressing cell line, 2.5X10 CHO-K1-CEACAM5 (full) cells were first added to a 5 mL round FACS tube. 5 / well and centrifuged at 300xg for 3 minutes, then the supernatant was removed. After that, CEACAM5 antibodies purified from single clones (hybridoma cells) were prepared by diluting 1:100 in 1X DPBS (gibco), dispensed 100 μl each into tubes, and reacted for 30 minutes at 4℃ in a light-protected state. After that, each tube was washed with 2 mL of 1X DPBS (gibco), and the washing process was repeated twice. Alexa Fluor647-anti-mouse IgG, Fcγ, or PE anti-mouse IgG (Biolegend) was prepared by diluting 1:100 in 1X DPBS, dispensed 100 μl each into tubes, and reacted for 30 minutes at 4℃ in a light-protected state. After the reaction, each tube was washed with 2 mL of 1X DPBS (gibco), and the washing process was repeated twice. 250 μl of 1X DPBS was added to each tube, and the binding of antibodies was confirmed using a FACS Canto II or FACS Verse I device.

[0317] As a result, as shown in Figures 4c to 4h, all antibodies purified from subclones 36B5H8, 81E3A8, 83G8D11, 94E12E2, 37B6H9, and 45A2E10 were confirmed to have high binding affinity to CEACAM5-expressing cell lines.

[0318]

[0319] The amino acid sequences of antibody clones that have been confirmed to bind to CEACAM5 are as follows. Table 1 below shows the heavy and light chain CDR amino acid sequences of the antibodies, and Table 2 shows the heavy chain variable region and light chain variable region amino acid sequences.

[0320]

[0321] [Table 1]

[0322]

[0323] [Table 2]

[0324]

[0325] Example 4: Confirmation of binding affinity of selected antibodies to CEACAM5 protein

[0326] Hereinafter, the binding affinity to a CEACAM5 recombinant protein according to one embodiment of the present invention will be described in detail with reference to FIG. 5.

[0327]

[0328] First, to evaluate the binding affinity of anti-CEACAM5 antibodies purified from the culture medium of a single clone to the CEACAM5 protein, CEACAM5 recombinant protein (ACROBiosystems) was prepared at a concentration of 0.5 mg / mL. The prepared protein solution was dispensed into a designated 384-well plate (Nunc) at 30 mL / well and incubated at 4 °C for 16 h to perform coating. The coated microplate was then washed four times with 100 mL / well of PBST (DPBS + 0.05% Tween 20, Dynebio).

[0329] 1x PBS containing 3% BSA was added to the microplate at 80 mL / well and incubated at room temperature for 2.5 hours. The microplate was then washed four times with 100 mL / well of PBST (DPBS + 0.05% Tween 20, Dynebio).

[0330] The purified antibody was serially diluted threefold with DPBS containing 0.5% FBS to prepare concentrations ranging from 0.0004 nM to 200 nM. Each dilution solution was added to the designated wells of a microplate at 30 mL / well and reacted at room temperature for 1 hour. The microplate was washed four times with 100 mL / well of PBS-T (1x PBS, 0.05% Tween-20).

[0331] HRP-anti-mouse IgG, Fcg (Jackson Immuno Research), which binds to mouse antibodies, was diluted to a concentration of 0.16 mg / mL, placed in a microplate at 30 mL / well, reacted at room temperature for 1 hour, and then washed four times by adding 100 mL / well of PBS-T (1x PBS, 0.05% Tween-20) to the microplate.

[0332] TMB substrate solution was added to the microplate at 30 mL / well and reacted at room temperature for 10 minutes. Then, 0.16 M sulfuric acid (stop solution, Invitrogen) was added to the microplate at 30 mL / well and the absorbance was measured at 450 nm.

[0333]

[0334] As a result, as shown in Fig. 5, the binding affinity of antibodies purified from the obtained subclones 36B5H8, 45A2E10, 81E3A8, and 83G8D11 to the CEACAM5 full protein was confirmed, and as a result, it was confirmed that all antibodies bind to CEACAM5.

[0335]

[0336] Example 5: Humanized Antibodies

[0337] To humanize the selected antibodies, homology modeling was performed on the Fv region of the parental antibody. The sequence of the parental antibody was searched in the IgBLAST database to identify the human antibody sequence with the highest homology. The CDR sequences of the parental antibody were grafted onto the corresponding human antibody sequences to design 3-4 VH sequences and 3-4 VL sequences. The human antibody sequences used for grafting were selected by comparing them to the parental antibody sequence based on factors such as CDRs, conserved residues, loop interactions, and proximity of the foundation region. Afterwards, back mutations were applied to residues that differed between the parental antibody and the humanized antibody to design the final humanized antibody sequence.

[0338]

[0339] The humanized antibodies produced have the same CDR sequences as the parent antibodies, and the amino acid sequences of the variable regions are as follows.

[0340]

[0341] [Table 3]

[0342]

[0343] The underlined sequences above represent the amino acid sequences of CDR1, CDR2, and CDR3, in that order.

[0344]

[0345] Example 6: Confirmation of binding affinity of selected antibodies to CEACAM5-expressing cells

[0346] Hereinafter, the binding affinity of a CEACAM5 antibody purified as a single clone according to one embodiment of the present invention to A549 or CHO-K1 cell lines expressing CEACAM5 will be described in detail through FIGS. 6a to 6d.

[0347]

[0348] First, A549-CEACAM5 or CHO-K1-CEACAM5 cell lines expressing CEACAM5 were seeded in 96-well plates at 3x10 6 Cells / well were added, centrifuged at 1500 rpm for 3 minutes, and the supernatant was removed. Antibodies at various concentrations (0.008 nM - 100 nM or 0.0002 μg / mL to 45 μg / mL) were diluted with DPBS solution, dispensed at 50 μL / well onto A549-CEACAM5 or CHO-K1-CEACAM5 cell plates, and reacted at 4°C for 30 minutes. Each well was washed with 250 μL of 1X PBS, and the washing process was repeated twice.

[0349] A solution containing Alexa Flour 647-conjugated anti-mouse or human IgG Fcγ secondary antibody (Jackson ImmunoResearch) diluted in DPBS to a concentration of 15 μg / ml was dispensed into the cells at 50 μL / well and incubated at 4°C for 30 minutes in a light-protected state. Each well was washed with 250 μL of 1X PBS, and the washing process was repeated twice.

[0350] 250 μL / well of PBS was dispensed and the degree of antibody binding was confirmed using a flow cytometer (FACS Verse II or MACSQuant®Analyzer 10).

[0351] The binding affinity of antibodies purified from the subclones 36B5H8, 45A2E10, 81E3A8, 37B6H9, 83G8D11, 94E12E2, h37B6H9, h36B5H8, h83G8-16, and h81E3-06 secured as above to the CEACAM5-expressing cell line was confirmed, and as a result, as shown in Figures 6a to 6d, it was confirmed that all of them had high binding affinity to CEACAM5.

[0352]

[0353] Example 7: Identification of the epitope of the h81E3-06 antibody

[0354] Hereinafter, the results of epitope identification of the h81E3-06 antibody binding to human CEACAM5 protein are described with reference to FIG. 7. The h81E3-06 antibody comprises a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6.

[0355]

[0356] To characterize the molecular interface between the h81E3-06 antibody and the human CEACAM5 full-length protein complex, the protein complex was first incubated with a deuterated cross-linker and subjected to multiple enzymatic digestions to determine the epitopes of human CEACAM-5 full-length and the antibody HU1134-81E3hVH1-hVL2 at high resolution. After concentrating the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry, and the generated data were analyzed using dedicated software.

[0357] The mixture of CEACAM-5 / HU1134-81E3hVH1-hVL2 was prepared by preparing the following volumes and concentrations (CEACAM5, 10 μL / 4 uM, HU1134-81E3hVH1-hVL2 10 μL / 2 uM. The mixture of 2 uM of CEAMCAM and 1.0 uM of HU1134-81E3hVH1-hVL2 was prepared in a final volume of 20 μL).

[0358] Afterwards, cross-linking, reduction / alkylation, and proteolysis using four enzymes (trypsin, chymotrypsin, elastase, and thermolysin) were performed according to CovalX SOP #PROT_010621_A, and 1 μl of each peptide solution generated by proteolysis was loaded onto the nano-liquid chromatography system, and liquid chromatography was performed according to CovalX SOP #LC_150621_A.

[0359] The nLC chromatography system was matched to an Orbitrap Q-Exactive Plus mass spectrometer, MS analysis was performed according to CovalX SOP #MS_140621_A, and the MS and MS / MS data generated from data analysis were analyzed according to CovalX SOP #Data_200621_A.

[0360]

[0361] Referring to FIG. 7 and the sequence of the Human CEACAM5 N domain and the A1 domain sequence below, the epitopes associated with the binding of the h81E3-06 antibody analyzed in the sequence of the Human CEACAM5 N domain and the sequence of the A1 domain were identified as H, H, S, Y, T, T, Y in the Human CEACAM5 N domain and S, R in the A1 domain.

[0362]

[0363] N domain

[0364] KLTIESTPFNVAEGKEVLLLV'H'NLPQ'H'LFGYSWYKGERVDGNRQIIG'Y'VIG'T'QQA'T'PGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRV'Y'PEL

[0365]

[0366] A1 domain

[0367] PKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPV'S'A'R'RSDSVILN

[0368]

[0369] Example 8: Antibody internalization assay

[0370] Antibody internalization analysis was performed as follows. Tumor cells were cultured in 96-well microplates at a concentration of 10,000 cells / well for 24 hours, and then labeled with antibodies at a molar ratio of 1:3 using Incucyte® Fabfluor-pH Reagent (Essen BioScience, Mouse IgG1: Cat. 4723, IgG2a: Cat. 4750, IgG2b: Cat. 4751). The cells were incubated at 37°C in a light-protected environment for 15 minutes, and then the labeled antibodies were added to each well of the cultured tumor cells at a 2X concentration. The plates were measured every 1-2 hours for 36 hours in an Incucyte® Live-Cell Analysis System and analyzed using Incucyte® Software. The surface area of ​​the red-stained cells observed at each time point, i.e., the rate at which the antibody penetrated into a specific surface and was internalized, was calculated, and the Area Under the Curve (AUC) of the measured graph was calculated using Prism10 software.

[0371] The biparatopic antibodies used were h81E-m83G and h81E-h94E.

[0372] h81E-m83G comprises a heavy chain variable region (including a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3) and a light chain variable region (including a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6) of the above-described h81E3-06, and a heavy chain variable region (including a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9) and a light chain variable region (including a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 11) of the above-described 83G8D11. CDR2, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12.

[0373] h81E-h94E comprises a heavy chain variable region (including a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3) and a light chain variable region (including a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6) of the above-described h81E3-06, and a heavy chain variable region (including a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15) and a light chain variable region (including a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 17) of the above-described h94E12-23. light chain CDR2, and an amino acid sequence represented by SEQ ID NO: 18).

[0374]

[0375] The biparatopic antibodies used were prepared by cloning the gene sequence encoding the amino acid sequence of the antibody, expressing it in CHO cells, and purifying it. To produce biparatopic antibodies with precisely combined antibody chains, plasmids containing the antibody genes were cloned using the known CrossMab and Knob-into-Hole (KiH) technologies. The constructed plasmids were transformed into CHO-S cells and cultured for 4–6 days under conditions of 37°C, 8% CO₂, and 120 rpm. The supernatant of the cultured CHO-S cells was recovered, and the antibodies were purified through Protein A affinity chromatography and hydrophobic interaction chromatography (HIC). The purified antibodies were confirmed to have a purity of 95% or higher through SDS-PAGE and SEC-HPLC analyses.

[0376]

[0377] As shown in Figures 8a and 8b, the biparatopic antibody-ADC treatment group showed a significantly higher internalization effect than the control and monoclonal antibody treatment groups in all cancer cell lines. In particular, in the pancreatic cancer cell line HPAC, treatment with h81E-m83G and h81E-h94E showed a high increase in internalization of approximately 280% and 300%, respectively, compared to treatment with h81E3-06 (also referred to as h81E) alone, and in HPAF-II, similarly, an increase of approximately 380% and 390% compared to treatment with h81E3-06 alone was shown. In the gastric cancer cell line MKN-45, both ADCs showed the strongest effect, showing a high increase in internalization of approximately 550% and 580%, respectively, compared to treatment with h81E3-06 alone.

[0378] Significant effects were also observed in colon cancer cell lines, with h81E-m83G and h81E-h94E showing increases of approximately 380% and 400%, respectively, compared to h81E3-06 alone in H3122, and relatively lower but still significant increases of approximately 860% and 650% in LS174T.

[0379] These results suggest that the biparatopic antibody of the present invention can exert a potent and selective internalization effect in various cancer types.

[0380]

[0381] Example 9: Measurement of cytotoxicity of biparatopic ADC

[0382] Hereinafter, with reference to FIGS. 9a and 9b, the results of measuring cell viability according to the concentration of ADC when the biparatopic antibody according to the present invention is administered in the form of ADC will be described.

[0383]

[0384] To measure the in vitro tumor cell killing effect of two types of biparatopic antibody ADC materials (81E / 83G-DXd, 81E / 94E-DXd), the results were measured using NKN-45, a gastric cell line expressing CEACAM5, LS174T, a colon cancer cell line, and H1650, a cell line that does not express CEACAM5.

[0385] Each biparatopic antibody-ADC drug was manufactured in the form of a bispecific antibody-ADC through Wuxi-XDC, and the payload Dxd (Exatecan derivative for ADC; Cat. No. HY-13631D) was purchased from MedChemExpress.

[0386] First, each tumor cell line was cultured in a 96-well plate at a density of approximately 1,000-2,000 cells / well, and after 24 hours, the cells were treated with various concentrations (1000, 200, 40, 8, 1.6, 0.32, 0.06, 0.01, 0.002, 0 nM) of biparatopic ADC drugs and payloads. After reacting for 6 days, the cell death effect was measured using a CellTiter Glo Luminescent Cell Viability Assay kit (Promega No. G7571) in a microplate reader (EG&G Berthold, Centro XS3 LB960).

[0387] The IC50 value was calculated as the ratio of the degree of tumor cell death to the concentration at which the drug was not administered (0 nM; 100%) (GraphicPad Prism10 software).

[0388]

[0389] As a result, referring to Figures 9a and 9b, the killing effect (IC50) of the CEACAM5 biparatopic antibody-ADC against the tumor cell line MKN45 was measured to be 0.3 nM and 0.2 nM for 81E / 83G-Dxd and 81E / 94E, respectively, and showed a higher cell killing effect (IC50 = 1.5 nM) than the test group treated with the payload alone without the biparatopic antibody conjugation. When the cell killing effect was measured against the lung cancer cell line H1650 that does not express CEACAM5, the payload alone without the biparatopic antibody conjugation showed a cell killing effect (IC50 = 8.0 nM), whereas the experimental group treated with the biparatopic antibody conjugate did not show cytotoxicity.

[0390] This suggests that when the biparatopic antibody of the present invention is used in conjunction with a cytotoxic payload, the payload-induced toxicity to normal cells is reduced. For the colon cancer cell line LS174T, a payload-only treatment group was not included.

[0391]

[0392]

[0393] Example 10: Measurement of the tumor inhibitory effect of biparatopic ADCs

[0394] Hereinafter, with reference to Figures 10a and 10b, changes in tumor volume and body weight in mice over time are described when the biparatopic antibody according to the present invention and the conventionally known tusamitamab were administered in the form of ADC. Tusamitamab is a monoclonal antibody that has been confirmed to exhibit excellent antitumor activity in CEACAM5-expressing solid tumors through clinical studies.

[0395]

[0396] The efficacy of three types of antibody ADC materials (Tusamitamab-DM4, 81E / 83G-DXd, 81E / 94E-DXd) was evaluated with a single dose of 5 mg / kg in a NOG mouse model subcutaneously implanted with the lung cancer cell line H3122. Seven mice were used in each group, and tumor size and body weight of each mouse were measured daily using a caliper for 5 weeks after administration.

[0397] First, H3122 cells were thawed and cultured for 2 weeks in culture medium (RPMI-1640 medium-10% heat-inactivated FBS-1% antibiotic-antimycotic) at 37℃ and 5% CO2, and subcultured at 2-3 day intervals. The cultured cells were confirmed to be free of bacterial, yeast, and mycoplasma infection and used for transplantation. The cultured cancer cells were washed with PBS, harvested using trypsin-EDTA, and prepared by mixing with cold salin so that 5x106 cells / 100 μl / mouse could be transplanted per mouse. Before inoculation into mice, the right flank was disinfected with a 70% alcohol swab under isoflurane inhalation anesthesia, and the prepared cells were subcutaneously transplanted into the right flank of a 6-week-old female BALB / c-nude mouse that had been stabilized for 1 week using a disposable syringe. Care was taken to prevent the tumor cell suspension from leaking out from the injection site during transplantation. Afterwards, body weight, tumor formation, and tumor growth were periodically observed, and the tumor volume was calculated using the measured tumor length using the following formula (volume = (a 2 b)*0.532, a is the short diameter, b is the long diameter), and the tumor volume is 200±20 mm 3 The mice were group separated.

[0398] The test substance was prepared by thawing and diluting it in vehicle (20 mM Histidine, 8% Sucrose, pH 6.0). After group separation, the drug was administered to the mice in each group at the specified dose and method, and both the test substance and the positive control group were administered once according to the dosing schedule and observed for 5 weeks.

[0399] As a result, referring first to Fig. 10a, compared to the vehicle group added as a control group in the experiment, a significant tumor suppression effect was observed in the three mouse groups administered the ADC drug, and in particular, the most excellent tumor suppression effect was confirmed in 81E / 83G and 81E / 94E produced biparatopically. Referring further to Fig. 10b, the toxicity of the antibody ADC drug was verified by measuring the body weight loss of mice administered the drug, and as a result, no body weight loss was observed in any mouse group. This confirmed that the administered drugs did not exhibit toxic effects on the mice.

[0400] Although the embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be implemented without departing from the technical spirit of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain it, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are exemplary in all aspects and not restrictive. The protection scope of the present invention should be interpreted by the claims below, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims

1. An antibody that binds to two or more different epitopes on CEACAM5, (1) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 1; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, A heavy chain CDR3 comprising an amino acid sequence represented by sequence number 3, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6; (2) Heavy chain CDR1 comprising the amino acid sequence represented by sequence number 7; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12; (3) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 13; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 18; (4) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 19; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 22, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 24, (5) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 25; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 27, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 28, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 30; or (6) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 31; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 33, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 34, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 36 An antibody comprising, or an antigen-binding fragment thereof.

2. An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence represented by SEQ ID NO:

6.

3. An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence represented by SEQ ID NO:

12.

4. An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence represented by SEQ ID NO:

18.

5. An antibody that binds to two or more different epitopes on CEACAM5, (1) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (2) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (3) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (4) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 43, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 44, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (5) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 46, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; or (6) A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 47, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 48, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto. An antibody comprising, or an antigen-binding fragment thereof.

6. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions.

7. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions.

8. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions.

9. In any one of paragraphs 1 to 8, (1) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 1; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, A heavy chain CDR3 comprising an amino acid sequence represented by sequence number 3, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6; (2) Heavy chain CDR1 comprising the amino acid sequence represented by sequence number 7; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12; (3) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 13; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 18; (4) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 19; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 20, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 22, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 23, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 24, (5) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 25; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 27, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 28, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 30; or (6) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 31; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 33, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 34, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 35, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 36 An antibody, or an antigen-binding fragment thereof, additionally comprising:

10. In paragraph 9, A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6; and A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12 An antibody or antigen-binding fragment thereof comprising:

11. In paragraph 9, A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6; and A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 18 An antibody or antigen-binding fragment thereof comprising:

12. In any one of paragraphs 1 to 8, (1) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (2) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (3) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (4) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 43, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 44, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; (5) a heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 46, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; or (6) A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 47, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 48, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto. An antibody, or antigen-binding fragment thereof, additionally comprising:

13. In paragraph 12, A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; and A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto. An antibody or antigen-binding fragment thereof comprising:

14. In paragraph 15, A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 38, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; and A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 42, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto. An antibody or antigen-binding fragment thereof comprising: 15.(1) Heavy chain CDR1 comprising the amino acid sequence represented by sequence number 7, A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 8, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 9, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 11, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 12; or (2) Heavy chain CDR1 comprising an amino acid sequence represented by sequence number 25; A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 26, A heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 27, A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 28, A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29, and A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO:

30. An antibody that specifically binds to CEACAM5, or an antigen-binding fragment thereof. 16.(1) A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 40, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions; or (2) A heavy chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions, and a light chain variable region sequence comprising an amino acid sequence represented by SEQ ID NO: 46, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity thereto and / or having one or more conservative amino acid substitutions thereto. An antibody comprising the antibody, or an antigen-binding fragment thereof.

17. An antibody or antigen-binding fragment thereof that does not substantially bind to soluble CEACAM5 according to any one of claims 1 to 16.

18. An antibody drug conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17.

19. An antibody drug conjugate that exhibits greater internalization into CEACAM5-expressing cells compared to a corresponding antibody drug conjugate comprising a bivalent monospecific antibody in claim 18.

20. An engineered cell comprising an antigen-binding fragment of an antibody according to any one of claims 1 to 17.

21. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, an antibody-drug conjugate according to claim 18 or 19, or a cell according to claim 20.

22. A pharmaceutical composition according to claim 21, wherein the cancer is a solid cancer.

23. A pharmaceutical composition according to claim 22, wherein the solid cancer is selected from the group consisting of colon cancer, pancreatic cancer, lung cancer, stomach cancer, hepatocellular carcinoma, breast cancer, and thyroid cancer.

24. A method for preventing or treating cancer, comprising administering a pharmaceutical composition according to Article 22 to a subject in need of cancer treatment.

25. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17.

26. An expression vector comprising a nucleic acid according to Article 25.

27. A host cell comprising an expression vector according to Article 26.

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