Novel Anti-CDCP1 humanized antibody and use thereof

Abstract

The present invention relates to a novel anti-CDCP1 antibody or an antigen-binding fragment thereof. The anti-CDCP1 antibody or antigen-binding fragment thereof is an antibody capable of specifically binding to human CDCP1 and capable of cellular internalization. Accordingly, the antibody or antigen-binding fragment thereof can be effectively used for preventing or treating cancer.

Description

Novel anti-CDCP1 humanized antibodies and uses thereof

[0001] This application claims priority to Korean Patent Application No. 10-2024-0053487, filed April 22, 2024, and Korean Patent Application No. 10-2025-0010708, filed January 23, 2025, the entire disclosures of which are incorporated herein by reference.

[0002] The present invention relates to a novel anti-CDCP1 antibody or binding fragment thereof and a pharmaceutical use thereof, and more particularly, to an anti-CDCP1 antibody or antigen-binding fragment thereof that specifically binds only to CDCP-1 of cancer cells with high affinity, a nucleic acid molecule encoding the anti-CDCP1 antibody or antigen-binding fragment thereof, a recombinant vector or transformant comprising the nucleic acid molecule, a method for producing the anti-CDCP1 antibody or antigen-binding fragment thereof, a pharmaceutical composition for preventing or treating cancer comprising the anti-CDCP1 antibody or antigen-binding fragment thereof as an active ingredient, a method for preventing or treating cancer comprising the anti-CDCP1 antibody or antigen-binding fragment thereof, and a kit for detecting CDCP1 or diagnosing cancer comprising the anti-CDCP1 antibody or antigen-binding fragment thereof.

[0003] CDCP1 (CUB-domain containing protein 1), also known as CD318, SIMA135, TRASK, and gp140, is a type 1 transmembrane glycoprotein that is upregulated in malignant tumors of the breast, lung, colorectal, ovarian, renal, hepatic, pancreatic, and hematopoietic systems. Specifically, human CDCP1 is approximately 836 amino acids long and contains three CUB domains: the complement C1r / C1s, Uegf, and Bmp1 domains. Increased CDCP1 expression has been associated with cancer progression. CDCP1, located primarily on the cell surface, is positioned on key oncogenic and metastatic signaling cascades, including the SRC / PKCδ, PI3K / AKT, WNT and RAS / ERK axes, the oxidative pentose phosphate pathway and fatty acid oxidation, and plays a crucial role in cancer cell survival and growth, metastasis and therapeutic resistance (Shion A. Lim.et al.,Targeting a proteolytic neoepitope on CUB domain containing protein 1(CDCP1) for RAS-driven cancers, 2022).

[0004] Therefore, there is a need to develop an effective treatment that is specific to CDCP1, has no side effects, and is effective for the detection and treatment of various cancers.

[0005] [Prior Art Literature]

[0006] [Non-patent literature]

[0007] (Non-patent Document 1) Shion A. Lim. et al., Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers, 2022

[0008] Accordingly, the present inventors conducted research to develop an anti-CDCP1 antibody with excellent efficacy for treating various cancer diseases, and as a result, developed a novel anti-CDCP1 antibody that can be internalized into cells.

[0009] Accordingly, it is an object of the present invention to provide a novel anti-CDCP1 antibody that binds to CDCP1 with high affinity and can be internalized into cells expressing CDCP1.

[0010] Another object of the present invention is to provide a nucleic acid molecule encoding the anti-CDCP1 antibody.

[0011] Another object of the present invention is to provide a vector comprising the nucleic acid molecule and a host cell comprising the vector.

[0012] Another object of the present invention is to provide a method for producing an anti-CDCP1 antibody using the host cell.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the above-described anti-CDCP1 antibody as an active ingredient.

[0014] Another object of the present invention is to provide a method for preventing or treating cancer using the anti-CDCP1 antibody described above.

[0015] Another object of the present invention is to provide a use of the above-mentioned anti-CDCP1 antibody for the manufacture of a medicament for preventing or treating cancer.

[0016] Another object of the present invention is to provide a kit for detecting CDCP1 using the anti-CDCP1 antibody described above.

[0017] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0018] To solve the above-described problem, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 (CUB domain containing protein 1), comprising any one of the following (i) to (vii):

[0019] (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (TISSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 (HADYVDVVFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6 (QQWSSNPPT);

[0020] (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (TISSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7 (HADYMDVAFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0021] (iii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (TISSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9 (HADYMDVGFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0022] (iv) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10 (TISSGGRYTYYPDSVEG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11 (HADYVDVAFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0023] (v) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10 (TISSGGRYTYYPDSVEG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12 (HADYVDVGFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0024] (vi) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13 (TTSSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7 (HADYMDVAFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT); or

[0025] (vii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14 (SQGMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15 (TISVTGRYLYYADNVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16 (HADYQDVSFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17 (QQWQMEPPT).

[0026] In the present invention, the antibody or antigen-binding fragment thereof may include any one of the following (i) to (ix):

[0027] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region of SEQ ID NO: 19;

[0028] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21;

[0029] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22;

[0030] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0031] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0032] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0033] (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0034] (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; or

[0035] (ix) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30.

[0036] In the present invention, the Fc domain of the antibody may comprise: (a) a wild-type IgG Fc; or (b) an Fc domain variant comprising one or more amino acid substitutions that reduce binding to an Fc receptor.

[0037] In the present invention, the Fc domain variant may comprise one or more amino acid substitutions that reduce binding to an Fcγ receptor.

[0038] In the present invention, the Fc domain variant may be an Fc variant comprising L235A and G237A amino acid substitutions, an Fc variant comprising L234A and L235E amino acid substitutions, or an Fc variant comprising L234A, L235A, and P329G amino acid substitutions, and the residues may be numbered according to the EU index of Kabat.

[0039] In addition, the present invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to the aforementioned CDCP1.

[0040] In addition, the present invention provides a recombinant vector comprising the nucleic acid molecule and a transformant comprising the recombinant vector.

[0041] Additionally, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CDCP1, comprising the following steps (a) and (b): (a) culturing the transformant described above; and (b) recovering the antibody or an antigen-binding fragment thereof that specifically binds to CDCP1 from the culture.

[0042] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer expressing CDCP1, comprising an antibody or an antigen-binding fragment thereof that specifically binds to the aforementioned CDCP1 as an active ingredient.

[0043] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to the aforementioned CDCP1.

[0044] Additionally, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the aforementioned CDCP1 for use in preventing or treating cancer.

[0045] The present invention also provides the use of an antibody or antigen-binding fragment thereof that specifically binds to the aforementioned CDCP1 for the manufacture of a drug for preventing or treating cancer.

[0046] In the present invention, the cancer may be selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, vulvar cancer, breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, skin cancer, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, endocrine cancer, and sarcoma.

[0047] In addition, the present invention provides a kit for detecting CDCP1 comprising an antibody or an antigen-binding fragment thereof that specifically binds to the aforementioned CDCP1.

[0048] The anti-CDCP1 antibody or antigen-binding fragment thereof of the present invention specifically binds to CDCP1 and has strong binding affinity. Furthermore, the antibody is capable of cell internalization and exhibits excellent stability under pH and temperature conditions, so the anti-CDCP1 antibody or antigen-binding fragment thereof can be useful for preventing, treating, or diagnosing cancers mediated by CDCP1.

[0049] Figure 1a shows the CD34 of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention. + To confirm the ability to bind to CDCP1 expressed in cells, expression in CD34+ cells was confirmed by FACS using commercially available anti-CDCP1 antibodies and anti-CD34 antibodies.

[0050] Figure 1b shows the CD34 of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] prepared in the present invention. + The ability to bind to CDCP1 expressed in cells was confirmed by FACS.

[0051] Figure 2a shows the results of ELISA confirming the binding ability of anti-CDCP1 antibodies [2F9 chimeric antibody and its humanized antibodies (2F9-679 and 2F9-700)] manufactured in the present invention to the CDCP1 antigen.

[0052] Figure 2b is a graph representing Figure 2a (x-axis: concentration, y-axis: absorbance).

[0053] Figure 2c shows the results of ELISA confirming the binding ability of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention to the CDCP1 antigen.

[0054] Figure 3a shows the results of confirming the binding ability of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention to human CDCP1 using a surface plasmon resonance assay (SPR assay).

[0055] Figure 3b shows the results of SPR assay confirming the binding ability of anti-CDCP1 antibodies [2F9 chimeric antibody and its humanized antibodies (2F9-679 and 2F9-700)] manufactured in the present invention to CDCP1 under each pH condition.

[0056] Figure 3c shows the results of SPR assay to confirm the binding ability of anti-CDCP1 antibodies [affinity matured antibodies (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention to CDCP1 under each pH condition.

[0057] Figure 4a shows the cell surface binding results of the anti-CDCP1 antibodies [2F9 chimeric antibody and its humanized antibodies (2F9-679, 2F9-700)] manufactured in the present invention in the HCT116 cell line.

[0058] Figure 4b shows the cell surface binding results of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention in the CAPAN-1 cell line.

[0059] Figure 4c shows the cell surface binding results of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention in the PANC-1 cell line.

[0060] Figure 4d shows the cell surface binding results of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention in the MCF-7 cell line.

[0061] Figures 5a and 5b show the results of confirming internalization of anti-CDCP1 antibodies [2F9 chimeric antibody and its humanized antibodies (2F9-679, 2F9-700)] manufactured in the present invention by culturing them in HCT116 cells for 4 hours (Figure 5a) or 24 hours (Figure 5b).

[0062] Figures 5c and 5d show the results of confirming internalization of anti-CDCP1 antibodies manufactured in the present invention [2F9 chimeric antibody and its humanized antibodies (2F9-679, 2F9-700)] in CAPAN-1 cell lines after culturing for 4 hours (Figure 5c) or 24 hours (Figure 5d).

[0063] Figures 5e and 5f show the results of confirming internalization of anti-CDCP1 antibodies manufactured in the present invention [2F9 chimeric antibody and its humanized antibodies (2F9-679, 2F9-700)] in HCC1954 cell lines after culturing for 4 hours (Figure 5e) or 24 hours (Figure 5f).

[0064] Figure 5g is a table summarizing the results of Figures 5a to 5f.

[0065] Figure 5h shows the results of confirming internalization of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention in the CAPAN-1 cell line after culturing for 4 hours or 24 hours.

[0066] Figure 5i shows the results of confirming internalization of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13, and M14)] manufactured in the present invention in the PANC-1 cell line after culturing for 4 hours or 24 hours.

[0067] Figure 5j shows the results of confirming internalization of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention by culturing it in the MCF-7 cell line for 4 hours or 24 hours.

[0068] Figure 6a shows the results of confirming the stability of the anti-CDCP1 antibody [2F9 chimeric antibody and its humanized antibody (2F9-679, 2F9-700)] manufactured in the present invention against freezing and thawing by SDS-PAGE.

[0069] Figure 6b shows the results of confirming the stability of the anti-CDCP1 antibody [2F9 chimeric antibody and its humanized antibody (2F9-679, 2F9-700)] manufactured in the present invention against freezing and thawing using high-performance liquid chromatography.

[0070] Figure 6c shows the results of confirming the stability of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention against freezing and thawing using high-performance liquid chromatography.

[0071] Figure 6d shows the results of ELISA to confirm the binding ability of anti-CDCP1 antibodies [affinity matured antibodies (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention to CDCP1 after freezing and thawing.

[0072] Figure 7a shows the results of confirming the thermal stability of the anti-CDCP1 antibody [2F9 chimeric antibody and its humanized antibody (2F9-679, 2F9-700)] manufactured in the present invention through PTS analysis (protein thermal shift™).

[0073] Figure 7b shows the results of confirming the thermal stability of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention through PTS analysis.

[0074] Figure 8 shows the results of measuring surface binding of anti-CDCP1 antibodies [2F9 chimeric antibody and its humanized antibodies (2F9-679 and 2F9-700)] manufactured in the present invention on the MDA-MB-453 cell line, and the results are compared with cells in which CDCP1 expression was reduced in the same cell line.

[0075] Figure 9a shows the results of confirming the cell migration inhibitory effect of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention on the MDA-MB-231 cell line.

[0076] Figure 9b shows the results of Western blot analysis of CDCP1 protein degradation through internalization of CDCP1 following antibody treatment in cells that confirmed the cell migration inhibitory effect of the anti-CDCP1 antibody [affinity matured antibody (AM h2F9 M4, M6, M9, M10, M13 and M14)] manufactured in the present invention on the MDA-MB-231 cell line.

[0077] Definition of Terms

[0078] The term "antibody" as used herein refers to immunoglobulin molecules and multimers thereof having a structure in which one light chain is linked to each of two heavy chains linked to each other by a disulfide bond. The light chain comprises two domains: one variable domain (light chain variable domain, VL) and one constant domain (light chain constant domain, CL). The heavy chain comprises four domains: one variable domain (heavy chain variable domain, VH) and three constant domains (heavy chain constant domains, CH; CH1, CH2, and CH3). The constant domains of the light and heavy chains serve to impart biological properties such as binding between light and / or heavy chains, secretion, complement binding, and binding to Fc receptors (FcR), while the variable domains of the light and heavy chains determine recognition and binding specificity for antigens.

[0079] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the possible exception of variant antibodies, including those containing naturally occurring mutations or those arising during the production of the monoclonal antibody preparation, which mutations are generally present in minor amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0080] The term "monospecific" antibody, as used herein, refers to an antibody having more than one binding site, each of which binds to the same epitope of the same antigen.

[0081] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of native antibodies. "Native antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), called heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), called the variable light domain or light chain variable domain, followed by a light chain constant domain (CL), called the light chain constant region. The heavy chains of antibodies can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further classified into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0082] As used herein, the term "antigen-binding fragment" means any polypeptide or glycoprotein comprising a portion of an intact antibody, particularly an antigen-binding site or variable region of an intact antibody. Such antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical digestion of an intact antibody, and examples of such antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv-Fc, scFab-Fc, diabodies, minibodies, scAbs, dAbs, half-IgGs or combinations thereof, as well as bispecific and multispecific antibodies formed therefrom.

[0083] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies as described herein, as well as production in recombinant host cells, such as E. coli or phage.

[0084] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, which contain the heavy and light chain variable domains and the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain. A Fab' fragment differs from a Fab fragment in that it adds several residues to the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domains have a free thiol group. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and part of the Fc region. As used herein, the "F(ab')2 fragment" comprises, as described above, two light chains and two heavy chains comprising a variable region, CH1, and a portion of a constant region between the CH1 and CH2 domains, thereby forming an intrachain disulfide bond between the two heavy chains. Accordingly, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are linked to each other by a disulfide bond therebetween.

[0085] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. Two different chain compositions of crossover Fab molecules are possible and are included in the anti-CDCP1 antibodies of the present invention: on the one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Such a crossover Fab molecule is also referred to as CrossFab (VLVH). In contrast, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). Such a crossover Fab molecule is also called CrossFab(CLCH1).

[0086] A "single chain Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following sequences from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide having at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized by a natural disulfide bond between the CL domain and the CH1 domain. Additionally, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0087] A "crossover single chain Fab fragment" or "x-scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following sequences from N-terminus to C-terminus: (a) VH-CL-linker-VLCH1 and (b) VL-CH1-linker-VH-CL; wherein the VH and VL together form an antigen-binding domain that specifically binds to an antigen, and wherein the linker is a polypeptide having at least 30 amino acids. Additionally, these x-scFab molecules can be further stabilized by formation of an interchain disulfide bond through insertion of a cysteine ​​residue (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0088] An "Fv region" is an antibody that includes the variable regions of each heavy and light chain, but not the constant regions. An scFv is an Fv linked by a flexible linker. An scFv-Fc is an Fc linked to an scFv. A diabody comprises two molecules of scFv. A "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, the regions are linked to a short linker peptide having from 10 to about 25 amino acids. The linker may be glycine-rich for flexibility and serine or threonine-rich for solubility, and may link the N-terminus of the VH to the C-terminus of the VL, or vice versa. These proteins retain the specificity of the native immunoglobulin despite the deletion of the constant region and the introduction of a linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0089] A "short-chain antibody (scAb)" is a single polypeptide chain comprising one heavy chain variable region or one light chain constant region, with the heavy and light chain variable regions connected by a flexible linker. For single-chain antibodies, see, for example, U.S. Patent No. 5,260,203, which is incorporated herein by reference.

[0090] A "domain antibody (dAb)" is an immunologically functional immunoglobulin fragment comprising only the variable region of a heavy chain or the variable region of a light chain. In one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of such a bivalent domain antibody may target the same or different antigens.

[0091] The term "full-length IgG" according to the present invention is defined as comprising essentially complete IgG, but does not necessarily have all the functions of a complete IgG. For the avoidance of doubt, a full-length IgG contains two heavy chains and two light chains. Each chain contains constant (C) and variable (V) regions, which can be divided into domains designated CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens through the variable region domains contained in the Fab portion, and after binding, can interact with cells and molecules of the immune system through the constant domains, mostly through the Fc portion. The terms 'variable region domain', 'variable region', 'variable domain', 'VH / VL pair', 'VH / VL', 'Fab portion', 'Fab arm', 'Fab' or 'arm' are used interchangeably herein. A full-length antibody according to the present invention includes an IgG molecule that may have mutations that provide the desired characteristics. Such mutations must not result in the deletion of a significant portion of any region. However, an IgG molecule in which one or more amino acid residues are deleted without substantially altering the binding properties of the resulting IgG molecule is included within the term "full-length IgG". For example, such an IgG molecule may have one or more deletions of 1 to 10 amino acid residues, preferably in a non-CDR region, wherein the deletion of the amino acid is not essential for the binding specificity of the IgG.

[0092] As used herein, the term "heavy chain" refers to a full-length heavy chain and antigen-binding fragments thereof, including a variable region (VH) and three constant regions, CH1, CH2, and CH3. Heavy chain constant regions (CH) have different amino acid compositions and sequences, and thus possess different types of antigenicity. Therefore, immunoglobulins can be classified into five categories and referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. In addition, depending on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same type of immunoglobulin can be classified into different subtypes. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4.

[0093] As used herein, the term "light chain" refers to two types, λ and κ, consisting of approximately 211 to 217 amino acids. Each human antibody has exactly one such chain. The light chain is composed of a continuous variable region (VL) and a constant region (CL). The light chain may include both a full-length light chain and an antigen-binding fragment thereof. Any CL and CH1 regions of these antibody classes may be used in the present disclosure.

[0094] The above "variable region" refers to the region of an antibody to which an antigen binds. The variable region includes three hypervariable regions called complementarity determining regions (CDRs) and four framework regions (FRs).

[0095] As used herein, the term "complementarity determining region (CDR)" refers to a region among the variable regions of an antibody that confers binding specificity to an antigen. The CDR primarily plays a role in binding to an epitope of the antigen. The heavy chain and the light chain each contain three complementarity determining regions. The CDRs of each chain are typically called CDR1, CDR2, and CDR3, sequentially starting from the N-terminus, and are identified by the chain on which the particular CDR is located. The FRs of each chain are typically called FR1, FR2, FR3, or FR4, sequentially starting from the N-terminus, and are identified by the chain on which the particular FR is located.

[0096] In naturally occurring antibodies, the six "complementarity determining regions", or "CDRs," present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain because the antibody adopts a three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops that connect them, and in some cases, form part of the β-sheet structure. Thus, the framework region functions to form a scaffold that positions the CDRs in the correct orientation by interchain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to epitopes on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDR and framework regions, respectively, for any given heavy or light chain variable region can be readily identified by those skilled in the art, as these are precisely defined (see www.bioinf.org.uk: Dr. Andrew CR Martin's Group; "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987)).

[0097] Where there are two or more definitions for a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless explicitly stated to the contrary. As a specific example, the term "complementarity determining region" ("CDR") is used to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are described in Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196: 901-917 (1987), the entire contents of which are incorporated herein by reference. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nonetheless, the application of the definitions to refer to CDRs of an antibody or variant thereof is intended to be within the scope of the terms defined and used herein. Appropriate amino acid residues comprising the CDRs defined by each of the references cited above are provided in Table 1 below for comparison. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine whether a residue comprises a particular CDR by considering the variable region amino acid sequence of the antibody.

[0098] KabatchotiaCDR-H131-3526-32CDR-H150-6552-58CDR-H195-10295-102CDR-L124-3426-32CDR-L250-5650-52CDR-L389-9791-96

[0099] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. One skilled in the art can unambiguously assign the "Kabat numbering" system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system presented in Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0100] The antibodies disclosed herein may be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

[0101] As used herein, the term "Fc region" refers to the Fc domain of an immunoglobulin. The Fc region refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include the variable regions of the heavy and light chains of the immunoglobulin, the heavy chain constant region 1 (CH1), and the light chain constant region (CL).

[0102] The Fc region may be a wild-type Fc domain or an Fc domain fragment. For example, the Fc region fragment may have lysine (K) deleted from the C-terminus. Alternatively, the Fc region fragment may contain only CH3. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM. Additionally, it may be IgG1, IgG2, IgG3, or IgG4, which are subclasses of IgG, or IgA1 or IgA2, which are subclasses of IgA.

[0103] The heavy chain constant region of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0104] As used herein, the term "antigen" refers to a structure capable of selectively binding to an antibody. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. Specifically, an antigen may be a polypeptide or a protein present on the cell surface or within a cell.

[0105] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational configuration comprised of different regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, the surface of virus-infected cells, the surface of other diseased cells, the surface of immune cells, in serum-free blood, and / or in the extracellular matrix (ECM). Unless otherwise specified, a protein useful as an antigen herein can be any native form of a protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human or mouse protein. When referring to a specific protein herein, the term encompasses the "full-length," unprocessed protein, as well as all forms of the protein produced by processing in cells. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0106] "Specific binding" means that binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody or antibody fragment to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0107] "Affinity" or "binding affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). Thus, equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by routine methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0108] According to one embodiment of the present invention, the anti-CDCP1 antibody of the present invention has an IC of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -7 M or less, for example 10 -7 M to 10 -13 M, for example 10 -9 M to 10 -13 It has a dissociation constant (Kd) of M).

[0109] The term "high affinity" of an antibody refers to its ability to bind to its target antigen at a rate of 10 -9 M or less, preferably 10 -10 Refers to antibodies with a Kd of M or less.

[0110] The term "mouse" antibody is intended to encompass antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of the present disclosure may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by in vivo complementary somatic mutagenesis).

[0111] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0112] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0113] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.

[0114] A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from the constant region of the original antibody to produce the properties of the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0115] A "human" antibody is one that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0116] The term "linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Any peptide linker known in the art can be used as the linker. The peptide linker separates the light chain variable domain and the heavy chain variable domain by a sufficient distance to allow each variable domain to fold into the appropriate secondary and tertiary structures. The sequence of a suitable peptide linker can be selected by considering the following factors: (a) the ability to have a flexible extended conformation; (b) the ability to not create secondary structures that interact with the epitope; and (c) the absence of hydrophobic residues or charged residues that can react with the epitope. Preferred peptide linkers include Gly, Glu, Asn, Lys, Ser, and Pro residues. Other neutral amino acids, such as Thr and Ala, can also be included in the linker sequence. The linker sequence may consist of 1-50 amino acid residues, preferably 10-20 amino acid residues.

[0117] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage by which the amino acid residues in the candidate sequence are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the compared sequences. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package, version 36.3.8c or later, in conjunction with a BLOSUM50 comparison matrix. The FASTA program package is described in WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WR Pearson (1996), "Effective Protein Sequence Comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at http: / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml. Alternatively, see http: / fasta.bioch.virginia.edu / fasta_www2 / index.You can compare sequences using a public server accessible from cgi, and use the ggsearch(global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup=2) to perform a global alignment rather than a local one. The percent amino acid identity (%) is provided in the output alignment header.

[0118] As used herein, the term "polypeptide" is intended to encompass not only a singular "polypeptide" but also a plural "polypeptides," and refers to a molecule comprising monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are encompassed within the definition of "polypeptide," and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a designated nucleic acid sequence. They may be produced by any means, including chemical synthesis. The term "polypeptide" also encompasses variants and derivatives of polypeptides. Furthermore, "polypeptide fragment" refers to a polypeptide having a deletion of the amino-terminal amino acid sequence, a deletion of the carboxyl-terminal amino acid sequence, and / or an internal deletion, compared to the full-length protein. Such fragments may also contain modified amino acids compared to the full-length protein.In one embodiment, the fragment can be about 5 to 900 amino acids in length, for example at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more amino acids in length. For the purposes of the present invention, useful polypeptide fragments include immunologically functional fragments of antibodies comprising an antigen-binding domain. For CDCP1 binding antibodies, such useful fragments include, but are not limited to, all or part of an antibody chain comprising one, two or three heavy or light chain CDR sequences, or a variable or constant region of a heavy or light chain.

[0119] As used herein, the term "recombinant" with respect to a polypeptide or polynucleotide means a form of a polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which can be formed by combining polynucleotides or polypeptides that do not normally exist together.

[0120] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of positions shared by the sequences that are identical or homologous. An "unrelated" or "non-homologous" sequence shares less than 40% identity, and preferably less than 25% identity, with one of the sequences of the present disclosure.

[0121] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a given percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that when two sequences are aligned, the bases (or amino acids) are the same by that percentage.

[0122] The term "polynucleotide" refers to an isolated nucleic acid molecule or structure, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise conventional phosphodiester linkages or non-conventional linkages (e.g., amide linkages, such as those found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0123] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, obtained from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Additional examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. An isolated polynucleotide generally includes a polynucleotide molecule contained in a cell containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from the natural chromosomal location. Isolated RNA molecules include the in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include synthetically produced molecules. Additionally, the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, ribosome binding site, or transcription terminator. The term "isolated" as used herein also refers to a nucleic acid or peptide being substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "isolated" is also used herein to refer to a cell or polypeptide being separated from other cellular proteins or tissues. Isolated polypeptides are meant to include both purified polypeptides and recombinant polypeptides.

[0124] The term "expression cassette" refers to a polynucleotide produced recombinantly or synthetically using a series of specified nucleic acid elements that enable transcription of a specific nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector comprises, among other things, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding an antibody of the invention.

[0125] As used herein, the term "vector" refers to a material for carrying or expressing a nucleic acid sequence comprising a nucleic acid sequence encoding a multispecific fusion protein (e.g., an antibody) as described herein. Specifically, vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes.

[0126] Hereinafter, the present invention will be described in detail.

[0127] An antibody or antigen-binding fragment thereof that specifically binds to CDCP1

[0128] One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 (CUB domain containing protein 1), comprising any one of the following (i) to (vii):

[0129] (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (TISSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 (HADYVDVVFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6 (QQWSSNPPT);

[0130] (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (TISSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7 (HADYMDVAFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0131] (iii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (TISSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9 (HADYMDVGFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0132] (iv) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10 (TISSGGRYTYYPDSVEG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11 (HADYVDVAFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0133] (v) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10 (TISSGGRYTYYPDSVEG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12 (HADYVDVGFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT);

[0134] (vi) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (SYAMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13 (TTSSGGRYTYYPDSVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7 (HADYMDVAFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (QQWSSDPPT); or

[0135] (vii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14 (SQGMS), a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15 (TISVTGRYLYYADNVKG), and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16 (HADYQDVSFDF), and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (SASSSANFMH), a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (DTSKLAS), and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17 (QQWQMEPPT).

[0136] Additionally, the antibody may comprise the following heavy chain variable region and light chain variable region:

[0137] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region of SEQ ID NO: 19;

[0138] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21;

[0139] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22;

[0140] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0141] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0142] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0143] (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24;

[0144] (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; or

[0145] (ix) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30.

[0146] Additionally, the antibody may comprise a heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18, 20, 23, 25, 26, 27, or 28; and a light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19, 21, 22, 24, or 30.

[0147] As used herein, the term "CDCP1 (CUB-domain containing protein 1)", also known as CD318, SIMA135, TRASK, and gp140, is a type 1 transmembrane glycoprotein that is upregulated in malignant tumors of the breast, lung, colorectal, ovarian, renal, hepatic, pancreatic, and hematopoietic systems. Specifically, human CDCP1 consists of a full-length of approximately 836 amino acids and has three CUB domains (complement C1r / C1s, Uegf, and Bmp1 domains). Increased CDCP1 is known to be associated with cancer progression. CDCP1, which is mainly located on the cell surface, is located at the nexus of major oncogenesis and signaling cascades, including the SRC / PKCδ, PI3K / AKT, WNT, and RAS / ERK axes, the pentose phosphate pathway, and fatty acid oxidation, and thus plays an important role in cancer cell survival and growth, metastasis, and therapeutic resistance.

[0148] In the present invention, the CDCP1 may be derived from mammals without limitation, including primates such as humans and monkeys, and rodents such as rats and mice.

[0149] The term "anti-CDCP1 antibody" as used herein refers to an antibody capable of binding to CDCP1, and may be described herein interchangeably with "antibody specific for CDCP1" or "antibody that specifically binds to CDCP1." In this case, the anti-CDCP1 antibody can specifically bind to a CDCP1 fragment. "Specifically binding to CDCP1" may mean having affinity for CDCP1 or an antigen-binding fragment thereof.

[0150] According to one embodiment of the present invention, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to produce an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0151] According to a specific embodiment of the present invention, the Fc domain of the antibody may comprise the following (a) or (b):

[0152] (a) wild-type IgG Fc; or

[0153] (b) An Fc domain variant comprising one or more amino acid substitutions that reduce binding to an Fc receptor.

[0154] The anti-CDCP1 antibody of the present invention may have its Fc domain modified to reduce Fc receptor binding and / or effector function.

[0155] Specifically, the Fc domain of an anti-CDCP1 antibody according to the present invention may comprise one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor. For example, the Fc domain is of the human IgG1 subclass, including an Fc variant comprising amino acid substitutions L235A and G237A; an Fc variant comprising amino acid substitutions L234A and L235E; or an Fc variant comprising amino acid substitutions L234A, L235A, and P329G. Residues may be numbered according to the EU index of Kabat.

[0156] The Fc domain confers favorable pharmacodynamic properties to the anti-CDCP1 antibody of the present invention, including a long serum half-life that contributes to good accumulation in target tissues and a favorable tissue-to-blood distribution ratio. However, this may simultaneously lead to undesirable targeting of the anti-CDCP1 antibody of the present invention to cells expressing Fc receptors, rather than to desired antigen-bearing cells. The Fc domain of the anti-CDCP1 antibody according to one embodiment of the present invention exhibits reduced binding affinity for Fc receptors and / or reduced effector function compared to a native IgG Fc domain, particularly an IgG1 Fc domain. Preferably, the Fc domain may be an IgG1 Fc domain or a variant thereof.

[0157] According to a specific embodiment of the present invention, the Fc domain exhibits a binding affinity for an Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%, compared to a native IgG1 Fc domain (or an antigen-binding molecule of the present invention comprising a native IgG1 Fc domain), and / or exhibits an effector function of less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%, compared to a native IgG1 Fc domain. In one embodiment, the Fc domain does not substantially bind to and / or induce an effector function of an Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In yet another embodiment, the Fc receptor is an activating Fc receptor. In another embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, preferably human FcγRIIIa. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In another embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain exhibits greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90%, of the binding affinity for FcRn of a native IgG1 Fc domain.

[0158] According to one embodiment of the present invention, the Fc domain can be engineered to have reduced binding affinity and / or reduced effector function for an Fc receptor, compared to a non-engineered Fc domain (wild-type Fc). In one embodiment, the Fc domain of an anti-CDCP1 antibody of the present invention can comprise one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain for an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. The amino acid mutations can reduce the binding affinity of the Fc domain for an Fc receptor. For example, the amino acid mutations can reduce the binding affinity of the Fc domain for an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In a specific embodiment, an anti-CDCP1 antibody of the invention comprising an engineered Fc domain may exhibit a binding affinity for an Fc receptor of less than 20%, particularly less than 10%, and more particularly less than 5%, compared to an anti-CDCP1 antibody of the invention comprising a non-engineered Fc domain. In one embodiment, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In yet another embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, preferably human FcγRIIIa. In one embodiment, the binding affinity for complement components, particularly binding affinity for C1q, may also be reduced. In another embodiment, the binding affinity for neonatal Fc receptor (FcRn) may be reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain for the receptor, is achieved when the Fc domain exhibits a binding affinity for FcRn that is greater than about 70% of that of the unengineered form of the Fc domain.The Fc domain, or the anti-CDCP1 antibody of the present invention comprising the Fc domain, may exhibit an affinity of greater than about 80% and even greater than about 90%.

[0159] In some embodiments of the invention, the Fc domain of an anti-CDCP1 antibody of the invention is designed to have reduced effector function compared to a non-engineered Fc domain. Such reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced induction of apoptosis by direct neuronal delivery, reduced maturation of dendritic cells, or reduced T cell priming.

[0160] Antibodies with reduced effector function may comprise those having substitutions at one or more of Fc region residues 234, 235, 237, and 329. In one specific embodiment, the Fc domain may comprise the amino acid substitutions L235A and G237A (“LAGA”), L234A, L235A, and P329G (“LALAPG”), or the amino acid substitutions L234A and L235E (“LALE”).

[0161] Binding to Fc receptors can be readily measured, for example, by surface plasmon resonance (SPR) using standard equipment such as an ELISA or BIAcore instrument (GE Healthcare), and the Fc receptors themselves can be obtained by recombinant expression. The binding affinity of an Fc domain or a cell-activating antibody comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a specific Fc receptor, such as the human NK cell-expressed FcγIIIa receptor. The effector function of an Fc domain or an anti-CDCP1 antibody of the invention comprising an Fc domain can be measured by methods known in the art.

[0162] In the present invention, the anti-CDCP1 antibody may have its amino acid sequence altered to improve its binding affinity and / or other biological properties. Such alterations include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0163] Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0164] According to a specific embodiment of the present invention, the heavy chain of the antibody may comprise or consist of an amino acid sequence of SEQ ID NO: 46, 48, 51, 53, 54, 55, 56 or 57.

[0165] The amino acid sequences of the above sequence numbers 46, 48, 51, 53, 54, 55, 56 and 57 are as follows.

[0166] MGWSCIILFLVATATGVHSEVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISSGGRYTYYPDSVKGRFTISRDNAKNTLYLQVSSLRSEDTAMYYCAGHADYVDVVFDFWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 46)

[0167] MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYVDVVFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 48)

[0168] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHADYMDVAFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 51)

[0169] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYMDVGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 53)

[0170] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYVDVAFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 54)

[0171] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYVDVGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 55)

[0172] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTTSSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYMDVAFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 56)

[0173] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSQGMSWVRQAPGKGLEWVSTISVTGRYLYYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHADYQDVSFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 57)

[0174] According to a specific embodiment of the present invention, the light chain of the antibody may comprise or consist of the amino acid sequence of SEQ ID NO: 47, 49, 50, 52 or 58.

[0175] The amino acid sequences of the above sequence numbers 47, 49, 50, 52 and 58 are as follows.

[0176] MGWSCIILFLVATATGVHSQIVLTQSPAIMSASPGEKVTTMTCSASSSANFMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 47)

[0177] MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 49)

[0178] MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPPTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50)

[0179] EIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSDPPTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 52)

[0180] EIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYCQQWQMEPPTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 58)

[0181] Considering the mutations having the above-described biological equivalent activity, the anti-CDCP1 antibody of the present invention and the nucleic acid molecule encoding the same are interpreted to also include sequences that show substantial identity with the sequences listed in the sequence listing. The substantial identity means a sequence that shows at least 60% identity, more preferably 70% identity, even more preferably 80% identity, and most preferably at least 90% identity when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0182] A polynucleotide encoding an antibody or an antigen-binding fragment thereof

[0183] Another aspect of the present invention provides a polynucleotide encoding an anti-CDCP1 antibody or an antigen-binding fragment thereof. The anti-CDCP1 antibody and antigen-binding fragment thereof are the same as described above.

[0184] Specifically, the polynucleotide encoding the heavy chain variable region of the anti-CDCP1 antibody may include a base sequence encoding an amino acid sequence of SEQ ID NO: 18, 20, 23, 25, 26, 27, 28, or 29. The polynucleotide encoding the light chain variable region of the anti-CDCP1 antibody may include a base sequence encoding an amino acid sequence of SEQ ID NO: 19, 21, 22, 24, or 30.

[0185] According to a specific embodiment of the present invention, the polynucleotide encoding the heavy chain variable region of the anti-CDCP1 antibody may comprise or consist of a base sequence of SEQ ID NO: 59, 61, 64, 66, 67, 68, 69 or 70, but is not limited thereto.

[0186] The base sequence of the above sequence number 59, 61, 64, 66, 67, 68, 69 or 70 is as follows.

[0187] GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGAAACCTGGCGGCAGCCTGAAGCTGTCCTGCGCCGCTTCTGGCTTCACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGACCCCTGAGAAGCGGCTGGAATGGGTGGCCACAATCAGCAGCGGCGGACGCTACACCTACTAC CCCGATTCCGTGAAGGGCAGATTTACCATCTCTCGGGACAACGCCAAGAACACCCTGTACCTGCAGGTGTCTTCTCTGAGATCCGAGGATACCGCTATGTACTATTGTGCTGGCCACGCCGACTACGTGGACGTGGTGTTCGACTTCTGGGGACAGGGCACCACACTGACCGTGTCCTCT (SEQ ID NO: 59)

[0188] GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGCAGCCTGGAGGATCTCTGCGGCTGTCTTGTGCTGCTAGCGGCTTCACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAAGCTCCTGGCAAGGGCCTGGAATGGGTGTCCACCATCTCCTCTGGCGGCAGATACACCTACTATCCCGATTCCGTGAAAGGCCGCTTTACAATCTCTCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGATACCGCCGTGTACTACTGCGCCGGCCACGCCGACTACGTGGACGTGGTGTTCGACTTCTGGGGCCAGGGCACCACAGTGACCGTGTCTAGC (서열번호 61)

[0189] GAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCAGCCACGCCGATTACATGGATGTGGCCTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC (서열번호 64)

[0190] GAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACATGGATGTGGGgTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC (서열번호 66)

[0191] GAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACGTGGATGTGGCGTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC (서열번호 67)

[0192] GAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACGTGGATGTGGGGTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC (서열번호 68)

[0193] GAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCACcAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACATGGATGTGGCgTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC (서열번호 69)

[0194] GAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCCAGGGCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCGTGACCGGCCGCTACCTGTACTAC GCTGATAACGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCAGCCACGCCGATTACCAGGATGTGAGCTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC (SEQ ID NO: 70)

[0195] According to a specific embodiment of the present invention, the polynucleotide encoding the light chain variable region of the anti-CDCP1 antibody may comprise or consist of the base sequence of SEQ ID NO: 60, 62, 63, 65 or 71, but is not limited thereto.

[0196] CAAATCGTGCTAACTCAAAGTCCAGCAATCATGTCTGCTTCTCCTGGCGAGAAGGTGACCATGACCTGTTCTGCCTCCAGCAGCGCCAACTTCATGCACTGGTATCAGCAAAAGTCCGGCACCTCTCCCAAGCGGTGGATCTACGACACCTCCAAGCTG GCCTCTGGCGTGCCTGCCAGATTTAGCGGCTCTGGCTCCGGCACATCCTACTCCCTGACCATCTCCTCTATGGAAGCCGAGGATGCTGCTACCTACTACTGCCAGCAGTGGTCCTCCAACCCTCCAACCTTCGGCGGAGGAACAAAACTGGAGATCAAG (SEQ ID NO: 60)

[0197] GAAATCGTGCTAACTCAAAGTCCAGCAACTCTGTCCCTGTCTCCTGGCGAGAGAGCCACACTGAGCTGTTCTGCTTCTTCCTCTGCCAACTTCATGCACTGGTATCAGCAAAAACCCGGCCAGGCTCCTCGGCGGCTGATCTACGACACCTCCAAGCTGGCCTCTGGCATCCCTGCTAGATTCTCCGGCTCCGGATCTGGCACCGACTACACCCTGACCATCTCCAGCCTGGAACCTGAGGATTTTGCCGTGTACTACTGCCAGCAGTGGTCCTCCAACCCTCCAACCTTCGGCGGCGGCACCAAGCTCGAGATCAAG (서열번호 62)

[0198] GAAATCGTGCTAACTCAAAGTCCAGCAACTCTCAGCCTGTCTCCTGGCGAGCGGGCTACCCTGTCCTGCTCCGCCTCCAGCTCTGCCAACTTCATGCACTGGTACCAGCAGAAACCAGGCCAGGCTCCTAGACGGCTGATCTACGACACCTCCAAGCTGGCCTCTGGCATCCCTGCTAGATTCTCCGGCTCTGGATCTGGAACAGATTTTACCCTGACCATCTCCTCTCTGGAACCCGAGGACTTCGCCGTGTACTACTGTCAACAGTGGTCCTCCAACCCTCCTACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG (서열번호 63)

[0199] GAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTG GCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGAGCAGCGACCCGCCGACCTTCGGCGGTGGCACCAAACTGGAGATCAAG (SEQ ID NO: 65)

[0200] GAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTG GCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGCAGATGGAGCCGCCTACCTTCGGCGGTGGCACCAAACTGGAGATCAAG (SEQ ID NO: 71)

[0201] In the present invention, the polynucleotide encoding the heavy chain of the anti-CDCP1 antibody may include a base sequence encoding an amino acid sequence of SEQ ID NO: 46, 48, 51, 53, 54, 55, 56, or 57. The polynucleotide encoding the light chain variable region of the anti-CDCP1 antibody may include a base sequence encoding an amino acid sequence of SEQ ID NO: 47, 49, 50, 52, or 58.

[0202] According to a specific embodiment of the present invention, the polynucleotide encoding the heavy chain of the anti-CDCP1 antibody may comprise or consist of a base sequence of SEQ ID NO: 72, 74, 77, 79, 80, 81, 82 or 83, but is not limited thereto.

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] According to a specific embodiment of the present invention, the polynucleotide encoding the light chain of the anti-CDCP1 antibody may comprise or consist of the base sequence of SEQ ID NO: 73, 75, 76, 78 or 84, but is not limited thereto.

[0212] atgggctggtcatgtattattctgtttctggtcgcaactgctacaggggtccatagtCAAATCGTGCTAACTCAAAGTCCAGCAATCATGTCTGCTTCTCCTGGCGAGAAGGTGACCATGACCTGTTCTGCCTCCAGCAGCGCCAACTTCATGCACTGGTATCAGCAAAAGTCCGGCACCTCTCCCAAGCGGTGGATCTACGACACCTCCAAGCTGGCCTCTGGCGTGCCTGCCAGATTTAGCGGCTCTGGCTCCGGCACATCCTACTCCCTGACCATCTCCTCTATGGAAGCCGAGGATGCTGCTACCTACTACTGCCAGCAGTGGTCCTCCAACCCTCCAACCTTCGGCGGAGGAACAAAACTGGAGATCAAGaggacagtggccgccccaagcgtgttcatctttcccccttccgacgagcagctgaagtctggcaccgccagcgtggtgtgcctgctgaacaacttctaccctcgggaggccaaggtccagtggaaggtggataacgccctgcagtctggcaatagccaggagtccgtgaccgagcaggactctaaggatagcacatattccctgtctagcaccctgacactgagcaaggccgattacgagaagcacaaggtgtatgcctgtgaagtcacccatcaggggctgtcatcacccgtcactaagtcattcaatcgcggagaatgc (서열번호 73)

[0213] atgggctggtcatgtattattctgtttctggtcgcaactgctacaggggtccatagtGAAATCGTGCTAACTCAAAGTCCAGCAACTCTGTCCCTGTCTCCTGGCGAGAGAGCCACACTGAGCTGTTCTGCTTCTTCCTCTGCCAACTTCATGCACTGGTATCAGCAAAAACCCGGCCAGGCTCCTCGGCGGCTGATCTACGACACCTCCAAGCTGGCCTCTGGCATCCCTGCTAGATTCTCCGGCTCCGGATCTGGCACCGACTACACCCTGACCATCTCCAGCCTGGAACCTGAGGATTTTGCCGTGTACTACTGCCAGCAGTGGTCCTCCAACCCTCCAACCTTCGGCGGCGGCACCAAGCTCGAGATCAAGaggacagtggccgccccaagcgtgttcatctttcccccttccgacgagcagctgaagtctggcaccgccagcgtggtgtgcctgctgaacaacttctaccctcgggaggccaaggtccagtggaaggtggataacgccctgcagtctggcaatagccaggagtccgtgaccgagcaggactctaaggatagcacatattccctgtctagcaccctgacactgagcaaggccgattacgagaagcacaaggtgtatgcctgtgaagtcacccatcaggggctgtcatcacccgtcactaagtcattcaatcgcggagaatgc (서열번호 75)

[0214] atgggctggtcatgtattattctgtttctggtcgcaactgctacaggggtccatagtGAAATCGTGCTAACTCAAAGTCCAGCAACTCTCAGCCTGTCTCCTGGCGAGCGGGCTACCCTGTCCTGCTCCGCCTCCAGCTCTGCCAACTTCATGCACTGGTACCAGCAGAAACCAGGCCAGGCTCCTAGACGGCTGATCTACGACACCTCCAAGCTGGCCTCTGGCATCCCTGCTAGATTCTCCGGCTCTGGATCTGGAACAGATTTTACCCTGACCATCTCCTCTCTGGAACCCGAGGACTTCGCCGTGTACTACTGTCAACAGTGGTCCTCCAACCCTCCTACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGaggacagtggccgccccaagcgtgttcatctttcccccttccgacgagcagctgaagtctggcaccgccagcgtggtgtgcctgctgaacaacttctaccctcgggaggccaaggtccagtggaaggtggataacgccctgcagtctggcaatagccaggagtccgtgaccgagcaggactctaaggatagcacatattccctgtctagcaccctgacactgagcaaggccgattacgagaagcacaaggtgtatgcctgtgaagtcacccatcaggggctgtcatcacccgtcactaagtcattcaatcgcggagaatgc (서열번호 76)

[0215] GAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTGGCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGAGCAGCGACCCGCCGACCTTCGGCGGTGGCACCAAACTGGAGATCAAGCGCACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (서열번호 78)

[0216] GAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTG GCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGCAGATGGAGCCGCCTACCTTCGGCGGTGGCACCAAACTGGAGATCAAGC GCACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 84)

[0217] In addition, if the polynucleotide encodes the same polypeptide, one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. When producing a polynucleotide sequence by chemical synthesis, synthetic methods widely known in the art can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and examples thereof include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on solid supports.

[0218] According to one specific example, the polynucleotide encoding the heavy chain variable region has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 18, 20, 23, 25, 26, 27, 28, or 29, or a base sequence of SEQ ID NO: 46, 48, 51, 53, 54, 55, 56, or 57. It may contain a base sequence.

[0219] In one specific example, the polynucleotide encoding the light chain variable region can comprise a polynucleotide encoding an amino acid sequence of SEQ ID NO: 19, 21, 22, 24 or 30, or a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a base sequence of SEQ ID NO: 47, 49, 50, 52 or 58.

[0220] According to one specific example, the polynucleotide encoding the heavy chain has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 46, 48, 51, 53, 54, 55, 56, or 57, or a base sequence of SEQ ID NO: 72, 74, 77, 79, 80, 81, 82, or 83. It may contain a base sequence.

[0221] In one specific example, the polynucleotide encoding the light chain can comprise a polynucleotide encoding an amino acid sequence of SEQ ID NO: 47, 49, 50, 52, or 58, or a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a base sequence of SEQ ID NO: 73, 75, 76, 78, or 84.

[0222] The polynucleotide may additionally include a signal sequence or a leader sequence. The term "signal sequence" as used herein refers to a nucleic acid encoding a signal peptide that directs the secretion of a target protein. The signal peptide is cleaved after translation in the host cell. Specifically, the signal sequence of the present invention is a polynucleotide encoding an amino acid sequence that initiates the movement of a protein across the endoplasmic reticulum (ER) membrane.

[0223] Signal sequences are well-characterized in the art and typically contain 16 to 30 amino acid residues, although they may contain more or fewer. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during movement of the immature polypeptide.

[0224] After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. At this time, the signal sequence may be a secretion signal sequence of tPa (tissue Plasminogen Activation), HSV gDs (signal sequence of herpes simplex virus glycoprotein D), or growth hormone. Preferably, a secretion signal sequence used in higher eukaryotic cells including mammals can be used. In addition, it can be used by substituting a codon with a high expression frequency in the host cell. In one specific example, the signal sequence may include the amino acid sequence of SEQ ID NO: 13.

[0225] vector loaded with polynucleotides

[0226] Another aspect of the present invention provides a vector loaded with a polynucleotide encoding the anti-CDCP1 antibody or an antigen-binding fragment thereof. Specifically, the vector may include a polynucleotide encoding a heavy chain region of the anti-CDCP1 antibody or an antigen-binding fragment thereof. In this case, the polynucleotide may include a base sequence encoding a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 18, 20, 23, 25, 26, 27, 28, or 29. The vector may include a polynucleotide encoding a light chain region of the anti-CDCP1 antibody. In this case, the polynucleotide may include a base sequence encoding a light chain variable region comprising an amino acid sequence of SEQ ID NO: 19, 21, 22, 24, or 30.

[0227] The vector can be introduced into a host cell and recombined and integrated into the host cell genome. Alternatively, the vector is understood to be a nucleic acid vehicle comprising a polynucleotide sequence capable of autonomously replicating as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0228] Specifically, the vector may be a plasmid DNA, phage DNA, etc., and may be a commercially developed plasmid (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (e.g., pUB110, pTP5, etc.), a yeast-derived plasmid (e.g., Yep13, Yep24, Ycp50, etc.), a phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.), or an insect virus vector (e.g., baculovirus, etc.). There is. Since the protein expression amount and formula of the above vector differ depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0229] As used herein, the term "gene expression" or "expression" of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may include a human cytomegalovirus (CMV) promoter to promote continuous transcription of the gene of interest in mammalian cells, and a polyadenylation signal sequence to increase the steady-state level of RNA after transcription.

[0230] Transformed cells

[0231] Another aspect of the present invention provides a transformed cell into which an expression vector comprising a polynucleotide encoding the anti-CDCP1 antibody or an antigen-binding fragment thereof has been introduced.

[0232] As used herein, the term "transformed cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. Such transformed cells can be produced by introducing the vector into a host cell and transforming it. Furthermore, the polynucleotide contained in the vector can be expressed to produce an antibody according to the present invention.

[0233] The above transformation can be performed by various methods. As long as the antibody of the present invention can be produced, it is not particularly limited thereto. Specifically, the transformation method may be CaCl2 precipitation, Hanahan method which increases efficiency by using a reducing substance called DMSO (dimethyl sulfoxide) in CaCl2 precipitation, electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fiber, Agrobacterium-mediated transformation, PEG-based transformation, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation. In addition, the target substance can be delivered into the cell using virus particles by means of infection. In addition, the vector can be introduced into the host cell by gene bombardment, etc.

[0234] In addition, the host cell used for producing the transformed cell is not particularly limited as long as it can produce the antibody of the present invention or an antigen-binding fragment thereof. Specifically, the host cell may include, but is not limited to, a prokaryotic cell, a eukaryotic cell, a mammal, a plant, an insect, a fungus, or a cell of cellular origin. An example of the prokaryotic cell may be Escherichia coli. In addition, an example of the eukaryotic cell may be yeast. In addition, the mammalian cell may be, but is not limited to, CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, or HEK293T cells. Any cell known to those skilled in the art that can be used as a mammalian host cell may be used.

[0235] In addition, in order to optimize the properties of the multi-specific fusion protein according to the present invention as a therapeutic agent or for other purposes, the glycosylation-related genes of the host cell can be manipulated using a method known to those skilled in the art to adjust the sugar chain pattern of the antibody (e.g., sialic acid, fucosylation, glycosylation).

[0236] Method for producing an antibody or antigen-binding fragment thereof that specifically binds to CDCP1

[0237] Another aspect of the present invention provides a method for preparing the anti-CDCP1 antibody or antigen-binding fragment thereof, comprising the following steps (a) and (b):

[0238] (a) a step of culturing the above-mentioned transformant; and

[0239] (b) A step of recovering an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 from the culture.

[0240] Transformants for antibody production can be cultured using appropriate media and culture conditions known in the art. These culture processes can be easily adjusted by those skilled in the art for the selected strain. Cell culture can be categorized into suspension and adherent cultures based on cell growth patterns, and batch, fed-batch, and continuous culture methods based on culture method. The culture medium used must adequately meet the requirements of the specific strain.

[0241] The medium used for culturing animal cells contains various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These carbon sources can be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL), and soybean meal; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources can be used alone or in combination. The above-mentioned medium may contain, as personnel, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salt. It may also contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included.

[0242] During cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be appropriately added to the culture to adjust the pH of the culture. Furthermore, foaming can be suppressed during cultivation using antifoaming agents such as fatty acid polyglycol esters. Furthermore, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain an aerobic state. The culture temperature is usually between 20°C and 45°C, preferably between 25°C and 40°C.

[0243] Antibodies obtained by culturing transformants can be used in an unpurified state, or can be further purified to high purity using various conventional methods, such as dialysis, salt precipitation, and chromatography. Among these, the method using chromatography is the most commonly used, and the type and order of columns can be selected from ion exchange chromatography, size exclusion chromatography, and affinity chromatography, depending on the characteristics of the antibody, the culture method, etc.

[0244] Pharmaceutical compositions, therapeutic methods and uses

[0245] Another aspect of the present invention provides an anti-CDCP1 antibody or an antigen-binding fragment thereof for use in the prevention or treatment of cancer, and a pharmaceutical composition for the prevention or treatment of cancer comprising the same as an active ingredient. The anti-CDCP1 antibody or antigen-binding fragment thereof is the same as described above.

[0246] The anti-CDCP1 antibody or antigen-binding fragment thereof according to the present invention specifically binds only to CDCP-1 of cancer cells while not binding to CDCP1 of normal cells, and is therefore useful for preventing, improving, or treating cancer expressing CDCP-1.

[0247] The term "cancer" as used herein refers to a general term for diseases caused by cells that have aggressive characteristics in which cells divide and proliferate in defiance of normal growth limits, invasive characteristics in which cells invade surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body, and is used with the same meaning as malignant tumor.

[0248] The above cancer may be selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, vulvar cancer, breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, skin cancer, brain and spinal tumor, brain cancer, thymoma, mesothelioma, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, endocrine cancer and sarcoma, but may be included without limitation as long as it expresses CDCP-1.

[0249] The term “prevention” as used herein refers to any act of inhibiting the occurrence of cancer or delaying its onset by administering the pharmaceutical composition.

[0250] The above “treatment” refers to any act of improving or beneficially changing the symptoms of cancer by administering the above pharmaceutical composition.

[0251] The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0252] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound. The carrier is used to mean an excipient, diluent, or auxiliary. The carrier may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, saline, a buffer such as PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or a combination thereof.

[0253] The pharmaceutical composition described above may be prepared in any dosage form according to conventional methods. The composition may be formulated, for example, as a parenteral formulation. In addition, the composition may be prepared as a systemic formulation or a topical formulation. When the pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration agent, a nasal inhalant, or a suppository using a suitable carrier according to a method known in the art. Injectable formulations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized formulations. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Meanwhile, injectable formulations may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Formulation of pharmaceutical compositions is well known in the art, and reference can be made to references such as Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0254] In the pharmaceutical composition, as long as the antibody or antigen-binding fragment thereof can exhibit anticancer activity, it may be included in any amount (effective amount) depending on the intended use, formulation, compounding purpose, etc. Here, the "effective amount" refers to the amount of the effective ingredient that can induce an anticancer effect. Such an effective amount can be experimentally determined within the scope of a person skilled in the art. The pharmaceutical composition may contain the antibody or antigen-binding fragment thereof as an effective ingredient in an amount of about 0.5 μg to about 2 g, about 1 μg to about 1 g, about 10 μg to about 500 mg, about 100 μg to about 100 mg, or about 1 mg to about 50 mg per pharmaceutical composition.

[0255] Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also influence efficacy. Therefore, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and enhanced efficacy, and can be measured by comparing parameters such as clearance rate and the treatment or improvement of cancer in test animals or human subjects.

[0256] The antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, may be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

[0257] The term "administration" as used herein means introducing a given substance into a subject by an appropriate method, and the route of administration of the composition may be any common route as long as it can reach the target tissue. Examples include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, or rectal administration.

[0258] Here, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route and excretion rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. Specifically, the therapeutically effective amount refers to an amount of a drug that is effective in treating cancer.

[0259] The dosage of the pharmaceutical composition may be, for example, in the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg for adults. The administration may be once a day, twice to 24 times a day, once to twice every three days, once to six times a week, once to 10 times every two weeks, once to 15 times every three weeks, once to three times every four weeks, or once to 12 times a year. However, the dosage may increase or decrease depending on the route of administration, severity of the disease, gender, weight, age, etc., and thus the scope of the present invention is not limited thereto.

[0260] The subjects to which the above pharmaceutical composition can be applied (prescribed) are mammals and humans, and humans are particularly preferred.

[0261] The above antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, may be administered as an individual therapeutic agent or in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, or may be administered singly or in multiple doses. In this case, the other therapeutic agent may additionally include any compound or natural extract that has already been proven safe and known to have anticancer activity, in order to enhance or reinforce anticancer activity. Taking all of the above factors into consideration, it is important to administer an amount that achieves the maximum effect with the minimum amount of side effects or without side effects, and this can be readily determined by those skilled in the art.

[0262] Another aspect of the present invention provides a use of an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 for the prevention or treatment of cancer.

[0263] Another aspect of the present invention provides the use of an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 for the manufacture of a medicament for preventing or treating cancer.

[0264] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject an antibody or antigen-binding fragment thereof that specifically binds to CDCP1, and a pharmaceutical composition comprising the same. In this case, the antibody, antigen-binding fragment, pharmaceutical composition, cancer, and prevention and treatment are the same as those described above.

[0265] The subject may be a mammal, such as a human, mouse, rat, guinea pig, hamster, rabbit, chicken, egg, monkey, ape, cow, horse, pig, dog, sheep, goat, or cat, and preferably a human. In addition, the subject may be an individual suffering from or likely to suffer from cancer. This term does not indicate a specific age or gender. Therefore, it is intended to include adults and newborns, as well as fetuses, whether female or male. A patient refers to an individual suffering from a disease or disorder. The term patient includes human and veterinary subjects.

[0266] The preferred dosage of the antibody or antigen-binding fragment thereof varies depending on the patient's condition and weight, the extent of the disease, the drug form, the route and duration of administration, and can be appropriately selected by those skilled in the art. The dosage may be, for example, in the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg for adults. The administration may be administered once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year. In addition, the method may further comprise a step of administering an anticancer agent to the subject. The anticancer agent may be administered simultaneously, separately, or sequentially with the antibody or antigen-binding fragment thereof.

[0267] CDCP1 detection kit and CDCP1 detection method

[0268] Another aspect of the present invention provides a kit for detecting CDCP1 comprising the anti-CDCP1 antibody or an antigen-binding fragment thereof and a method for detecting CDCP1 using the anti-CDCP1 antibody or an antigen-binding fragment thereof.

[0269] The CDCP1 detection kit according to the present invention can be applied to biological samples to diagnose cancer expressing CDCP1. Accordingly, the present invention provides a kit for diagnosing cancer, particularly cancer expressing CDCP1, comprising the aforementioned anti-CDCP1 antibody or antigen-binding fragment thereof.

[0270] The kit of the present invention can be manufactured to be suitable for various immunoassays or immunostaining. The immunoassays or immunostaining include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, radioimmunoassay (RIA), and protein chips.

[0271] The kit of the present invention may additionally include tools or reagents known in the art for use in immunological analysis in addition to the anti-CDCP1 antibody or antigen-binding fragment thereof.

[0272] Tools or reagents used in immunological analysis may include suitable carriers or supports, labels capable of generating detectable signals, solubilizers, detergents, and stabilizers. Suitable carriers may also include, but are not limited to, a substrate capable of measuring enzyme activity when the label is an enzyme, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, and a reaction stopper.

[0273] The anti-CDCP1 antibody or antigen-binding fragment thereof included in the kit of the present invention can preferably be immobilized on a suitable carrier or support using various methods as disclosed in the literature, and examples of suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polyesterine, gabbro, filter paper, ion exchange resin, plastic film, plastic tube, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or magnetic particles. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (inside a test tube or well), planar (sheet, test strip).

[0274] Labels capable of generating a detectable signal enable qualitative or quantitative measurement of the formation of antigen-antibody complexes, and examples of such labels include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes. Enzymes that can be used include β-glucuronidase, β-D-glucosidase, urease, peroxidase (such as horseradish peroxidase), alkaline phosphatase, acetylcholinesterase, glycose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, and luciferase. Fluorescent substances that can be used include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorsine isothiocyanate. Ligands include biotin derivatives, and luminescent substances include acridinium esters and luciferin. Microparticles include colloidal gold and colored latex, and redox molecules include ferrocene, ruthenium complexes, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, and hydroquinone. Radioisotopes include 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, etc. However, in addition to those exemplified above, any that can be used in immunological analysis can be used.

[0275] As an enzyme chromogenic substrate, for example, when horseradish peroxidase (HRP) is selected as an enzyme label, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, o-dianisidine, or 3,3-dimethoxybenzidine can be used as a substrate. In addition, when alkaline phosphatase is selected as an enzyme label, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, or p-nitrophenyl phosphate can be used as a substrate. Additionally, when β-D-galactosidase is selected as an enzyme marker, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indole-β-D-galactopyranoside can be used as a substrate. In addition, various enzymes and enzyme chromogenic substrates known in the art can be used.

[0276] A CDCP1 detection method according to one embodiment of the present invention may detect CDCP1 protein in a biological sample or a mammal, including a human. Specifically, the CDCP1 detection method according to the present invention may include a step of detecting a CDCP1 antigen-antibody complex using the aforementioned anti-CDCP1 antibody or antigen-binding fragment thereof.

[0277] In the present invention, the sample is a biological sample, and may include, but is not limited to, tissue, cell, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems. For example, the biological sample may be isolated from a mammal, including a human, having a disease in which CDCP1 is overexpressed or a disease related to CDCP1, or may be isolated from an animal model that overexpresses CDCP1 protein or an animal model of a disease related to CDCP1, but is not limited thereto. These biological samples may be reacted with the anti-CDCP1 antibody of the present invention or an antigen-binding fragment thereof, with or without manipulation, to confirm the presence or absence of CDCP1 protein.

[0278] The mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0279] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0280] [Manufacturing Example 1]

[0281] 1-1. Production of parent antibodies that specifically bind to CDCP1

[0282] The parent antibody of the present invention was prepared by performing the following steps.

[0283] (A) transforming a host cell with a vector containing a nucleic acid fragment encoding an anti-CDCP1 antibody; (B) culturing the host cell under conditions suitable for expression of the antibody molecule; and (C) recovering and purifying the antibody from the culture of the host cell.

[0284] First, a vector encoding an antibody was transiently transfected into cultured cells. Specifically, ExpiCHO-S cells (ThermoFisher) were inoculated into a culture flask containing CHO cell expression medium (ThermoFisher) and cultured at 37°C, 120 rpm, and 8% CO2 to induce 6 x 10 6 The cells were prepared at a density of 10 cells / mL. The prepared ExPiCHO-S cells, the anti-CDCP1 antibody expression vector, and the reagents of the transfection kit were co-cultured for transfection. Feed supplements were added on days 1 and 5 after transfection, and the culture conditions were changed to 37°C and 5% CO2. The culture medium containing the antibody was harvested after 10 to 12 days, and the antibody was purified from the supernatant obtained by centrifugation and filtration, or the supernatant was frozen and stored.

[0285] Thereafter, the antibody was recovered from the culture and purified. Purification of the anti-CDCP1 antibody was performed by centrifuging the cell culture, filtering it using a 0.2 μm PES filter, and then using a protein A (Cytiva, PrismA) column. The buffer of the purified sample was exchanged with PBS, pH 7.4. Thereafter, size exclusion chromatography was used to detect soluble high-molecular-weight forms (aggregates) and low-molecular-weight hydrolysis products in the composition. This method was performed using TSKgel Super SW 3000SW 4.6 x 300 mm 4 μm (Tosoh Bioscience, 18675). Intact monomers, aggregates, and fragments were separated through isocratic elution, and the separation was performed under the conditions of 50 mM potassium phosphate, 150 mM potassium chloride, pH 6.8 as the mobile phase, and detection was performed at 280 nm.

[0286] Subsequently, through sequence analysis of the antibodies that showed binding affinity, three different antibodies (named '2F9-chimeric', '2F9-679', and '2F9-700', respectively) having complementarity-determining regions (CDRs) in the heavy chain and heavy chain variable regions were discovered, as shown in Table 2. The amino acid sequences of the full-length antibodies including the heavy chain variable region (VH) and light chain variable region (VL) of each antibody are listed in Tables 3 to 5, and the base sequences are listed in Tables 6 to 8.

[0287] The following antibodies can be used interchangeably with 2F9-chimeric or 2F9-chimeric antibody, 2F9-679 or 2F9-679 antibody, 2F9-700 or 2F9-700 antibody.

[0288] Antibody name CDR amino acid sequence SEQ ID NO. 2F9 chimeric 2F9-679 2F9-700 HCDR1 SYAMS 1 HCDR2 TISSGGRYTYYPDSVKG 2 HCDR3 HADYVDVVFDF 3 LCDR1 SASSSANFMH 4 LCDR2 DTSKLAS 5 LCDR3 QQWSSNPPT 6

[0289] 2F9-chimeric아미노산 서열서열번호HC신호 펩타이드MGWSCIILFLVATATGVHS31VHEVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISSGGRYTYYPDSVKGRFTISRDNAKNTLYLQVSSLRSEDTAMYYCAGHADYVDVVFDFWGQGTTLTVSS18CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32힌지EPKSCDKTHTCPPCP33CH2APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK34CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35LC신호 펩타이드MGWSCIILFLVATATGVHS31VLQIVLTQSPAIMSASPGEKVTMTCSASSSANFMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK19CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0290] 2F9-679아미노산 서열서열번호HC신호 펩타이드MGWSCIILFLVATATGVHS31VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYVDVVFDFWGQGTTVTVSS20CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32힌지EPKSCDKTHTCPPCP33CH2APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK34CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35LC신호 펩타이드MGWSCIILFLVATATGVHS31VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIK21CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0291] 2F9-700아미노산 서열서열번호HC신호 펩타이드MGWSCIILFLVATATGVHS31VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYVDVVFDFWGQGTTVTVSS20CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32힌지EPKSCDKTHTCPPCP33CH2APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK34CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35LC신호 펩타이드MGWSCIILFLVATATGVHS31VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIK22CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0292]

[0293]

[0294]

[0295] 1-2. Affinity maturation of parent antibody

[0296] Based on the confirmed 2F9-679 antibody sequence, humanized antibody sequences were obtained through antibody humanization. Subsequently, to enhance antigen binding affinity, additional sequences of antibodies M4, M6, M9, M10, M13, and M14 were obtained through affinity maturation. Based on the obtained sequences, cloning was performed into the PhiC31 vector (SBI, FC600A-1-SBI).

[0297] The sequences of antibodies M4, M6, M9, M10, M13, and M14 were prepared through gene synthesis, and were inserted into the PhiC31 vector using restriction enzymes Nhe I (New England Biolabs, R3131S) and Hind III (New England Biolabs, R3104S). The restriction enzyme reaction was mixed with 10X Cutsmart Buffer to adjust the total reaction volume to 50 μL, and then performed in a 37°C incubator (Biofree) for 5 hours.

[0298] After the reaction, the proper function of the restriction enzyme was confirmed through 1% agarose gel electrophoresis. T4 ligase (New England Biolabs, M0202S) and 10X T4 DNA ligase buffer were added to the prepared sample, and the total volume was adjusted to 20 μL. The reaction was performed at 25°C for 2 hours to allow the antibody gene to be linked to the vector.

[0299] The combined vector was transformed into competent E. coli DH5a cells (RBC, RH718), plated on kanamycin LB agar plates, and cultured at 37°C for 16 hours to confirm the formation of single colonies. Using the formed single colonies, DNA was obtained by Mini-prep (Qiagen, 27106), and after DNA digestion with a restriction enzyme, the size of the inserted gene was confirmed through electrophoresis. The final sequence was then verified through sequencing.

[0300] The vector prepared above was cultured in 3 mL LB medium supplemented with antibiotics for 8 hours, then transferred to 250 mL LB medium supplemented with the same antibiotics and cultured for 16 hours. Plasmid DNA was extracted using a Maxi-prep kit (Macherey-Nagel, 740414.50), and the DNA was filtered using a 0.22 μm PES filter. The filtered DNA was then introduced into cells in the next step. The day before transfection, ExpiCHO (Gibco) cells were cultured at 3 X 10 in ExpiCHO Expression Medium (Gibco, A2910002). 6 ~ 4 Х 10 6 After adjusting the concentration to 10 viable cells / mL, the cells were cultured for 1 day under conditions of 8% CO2, 37°C, and 120 rpm.

[0301] On the day of DNA transfection, 7 Х 10 6 ~ 10 Х 10 6 Cells with a viability of 95% or more were plated in fresh medium at a concentration of 6 Х 10 viable cells / mL. 6The cells were diluted to 1 viable cell / mL. The ExpiFectamine CHO transfection kit (Gibco, A29129) and Optipro SFM (Gibco, 12309019) were used for transfection into the prepared ExpiCHO cells, and DNA was transfected at a concentration of 1 μg / mL. After transfection, supplements were added on the first day, and the culture conditions were changed to 5% CO2, 32°C, and 120 rpm. Supplements were added again on the fifth day, and production was completed after culturing for 10 to 12 days. To secure the culture medium after completion of production, the culture medium was transferred to a centrifugation-only container and centrifuged at 4°C, 8000 rpm for 30 minutes. Afterwards, the supernatant was mixed with diatomaceous earth and filtered through a 0.2 μm PES filter to remove suspended matter, and the culture solution was purified to obtain M4, M6, M9, M10, M13, and M14 antibodies, respectively. The amino acid sequences of the heavy chain CDR1-3 (HCDR1, HCDR2, HCDR3) and light chain CDR1-3 (LCDR1, LCDR2, LCDR3) of the six anti-CDCP-1 antibodies obtained through affinity maturation of the parent antibody are shown in Table 9, the amino acid sequences and base sequences of the heavy chain variable region (VH) and the light chain variable region (VL) are shown in Tables 10 and 11, respectively, the amino acid sequences of the full-length antibodies are shown in Tables 12 to 17, and the base sequences of the full-length antibodies are shown in Tables 18 to 23.

[0302] The following antibodies can be used interchangeably with M4 or M4 antibody, M6 or M6 antibody, M9 or M9 antibody, M10 or M10 antibody, M13 or M13 antibody and M14 or M14 antibody.

[0303] 항체명CDR아미노산 서열서열번호A.M h2F9 M4(LAGA)HCDR1SYAMS1HCDR2TISSGGRYTYYPDSVKG2HCDR3HADYMDVAFDF7LCDR1SASSSANFMH4LCDR2DTSKLAS5LCDR3QQWSSDPPT8A.M h2F9 M6(LAGA)HCDR1SYAMS1HCDR2TISSGGRYTYYPDSVKG2HCDR3HADYMDVGFDF9LCDR1SASSSANFMH4LCDR2DTSKLAS5LCDR3QQWSSDPPT8A.M h2F9 M9(LAGA)HCDR1SYAMS1HCDR2TISSGGRYTYYPDSVEG10HCDR3HADYVDVAFDF11LCDR1SASSSANFMH4LCDR2DTSKLAS5LCDR3QQWSSDPPT8A.M h2F9 M10(LAGA)HCDR1SYAMS1HCDR2TISSGGRYTYYPDSVEG10HCDR3HADYVDVGFDF12LCDR1SASSSANFMH4LCDR2DTSKLAS5LCDR3QQWSSDPPT8A.M h2F9 M13(LAGA)HCDR1SYAMS1HCDR2TTSSGGRYTYYPDSVKG13HCDR3HADYMDVAFDF7LCDR1SASSSANFMH4LCDR2DTSKLAS5LCDR3QQWSSDPPT8A.M h2F9 M14(LAGA)HCDR1SQGMS14HCDR2TISVTGRYLYYADNVKG15HCDR3HADYQDVSFDF16LCDR1SASSSANFMH4LCDR2DTSKLAS5LCDR3QQWQMEPPT17

[0304] Antibody domain amino acid sequence AM h2F9 M4(LAGA)VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHADYMDVAFDFWGQGTTVTVSS23VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSDPPTFGGGTKLEIK24A.M h2F9 M6(LAGA)VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYPPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCAGHADYMDVGFDFWGQGTTVTVSS25VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYCQQWSSDPPTFGGGTKLEIK24A.M h2F9 M9(LAGA)VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYPPDSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYVDVAFDFWGQGTTVTVSS26VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYCQQWSSDPPTFGGGTKLEIK24A.M h2F9 M10(LAGA)VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISSGGRYTYYPDSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYVDVGFDFWGQGTTVTVSS27VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSDPPTFGGGTKLEIK24A.M h2F9 M13(LAGA)VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTTSSGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGHADYMDVAFDFWGQGTTVTVSS28VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSDPPTFGGGTKLEIK24A.M h2F9 M14(LAGA)VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSQGMSWVRQAPGKGLEWVSTISVTGRYLYYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHADYQDVSFDFWGQGTTVTVSS29VLEIVLTQSPATLSLSPGERATLSCSASSSANFMHWYQQKPGQAPRRLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWQMEPPTFGGGTKLEIK30.

[0305] 항체명영역염기서열서열번호A.M h2F9 M4(LAGA)VHGAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCAGCCACGCCGATTACATGGATGTGGCCTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC64VLGAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTGGCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGAGCAGCGACCCGCCGACCTTCGGCGGTGGCACCAAACTGGAGATCAAG65A.M h2F9 M6(LAGA)VHGAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACATGGATGTGGGgTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC66VLGAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTGGCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGAGCAGCGACCCGCCGACCTTCGGCGGTGGCACCAAACTGGAGATCAAG65A.M h2F9 M9(LAGA)VHGAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACGTGGATGTGGCGTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC67VLGAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTGGCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGAGCAGCGACCCGCCGACCTTCGGCGGTGGCACCAAACTGGAGATCAAG65A.M h2F9 M10(LAGA)VHGAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACGTGGATGTGGGGTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC68VLGAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTGGCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGAGCAGCGACCCGCCGACCTTCGGCGGTGGCACCAAACTGGAGATCAAG65A.M h2F9 M13(LAGA)VHGAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCACcAGCAGCGGTGGCCGCTACACCTACTACCCGGATAGCGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCGGCCACGCCGATTACATGGATGTGGCgTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC69VLGAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTGGCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGAGCAGCGACCCGCCGACCTTCGGCGGTGGCACCAAACTGGAGATCAAG65A.M h2F9 M14(LAGA)VHGAAGTGCAACTGGTGGAGAGCGGCGGTGGCCTCGTGCAGCCCGGCGGTAGCCTGCGCCTGAGCTGCGCCGCGAGCGGCTTCACCTTCAGCAGCCAGGGCATGAGCTGGGTGCGCCAAGCCCCAGGCAAAGGCCTGGAATGGGTGAGCACCATCAGCGTGACCGGCCGCTACCTGTACTACGCTGATAACGTGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTACTACTGCGCCAGCCACGCCGATTACCAGGATGTGAGCTTCGATTTCTGGGGCCAAGGCACCACAGTGACCGTCAGCAGC70VLGAAATTGTGCTCACACAGTCCCCGGCCACACTGAGCCTCTCACCTGGTGAACGCGCTACTCTGAGCTGCTCCGCGAGCAGCAGCGCCAACTTCATGCACTGGTATCAGCAGAAACCGGGCCAAGCCCCGCGTCGCCTGATCTACGATACAAGCAAACTGGCGAGCGGCATCCCGGCACGCTTCAGCGGCAGCGGCAGCGGCACCGATTACACCCTGACAATCAGCAGCCTCGAACCGGAAGATTTCGCCGTCTACTACTGTCAGCAGTGGCAGATGGAGCCGCCTACCTTCGGCGGTGGCACCAAACTGGAGATCAAG71.

[0306] Antibodies명오역아미노산 서이서엄번호AM h2F9 M6(LAGA)VHFR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS37CDR1SYAMS1FR2WVRQAPGKGLEWVS38CDR2TISSGGRYTYYPDSVKG2FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYCAG39CDR3HADYMDVGF DF9FR4WGQGTTVTVSS40CHCH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32한지EPKSCDKTHTCPPCP33CH2 (READ)APEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK41CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENFLYKTTPPVLDSDGSF SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35VLFR1EIVLTQSPATLSLSPGERATLSC42CDR1SASSSANFMH4FR2WYQQKPGQAPRRLIY43CDR2DTSKLAS5FR3GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC44CDR3QQWSSDPPT8FR4FGGGTKLEIK45CLCL KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0307] Antibody name domain amino acid sequence sequence number AM h2F9 M9(LAGA)VHFR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS37CDR1SYAMS1FR2WVRQAPGKGLEWVS38CDR2TISSGGRYTYYPDSVEG10FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG39CDR3HADYVDVAFDF11FR4WGQGTTVTVSS40CHCH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32hingeEPKSCDKTHTCPPCP33CH2 (LAGA)APEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAK41CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35VLFR1EIVLTQSPATLSLSPGERATLSC42CDR1SASSSANFMH4FR2WYQQ KPGQAPRRLIY43CDR2DTSKLAS5FR3GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC44CDR3QQWSSDPPT8FR4FGGGTKLEIK45CLCL KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0308] Antibody name domain amino acid sequence sequence number AM h2F9 M10(LAGA)VHFR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS37CDR1SYAMS1FR2WVRQAPGKGLEWVS38CDR2TISSGGRYTYYPDSVEG10FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG39CDR3HADYVDVGFDF12FR4WGQGTTVTVSS40CHCH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32hingeEPKSCDKTHTCPPCP33CH2 (LAGA)APEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAK41CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35VLFR1EIVLTQSPATLSLSPGERATLSC42CDR1SASSSANFMH4FR2WYQQ KPGQAPRRLIY43CDR2DTSKLAS5FR3GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC44CDR3QQWSSDPPT8FR4FGGGTKLEIK45CLCL KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0309] Antibody name domain amino acid sequence sequence number AM h2F9 M13(LAGA)VHFR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS37CDR1SYAMS1FR2WVRQAPGKGLEWVS38CDR2TTSSGGRYTYYPDSVKG13FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG39CDR3HADYMDVAFDF7FR4WGQGTTVTVSS40CHCH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32hingeEPKSCDKTHTCPPCP33CH2 (LAGA)APEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAK41CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35VLFR1EIVLTQSPATLSLSPGERATLSC42CDR1SASSSANFMH4FR2WYQQ KPGQAPRRLIY43CDR2DTSKLAS5FR3GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC44CDR3QQWSSDPPT8FR4FGGGTKLEIK45CLCL KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0310] Antibody name domain amino acid sequence sequence number AM h2F9 M14(LAGA)VHFR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS37CDR1SQGMS14FR2WVRQAPGKGLEWVS38CDR2TISVTGRYLYYADNVKG15FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS39CDR3HADYQDVSFDF16FR4WGQGTTVTVSS40CHCH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV32hingeEPKSCDKTHTCPPCP33CH2 (LAGA)APEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAK41CH3GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK35VLFR1EIVLTQSPATLSLSPGERATLSC42CDR1SASSSANFMH4FR2WYQQK PGQAPRRLIY43CDR2DTSKLAS5FR3GIPARFSGSGSGTDYTLTISSLEPEDFAVYC44CDR3QQWQMEPPT17FR4FGGGTKLEIK45CLCL KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC36

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317] [Example 1]

[0318] Confirmation of binding to CDCP1 expressed in CD34+ cells

[0319] 1-1. Confirmation of CDCP1 expression in CD34+ cells

[0320] CD34 used in this experiment + To confirm whether CDCP1 is expressed in cells, FACS was performed using commercially available anti-CDCP1 antibodies (Biolegend, Cat#324017) and anti-CD34 antibodies (BD, Cat#561209), and as shown in Figure 1a, it was confirmed that CDCP1 was expressed in CD34+ cells.

[0321] 1-2. Confirmation of the binding ability of anti-CDCP1 antibodies to CD34+ cells expressing CDCP1.

[0322] CD34, in which CDCP1 expression was confirmed in Example 1-1 + FACS was performed to confirm the binding of the parent antibody (2F9-679) and affinity matured antibody (AM h29F M4, M6, M9, M10, M13, and M14) clones to cells. As confirmed in Fig. 1b, it was confirmed that the parent antibody (2F9-679) and its affinity matured antibodies (AM h29F M4, M6, M9, M10, M13, and M14) of the present invention did not bind to CDCP1 of normal cells.

[0323] [Example 2]

[0324] Confirmation of binding affinity of anti-CDCP1 antibodies to CDCP1 antigen

[0325] 2-1. Confirmation of the binding ability of anti-CDCP1 antibodies to CDCP1 using ELISA

[0326] To determine the binding capacity of the 2F9-chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, to CDCP1, 50 ng of CDCP1 antigen (Acro Biosystems, CD1-H52H6) was added to each well and the antigen was coated. The amino acid sequence information of the CDDP1 antigen used is as shown in SEQ ID NO: 120.

[0327] CDCP-1 antigen (Accession # Q9H5V8-1, Phe30-Thr667)

[0328] FEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKM QEGVKMALHLPWFHPRNVSGFSIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSLQGCDQDAQSPGILRLQFQVLVQHPQNESNKIYVVD LSNERAMSLTIEPRPVKQSRKFVPGCFVCLESRTCSSNTLTSGSKHKISFLCDDLTRLWMNVEKTISCTDHRYCQRKSYSLQVPSDILHLPVELHDFSWKLLVPKDRLSLVLVPAQKLQQHTHEKPCNTSFSYLVASAIPSQDLYFGSFCPGGSIKQI QVKQNISVTLRTFAPSFQQEASRQGLTVSFIPYFKEEGVFTVTPDTKSKVYLRTPNWDRGLPSLTSVSWNISVPRDQVACLTFFKERSGVVCQTGRAFMIIQEQRTRAEEIFSLDEDVLPKPSFHHHSFWVNISNCSPTSGKQLDLLFSVTLTPRTVDLT (SEQ ID NO: 120)

[0329] In addition, to confirm the binding ability of antibodies (AM h2F9 M4, M6, M9, M10, M13, and M14) obtained through affinity maturation of the humanized antibody 2F9-679 to CDCP1, 100 ng of CDCP1 antigen (Acro Biosystems, CD1-H52H6) was added to each well to coat the antigen. Each well was blocked by treating it with a PBS solution containing 5% BSA (bovine serum albumin). After blocking, each anti-CDCP1 antibody diluted to a certain concentration was treated to each well and reacted for 60 minutes.

[0330] Afterwards, a secondary antibody recognizing anti-CDCP1 antibody (Peroxidase AffiniPure™ F(ab')2Fragment Goat Anti-Human IgG(H+L), Jackson ImmunoResearch, 109-036-088) was added to each well and reacted for 60 minutes. After the reaction, the color was developed with TMB (BD OptEIA™ TMB Substrate Reagent Set, BD Biosciences, 555214) solution for 10 minutes, and the absorbance (450 nm) of the reaction product was measured using a Microplate Reader (BMG LABTECH).

[0331] As a result, as shown in Figures 2a to 2c, it was confirmed that the binding of the nine anti-CDCP1 antibodies increased in a concentration-dependent manner of the coated antigen.

[0332] 2-2. Confirmation of the binding ability of anti-CDCP1 antibodies to CDCP1 using SPR analysis.

[0333] The binding ability of six affinity matured antibodies (AM h2F9 M4, M6, M9, M10, M13, and M14) was analyzed by SPR analysis under human CDCP-1 antigen binding conditions. The binding ability was analyzed by SPR (Surface plasmon resonance) analysis. After immobilizing the CDCP1 protein used for antibody production on a CM5 chip (Cytiva, 29149603) using Biacore 1K+ (Cytiva, USA), the affinity for CDCP1 when each of the six monoclonal antibodies was flowed at 30 μL per minute was measured. D The values ​​were analyzed using the Biacore Insight Control & Evaluation Software program. D The value is the Kd divided by the Ka, and a lower value indicates a stronger binding ability to the target. As confirmed in Figure 3a, all six antibodies bind to the human CDCP-1 antigen with high affinity, and M13 and M14 showed particularly high affinity.

[0334] Additionally, the binding ability of the 2F9 chimeric antibody and its humanized antibodies 2F9-679 and 2F9-700, and antibodies (AM h2F9 M4, M6, M9, M10, M13, and M14) obtained through affinity maturation of the humanized antibody 2F9-679 was analyzed through SPR (Surface plasmon resonance) analysis under pH 5.5, pH 6.5, and pH 7.4 conditions. The analysis method is as described above.

[0335] As a result, as shown in Fig. 3b, the 2F9 chimeric antibody and its humanized antibodies (2F9-679 and 2F9-700) were able to confirm the same or higher level of binding ability in low pH conditions of pH 5.5 and pH 6.5 compared to the normal pH condition of pH 7.4.

[0336] In addition, as confirmed in Fig. 3c, in the case of affinity matured antibodies, except for AM h2F9 M10, the remaining antibodies (AM h2F9 M4, M6, M9, M13, and M14) showed similar or slightly reduced levels of affinity in low pH environments of pH 5.5 and pH 6.5 compared to normal pH environment of pH 7.4, and in the case of M10 antibody, a certain level of reduced affinity was shown in low pH environments of pH 5.5 and pH 6.5 compared to normal pH environment of pH 7.4. Consequently, it can be seen that the above 9 antibodies can maintain a certain affinity even in a tumor-like environment.

[0337] 2-3. Confirmation of the binding ability of 2F9 chimeric antibody and its humanized antibody to the surface of HCT116 (colon cancer) cell line.

[0338] Surface binding of the 2F9 chimeric antibody and its humanized antibodies (2F9-679 and 2F9-700) was confirmed on HCT116 cells. Surface binding was measured by treating the cell line with 0.1 μg / mL or 1 μg / mL of the antibody.

[0339] HCT116 cells were seeded at 1 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5 After diluting to a concentration of 10 cells / mL, the solution was cultured and prepared. Meanwhile, 0.1 μg / mL and 1 μg / mL anti-CDCP1 antibodies were added to the prepared HCT116 cells and incubated at 4°C for 1 hour to confirm cell surface binding. At this time, human IgG1 was used as a negative control.

[0340] As a result, as shown in Fig. 4a, when comparing the 2F9 chimeric antibody with the 2F9 humanized antibody, it can be confirmed that the same binding level is maintained. Therefore, it can be seen that the complementary determining region (CDR) of the chimeric antibody and its humanized antibody are identical.

[0341] 2-4. Confirmation of the binding ability of affinity-matured antibodies to the surfaces of CAPAN-1 (pancreatic cancer), PANC-1 (pancreatic cancer), and MCF-7 (breast cancer) cell lines.

[0342] To confirm surface binding of six affinity matured antibodies (AM h2F9 M4, M6, M9, M10, M13, M14) on CAPAN-1 (pancreatic cancer), PANC-1 (pancreatic cancer), and MCF-7 (breast cancer) cell lines, the cell lines were treated with each antibody at different concentrations (0.04 μg / mL, 0.2 μg / mL, 1 μg / mL, and 5 μg / mL) and surface binding was measured.

[0343] CAPAN-1, PANC-1, and MCF-7 cells were seeded at 5 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5 After diluting to a concentration of 10 cells / mL, the cells were cultured and prepared. Meanwhile, each antibody was added to the prepared CAPAN-1, PANC-1, and MCF-7 cells at different concentrations (0.04 μg / mL, 0.2 μg / mL, 1 μg / mL, and 5 μg / mL), and cell surface binding was confirmed after incubation at 4°C for 1 hour. At this time, human IgG1 was used as a negative control.

[0344] As confirmed in Figures 4b to 4d, each antibody was saturated at a concentration of 0.2 μg / ml or higher in CAPAN-1 and PANC-1 cell lines, and since the MCF-7 cell line does not express the human CDCP-1 antigen, each antibody did not bind to the cell line surface despite the high concentration level. Through the above results, it was confirmed that the six types of affinity-matured antibodies exhibited specific binding affinity to the surface of cell lines expressing human CDCP-1.

[0345] [Example 3]

[0346] Confirmation of internalization of anti-CDCP1 antibodies

[0347] 3-1. Confirmation of internalization of 2F9 chimeric antibody and its humanized antibody in HCT116 (colon cancer) cells.

[0348] The internalization ability of 2F9 chimeric antibody and its humanized antibodies (2F9-679 and 2F9-700) into HCT116 (colon cancer cell line) cells was confirmed.

[0349] HCT116 cells were seeded at 1 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5 After diluting to a concentration of 10 cells / mL, the cells were cultured and prepared. Meanwhile, antibodies were added to the prepared HCT116 cells at concentrations of 0.1 μg / mL and 1 μg / mL, respectively, and incubated at 37°C for 4 or 24 hours to observe the internalization of the antibodies in the cells. At this time, human IgG1 was used as a negative control.

[0350] As a result, as shown in Fig. 5a, when treated with antibodies at a concentration of 0.1 μg / mL for 4 hours, the internalization degrees of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, were similar. Furthermore, when treated with antibodies at a concentration of 1 μg / mL for 4 hours, the internalization degrees of the 2F9 chimeric antibody and the 2F9 humanized antibodies, 2F9-679 and 2F9-700, were similar.

[0351] In addition, as shown in Fig. 5b, when treated with antibodies at a concentration of 0.1 μg / mL for 24 hours, the degree of internalization of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, was similar, and when treated with 1 μg / mL of antibody for 24 hours, the internalization pattern was similar to that when treated with 0.1 μg / mL of antibody.

[0352] Therefore, it was confirmed that the degree of cellular internalization of the 2F9 chimeric antibody and 2F9 humanized antibody in the above cell lines was similar.

[0353] 3-2. Confirmation of internalization of 2F9 chimeric antibody and its humanized antibody in CAPAN-1 (pancreatic cancer) cells.

[0354] The internalization ability of 2F9 chimeric antibody and 2F9 humanized antibody (2F9-679 and 2F9-700) into CAPAN-1 cells (pancreatic cancer cell line) was confirmed.

[0355] CAPAN-1 cells were seeded at 1 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 20% ​​FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5After diluting to the cell / sample concentration, the cells were cultured and prepared. Meanwhile, anti-CDCP1 antibodies were incubated at 0.1 μg / mL and 1 μg / mL at 37°C for 4 or 24 hours, respectively, to observe internalization. Human IgG1 was used as a negative control.

[0356] As a result, as shown in Fig. 5c, when treated with antibodies at a concentration of 0.1 μg / mL, the internalization degrees of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, were similar. Furthermore, when treated with antibodies at a concentration of 1 μg / mL for 4 hours, the internalization degrees of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, were similar.

[0357] In addition, as shown in Fig. 5d, when treated with 0.1 μg / mL of 2F9 antibody for 24 hours, the degree of internalization of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, was similar, and when treated with 1 μg / mL of antibody for 24 hours, the internalization pattern was similar to when treated with 0.1 μg / mL of antibody for 24 hours.

[0358] Therefore, it was confirmed that the degree of cellular internalization of the 2F9 chimeric antibody and 2F9 humanized antibody in the above cell lines was similar.

[0359] 3-3. Confirmation of internalization of 2F9 chimeric antibody and its humanized antibody into HCC1954 (breast cancer) cells.

[0360] The internalization ability of 2F9 chimeric antibody and 2F9 humanized antibody (2F9-679 and 2F9-700) into HCC1954 cells (breast cancer cell line) was confirmed.

[0361] HCC1954 cells were seeded at 1 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5 After diluting to the cell / sample concentration, the cells were cultured and prepared. Meanwhile, anti-CDCP1 antibodies were incubated at 0.1 μg / mL and 1 μg / mL at 37°C for 4 or 24 hours, respectively, to observe internalization. Human IgG1 was used as a negative control.

[0362] As a result, as shown in Fig. 5e, when treated with antibodies at a concentration of 0.1 μg / mL for 4 hours, the internalization degrees of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, were similar. Furthermore, when treated with antibodies at a concentration of 1 μg / mL for 4 hours, the internalization degrees of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, were similar.

[0363] Furthermore, as shown in Fig. 5f, when treated with antibodies at a concentration of 0.1 μg / mL for 24 hours, the internalization degrees of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, were similar. Furthermore, when treated with antibodies at a concentration of 1 μg / mL for 24 hours, the internalization degrees of the 2F9 chimeric antibody and its humanized antibodies, 2F9-679 and 2F9-700, were similar.

[0364] Therefore, it was confirmed that the degree of cellular internalization of 2F9 chimeric and 2F9 humanized antibodies was similar in the above cell lines.

[0365] Figure 5g is a summary of the degree of internalization according to the concentration (0.1 μg / mL, 1 μg / mL) and time (4 hours, 24 hours) of the antibodies of Figures 5a to 5f.

[0366] 3-4. Confirmation of internalization of affinity-matured antibodies against CAPAN-1 and PANC-1 (pancreatic cancer) cells.

[0367] CAPAN-1 and PANC-1 cells were seeded at 1 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5 After diluting to a concentration of 10 cells / mL, the cells were cultured and prepared. The prepared CAPAN-1 and PANC-1 cells were treated with affinity-matured antibodies [AM h2F9 M4, M6, M9, M10, M13, M14] at a concentration of 1 μg / mL, respectively, and incubated at 37°C for 4 or 24 hours to observe the internalization of the antibodies in the cells. At this time, human IgG1 was used as a negative control.

[0368] As a result, as shown in Figures 5h and 5i, when cultured for 24 hours, each antibody showed internalization of more than 90%, confirming that almost all antibodies were internalized within 24 hours.

[0369] 3-5. Confirmation of internalization of affinity-matured antibodies into MCF-7 (breast cancer cell line) cells.

[0370] MCF-7 cells were seeded at 1 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5After diluting to a concentration of 10 cells / mL, the cells were cultured and prepared. The prepared MCF-7 cells were treated with affinity-matured antibodies [AM h2F9 M4, M6, M9, M10, M13, M14] at a concentration of 1 μg / mL and incubated at 37°C for 4 or 24 hours to observe the internalization of the antibodies in the cells. At this time, human IgG1 was used as a negative control.

[0371] As confirmed in Fig. 5j, since MCF-7 cells do not express human CDCP-1 antigen, each antibody was not internalized at all not only at the beginning of the incubation at 4 hours but also after 24 hours.

[0372] The above results indicate that each antibody is specifically internalized in a cell line expressing the human CDCP-1 antigen.

[0373] [Example 4]

[0374] Confirmation of the stability of anti-CDCP1 antibodies during freezing and thawing

[0375] 4-1. Stability verification through SDS-PAGE

[0376] The stability of the 2F9 chimeric antibody and its humanized antibodies (2F9-679, 2F9-700) was confirmed by repeating freezing and thawing once to four times using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

[0377] #18: 2F9 Chimeric 5 μg

[0378] #19: 2F9 Humanized #679 5 μg, Freezing 0 times

[0379] #20: 2F9 Humanized #700 5 μg, Freezing 0 times

[0380] #21: 2F9 Humanized #679 5 μg, Freezing once

[0381] #22: 2F9 Humanized #700 5 μg, Freezing once

[0382] #23: 2F9 Humanized #679 5 μg, Freezing 2 times

[0383] #24: 2F9 Humanized #700 5 μg, Freezing twice

[0384] #25: 2F9 Humanized #679 5 μg, Freezing 3 times

[0385] #26: 2F9 Humanized #700 5 μg, Freezing 3 times

[0386] #27: 2F9 Humanized #679 5 μg, Freezing 4 times

[0387] #28: 2F9 Humanized #700 5 μg, Freezing 4 times

[0388] As a result, as shown in Fig. 6a, when each antibody was repeatedly frozen and thawed up to the 4th time, it was confirmed that no denatured low-molecular or high-molecular substance was generated compared to the 0th time.

[0389] 4-2. Stability verification using high-performance liquid chromatography

[0390] The stability of the 2F9 chimeric antibody and its humanized antibodies (2F9-679, 2F9-700) was analyzed by high performance liquid chromatography (HPLC) after repeating freezing and thawing once to four times.

[0391] As a result, as shown in Fig. 6b, when each antibody was frozen and thawed up to the 4th time, it was confirmed that no denatured low-molecular or high-molecular substance was generated compared to the 0th time.

[0392] Additionally, the stability of the affinity matured antibodies [AM h2F9 M4, M6, M9, M10, M13, M14] was analyzed by HPLC by repeating freezing and thawing 0, 1, 3, and 5 times.

[0393] As a result, as confirmed in Fig. 6c, it was observed that all six types of antibodies maintained a stable state without significant change even after repeating freezing and thawing cycles 0 to 5 times.

[0394] 4-3. Confirmation of binding ability to CDCP1 using ELISA

[0395] After repeating 0, 1, 3, and 5 freeze-thaw cycles with affinity matured antibodies [AM h2F9 M4, M6, M9, M10, M13, M14], the binding ability of the antibodies to human anti-CDCP-1 was analyzed by ELISA. For ELISA analysis, 100 ng of CDCP-1 antigen (Acro Biosystems, CD1-H52H6) was added to each well to coat the antigen. Each well was blocked with a PBS solution containing 5% BSA (Bovine Serum Albumin). After blocking, affinity matured antibodies [AM h2F9 M4, M6, M9, M10, M13, M14] diluted to a certain concentration were treated to each well and reacted for 60 minutes. Afterwards, the secondary antibody recognizing the affinity-matured antibody [Peroxidase AffiniPure™ F(ab')2Fragment Goat Anti-Human IgG(H+L), Jackson ImmunoResearch, 109-036-088] was treated to each well and reacted for 60 minutes. After the reaction, the color was developed with TMB (BD OptEIA™ TMB Substrate Reagent Set, BD Biosciences, 555214) solution for 10 minutes, and the absorbance (450 nm) of the reaction product was measured using a Microplate Reader (BMG LABTECH).

[0396] The EC50 value indicates the concentration at which the antibody binds to the antigen by more than 50%, and a lower concentration indicates better binding strength.

[0397] As a result, as confirmed in Fig. 6d, all six antibodies were found to bind to the human CDCP-1 antigen in a concentration-dependent manner, and were found to remain stable without significant changes in EC50 values ​​even after repeated freezing and thawing cycles.

[0398] [Example 5]

[0399] Confirmation of thermal stability of anti-CDCP1 antibodies using PTS analysis

[0400] Anti-CDCP1 antibodies [2F9 chimeric antibody, its humanized antibodies (2F9-679 and 2F9-700) and affinity matured antibodies (AM h2F9 M4, M6, M9, M10, M13 and M14)] were exposed to temperatures ranging from low to high temperatures, and the denaturation of the antibodies was measured and confirmed using protein thermal shift™ (PTS) analysis.

[0401] As a result, as shown in Fig. 7a, the first denaturation occurred at approximately 68°C for each antibody. In addition, the 2F9 chimeric antibody underwent a second denaturation at approximately 75°C, and the second denaturation temperature of the 2F9 humanized antibodies (2F9-679 and 2F9-700) was not measured. In addition, as shown in Fig. 7b, the six affinity-matured antibodies were confirmed to denature between 67°C and 68°C, and did not show a significant difference in thermal stability.

[0402] [Example 6]

[0403] Surface binding ability of anti-CDCP1 antibodies to the MDA-MB-453 cell line, a CDCP1-expressing cell line, and the K / D cell line, which inhibits CDCP1 expression in these cells.

[0404] Anti-CDCP1 antibodies [2F9 chimeric antibody and its humanized antibodies (2F9-679 and 2F9-700)] were prepared at 0.1 μg / mL or 1 μg / mL, respectively, and the cell surface binding ability of the anti-CDCP1 antibodies was confirmed in MDA-MB-453, a CDCP1-expressing cell line. Antibody binding ability was compared in scrambled MDA-MB-453 cells with normal CDCP1 expression and CDCP1 knockdown (K / D) MDA-MB-453 cells in which CDCP1 expression was inhibited.

[0405] MDA-MB-453 cells were seeded at 1 x 10 in growth medium (RPMI 1640 medium with HEPES, Cytiva, SH30255.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5 After diluting to a concentration of 10 cells / mL, the solution was cultured and prepared. Meanwhile, 0.1 μg / mL and 1 μg / mL anti-CDCP1 antibodies were added to scrambled MDA-MB-453 cells with normal CDCP1 expression and CDCP1 knockdown (K / D) MDA-MB-453 cells with suppressed CDCP1 expression, and cell surface binding was confirmed after incubation at 4°C for 1 hour. At this time, human IgG1 was used as a negative control.

[0406] As a result, as shown in Fig. 8, it was confirmed that the binding ability of CDCP1 K / D was significantly reduced compared to the binding ability of scrambled MDA-MB-453.

[0407] [Example 7]

[0408] Confirmation of inhibition of cell migration in MDA-MB-231 cell line, a CDCP1-expressing cell line, by anti-CDCP1 antibody.

[0409] Anti-CDCP1 antibodies [2F9 chimeric antibody (parent antibody) and its humanized affinity matured antibodies (AM h2F9 M4, M6, M9, M10, M13, and M14)] were prepared at 1 μg / mL, and the inhibition of cell migration ability by anti-CDCP1 antibodies was confirmed in MDA-MB-231, a CDCP1-expressing cell line. MDA-MB-231 cells with normal CDCP1 expression were treated with each anti-CDCP1 antibody, and the inhibitory ability of each antibody was compared and evaluated compared to the isotype IgG1 antibody.

[0410] MDA-MB-231 cells were seeded at 2 x 10 in growth medium (DMEM, Cytiva, SH30243.01) containing 10% FBS (HyClone™ Characterized Fetal Bovine Serum, Cytiva, SH30919.03). 5 After diluting to a concentration of 10 cells / mL, the cells were cultured and prepared. After 24 hours of culture, the cells were washed twice with serum starvation medium and cultured for an additional 16 hours. 1 μg / mL anti-CDCP1 antibody was added to MDA-MB-231 cells cultured for an additional 16 hours in serum starvation medium, and cell migration ability was confirmed after 24 hours of culture. Human IgG1 was used as a negative control.

[0411] As a result, as shown in Figures 9a and 9b, it was confirmed that the cell migration ability by anti-CDCP1 antibody was significantly reduced compared to IgG1, and the expression of CDCP1 was also

[0412] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CDCP1 (CUB domain containing protein 1), comprising any one of the following (i) to (vii): (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (vi) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; or (vii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

17.

2. In paragraph 1, (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region of SEQ ID NO: 19; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24; or (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; An antibody or antigen-binding fragment thereof that specifically binds to CDCP1, comprising:

3. An antibody or antigen-binding fragment thereof that specifically binds to CDCP1, comprising a heavy chain comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 18, 20, 23, 25, 26, 27, or 28; and a light chain comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 19, 21, 22, 24, or 30.

4. In paragraph 1, the Fc domain of the antibody: (a) wild-type IgG Fc; or (b) an Fc domain variant comprising one or more amino acid substitutions that reduce binding to an Fc receptor; An antibody or antigen-binding fragment thereof that specifically binds to CDCP1, comprising:

5. An antibody or antigen-binding fragment thereof that specifically binds to CDCP1, wherein the Fc domain variant comprises one or more amino acid substitutions that reduce binding to an Fcγ receptor.

6. An antibody or antigen-binding fragment thereof that specifically binds to CDCP1, wherein the Fc domain variant is an Fc variant comprising amino acid substitutions L235A and G237A, an Fc variant comprising amino acid substitutions L234A and L235E, or an Fc variant comprising amino acid substitutions L234A, L235A, and P329G, wherein the residues are numbered according to the EU index of Kabat.

7. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to CDCP1 according to any one of claims 1 to 6.

8. A recombinant vector comprising the nucleic acid molecule of paragraph 7.

9. A transformant containing a recombinant vector according to Article 8.

10. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to CDCP1, comprising the following steps (a) and (b): (a) a step of culturing the transformant of paragraph 9; and (b) A step of recovering an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 from the culture.

11. A pharmaceutical composition for preventing or treating cancer, comprising an antibody or an antigen-binding fragment thereof that specifically binds to CDCP1 according to any one of claims 1 to 6 as an active ingredient.

12. A pharmaceutical composition for preventing or treating cancer, wherein the cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, vulvar cancer, breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, skin cancer, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, endocrine cancer, and sarcoma.

13. A kit for detecting CDCP1 comprising an antibody or an antigen-binding fragment thereof that specifically binds to CDCP1 according to any one of claims 1 to 6.

14. A method for preventing or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to CDCP1 according to any one of claims 1 to 6.

15. A method for preventing or treating cancer in claim 14, wherein the cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, vulvar cancer, breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, skin cancer, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, endocrine cancer, and sarcoma.

16. Use of an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 according to any one of claims 1 to 6 for the manufacture of a drug for preventing or treating cancer.

17. The use according to claim 16, wherein the cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, vulvar cancer, breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, skin cancer, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, endocrine cancer, and sarcoma.

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