ANTIBODY AGAINST CEACAM5 AND ITS APPLICATION

Abstract

The present specification relates to an anti-CEACAM5 antibody or binding fragment thereof that specifically binds to CEACAM5 remaining on the surface of cells without binding to sCEACAM 5 present in the form of soluble CEACAM5 (sCEACAM5) in blood. Therefore, the antibody of the present invention can be delivered in an increased amount to solid tumor tissues, thus being effective as a therapeutic agent targeting cancer cells expressing CEACAM5.

Description

Anti-CEACAM5 antibodies and uses thereof

[0001] The present invention relates to anti-CEACAM5 antibodies and uses thereof.

[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 subfamily proteins: the carcinoembryonic antigen-related cell adhesion protein (CEACAM) subfamily and the pregnancy-specific glycoprotein subfamily (Kammerer & Zimmermann, BMC Biology 2010, 8:12).

[0003] The extracellular domains of CEACAM family members consist of repeated immunoglobulin-like (Ig-like) domains, which are classified into three types, A, B, and N, based on sequence homology. CEACAM5 contains seven of these domains: N, A1, B1, A2, B2, A3, and B3. CEACAMs are involved in various cellular functions, regulating cell growth and differentiation through signaling based on their cell-cell adhesion functions, and playing a crucial role in insulin homeostasis, angiogenesis, and immune regulation. Members of the CEACAM family are implicated in various pathophysiological roles, including their role as receptors for microbial pathogens. They play a crucial role in carcinogenesis, particularly in cancer detection, progression, and metastasis.

[0004] Meanwhile, CEACAM5 (referred to as CEA, also known as CD66e) is a glycoprotein with a molecular weight of approximately 180 kDa. CEACAM5, which contains seven domains, is anchored to the cell membrane via glycosyl-phosphatidyl-inositol (GPI). The seven domains include a single N-terminal immunoglobulin (Ig) variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to Ig constant domains. CEACAM5 was initially thought to be a protein expressed in fetal tissues, but has recently been identified in many normal adult tissues. Furthermore, overexpression of CEACAM5 has been observed in several types of cancer, and further studies have shown that its overexpression is associated with several malignancies and often correlates with a poor prognosis.

[0005] Furthermore, CEACAM5 has been found to be overexpressed in various malignant tumors, including breast, pancreatic, ovarian, colon, lung, and gastric tumors, and has been implicated in tumor invasion and metastasis. Therefore, CEACAM5 is increasingly recognized as a tumor-associated antigen useful for targeted therapy.

[0006] The background technology of the invention has been prepared to facilitate a better understanding of the present invention. It should not be construed as an admission that the matters described in the background technology of the invention constitute prior art.

[0007] Meanwhile, while advances have been made in the field of immunotherapy, there is still a continuing need for new treatments that are more effective, have longer lasting effects, incorporate novel targets, and can act as single agents or in combination with known therapies to ultimately produce long-lasting responses in cancer patients.

[0008] There remains an unmet need to provide humanized antibodies that recognize specific tumor-specific proteins that are safer, more efficacious, and can be used diagnostically and therapeutically in diseases involving tumor-specific protein expression or activation.

[0009] Meanwhile, CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5) is preferentially overexpressed in some solid tumors, such as colon cancer, pancreatic cancer, lung cancer, gastric cancer, hepatocellular carcinoma, breast cancer, and thyroid cancer, and is particularly highly expressed on the surface of colon, gastric, lung, and uterine tumor cells.

[0010] Accordingly, the inventors of the present invention focused on CEACAM5 to overcome the aforementioned limitations. More specifically, CEACAM5 is a member of the carcinoembryonic antigen (CEA) gene family, which belongs to the immunoglobulin (Ig) superfamily and is also known as cluster 66e of differentiation (CD66e).

[0011] In addition, a significant amount of CEACAM5 expressed on the cell surface is shed from the cell surface and exists in the blood in the form of soluble CEACAM5 (sCEACAM5), and the concentration of sCEACAM5 in the serum increases rapidly in cancer patients and can reach up to 2 μg / ml. Therefore, antibody therapeutics against anti-CEACAM5 have been studied steadily for a long time, but sCEACAM5 present in the blood is considered to be a factor that neutralizes the administered antibody, reduces the amount of antibody delivered to solid cancer tumor tissue, and consequently inhibits the clinical efficacy of anti-CEACAM5 antibody therapeutics.

[0012] Accordingly, the inventors of the present invention confirmed the possibility of a therapeutic agent targeting cancer cells expressing CEACAM5 by developing an antibody that specifically binds to an epitope on CEACAM5 that remains on the cell surface without being detached from the cell surface during antibody screening.

[0013] More specifically, the inventors of the present invention confirmed the possibility of a therapeutic agent targeting cancer cells expressing CEACAM5 by using an antibody that specifically binds to an epitope on CEACAM5 remaining on the cell surface of the present invention, thereby increasing the amount of antibody delivered to solid cancer tumor tissue by specifically binding to an epitope on CEACAM5 remaining on the cell surface without substantially binding to sCEACAM5 that is detached from the cell surface and exists in the blood in the form of soluble CEACAM5 (sCEACAM5).

[0014] Finally, the inventors of the present invention developed an antibody that binds to an epitope on CEACAM5 remaining on the cell surface described above without substantially binding to sCEACAM5 existing in the form of soluble CEACAM5 (sCEACAM5).

[0015] At this time, the antibody of the present invention can bind to epitopes other than the A3 domain and / or B3 domain of CEACAM5.

[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] To solve the above-described problem, an anti-CEACAM5 antibody or antigen-binding fragment thereof according to one embodiment of the present invention is provided.

[0018] According to a feature of the present invention, the anti-CEACAM5 antibody of the present invention or an antigen-binding fragment thereof is a humanized antibody, and the antibody of the present invention is capable of binding to the CEACAM5 A3 domain and B3 domain represented by SEQ ID NO: 48. However, it should not be understood that the antibody of the present invention does not bind to other domains of CEACAM5.

[0019] Specifically, the antibody of the present invention can specifically bind to an epitope on CEACAM5 remaining on the cell surface without binding to sCEACAM5, which is detached from the cell surface and exists in the blood in the form of soluble CEACAM5 (sCEACAM5), and more specifically, to the CEACAM5 A3 domain and B3 domain, represented by SEQ ID NO: 48, which are predicted to be the portion of the membrane protein remaining after CEACAM5 is cleaved, but does not have to bind to other domains of CEACAM5.

[0020] Therefore, when using the anti-CEACAM5 antibody of the present invention or an antigen-binding fragment thereof, it can be used as a therapeutic agent targeting cancer cells expressing CEACAM5 by binding to CEACAM5 remaining on the cell surface without being detached from the cell surface during antibody screening.

[0021] In addition, in order to solve the above-described problem, a pharmaceutical composition for preventing or treating cancer comprising an anti-CEACAM5 antibody or an antigen-binding fragment thereof according to an embodiment of the present invention as an active ingredient is provided, and in addition, an anticancer adjuvant, a composition for inhibiting the proliferation of CEACAM5-expressing tumor cells, an antibody-drug conjugate, and a pharmaceutical composition for preventing or treating cancer comprising an antibody-drug conjugate as an active ingredient may be provided.

[0022] In addition, in order to solve the problem as described above, a method for preventing or treating cancer in a subject is provided, comprising a step of administering to the subject a pharmaceutical composition for preventing or treating cancer comprising an anti-CEACAM5 antibody or an antigen-binding fragment thereof as an active ingredient according to one embodiment of the present invention.

[0023] Specific details of other embodiments are included in the detailed description and drawings.

[0024] The present invention can increase the amount of antibody delivered to solid cancer tumor tissue by providing an antibody that specifically binds to an epitope on CEACAM5 remaining on the cell surface.

[0025] More specifically, the present invention provides an antibody that specifically binds to an epitope on CEACAM5 remaining on the cell surface, thereby preventing the antibody from being neutralized by soluble CEACAM5 (sCEACAM5) present in the blood and increasing the amount of antibody delivered to solid cancer tumor tissue, thereby further improving the clinical efficacy of an anti-CEACAM5 antibody therapeutic agent.

[0026] Furthermore, when using an antibody that specifically binds to an epitope on CEACAM5 remaining on the cell surface of the present invention, the therapeutic effect using the antibody can be maintained for a longer period of time since the antibody is not neutralized by soluble CEACAM5 (sCEACAM5) present in the blood, and thus a greater immune effect can be achieved.

[0027] The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0028] Figure 1 is a diagram showing the results of purifying and obtaining a CEACAM5 recombinant protein (full length) produced in a cell after transfection of a CHO-K1 cell according to one embodiment of the present invention.

[0029] Figure 2 is a diagram showing the process of immunizing a mouse with a CEACAM5 recombinant protein (full length) according to one embodiment of the present invention.

[0030] Figure 3 is a diagram showing the results of immunizing a mouse with a CEACAM5 recombinant protein (full length) according to one embodiment of the present invention.

[0031] Figure 4a is a diagram showing the results of screening for a parental clone that binds to the CEACAM5 full-length protein.

[0032] Figure 4b is a diagram showing the results of subclone selection that binds to the CEACAM5 full-length protein.

[0033] Figure 5 is a diagram showing the binding ability of purified antibodies to CHO-K1, CHO-K1-CEACAM5 (full), and CHO-K1-CEACAM5 (A3-B3) cells and six single antibody clones (hybridoma cells).

[0034] FIG. 6 is a diagram showing the results of purifying and obtaining a CEACAM5 recombinant protein (A3 domain and B3 domain) produced in a cell after transfection of a CHO-K1 cell according to one embodiment of the present invention.

[0035] Figure 7 is a diagram showing the results of immunizing a mouse with a CEACAM5 recombinant protein (A3 domain and B3 domain) according to one embodiment of the present invention.

[0036] Figure 8a is a diagram showing the results of screening of parental clones that bind to CEACAM 5 A3 domain and B3 domain proteins.

[0037] Figure 8b is a diagram showing the results of subclone selection that binds to CEACAM 5 A3 domain and B3 domain proteins.

[0038] Figures 9a and 9b are diagrams showing the binding ability of a CEACAM5 antibody purified as a single clone according to one embodiment of the present invention to the CEACAM5 full-length protein.

[0039] Figures 10a and 10b are diagrams showing the binding ability of a CEACAM5 antibody purified as a single clone according to one embodiment of the present invention to an A549 cell line expressing CEACAM5.

[0040] Figures 11a and 11b are diagrams showing the blocking effect of a CEACAM5 antibody according to one embodiment of the present invention due to soluble CEACAM5.

[0041] Figure 12a is a diagram showing the analysis of the binding strength of an antibody (81E3A8) produced by performing a humanization process according to one embodiment of the present invention.

[0042] Figure 12b is a diagram comparing the binding ability of an antibody (81E3A8) produced by performing a humanization process according to one embodiment of the present invention to a CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5 and a CHO-k1 (CHO-CEACAM8) cell line expressing CEACAM8.

[0043] Figure 12c is a diagram showing the binding ability of an antibody (81E3A8) produced by performing humanization work according to one embodiment of the present invention to H3122, KATO III, and MKN45 cell lines expressing CEACAM5.

[0044] Figures 13a and 13b are diagrams showing the results of analyzing the binding affinity of an antibody (45A2E10) produced by performing humanization work according to one embodiment of the present invention.

[0045] Figure 14 is a diagram showing the binding ability of an antibody (45A2E10) produced by performing humanization work according to one embodiment of the present invention to a CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5.

[0046] Figure 15 is a diagram showing the binding affinity of various clone antibodies (94E12E2) produced by performing humanization work according to one embodiment of the present invention, as confirmed by ELISA analysis.

[0047] Figure 16 is a diagram showing the binding affinity of various clone antibodies (94E12E2) produced by performing humanization work according to one embodiment of the present invention, as confirmed by flow cytometry.

[0048] Figures 17a and 17b are diagrams showing the analysis of the binding affinity of antibody (36B5H8) clones with modified sequences corresponding to post-translational modification (PTM) according to one embodiment of the present invention.

[0049] Figure 18 is a diagram showing the binding ability of selected antibodies using CHO-K1-CEACAM5 cells after humanization and post-translational modification (PTM) according to one embodiment of the present invention.

[0050] Figure 19a is a diagram showing the structure of a CEACAM5 (full length) antigen according to one embodiment of the present invention.

[0051] Figure 19b is a diagram showing the A3 domain and B3 domain of the CEACAM5 antigen according to one embodiment of the present invention.

[0052] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined solely by the scope of the claims.

[0053] 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 the heavy chain constant region genes μ, δγα, 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 differ. In addition, the IgG forms a very stable Y-shaped structure (molecular weight, approximately 150 kDa) composed only of 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.

[0054] The term "humanized antibody" as used herein refers to an antibody that is, in whole or in part, non-human in origin and has been modified to replace specific amino acids, for example, in the framework regions of the VH and VL domains, so as to evade or minimize an immune response in humans. The constant domains of a humanized antibody are in most cases human CH and CL domains.

[0055] Here, the term "heavy chain" as used herein, when used in relation to an antibody, may refer to any distinct type, for example, alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which generate the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, for example, IgG1, IgG2, IgG3, and IgG4.

[0056] 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.

[0057] 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).

[0058] 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; single-domain antibodies (e.g., VHH); 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 that include 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, i.e., linked VH-VL or single-chain Fv (scFv), having antigen-binding capacity and comprising amino acid sequences that are homologous or similar to the variable regions of immunoglobulin light and heavy chains.

[0059] As used herein, the term "Fab fragment" is a monovalent fragment having VL, VH, CL and CH domains; an "F(ab')2 fragment" is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; an "Fv fragment" has the VH and VL domains of a single arm of an antibody; and a "dAb fragment" has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain.

[0060] The terms "single-chain antibody" and "single-chain variable fragment (scFv)", as used herein, are used interchangeably and refer to an antigen-binding molecule in which the VL and VH regions are linked via a linker to form a continuous protein chain, wherein the linker is sufficiently long to allow the protein chain to fold itself and form a monovalent antigen-binding site. More specifically, scFv refers to a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together by a single-chain (usually serine, glycine) linker. A single-chain antibody has antigen-binding capacity and may be a single-chain composite polypeptide (linked VH-VL or single-chain Fv (scFv)) comprising amino acid sequences that are homologous or similar to the variable regions of an immunoglobulin light and heavy chain. Both the VH and VL may copy a monoclonal antibody sequence, or one or both of the chains may comprise a CDR-FR construct. Separate polypeptides similar to the variable regions of the light and heavy chains are joined by a polypeptide linker.

[0061] 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.

[0062] 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.

[0063] 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). Unless otherwise indicated, "avidity" as used herein refers to the intrinsic binding strength reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The binding of a molecule X to its partner Y can generally be represented by the dissociation constant (KD). Avidity 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.

[0064] The term "substantially does not bind" as used herein means that the binding domain of the present invention does not bind to an antigen or protein other than CEACAM5 present on the cell surface, and a target cell surface antigen, for example, soluble CEACAM5 (sCEACAM5), that is, when CEACAM5 present on the cell surface and an antigen or protein other than CEACAM5, and a target cell surface antigen, for example, soluble CEACAM5 (sCEACAM5) are present at the same time, the blocking effect of the CEACAM5 antibodies due to soluble CEACAM5 is less than about 60%, about 50% or less, about 20% or less, and more preferably about 15% or less.

[0065] The term “bispesific” as used herein means capable of recognizing and binding to two different antigens or epitopes.

[0066] The term "antibody-drug conjugate" as used herein refers to an antibody molecule coupled to a therapeutic agent or label, for example, a cytotoxic agent. An antibody molecule can be coupled directly or indirectly, for example, via a linker, to a non-antibody moiety. In the present specification, when an antibody or an antigen-binding fragment thereof is said to bind to CEACAM5, it can mean that it binds to an epitope on CEACAM5 that remains on the cell surface, for example, it does not substantially bind to an epitope on soluble CEACAM5 (sCEACAM5) that is detached from the cell surface and present in the blood, or binds at a significantly lower level, and in one embodiment of the present invention, it binds with high binding affinity to the A3 domain and B3 domain, which are expected to be the remaining portion of the membrane protein after CEACAM5 is cleaved, so that even when soluble CEACAM5 (sCEACAM5) is present together, the binding ability of the antibody or an antigen-binding fragment thereof to CEACAM 5 located on the tumor cell surface is not substantially inhibited. Additionally, without limitation, the antibody of the present invention may be capable of binding to epitopes other than the A3 domain and B3 domain of CEACAM5.

[0067] The term "conservative amino acid substitution" as used herein is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within the CDR(s) or within the framework region(s) of an antibody or antigen binding molecule (or fragment thereof) provided herein may be replaced with an amino acid residue having a similar side chain.

[0068] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their conventional meanings in the relevant art. The constant region is an antibody portion, e.g., the carboxyl-terminal portion of the light and / or heavy chains, that is not directly involved in binding the antibody to an antigen but may exhibit various effector functions, such as interactions with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0069] The term "epitope," as used herein, can mean a region of a particular antigen molecule to which a particular antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (a linear or contiguous epitope), or an epitope can be, for example, grouped together from two or more non-contiguous regions of a polypeptide or polypeptides (a conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled to mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art. Furthermore, peptide sequences derived from the epitope can be used alone or in combination with a carrier moiety to immunize animals and to produce additional polyclonal or monoclonal antibodies by applying methods known in the art. Isolated peptides derived from the epitope can be used in diagnostic methods for detecting antibodies and as therapeutic agents when inhibition of such antibodies is desired.

[0070] In one embodiment of the present invention, the antibody of the present invention can specifically bind to CEACAM5 represented by SEQ ID NO: 47, and more specifically, the region of a specific antigen molecule to which the antibody of the present invention can specifically bind may be, but is not limited to, the CEACAM 5 A3 domain and B3 domain comprising the amino acid sequence represented by SEQ ID NO: 48.

[0071] Sequence number 47 (CEACAM5)

[0072] KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI

[0073]

[0074] 서열번호 48 (CEACAM 5 A3 도메인 및 B3 도메인)

[0075] VSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLS

[0076] In addition, in a specific embodiment of the present invention, an anti-CEACAM5 antibody or antigen-binding fragment thereof that specifically binds to the CEACAM 5 A3 domain and B3 domain including the amino acid sequence represented by SEQ ID NO: 48 comprises (1) a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto. It may be an 81E3A8 antibody or an antigen-binding fragment thereof, comprising a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto,

[0077] In a specific embodiment of the present invention, an anti-CEACAM5 antibody or antigen-binding fragment thereof that specifically binds to the CEACAM 5 A3 domain and B3 domain including the amino acid sequence represented by SEQ ID NO: 48 comprises: (2) a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 14 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, and an amino acid sequence represented by SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto. It may be a 45A2E10 antibody or an antigen-binding fragment thereof, comprising a light chain CDR1 comprising a sequence, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 17 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto,

[0078] In a specific embodiment of the present invention, an anti-CEACAM5 antibody or antigen-binding fragment thereof that specifically binds to the CEACAM 5 A3 domain and B3 domain including the amino acid sequence represented by SEQ ID NO: 48 comprises: (3) a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 24 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, and an amino acid sequence represented by SEQ ID NO: 27 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto. It may be a 94E12E2 antibody or an antigen-binding fragment thereof, comprising a light chain CDR1 comprising a sequence, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 28 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 29 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto,

[0079] In a specific embodiment of the present invention, an anti-CEACAM5 antibody or antigen-binding fragment thereof that specifically binds to the CEACAM 5 A3 domain and B3 domain including the amino acid sequence represented by SEQ ID NO: 48 comprises: (4) a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 40 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 41 or 46 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 42 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, and an amino acid sequence represented by SEQ ID NO: 43 or a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto. The 36B5H8 antibody or antigen-binding fragment thereof may be, but is not limited to, a light chain CDR1 comprising an amino acid sequence having homology thereto, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 44 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology thereto.

[0080] In addition, in another specific embodiment of the present invention, the anti-CEACAM5 antibody or antigen-binding fragment thereof, which 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, may be a humanized antibody comprising any one of the framework sequences represented by SEQ ID NO: 7 or 11 in the heavy chain region, and any one of the framework sequences represented by SEQ ID NO: 8, 9, 10, and 12 in the light chain region,

[0081] In another embodiment, the anti-CEACAM5 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 may be a humanized antibody comprising any one of the framework sequences represented by SEQ ID NO: 19 or 21 in the heavy chain region, and any one of the framework sequences represented by SEQ ID NO: 20, 22, and 23 in the light chain region.

[0082] In another embodiment, (3) an anti-CEACAM5 antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 25, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 27, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 28, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 29 may be a humanized antibody comprising any one of the framework sequences represented by SEQ ID NOs: 30, 32, 34, 36, and 38 in the heavy chain region, and any one of the framework sequences represented by SEQ ID NOs: 31, 33, 35, 37, and 39 in the light chain region,

[0083] In another embodiment, the anti-CEACAM5 antibody or antigen-binding fragment thereof may be, but is not limited to, an anti-CEACAM5 antibody or antigen-binding fragment thereof, which comprises a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 40, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 41 or 46, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 42, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 43, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 44, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45, in which the heavy chain CDR2 is a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 46, the anti-CEACAM5 antibody or antigen-binding fragment thereof may be, but is not limited to, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 41 modified with a sequence corresponding to a post-translational modification (PTM) to a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 46.

[0084] The terms "peptide," "polypeptide," and "protein," as used herein, are used interchangeably herein and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A polypeptide, protein, or peptide must contain at least two amino acids, but there is no limitation on the maximum number of amino acids that may comprise the amino acid sequence of a protein or peptide. As used herein, the terms refer to both single chains, commonly also referred to as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to as proteins. "Polypeptide" includes, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. The term "polypeptide" includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0085] As used herein, the terms "transduction" and "transduced" refer to a method of introducing foreign DNA into a cell via a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentivirus vector, or any combination thereof.

[0086] The term "anticancer" as used herein includes "prevention" and "treatment," where "prevention" means any action that inhibits the proliferation of cancer or delays the progression of cancer by administering the anticancer agent, and "treatment" means any action that improves or beneficially changes the symptoms of cancer by administering the antibody of the present invention.

[0087] The term "cancer" as used herein may include, but is not limited to, at least one of pancreatic cancer, melanoma, lung cancer, and myeloma. Furthermore, any cancer or tumor that has CEACAM5 as a receptor and an abnormally functioning immune checkpoint pathway is not particularly limited, and may include solid tumors and hematological cancers. However, preferably, it may be lung cancer.

[0088] The term "cell therapy" as used herein refers to a pharmaceutical product used for preventive or therapeutic purposes through a series of actions that change the biological characteristics of living autologous, allogenic, or xenogenic cells by proliferating and selecting them in vitro to restore the function of cells and tissues. More specifically, it may be cytotoxic T cells or natural killer cells.

[0089] In addition, in one embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer comprising the anti-CEACAM5 antibody or binding fragment thereof of the present invention as an active ingredient may be used as a cell therapy agent comprising the anti-CEACAM5 antibody or binding fragment thereof as an active ingredient, but is not limited thereto. More specifically, in one embodiment, a pharmaceutical composition for preventing or treating cancer comprising the anti-CEACAM5 antibody or binding fragment thereof of the present invention as an active ingredient may be used in adaptive cell therapy (ACT) in which the anti-CEACAM5 antibody or binding fragment thereof is used. In this case, adaptive cell therapy (ACT) is a cell therapy method that collects immune cells in the body, strengthens them, or genetically modifies them, and then reintroduces them. Representative examples of this method for strengthening cellular immunity against cancer cells include cell therapy agents using tumor-infiltrating lymphocytes (TIL), T cell receptors (TCR), and chimeric antigen receptors (CAR).

[0090] The term "subject" as used herein may be a human or a non-human mammal, and means a subject suffering from a disease or a condition in which the disease can be alleviated, suppressed or treated by administering a pharmaceutical composition for preventing or treating cancer comprising the anti-CEACAM5 antibody or binding fragment thereof of the present invention as an active ingredient.

[0091] The term "administration" as used herein means introducing an effective amount of a substance into a subject by an appropriate method, and the route of administration of the composition comprising the anti-CEACAM5 antibody or antigen-binding fragment thereof of the present invention can be administered through a general route that can reach the target tissue. Specifically, it can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended use, and intravenous administration is preferred. In some cases, local administration may be preferable for administration to solid tumors to allow rapid and easy access of the antibody. The dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. A single dose can be administered in an amount of about 0.001 to 10 mg / kg on a daily or weekly basis. The effective dosage can be adjusted according to the opinion of the physician treating the patient.

[0092] As used herein, the terms "immunospecifically binds," "immunospecifically recognizes," "specifically binds," and "specifically recognizes" are similar terms and are used interchangeably in reference to antigen binding molecules and mean that a given molecule preferentially binds to an antigen (e.g., an epitope or immune complex) over non-specific binding, but not that it does not bind to antigens or epitopes other than the given antigen or epitope at all. For example, reference herein to "specifically binds to the A3 domain and the B3 domain" does not mean that the binding molecule (e.g., an antibody) must bind only to the CEACAM5 A3 domain and the B3 domain.

[0093] Example 1: Screening of antibodies binding to CEACAM5 (full length)

[0094] 1-1. CEACAM5 (full-length) antigen sequence analysis and protein purification

[0095] Hereinafter, CEACAM5 antigen sequence analysis and protein purification for production of anti-CEACAM5 antibodies according to one embodiment of the present invention will be described in detail with reference to FIG. 1.

[0096]

[0097] Figure 1 is a diagram showing the results of purifying and obtaining a CEACAM5 recombinant protein (full length) produced in a cell after transfection of a CHO-K1 cell according to one embodiment of the present invention.

[0098] Specifically, the amino acid sequence and DNA sequence of the Extracellular protein domain (ECD) of the human CEACAM5 protein (SEQ ID NO: 47, CEACAM5 full length amino acid sequence, Genebank NM_001291484.3) were confirmed, and the corresponding DNA gene was synthesized (GenscriptProbio).

[0099] The synthesized corresponding nucleotide sequence containing an amino acid His tag at the carbonyl-terminal of the protein amino acid sequence was transiently transfected into Expi293 cells, a cell line derived from human embryonic kidney, according to the manufacturer's instructions (Expi293 Expression System, ThermoFisher Scientific).

[0100] Afterwards, CEACAM5 full-length recombinant protein was purified from the expressed culture supernatant using Ni Sepharose affinity chromatography according to the manufacturer's instructions (GE Healthcare).

[0101]

[0102] 1-2. Confirmation of in vivo CEACAM5 (full-length) antibody production

[0103] Below, the in vivo CEACAM5 antibody production process in a mouse model is described in detail through Figures 2 and 3.

[0104]

[0105] Figure 2 is a diagram showing the process of immunizing a mouse with a CEACAM5 recombinant protein (full length) according to one embodiment of the present invention.

[0106] Figure 3 is a diagram showing the results of immunizing a mouse with a CEACAM5 recombinant protein (full length) according to one embodiment of the present invention.

[0107] First, referring to Fig. 2, the high binding 96-well microplate produced as described above was coated with 100 ul / well of CEACAM5 full-length recombinant protein at a concentration of 0.5 ul / ml, and then incubated at 4 degrees for 16 hours. 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 hour. After washing four times with PBS-T, the serum collected from the mouse was diluted by each dilution factor, added to the microplate at 100 ul / well, and reacted at room temperature for 2 hours. After washing four times with PBS-T, 100 ul / 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 ul / ml of TMB substrate solution was added, and reacted at room temperature for 10 minutes. Finally, 100 ul / ml of stop solution solution was added, and the absorbance was measured at 450 nm.

[0108] As a result, referring to Fig. 3, it was confirmed that CEACAM5 antibodies were well produced in all five mice.

[0109]

[0110] 1-3. Selection of a parental clone that binds to the full-length CEACAM5 protein

[0111] 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.

[0112]

[0113] Figure 4a is a diagram showing the results of screening for a parental clone that binds to the CEACAM5 full-length protein.

[0114] Hybridoma cells were produced by fusing mouse myeloma SP2 / 0 cells with mouse B cells, and cultured in 96-well plates (10 plates). Using the culture fluid obtained from the parental clones (1 parental clone / well) cultured in the division, clones that bind to full-length CEACAM5 were identified by ELISA, and also whether they nonspecifically bind to CEACAM1 and CEACAM6, which have similar homology. After selecting parental clones that bind to full-length CEACAM5 protein but not to other CEACAM proteins, subclones were selected.

[0115] Specifically, 100 μl / well of CEACAM5, CEACAM1, and CEACAM6 full-length recombinant proteins were coated at a concentration of 0.5 μl / ml in 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 PBS-T, mouse serum was diluted by each dilution factor and added to the microplate at 100 μl / well and reacted at room temperature for 2 hours. After washing four times with PBS-T, HRP-anti-mouse IgG, FcR (Jackson Immuno Research), which binds to mouse antibodies, was added at 100 μl / well and reacted at room temperature for 1 hour. After washing 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 was added and the absorbance was measured at 450 nm.

[0116] As a result, referring to Fig. 4a, among the parental clones, clones showing high binding affinity to CEACAM5 (full-length) and low binding affinity to CEACAM 1 and CEACAM 6 were selected, and it was confirmed that parental clones including 36-B5, 81-E3, and 94-E12 showed high binding affinity specifically to CEACAM5 (full-length).

[0117]

[0118] 1-4. Selection of subclones that bind to the full-length CEACAM5 protein

[0119] Below, subclone selection for selecting antibodies that specifically bind to CEACAM5 is described in detail with reference to FIG. 4b.

[0120]

[0121] Figure 4b is a diagram showing the results of subclone selection that binds to the CEACAM5 full-length protein.

[0122] Among the parental clones selected as in the above Examples 1-3, some were selected and separated into single clones, which were then cultured in 96-well plates. Using the culture fluid obtained from the single clones, clones that bind to full-length CEACAM5 were identified by ELISA, and also whether they nonspecifically bind to CEACAM1 and CEACAM6, which have similar homology, was confirmed.

[0123] Clones that bind to the CEACAM5 full-length protein but not to the CEACAM1 and CEACAM6 proteins were selected by conducting experiments in the same manner as in Example 1-3.

[0124] As a result, referring to Fig. 4b, it was confirmed that all subclones, including 36B5H8, 81E3A8, and 94E12E2 derived from the selected parent clone, did not bind to CEACAM 1 and CEACAM 6 proteins, but bound to CEACAM5 full-length protein.

[0125]

[0126] 1-5. Confirmation of the binding ability of antibodies to CHO-K1, CHO-K1-CEACAM5 (full), and CHO-K1-CEACAM5 (A3-B3) cells

[0127] Hereinafter, with reference to Fig. 5, the results of confirming the binding ability of purified antibodies in CHO-K1, CHO-K1-CEACAM5 (full), and CHO-K1-CEACAM5 (A3-B3) cells and six single antibody clones (hybridoma cells) are described.

[0128]

[0129] Figure 5 is a diagram showing the binding ability of purified antibodies to CHO-K1, CHO-K1-CEACAM5 (full), and CHO-K1-CEACAM5 (A3-B3) cells and six single antibody clones (hybridoma cells).

[0130] To select subclones that bind to the CEACAM5 A3 and B3 domains (Fig. 19b), which are domains predicted to remain in the membrane protein after CEACAM5 is cleaved, antibodies purified from six single-antibody clones (hybridoma cells) were added at 1 μg / 100 μl each to CHO-K1, CHO-K1-CEACAM5 (full), and CHO-K1-CEACAM5 (A3-B3) cells and reacted at 4°C for 30 min. Mouse IgG was used as a negative control and R&D product [FAB41281P] was used as a positive control. After washing with 1X PBS, 2 ml, PE-anti-mouse IgG was added at 1 μl / 100 μl to each sample and reacted at 4°C for 30 min in a light-protected state. After washing with 2 ml of 1X PBS, 300 μl of PBS was added to each sample, and the binding of antibodies was confirmed using a flow cytometer.

[0131] As a result, as shown in Fig. 5, among the subclones 98F8G11, 94E12E2, 83G8D11, 82H3A8, 81E3A8, and 36B5H8, 94E12E2, 83G8D11, 82H3A8, 81E3A8, and 36B5H8 were confirmed to have high binding affinity to the CEACAM5 full-length protein, and among them, 82H3A8, 81E3A8, and 36B5H8 were also confirmed to have high binding affinity to the A3 domain and B3 domain of CEACAM5.

[0132]

[0133] Example 2: Screening of antibodies binding to CEACAM5 (A3 domain and B3 domain)

[0134] 2-1. In vivo CEACAM5 (A3 domain and B3 domain) antigen sequence analysis and protein purification

[0135] Hereinafter, CEACAM5 antigen sequence analysis and protein purification for production of anti-CEACAM5 antibodies according to one embodiment of the present invention will be described in detail with reference to FIG. 6.

[0136]

[0137] FIG. 6 is a diagram showing the results of purifying and obtaining a CEACAM5 recombinant protein (A3 domain and B3 domain) produced in a cell after transfection of a CHO-K1 cell according to one embodiment of the present invention.

[0138] In order to obtain the CEACAM5 recombinant protein (A3 domain and B3 domain), in order to develop an antibody that recognizes the membrane protein remaining on the cell membrane surface after cleavage of CEACAM5, a gene containing only the A3 domain and B3 domain among the amino acid sequence and DNA sequence of the Extracellular protein domain (ECD) of human CEACAM5 protein (SEQ ID NO: 48, CEACAM5 A3-B3 amino acid sequence) was synthesized (GenscriptProbio).

[0139] The synthesized corresponding nucleotide sequence containing an amino acid His tag at the carbonyl-terminal of the protein amino acid sequence was transiently transfected into Expi293 cells, a cell line derived from human embryonic kidney, according to the manufacturer's instructions (Expi293 Expression System, ThermoFisher Scientific).

[0140] CEACAM5 (A3-B3) protein was purified from the expressed culture supernatant using Ni Sepharose affinity chromatography according to the manufacturer's instructions (GE Healthcare).

[0141]

[0142] 2-2. Confirmation of in vivo CEACAM5 (A3 domain and B3 domain) antibody production

[0143] Hereinafter, the process of producing in vivo CEACAM5 (A3 domain and B3 domain) antibodies in a mouse model will be described in detail with reference to FIGS. 2 and 7.

[0144]

[0145] Figure 2 is a diagram showing the process of immunizing a mouse with a CEACAM5 recombinant protein (full length) according to one embodiment of the present invention.

[0146] Figure 7 is a diagram showing the results of immunizing a mouse with a CEACAM5 recombinant protein (A3 domain and B3 domain) according to one embodiment of the present invention.

[0147]

[0148] Referring back to Figure 2, the CEACAM5 recombinant protein (A3 domain and B3 domain) was immunized into mice in the same manner as the process of immunizing mice with the CEACAM5 recombinant protein (full length) shown in Figure 2.

[0149] Specifically, the CEACAM5 (A3-B3) protein produced as described above was immunized three times in mice at 14-day intervals, and one week after the final immunization, serum was collected to confirm whether antibodies against CEACAM5 (A3-B3) were produced in the mouse body. The collected mouse serum was diluted 1,000 to 512,000 times with PBS, and the degree of CEACAM5 antibody production was confirmed through ELISA (ELISA antigen: CEACAM5 (496-685aa) / Coating Concentration: 0.5 ug / ml, 100 ul / well / Coating buffer: Phosphate buffered saline).

[0150] Referring to Figure 7, it was confirmed that CEACAM5 antibodies were well produced in all five mice that underwent immunization.

[0151]

[0152] 2-3. Selection of parental clones that bind to CEACAM5 A3 domain and B3 domain proteins

[0153] Below, the selection of parental clones binding to CEACAM 5 A3 domain and B3 domain proteins is described in detail with reference to FIG. 8a.

[0154]

[0155] Figure 8a is a diagram showing the results of screening of parental clones that bind to CEACAM 5 A3 domain and B3 domain proteins.

[0156] To select parental clones that bind to CEACAM5 A3 domain and B3 domain proteins, first, hybridoma cells were produced by fusing mouse myeloma cells, SP2 / 0 cells, and B cells obtained from mice, and then divided and cultured in 96-well plates (10 plates). Using the culture fluid obtained from the divided parental clones (1 parental clone / well), clones that bind to CEACAM5 A3 domain and B3 domain were confirmed by ELISA. In addition, it was confirmed whether they nonspecifically bind to CEACAM1 and CEACAM6, which have similar homology, and parental clones that bind to CEACAM5 A3-B3 proteins but not to CEACAM 1 and CEACAM 6 proteins were selected.

[0157] Specifically, 100 ul / well of CEACAM5, CEACAM1, and CEACAM6 full-length recombinant proteins were coated at a concentration of 0.5 ul / ml in a high binding 96-well microplate, and incubated at 4°C for 16 hours. 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 hour (blocking). After washing four times with PBS-T, mouse serum was diluted by each dilution factor and added to the microplate at 100 ul / well and reacted at room temperature for 2 hours. After washing four times with PBS-T, HRP-anti-mouse IgG, FcR (Jackson Immuno Research), which binds to mouse antibodies, was added at 100 ul / well and reacted at room temperature for 1 hour. Afterwards, the mixture was washed four times with PBS-T, TMB substrate solution was added at 100 ul / ml, and the mixture was reacted at room temperature for 10 minutes. Then, stop solution solution was added at 100 ul / ml, and the absorbance was measured at 450 nm.

[0158] As a result, referring to Fig. 8a, it was confirmed that the parental clones including 45-A2 showed high binding affinity specifically to the CEACAM5 A3-B3 protein and low binding affinity to the CEACAM 1 and CEACAM 6 proteins.

[0159]

[0160] 2-4. Selection of subclones that bind to CEACAM5 protein

[0161] Below, the selection of subclones binding to CEACAM 5 A3 domain and B3 domain proteins is described in detail with reference to FIG. 8b.

[0162]

[0163] Figure 8b is a diagram showing the results of subclone selection that binds to CEACAM 5 A3 domain and B3 domain proteins.

[0164] Among the parental clones selected as in Example 2-3, some were selected and separated into single clones, which were then cultured in 96-well plates. Using the culture fluid obtained from the single clones, clones that bind to CEACAM5 A3 domain and B3 domain proteins were identified by ELISA, and it was also confirmed whether they nonspecifically bind to CEACAM1 and CEACAM6, which have similar homology.

[0165] Clones that bind to CEACAM5 A3 domain and B3 domain proteins but not to CEACAM1 and CEACAM6 proteins were selected by conducting experiments in the same manner as in Example 2-3.

[0166] As a result, referring to Fig. 8b, it was confirmed that all subclones including 45A2E10 derived from the selected parent clone specifically bind to CEACAM5 A3 domain and B3 domain proteins, but not to CEACAM 1 and CEACAM 6 proteins.

[0167]

[0168] 2-5. Confirmation of binding ability of CEACAM5 antibody purified as a single clone to CEACAM5 full-length protein

[0169] Hereinafter, the binding ability of a CEACAM5 antibody purified as a single clone according to one embodiment of the present invention to the CEACAM5 full-length protein is described in detail with reference to FIGS. 9a and 9b.

[0170]

[0171] Figures 9a and 9b are diagrams showing the binding ability of a CEACAM5 antibody purified as a single clone according to one embodiment of the present invention to the CEACAM5 full-length protein.

[0172]

[0173] To confirm the binding ability of the CEACAM5 antibody purified as a single clone to the CEACAM5 full-length protein, 30 μl of CEACAM5 protein (Acrobiosystems) was dispensed per well of a 384-well microplate (Corning) at a concentration of 0.5 μg / ml, incubated at 4°C for 16 hours, and washed four times with 100 μL / well of PBST (DPBS + 0.05% Tween 20, Teknova). Then, 80 μL / well of 1X PBS buffer containing 3% FBS was added, incubated at room temperature for 2.5 hours, and washed four times with 100 μL / well of PBST as above. At this time, the antibody was prepared by diluting 3-fold to a concentration ranging from 200 nM to 0.0004 nM with DPBS (Bining buffer) containing 0.5% Fetal Bovin Serum (FBS, Cytiva). The antibodies prepared in this way were added to the CEACAM5-coated plate at 30 μL / well and incubated at room temperature for 1 hour. After washing the plate, HRP (horseradish peroxidase) anti-mouse IgG Fcγantibody, Jackson ImmunoResearch) was diluted to a concentration of 0.16 μg / mL (1:5000) in binding buffer and added to the plate at 30 μL / well and incubated at room temperature for 1 hour. After washing the plate in the same manner, TMB (3,3',5,5'-tetramethylbenzidine, Invitrogen) substrate was added to the plate at 30 μL / well and incubated in the dark for 10 minutes at room temperature. Finally 0.After adding 30 μL / well of 16 M sulfuric acid (stop solution, Invitrogen), the reaction was stopped by incubating in the dark for 10 minutes, and the absorbance was measured at 450 nm (Synergyhtx multimode reader, Biotek).

[0174] As a result, as shown in Figures 9a and 9b, it was confirmed that among the CEACAM5 antibodies purified as single clones of the present invention, the binding ability of the 36B5H8, 45A2E10, and 81E3A8 antibodies was particularly high.

[0175]

[0176] 2-6. Confirmation of binding ability of selected CEACAM5 antibodies to A549 cell line expressing CEACAM5

[0177] Hereinafter, the binding ability of a CEACAM5 antibody purified as a single clone according to one embodiment of the present invention to an A549 cell line expressing CEACAM5 is described in detail through FIGS. 10a and 10b.

[0178]

[0179] Figures 10a and 10b are diagrams showing the binding ability of a CEACAM5 antibody purified as a single clone according to one embodiment of the present invention to an A549 cell line expressing CEACAM5.

[0180] First, A549 cell line expressing CEACAM5 (A549-CEACAM5) was mixed with mouse antibodies at various concentrations (0.008 nM - 100 nM) in 1X PBS containing 3% FBS and incubated at 4°C for 2 hours. After washing twice in 1X PBS, secondary antibody of Alexa Flour 488-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) was mixed at a concentration of 5 ug / ml in 1X PBS containing 3% FBS and incubated at 4°C for 1 hour. After washing twice with cold PBS, fluorescence was measured on a flow cytometer (BD FACS Verse II) and MFI was analyzed using Flowjo software.

[0181] As a result, referring to Figures 10a and 10b, it was confirmed that the binding ability of the 81E3A8 and 45A2E10 antibodies to the A549 cell line was particularly excellent.

[0182]

[0183] 2-7. Confirmation of the blocking effect of selected CEACAM5 antibodies on soluble CEACAM5.

[0184] Hereinafter, the blocking effect of an antibody according to one embodiment of the present invention due to soluble CEACAM5 will be described in detail through FIGS. 11a and 11b.

[0185]

[0186] Figures 11a and 11b are diagrams showing the blocking effect of a CEACAM5 antibody according to one embodiment of the present invention due to soluble CEACAM5.

[0187] To confirm the blocking effect of CEACAM5 antibody due to soluble CEACAM5, first, A549 cell line expressing CEACAM5 (A549-CEACAM5) was mixed with mouse antibodies at various concentrations (0.008 nM - 100 nM) in 1X PBS containing 3% FBS. Then, cancer patient serum containing soluble CEACAM5 at a concentration of 100 ng / ml was added, incubated at 4°C for 2 hours, and washed twice in 1X PBS. Then, goat anti-mouse IgG conjugated with Alexa Flour 488 (Jackson ImmunoResearch) was mixed at a concentration of 5 ug / ml in 1X PBS containing 3% FBS, incubated at 4°C for 1 hour, and washed twice with cold PBS. Fluorescence was measured on a flow cytometer (BD FACS Verse II) and MFI was analyzed using Flowjo software. This value shows how much the tested antibody binds to the surface of the A549 cell line that expresses CEACAM5 on the cell surface. The blocking effect due to soluble CEACAM5 was expressed as a percentage by comparing the MFI values ​​of samples containing cancer patient serum containing soluble CEACAM5 (Sceacam5+) and samples without it (None).

[0188] Referring to FIGS. 11a and 11b, the CEACAM5 antibodies according to one embodiment of the present invention were found to have a blocking effect due to soluble CEACAM5 of less than 50% in a sample containing soluble CEACAM5, and in particular, in the case of the 81E3A8 antibody, the % ratio for the blocking effect due to soluble CEACAM5 was 11.88, confirming that it exhibited high binding ability to the A549 cell line expressing CEACAM5 even in a sample containing soluble CEACAM5.

[0189] That is, it was confirmed that the CEACAM5 antibodies according to one embodiment of the present invention did not substantially inhibit the binding ability of the antibodies to CEACAM5 located on tumor cells even in a sample containing soluble CEACAM5, and thus it was confirmed that they specifically bind to CEACAM5 present on the surface of tumor cells without substantially binding to soluble CEACAM5. This shows that the CEACAM5 antibodies according to the present invention can be used in a more effective and efficient manner for tumor treatment, which provides an important advantage in a cancer treatment method using a CEACAM5 antibody.

[0190]

[0191] 3-1. Humanization of antibody (81E3A8)

[0192] Hereinafter, an antibody (81E3A8) produced by performing a humanization process according to one embodiment of the present invention through FIG. 12a will be described in detail.

[0193]

[0194] Figure 12a is a diagram showing the analysis of the binding strength of an antibody (81E3A8) produced by performing a humanization process according to one embodiment of the present invention.

[0195] In order to analyze the binding affinity of the antibody (81E3A8) produced through humanization work, humanization work was performed on the 81E3A8 clone to produce humanized antibodies, and among them, five antibodies (81E3A8-02, 81E3A8-06, 81E3A8-10, 81E3A8-12, and 81E3A8-14) with excellent binding affinity were selected as shown in Fig. 12a. At this time, 81E3A8-01 corresponds to an antibody produced with a mouse sequence as a control, and the humanized antibodies of the clone antibodies were produced by combining the CDR 1 to 3 sequences of the clone antibodies differently, as shown in Tables 1 to 6 below.

[0196] CDR sequence of humanized 81E3A8 antibody AntibodyChain typessequenceSeq no.humanized 81E3A8antibodyVHCDR 1NFGMN1CDR 2WINTYTGKPTYDDDFKG2CDR 3EAGKDYAMDY3VLCDR 1SASSSVTFMH4CDR 2DTSKLAS5CDR 3QQWNNYPWT6

[0197] Frame work sequence of humanized 81E3A8-02 clone antibody Antibody Chain types sequence Seq no. humanized 81E3A8-02 antibody VHQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSS7VLEIVLTQSPATLSLSPGERATLSCSASSSVTFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDAAVYYCQQWNNYPWTFGGGTKVEIK8

[0198] Frame work sequence of humanized 81E3A8-06 clone antibody Antibody Chain types sequence Seq no. humanized 81E3A8-06 antibody VHQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSS7VLDIQLTQSPSTLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSLQPDDAATYYCQQWNNYPWTFGGGTKVEIK9

[0199] Frame work sequence of humanized 81E3A8-10 clone antibody Antibody Chain types sequence Seq no. humanized 81E3A8-10 antibody VHQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSS7VLEIVLTQSPATLSVSPGERATLSCSASSSVTFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTEYTLTISSMQSEDAAVYYCQQWNNYPWTFGGGTKVEIK10

[0200] Frame work sequence of humanized 81E3A8-12 clone antibody Antibody Chain types sequence Seq no. humanized 81E3A8-12 antibody VHQIQLVQSGAEVKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRVTMTLDTSTSTAYMELRSLRSDDMAVYYCAREAGKDYAMDYWGQGTLVTVSS11VLEIVLTQSPATLSVSPGERATLSCSASSSVTFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTEYTLTISSMQSEDAAVYYCQQWNNYPWTFGGGTKVEIK10

[0201] Frame work sequence of humanized 81E3A8-14 clone antibody Antibody Chain types sequence Seq no. humanized 81E3A8-14 antibody VHQIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGWINTYTGKPTYDDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAREAGKDYAMDYWGQGTLVTVSS7VLDIQLTQSPSSLSASVGDRVTITCSASSSVTFMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWNNYPWTFGGGTKVEIK12

[0202] The binding affinity of humanized antibodies was analyzed using a Biacore 8K instrument. First, antibodies were bound to a protein A chip at a concentration of 0.5 μg / ml, and then CEACAM5 recombinant protein was reacted at various concentrations ranging from 1.5625 to 200 nM. The binding affinity of the antibodies was analyzed and presented as shown in Figure 12a.

[0203]

[0204] 3-1. Confirmation of binding ability of antibody (81E3A8) produced through humanization process

[0205] Hereinafter, the binding ability of an antibody (81E3A8) produced by performing a humanization process according to one embodiment of the present invention through FIGS. 12b and 12c to a CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5 and a CHO-k1 (CHO-CEACAM8) cell line expressing CEACAM8 will be described in detail.

[0206]

[0207] Figure 12b is a diagram comparing the binding ability of an antibody (81E3A8) produced by performing a humanization process according to one embodiment of the present invention to a CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5 and a CHO-k1 (CHO-CEACAM8) cell line expressing CEACAM8.

[0208] Figure 12c is a diagram showing the binding ability of an antibody (81E3A8) produced by performing humanization work according to one embodiment of the present invention to H3122, KATO III, and MKN45 cell lines expressing CEACAM5.

[0209]

[0210] In order to confirm the binding ability of the antibody (81E3A8) produced by humanization according to one embodiment of the present invention to the CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5 and the CHO-k1 (CHO-CEACAM8) cell line expressing CEACAM8, the CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5 and the CHO-k1 (CHO-CEACAM8) cell line expressing CEACAM8 were first dispensed into a 96-well plate at 3x10^6 cells / well. Thereafter, humanized mAbs at various concentrations (0.025 nM - 500 nM) were diluted with DPBS solution, dispensed into the CHO-CEACAM5 cell plate at 50 μL / well, and incubated at 4°C for 20 minutes. The plate was washed twice with 250 μL / well of cold DPBS. (1500 RPM, 4℃, centrifugation for 3 minutes) Then, 50 μL / well of a solution of Alexa Flour 647-conjugated goat anti-human IgG Fcγ secondary antibody (Jackson ImmunoResearch) diluted in DPBS to a concentration of 15 μg / mL was dispensed onto the cells, and the plate was wrapped in foil to block light and incubated at 4℃ for 20 minutes. 250 μL / well of cold DPBS was washed twice. (1500 RPM, 4℃, centrifugation for 3 minutes) Finally, 250 μL / well of cold DPBS was dispensed, and the fluorescence was measured using a flow cytometer (MACSQuant®Analyzer 10, Miltenyi Biotec) and the MFI (geometric mean) was analyzed using a BD Flowjo.

[0211]

[0212] As a result, referring to FIG. 12b, it was confirmed that all five antibodies (81E3A8-02, 81E3A8-06, 81E3A8-10, 81E3A8-12, and 81E3A8-14) produced by performing humanization work according to one embodiment of the present invention had low binding ability to a cell line expressing CEACAM8, and high binding ability to a CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5.

[0213]

[0214] In addition, as a result of confirming the binding ability to H3122, KATO III, and MKN45 cell lines expressing CEACAM5 using the above method, it was confirmed that all antibodies (81E3A8-02, 81E3A8-06, 81E3A8-12, and 81E3A8-14) produced by performing the humanization process according to one embodiment of the present invention showed high binding ability.

[0215]

[0216] 3-2. Confirmation of binding ability of antibody (45A2E10) produced through humanization process

[0217] Hereinafter, the binding ability of an antibody (45A2E10) produced by performing a humanization process according to one embodiment of the present invention will be described in detail through FIGS. 13a and 13b.

[0218]

[0219] Figures 13a and 13b are diagrams showing the results of analyzing the binding affinity of an antibody (45A2E10) produced by performing humanization work according to one embodiment of the present invention.

[0220]

[0221] In order to analyze the binding affinity of various 45A2E10 clone antibodies produced through humanization work, first, 45A2E10 clones were humanized and produced, and among them, 7 antibodies (45A2E10-02, 45A2E10-03, 45A2E10-05, 45A2E10-06, 45A2E10-08, 45A2E10-09) showing binding affinity were selected. At this time, 45A2E10-01 corresponds to an antibody produced with a mouse sequence as a control, and the humanized antibodies of the clone antibodies have the same CDR 1 to 3 sequences of the clone antibodies as shown in Tables 7 to 13 below, and were produced by combining the frame work sequences differently.

[0222]

[0223] CDR sequence of humanized 45A2E10 antibody AntibodyChain typessequenceSeq no.humanized 45A2E10antibodyVHCDR 1NYGVH13CDR 2LIWAGGHTNYNSALMS14CDR 3EVRRDWYFDV15VLCDR 1RASENIYSYLT16CDR 2NAKILAE17CDR 3QHHFGTPFT18

[0224] Frame work sequence of humanized 45A2E10-02 clone antibody Antibody Chain types sequence Seq no. humanized 45A2E10-02 antibody VHQVQLQESGPGLVKPSETLSLTCTVSGGSISNYGVHWIRQPPGKGLEWIGLIWAGGHTNYNSALMSRVTISKDNSKSQISLKLSSVTAADTAVYYCAREVRRDWYFDVWGQGTMVTVSS19VLDIQMTQSPSSLSASVGDRVTITCRASENIYSYLTWYQQKPGKAPKLLIYNAKILAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHFGTPFTFGQGTKLEIK20

[0225] Frame work sequence of humanized 45A2E10-03 clone antibody Antibody Chain types sequence Seq no. humanized 45A2E10-03 antibody VHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSNYGVHWIRQPPGKGLEWIGLIWAGGHTNYNSALMSRVTISKDNSKSQISLKLSSVTAADTAVYYCAREVRRDWYFDVWGQGTMVTVSS21VLDIQMTQSPSSLSASVGDRVTITCRASENIYSYLTWYQQKPGKAPKLLIYNAKILAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHFGTPFTFGQGTKLEIK20

[0226] Frame work sequence of humanized 45A2E10-05 clone antibody Antibody Chain types sequence Seq no. humanized 45A2E10-05 antibody VHQVQLQESGPGLVKPSETLSLTCTVSGGSISNYGVHWIRQPPGKGLEWIGLIWAGGHTNYNSALMSRVTISKDNSKSQISLKLSSVTAADTAVYYCAREVRRDWYFDVWGQGTMVTVSS19VLDIQMTQSPSTLSASVGDRVTITCRASENIYSYLTWYQQKPGKAPKLLIYNAKILAEGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHHFGTPFTFGQGTKLEIK22

[0227] 인간화된 45A2E10-06 클론 항체의 Frame work 서열AntibodyChain typesequenceSeq no.humanized 45A2E10-06antibodyVHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSNYGVHWIRQPPGKGLEWIGLIWAGGHTNYNSALMSRVTISKDNSKSQISLKLSSVTAADTAVYYCAREVRRDWYFDVWGQGTMVTVSS21VLDIQMTQSPSTLSASVGDRVTITCRASENIYSYLTWYQQKPGKAPKLLIYNAKILAEGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHHFGTPFTFGQGTKLEIK22

[0228] 인간화된 45A2E10-08 클론 항체의 Frame work 서열AntibodyChain typesequenceSeq no.humanized 45A2E10-08antibodyVHQVQLQESGPGLVKPSETLSLTCTVSGGSISNYGVHWIRQPPGKGLEWIGLIWAGGHTNYNSALMSRVTISKDNSKSQISLKLSSVTAADTAVYYCAREVRRDWYFDVWGQGTMVTVSS19VLEIVMTQSPATLSVSPGERATLSCRASENIYSYLTWYQQKPGQAPRLLIYNAKILAEGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHHFGTPFTFGQGTKLEIK23

[0229] Frame work sequence of humanized 45A2E10-09 clone antibody AntibodyChain typesequenceSeq no.humanized 45A2E10-09antibodyVHQVQLQESGPGLVKPSQTLSLTCAVYGGSFSNYGVHWIRQPPGKGLEWIGLIWAGGHTNYNSALMSRVTISKDNSKSQISLKLSSVTAADTAVYYCAREVRRDWYF DVWGQGTMVTVSS21VLEIVMTQSPATLSVSPGERATLSCRASENIYSYLTWYQQKPGQAPRLLIYNAKILAEGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHHFGTPFTFGQGTKLEIK23

[0230] The binding affinity of humanized antibodies was analyzed using a Biacore 8K instrument. First, antibodies were bound to a protein A chip at a concentration of 1 μg / ml, and then reacted with CEACAM5 recombinant protein at a concentration of 200 nM. The binding affinity of these antibodies was analyzed, as shown in Figures 13a and 13b.

[0231]

[0232] 3-3. Confirmation of binding ability of antibody (45A2E10) produced through humanization process

[0233] Hereinafter, the binding ability of an antibody (45A2E10) produced by performing a humanization process according to one embodiment of the present invention through FIG. 14 to a CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5 will be described in detail.

[0234]

[0235] Figure 14 is a diagram showing the binding ability of an antibody (45A2E10) produced by performing humanization work according to one embodiment of the present invention to a CHO-k1 (CHO-CEACAM5) cell line expressing CEACAM5.

[0236] In order to confirm the binding ability of the antibody (45A2E10) produced by humanization according to one embodiment of the present invention to the CEACAM5-expressing CHO-k1 (CHO-CEACAM5) cell line, first, the CEACAM5-expressing CHO-k1 (CHO-CEACAM5) cell line was dispensed into a 96-well plate at 3x10^6 cells / well. Then, humanized mAbs at various concentrations (0.025 nM - 500 nM) were diluted with DPBS solution, dispensed into the CHO-CEACAM5 cell plate at 50 μL / well, and incubated at 4°C for 20 minutes. The plate was washed twice with 250 μL / well of cold DPBS. (1500 RPM, 4℃, centrifugation for 3 minutes) Then, 50 μL / well of a solution of Alexa Flour 647-conjugated goat anti-human IgG Fcγ secondary antibody (Jackson ImmunoResearch) diluted in DPBS to a concentration of 15 μg / mL was dispensed onto the cells, and the plate was wrapped in foil to block light and incubated at 4℃ for 20 minutes. 250 μL / well of cold DPBS was washed twice. (1500 RPM, 4℃, centrifugation for 3 minutes) Finally, 250 μL / well of cold DPBS was dispensed, and the fluorescence was measured using a flow cytometer (MACSQuant®Analyzer 10, Miltenyi Biotec) and the MFI (geometric mean) was analyzed using a BD Flowjo.

[0237]

[0238] As a result, referring to FIG. 14, it was confirmed that among the six antibodies produced by performing humanization work according to one embodiment of the present invention, the 45A2E10-08 antibody exhibited particularly high binding ability.

[0239]

[0240] 3-4. Confirmation of binding ability of antibody (94E12E2) produced through humanization process

[0241] Hereinafter, the binding ability of various clone antibodies (94E12E2) produced by performing humanization work according to one embodiment of the present invention through FIG. 15 will be described in detail.

[0242]

[0243] Figure 15 is a diagram showing the binding affinity of various clone antibodies (94E12E2) produced by performing humanization work according to one embodiment of the present invention, as confirmed by ELISA analysis.

[0244] First, the 94E12E2 clone was humanized to produce five humanized antibodies (94E12E2-02, 94E12E2-03, 94E12E2-04, 94E12E2-05, 94E12E2-06), and their binding affinity to the CEACAM5 recombinant protein was confirmed. 94E12E2-01 corresponds to an antibody produced using a mouse sequence as a control.

[0245]

[0246] CDR sequence of humanized 94E12E2 antibody AntibodyChain typessequenceSeq no.humanized 94E12E2antibodyVHCDR 1DNYMH24CDR 2WIDPENGDTEYDPKFQG25CDR 3ITMATPYPMDY26VLCDR 1SASTSVIYMH27CDR 2STSNLAS28CDR 3QQRSSYPLT29

[0247] Frame work sequence of humanized 94E12E2-02 clone antibody Antibody Chain types sequence Seq no. humanized 94E12E2-02 antibody VHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDNYMHWVRQAPGKGLEWVAWIDPENGDTEYDPKFQGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRITMATPYPMDYWGQGTLVTVSS30VLDIQMTQSPSSLSASVGDRVTITCSASTSVIYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQRSSYPLTFGQGTKVEIKR31

[0248] Frame work sequence of humanized 94E12E2-03 clone antibody Antibody Chain types sequence Seq no. humanized 94E12E2-03 antibody VHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDNYMHWVRQAPGKGLEWVAWIDPENGDTEYDPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVYYCNIITMATPYPMDYWGQGTLVTVSS32VLDIQMTQSPSSLSASVGDRVTITCSASTSVIYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQRSSYPLTFGQGTKVEIKR33

[0249] Frame work sequence of humanized 94E12E2-04 clone antibody Antibody Chain types sequence Seq no. humanized 94E12E2-04 antibody VHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDNYMHWVRQAPGKGLEWIGWIDPENGDTEYDPKFQGRATMSADTSKNTAYLQMNSLRAEDTAVYYCNIITMATPYPMDYWGQGTLVTVSS34VLDIQLTQSPSSLSASVGDRVTITCSASTSVIYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQRSSYPLTFGQGTKVEIKR35

[0250] Frame work sequence of humanized 94E12E2-05 clone antibody Antibody Chain types sequence Seq no. humanized 94E12E2-05 antibody VHEVQLVQSGAEVKKPGATVKISCKVSGFNIKDNYMHWVQQAPGKGLEWIGWIDPENGDTEYDPKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCNIITMATPYPMDYWGQGTLVTVSS36VLEIVLTQSPATLSLSPGERATLSCSASTSVIYMHWFQQKPGQAPRLWIYSTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPLTFGQGTKLEIKR37

[0251] 인간화된 94E12E2-06 클론 항체의 Frame work 서열AntibodyChain typesequenceSeq no.humanized 94E12E2-06antibodyVHQVQLVQSGAEVKKPGASVKVSCKASGFNIKDNYMHWVRQAPGQGLEWIGWIDPENGDTEYDPKFQGRATMTADTSISTAYMELSRLRSDDTAVYYCNIITMATPYPMDYWGQGTLVTVSS38VLEIVLTQSPDFQSVTPKEKVTITCSASTSVIYMHWFQQKPDQSPKLWIYSTSNLASGVPSRFSGSGSGTDYTLTINSLEAEDAATYYCQQRSSYPLTFGQGTKLEIKR39

[0252] To confirm the binding ability of the humanized 94E12E2 antibody, 30 μl of CEACAM5 protein (Acrobiosystems) was dispensed into each well of a 384-well microplate (Corning) at a concentration of 0.5 μg / ml, incubated at 4°C for 16 hours, and washed four times with 100 μL / well of PBST (DPBS + 0.05% Tween 20, Teknova). Afterwards, 80 μL / well of 1X PBS buffer containing 3% FBS was added, incubated at room temperature for 2.5 hours, and washed four times with 100 μL / well of PBST in the same manner as above. At this time, the antibody was prepared by diluting 3-fold to a concentration ranging from 200 nM to 0.0004 nM with DPBS (Bining buffer) containing 0.5% Fetal Bovin Serum (FBS, Cytiva). The antibodies prepared in this way were added to the CEACAM5-coated plate at 30 μL / well and incubated at room temperature for 1 hour. After washing the plate, HRP (horseradish peroxidase) anti-mouse IgG Fcγantibody, Jackson ImmunoResearch) was diluted to a concentration of 0.16 μg / mL (1:5000) in binding buffer and added to the plate at 30 μL / well and incubated at room temperature for 1 hour. After washing the plate in the same manner, TMB (3,3',5,5'-tetramethylbenzidine, Invitrogen) substrate was added to the plate at 30 μL / well and incubated in the dark for 10 minutes at room temperature. Finally, 30 μL / well of 0.16 M sulfuric acid (stop solution, Invitrogen) solution was added, incubated in the dark for 10 minutes to stop the reaction, and the absorbance was measured at 450 nm (Synergyhtx multimode reader, Biotek).

[0253] As a result, as shown in Figure 15, it was confirmed that among the antibodies produced through humanization, the 94E12E2-04 and 94E12E2-06 antibodies showed particularly higher binding ability.

[0254]

[0255] 3-5. Confirmation of binding ability of antibody (94E12E2) produced through humanization process

[0256] Hereinafter, the binding ability of various clone antibodies (94E12E2) produced by performing humanization work according to one embodiment of the present invention through FIG. 16 will be described in detail.

[0257]

[0258] Figure 16 is a diagram showing the binding affinity of various clone antibodies (94E12E2) produced by performing humanization work according to one embodiment of the present invention, as confirmed by flow cytometry.

[0259] In order to confirm the binding ability of the antibody (94E12E2) produced by humanization according to one embodiment of the present invention to the CEACAM5-expressing CHO-k1 (CHO-CEACAM5) cell line, 3x10^6 cells / well of the CEACAM5-expressing CHO-k1 (CHO-CEACAM5) cell line were first dispensed into a 96-well plate. Thereafter, humanized mAbs of various concentrations (0.025 nM - 500 nM) were diluted with DPBS solution, dispensed into the CHO-CEACAM5 cell plate at 50 μL / well, and incubated at 4°C for 20 minutes. The plate was washed twice with 250 μL / well of cold DPBS. (1500 RPM, 4℃, centrifugation for 3 minutes) Then, 50 μL / well of a solution of Alexa Flour 647-conjugated goat anti-human IgG Fcγ secondary antibody (Jackson ImmunoResearch) diluted in DPBS to a concentration of 15 μg / mL was dispensed onto the cells, and the plate was wrapped in foil to block light and incubated at 4℃ for 20 minutes. 250 μL / well of cold DPBS was washed twice. (1500 RPM, 4℃, centrifugation for 3 minutes) Finally, 250 μL / well of cold DPBS was dispensed, and the fluorescence was measured using a flow cytometer (MACSQuant®Analyzer 10, Miltenyi Biotec) and the MFI (geometric mean) was analyzed using a BD Flowjo.

[0260] As a result, as shown in Figure 16, it was confirmed that among the antibodies produced through humanization, the 94E12E2-04 antibody showed particularly higher binding ability.

[0261]

[0262] 4-1. Confirmation of binding ability of antibody (36B5H8) clones with modified sequences corresponding to post-translational modification (PTM)

[0263] Hereinafter, the binding ability of antibody (36B5H8) clones modified with a sequence corresponding to a post-translational modification (PTM) according to one embodiment of the present invention will be described in detail through FIGS. 17a and 17b.

[0264]

[0265] Figures 17a and 17b are diagrams showing the analysis of the binding affinity of antibody (36B5H8) clones with modified sequences corresponding to post-translational modification (PTM) according to one embodiment of the present invention.

[0266] To analyze the binding affinity of 36B5H8 clone antibodies with modified post-translational modification (PTM) parts, we first produced antibodies by modifying the parts predicted to undergo post-translational modification in the selected humanized 36B5H8 clone antibodies, and analyzed the binding affinity of these antibodies using a Biacore 8K instrument. First, after binding the antibodies to the CM5 chip, the CEACAM5 recombinant protein was reacted at various concentrations from 12.5 to 800 nM, and the binding affinity of these antibodies was analyzed. Here, 36B5H8-VH-3(4GS)-VL corresponds to an antibody produced with a mouse sequence as a control, and 36B5H8-VH-DA-VL corresponds to an antibody produced by modifying the post-translational modification part of the mouse sequence as a control. The sequence of the humanized and post-translationally modified (PTM) 36B5H8 antibody is identical to that of the humanized 36B5H8 antibody in VH CDR1, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, but some of the amino acid sequence of VH CDR2 is different.

[0267]

[0268] CDR sequence of humanized 36B5H8 antibody AntibodyChain typessequenceSeq no.humanized 36B5H8antibodyVHCDR 1GITFSRYAMS40CDR 2SISSDAITYYLDSVKG41CDR 3IYYYGSPFFDY42VLCDR 1KASQNVYTNVA43CDR 2SASNRYS44CDR 3QQYNNYPLFT45

[0269] Sequence of humanized and post-translationally modified (PTM) 36B5H8 antibody Antibody Chain types sequence Seq no. humanized 36B5H8 antibody VHCDR 1GITFSRYAMS40CDR 2SISSDAITYYLDAVKG46CDR 3IYYYGSPFFDY42VLCDR 1KASQNVYTNVA43CDR 2SASNRYS44CDR 3QQYNNYPLFT45

[0270] 4-2. Confirmation of binding ability of humanized and post-translationally modified (PTM) antibodies

[0271] Hereinafter, the binding ability of selected antibodies using CHO-K1-CEACAM5 cells after humanization and post-translational modification (PTM) according to one embodiment of the present invention is described in detail through FIG. 16.

[0272]

[0273] Figure 16 is a diagram showing the binding ability of selected antibodies using CHO-K1-CEACAM5 cells after humanization and post-translational modification (PTM) according to one embodiment of the present invention.

[0274] To confirm the binding of the selected antibodies using CHO-K1-CEACAM5 cells after humanization and post-translational modification (PTM), first, CHO-K1-CEACAM5 cells were incubated with 100 μl of 1X PBS at a concentration of 0 to 45 μg / ml each antibody and incubated at 4°C for 30 minutes. Then, 2 ml of 1X PBS was added, centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. Then, AF647-anti-human IgG, Fcr fragment binding antibody, which can confirm the primary antibody, was mixed at a concentration of 1 μg / ml in 100 μl of 1X PBS and incubated with the cells at 4°C for 30 minutes. Finally, 2 ml of 1X PBS was added to the cells, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and 300 μl of 1X PBS was added and measured using a flow cytometer.

[0275] As a result, as shown in Fig. 16, it was confirmed that all antibodies subjected to humanization and post-translational modification (PTM) work as in the present invention showed higher binding ability than antibody 36B5H8-VH-DA-VL, which was produced by modifying the post-translational modification portion of the mouse sequence as a control.

[0276]

[0277] According to the above results, the antibody according to one embodiment of the present invention, which targets CEACAM5 remaining on the cell surface, can have excellent tumor cell recognition ability in the tumor microenvironment by specifically binding to CEACAM5 remaining on the cell surface while not binding to soluble CEACAM5, and thus, the anticancer effect can be improved.

[0278]

[0279]

[0280] 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 against CEACAM5 or an antigen-binding fragment thereof that specifically binds to CEACAM5, comprising: (1) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, CDR3 of the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (2) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, CDR3 of the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18; (3) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, CDR3 of the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; or (4) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41 or 46, CDR3 of the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 42, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and CDR3 of the light chain comprising the amino acid sequence set forth in SEQ ID NO:

45.

2. The anti-CEACAM5 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-CEACAM5 antibody or antigen-binding fragment thereof specifically binds to CEACAM5 present on the cell surface, substantially without binding to soluble CEACAM5 (sCEACAM5), which does not bind to the cell surface.

3. An anti-CEACAM5 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-CEACAM5 antibody or antigen-binding fragment thereof is a humanized antibody.

4. An antibody-drug conjugate comprising an anti-CEACAM5 antibody or a binding fragment thereof according to claim 1 or 2 as an active ingredient.

5. A pharmaceutical composition for the prevention or treatment of a malignant tumor, comprising an antibody against CEACAM5 or a binding fragment thereof according to any one of claims 1 or 2 as an active ingredient.

6. A pharmaceutical composition for the prevention or treatment of a malignant tumor according to claim 5, wherein the malignant tumor is a solid malignant tumor.

7. A pharmaceutical composition for the prevention or treatment of a malignant tumor according to claim 6, wherein the solid malignant tumor is any of colon cancer, pancreatic cancer, lung cancer, stomach cancer, hepatocellular carcinoma, breast cancer, and thyroid cancer.

8. A pharmaceutical composition for the prevention or treatment of a malignant tumor according to claim 5, wherein the antibody against CEACAM5 or a binding fragment thereof is used as a cell therapy agent for adoptive cell therapy (ACT).

9. A composition for inhibiting the proliferation of CEACAM5-expressing tumor cells, comprising an antibody against CEACAM5 or a binding fragment thereof according to claim 1 or 2.

10. A nucleic acid encoding an antibody against CEACAM5 or a binding fragment thereof according to claim 1 or 2.

11. A recombinant expression vector containing the nucleic acid of claim 10.

12. A host cell transformed with a recombinant expression vector according to claim 11.

13. A method for preventing or treating a malignant tumor in an individual, comprising administering to the individual the pharmaceutical composition of claim 5.