CDCP1 antibody or antigen-binding fragment thereof

Abstract

To provide a CDCP1 antibody or an antigen-binding fragment thereof with novel functionality.SOLUTION: The present invention provides an antibody or an antigen-binding fragment thereof with binding affinity to the region A within the C-terminal domain of the extracellular region of CDCP1 protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a CDCP1 antibody or an antigen-binding fragment thereof, and the like. [Background technology]

[0002] CDCP1 (CUB domain containing protein 1) is a transmembrane protein, and its expression has been reported to be enhanced in various cancers. For this reason, antibodies capable of binding to the extracellular domain of CDCP1 have been developed as cancer therapeutics. For example, Patent Document 1 describes the production of an antibody CP13E10 against human CDCP1 and its derived antibodies. CP13E10 binds to the extracellular domain of human CDCP1 and can induce transient phosphorylation and internalization, and further temporarily activates intracellular signal transduction (Src-PKC). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2020 / 097336 Summary of the Invention [Problem to be solved by the invention]

[0004] Previously, the extracellular region of CDCP1 was thought to have a structure consisting of three CUB domains arranged in a row based on amino acid sequence information. However, as research progressed, it was confirmed that the extracellular region of human CDCP1 has a structure that is significantly different from that previously imagined, consisting of two new structural regions (the N-terminal domain (NTD) and the C-terminal domain (CTD)) arranged in a row (Figure 1). Furthermore, as research progressed, it was found that conventional antibodies intended for cancer therapy recognize the N-terminal domain of the extracellular region. It was then noted that different regions may have different functionality.

[0005] An objective of the present invention is to provide a CDCP1 antibody or an antigen-binding fragment thereof having a new function. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted intensive research and found that the above problems can be solved by an antibody or an antigen-binding fragment thereof that has binding affinity to region A in the C-terminal domain of the extracellular region of CDCP1 protein. Further research based on this finding led to the completion of the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. An antibody or an antigen-binding fragment thereof having binding affinity to region A in the C-terminal domain of the extracellular region of CDCP1 protein.

[0008] Item 2. The C-terminal domain, A region C1 from the N-terminus of the 344th amino acid to the 664th amino acid in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region C2 corresponding to the region C1 in the CDCP1 protein of an organism other than human; The antibody or antigen-binding fragment thereof according to item 1,

[0009] Item 3. The region A is A region A1 from the 450th amino acid to the 664th amino acid from the N-terminus of the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region A2 corresponding to the region A1 in the CDCP1 protein of an organism other than human; 3. The antibody or antigen-binding fragment thereof according to item 1 or 2,

[0010] Item 4. The region A is A region A11 from the N-terminus of the 500th amino acid to the 664th amino acid in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region B12 corresponding to the region A11 in the CDCP1 protein of an organism other than human; The antibody or antigen-binding fragment thereof according to any one of Items 1 to 3,

[0011] Item 5. The region A is A region A111 from the N-terminus of the 550th amino acid to the 664th amino acid in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region B112 corresponding to the region A111 in the CDCP1 protein of an organism other than human; 5. The antibody or antigen-binding fragment thereof according to any one of items 1 to 4,

[0012] Section 6.(a) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:3; and / or A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; A light chain variable region comprising antibody a; (b) A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; and / or A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12; A light chain variable region comprising an antibody b comprising: (c) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and / or A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18; A light chain variable region comprising including, antibody c; The antibody or antigen-binding fragment thereof according to any one of Items 1 to 5,

[0013] Item 7. The antibody a comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 19 or 20, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 21 or 22. The antibody b comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 23, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 24, or The antibody c comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 25, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 26, The antibody or antigen-binding fragment thereof according to item 6.

[0014] Item 8. A polynucleotide comprising a coding sequence for the antibody or antigen-binding fragment thereof according to any one of items 1 to 7.

[0015] Item 9. A cell containing the polynucleotide according to Item 8.

[0016] Item 10. A pharmaceutical composition comprising at least one selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of items 1 to 7, the polynucleotide according to item 8, and the cell according to item 9.

[0017] Item 11. The pharmaceutical composition according to Item 10, which is used for promoting cell proliferation, promoting tissue damage repair, or promoting tissue regeneration.

[0018] Item 12. The composition further comprises at least one selected from the group consisting of a growth factor and a polynucleotide comprising a coding sequence for a growth factor, or For use in combination with at least one selected from the group consisting of a growth factor and a polynucleotide comprising a coding sequence for the growth factor; Item 12. The pharmaceutical composition according to item 10 or 11.

[0019] Item 13. A reagent comprising at least one selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of items 1 to 7, the polynucleotide according to item 8, and the cell according to item 9.

[0020] Item 14. (P) A step of culturing CDCP1-expressing cells in a state in which the cells can be contacted with a test antibody for 3 hours or more; and (Q) A method for producing an antibody or an antigen-binding fragment thereof according to any one of items 1 to 7, comprising a step of selecting a test antibody when a test antibody and / or CDCP1 is accumulated on the cell membrane of the CDCP1-expressing cells after culture, or when enhancement or maintenance of CDCP1, enhancement or maintenance of phosphorylated CDCP1, activation of the Src-PKC pathway, induction of accumulation or increase of growth factor receptors, or activation of signal transduction after stimulation with a growth factor is observed in the CDCP1-expressing cells. Effect of the Invention

[0021] According to the present invention, it is possible to provide a CDCP1 antibody or an antigen-binding fragment thereof having a novel function. Use of the CDCP1 antibody of the present invention is expected to promote cell proliferation, promote repair of tissue damage, promote tissue regeneration, and the like. [Brief description of the drawings]

[0022] [Figure 1] The structure of human CDCP1 is shown. [Diagram 2] An example of size exclusion chromatography analysis of Test Example 4 is shown below. [Diagram 3] The binding sites on the CTD for antibody #25 and antibody #43 are shown. [Figure 4] The binding sites on the CTD for antibody #26 and antibody #43 are shown. [Diagram 5] The results of the analysis of intracellular localization in Test Example 5 are shown. The top row shows the results when antibody #14 was used, the middle row shows the results when antibody #25 was used, and the bottom row shows the results when antibody #43 was used. The bottom of each row shows the localization of CDCP1, and the top row shows the merge of the localization of CDCP1 and the localization of the test antibodies (antibodies #14, 25, and 43). The incubation time from the addition of the test antibodies is shown at the top of the photograph. [Figure 6] 1 shows the results of dimer formation analysis in Test Example 6. The vertical axis shows the amount of luminescence produced by luciferase. When dimer formation is promoted by an anti-CDCP1 antibody, a higher amount of luminescence is shown compared to the control. The horizontal axis shows the antibody used. IgG is the control. [Figure 7] The results of Western blot of Test Example 7 are shown. The antibody used for detection is shown on the left side of the photograph. The antibody added during culture is shown at the top of the photograph. The photograph on the left is for a case where the culture time after antibody addition was 24 hours. The numbers above the photograph on the right side indicate the culture time after antibody addition. [Figure 8] This shows the results of Western blotting in Test Example 8. The antibody used for detection is shown on the left side of the photograph. The antibody added during culture and the culture time after the growth factor was added are shown at the top of the photograph. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0024] 1. Antibody or antigen-binding fragment thereof In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof having binding affinity to region A in the C-terminal domain of the extracellular region of CDCP1 protein (also referred to herein as the "CDCP1 antibody of the present invention"). This will be described below.

[0025] The CDCP1 protein is an expression product of the CDCP1 gene and is expressed in an organism. The species from which the CDCP1 protein is derived is not particularly limited, and includes animals, such as various mammals, including humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Among these, humans and monkeys (especially cynomolgus monkeys) are preferred.

[0026] The amino acid sequences of CDCP1 proteins derived from various species are known or can be easily identified by identity analysis based on known amino acid sequences. Specifically, for example, an example of a human CDCP1 protein is a protein having the amino acid sequence shown in SEQ ID NO:27.

[0027] The CDCP1 protein may have amino acid mutations, such as substitutions, deletions, additions, insertions, etc., which may exist between individuals. Mutations are preferably substitutions, more preferably conservative substitutions.

[0028] "Conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution. Other conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0029] Preferable specific examples of the CDCP1 protein include the protein described in the following (a) and the protein described in the following (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 27, and (b) a protein having an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 27 and having the ability to interact with a growth factor receptor At least one selected from the group consisting of:

[0030] The "identity" of an amino acid sequence refers to the degree of amino acid sequence agreement between two or more comparable amino acid sequences. Thus, the higher the agreement between two amino acid sequences, the higher the identity or similarity between those sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87: 2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences" Proc Natl Acad Sci USA. 90: 5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of base sequences is also defined in the same manner as above.

[0031] In the above (b), the identity is more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.

[0032] An example of the protein described in (b) above is, for example, (b') a protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 27 and having the ability to interact with a growth factor receptor. Examples include:

[0033] In the above (b'), "plural" means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3.

[0034] The extracellular domain of the CDCP1 protein is a domain exposed outside the cell when the CDCP1 protein is arranged in a state where the CDCP1 protein penetrates the cell membrane, and is not particularly limited thereto. The extracellular domain of each CDCP1 protein is already known or can be easily determined using various transmembrane domain prediction tools (e.g., SOSUI: http: / / harrier.nagahama-i-bio.ac.jp / sosui / , etc.).

[0035] A specific example of the extracellular region is the region from the N-terminus to the 667th amino acid in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27. Specific examples of the extracellular region in other CDCP1 proteins include corresponding regions. Here, in this specification, the "corresponding region" refers to a corresponding region when, for example, the amino acid sequence of the human CDCP1 protein is compared with another CDCP1 amino acid sequence using a sequence analysis tool (FASTA, BLAST, etc.).

[0036] The extracellular domain of CDCP1 is composed of two domains rich in β-sheets and a linker that has no secondary structure connecting the two domains. These two domains are referred to as the N-terminal domain and the C-terminal domain from the N-terminus. The CDCP1 antibody of the present invention has binding affinity or binds to (preferably has specific binding affinity or specifically binds to) region A in this C-terminal domain. That is, the epitope of the CDCP1 antibody of the present invention is present in region A. Due to this binding affinity, the CDCP1 antibody of the present invention exerts a unique function different from that of conventional antibodies (antibodies that have binding affinity to the N-terminal domain), as shown in the test examples described below.

[0037] The C-terminal domain of CDCP1 can be identified by the analysis method shown in the later-described Test Example 1. The C-terminal domain is, for example, a region C1 from the N-terminus to the 344th to 664th amino acids in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or a region C2 corresponding to the region C1 in the CDCP1 protein of an organism other than human.

[0038] From the viewpoint of enabling the CDCP1 antibody of the present invention to exert its function more effectively, region A is: Preferably, A region A1 from the 450th amino acid to the 664th amino acid from the N-terminus of the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region A2 corresponding to the region A1 in the CDCP1 protein of an organism other than human; More preferably, A region A11 from the N-terminus of the 500th amino acid to the 664th amino acid in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region B12 corresponding to the region A11 in the CDCP1 protein of an organism other than human; More preferably, A region A111 from the N-terminus of the 550th amino acid to the 664th amino acid in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or and region B112, which corresponds to region A111 in the CDCP1 protein of an organism other than human.

[0039] The epitope of the CDCP1 antibody of the present invention may be a linear epitope or a conformational epitope. The number of amino acid residues constituting the epitope is not particularly limited, and is, for example, 40 or less, 35 or less, 6 to 30, 6 to 25, 6 to 20, 6 to 15, or 6 to 10.

[0040] The amino acids on the CDCP1 side to which the CDCP1 antibody of the present invention binds are particularly preferably D565-R566-E593-R594-V598-Q600-T646-S647-K649-Q650 or R603-F605-I607-E616-E617-I618-E623-D624 in the amino acid sequence shown in SEQ ID NO: 27, or amino acids corresponding to these amino acids in the CDCP1 protein of an organism other than humans.

[0041] In a particularly preferred embodiment, the CDCP1 antibody of the present invention is (a) A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:3; and / or (preferably, and) A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; A light chain variable region comprising antibody a; (b) A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; and / or (preferably, and) A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12; A light chain variable region comprising an antibody b comprising: (c) A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and / or (preferably, and) A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18; A light chain variable region comprising including, antibody c; It is.

[0042] Antibody a is preferably The antibody comprises a heavy chain variable region comprising an amino acid sequence that has 85% or more (preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) identity to the amino acid sequence shown in SEQ ID NO: 19 or 20, and / or a light chain variable region comprising an amino acid sequence that has 85% or more (preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more) identity to the amino acid sequence shown in SEQ ID NO: 21 or 22.

[0043] Antibody b is preferably It comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 23, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 24.

[0044] The antibody c is preferably It comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO:25, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO:26.

[0045] The term "antibody" is used to include monoclonal and polyclonal antibodies. The antibody may be of any isotype, such as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, IgM, etc. The antibody may be a human antibody or a non-human antibody. Examples of non-human antibodies include, but are not limited to, mouse antibodies, rat antibodies, rabbit antibodies, monkey antibodies, chimpanzee antibodies, etc. The antibody may be a chimeric antibody, such as a mouse-human chimeric antibody. The antibody may be a partially or fully humanized antibody. The CDCP1 antibody of the present invention is preferably a monoclonal antibody.

[0046] As used herein, the antigen-binding fragment of an antibody is not particularly limited as long as it contains heavy chain CDR1 to 3 and / or light chain CDR1 to 3, and examples thereof include Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, etc.

[0047] Fab comprises a heavy chain fragment containing a heavy chain variable region and CH1 in the heavy chain constant region, and a light chain containing a light chain variable region and a light chain constant region (CL), and has a structure in which the heavy chain variable region and the light chain variable region are associated by the above-mentioned non-covalent intermolecular interaction or are bonded by a disulfide bond. In Fab, CH1 and CL may be disulfide-bonded by the thiol groups of cysteine ​​residues present in each.

[0048] F(ab')2 has two pairs of the above-mentioned Fab, and has a structure in which the CH1s are bonded together via thiol groups of cysteine ​​residues contained therein via disulfide bonds.

[0049] A minibody is a structure in which two fragments, each of which is composed of a heavy chain variable region and a CH3 linked thereto, constituting the scFV described below, are associated with each other via non-covalent intermolecular interactions between the CH3s.

[0050] scFv-Fc is a structure in which two antibody fragments each containing the scFv, CH2, and CH3 described below are associated with each other through non-covalent intermolecular interactions between the CH3s, as in the minibody described above, and the thiol groups of the cysteine ​​residues contained in each CH3 are disulfide-bonded.

[0051] Fv is also called the minimum structural unit of an antibody, and is a structure in which a heavy chain variable region and a light chain variable region are associated with each other through non-covalent intermolecular interactions. In Fv, the thiol groups of cysteine ​​residues present in the heavy chain variable region and the light chain variable region may be disulfide-bonded.

[0052] An scFv has a structure in which the C-terminus of a heavy chain variable region and the N-terminus of a light chain variable region are linked by a linker, or a structure in which the N-terminus of a heavy chain variable region and the C-terminus of a light chain variable region are linked by a linker, and is also called a single-chain antibody.

[0053] Diabodies, triabodies, and tetrabodies are structures in which the above-mentioned scFvs form dimers, trimers, and tetramers, respectively, and associate in a structurally stable state through non-covalent intermolecular interactions between the variable regions, similar to Fvs.

[0054] The CDCP1 antibody of the present invention may be bound to or fused with other peptides, oligopeptides, or proteins. Examples of other peptides, oligopeptides, or proteins include albumin (e.g., serum albumin), protein tags (e.g., biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag), fluorescent proteins (e.g., GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, JRed, KillerRed, Cher y, HcRed, mPlum), luminescent proteins (e.g., luciferase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase), secretory signal sequences (e.g., Igκ signal sequence), protease recognition sequences (e.g., TEV protease recognition sequence), expression enhancing sequences, soluble sequences, multimerization domains (e.g., cartilage oligomeric matrix protein domain, leucine zipper domain, collagen-like domain, cholera toxin B subunit domain, tetrabrachion coiled core domain, reovirus σ1 protein domain, Hepatitis delta antigen domain), and the like. When the CDCP1 antibody of the present invention is bound to or fused with a multimerization domain, an antibody of the same species or a different species as the CDCP1 antibody of the present invention or an antigen-binding fragment thereof may be further bound to or fused to the multimerization domain to form a multimer (homomultimer or heteromultimer).

[0055] The CDCP1 antibody of the present invention may be chemically modified so long as the binding ability to region A in the C-terminal domain of the extracellular region of the CDCP1 protein is not significantly impaired.

[0056] The CDCP1 antibody of the present invention has a C-terminus that is a carboxyl group (-COOH), a carboxylate (-COO -), an amide group (-CONH2), or an ester group (-COOQ). Here, Q in the ester group may be, for example, a C group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0057] In the CDCP1 antibody of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester used may be, for example, the C-terminal ester described above.

[0058] In the CDCP1 antibody of the present invention, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1-6 Alkanoyl C 1-6 those protected by suitable protecting groups (e.g., C groups such as formyl and acetyl groups); those in which the N-terminal glutamine residue that may be cleaved in vivo is pyroglutamic-substituted; and those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) are protected by suitable protecting groups (e.g., C groups such as formyl and acetyl groups). 1-6 Alkanoyl group etc. 1-6 Also included are those protected by an acyl group, or conjugated proteins such as so-called glycoproteins to which a sugar chain is bound.

[0059] 2. Method for producing an antibody or an antigen-binding fragment thereof The CDCP1 antibody of the present invention can be produced in a variety of ways.

[0060] The CDCP1 antibody of the present invention can be produced, for example, by a method comprising the steps of culturing a host transformed with a polynucleotide containing the coding sequence of the CDCP1 antibody of the present invention (the polynucleotide of the present invention) and recovering a fraction containing the CDCP1 antibody of the present invention.

[0061] The polynucleotide of the present invention is not particularly limited as long as it contains the coding sequence of the CDCP1 antibody of the present invention. Preferably, the polynucleotide of the present invention contains the above coding sequence in a state capable of expressing the CDCP1 antibody of the present invention. The polynucleotide of the present invention may contain other sequences in addition to the above coding sequence. Examples of the other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, an origin of replication, and a drug resistance gene coding sequence that are located adjacent to the coding sequence of the CDCP1 antibody of the present invention. In addition, the polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (such as a vector).

[0062] Specific examples of the polynucleotide of the present invention include (I) a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDRs 1-3 of the CDCP1 antibody of the present invention, (II) a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDRs 1-3 of the CDCP1 antibody of the present invention, and (III) a nucleic acid comprising a base sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDRs 1-3 of the CDCP1 antibody of the present invention, and a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDRs 1-3 of the CDCP1 antibody of the present invention.

[0063] The host is not particularly limited, and examples thereof include insect cells, eukaryotic cells, mammalian cells, etc. Among these, mammalian cells such as HEK cells, CHO cells, NS0 cells, and SP2 / O cells are preferred from the viewpoint of more efficient expression of the antibody.

[0064] The methods for transformation, culture, and recovery are not particularly limited, and known methods for antibody production can be used.

[0065] After recovery, the CDCP1 antibody of the present invention may be purified, if necessary, by known methods for antibody production, such as chromatography and dialysis.

[0066] Polynucleotides such as DNA and RNA may be chemically modified as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphoric acid residue (phosphate) of each nucleotide may be replaced with a chemically modified phosphoric acid residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be replaced with -OR (R represents, for example, -CH3, -CH2CHOCH3, -CH2CH2NHC(NH)NH2, -CH2CONHCH3, or -CH2CH2CN). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. Furthermore, the phosphoric acid portion or the hydroxyl portion may be modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group, but is not limited thereto.

[0067] As another example, the CDCP1 antibody of the present invention is (P) culturing CDCP1-expressing cells in a state in which the cells can be contacted with a test antibody for 3 hours or more; and (Q) The antibody can be produced by a method including a step of selecting the test antibody when the test antibody and / or CDCP1 accumulates on the cell membrane of the CDCP1-expressing cells after culture, or when enhancement or maintenance of CDCP1, enhancement or maintenance of phosphorylated CDCP1, activation of the Src-PKC pathway, induction of accumulation or increase of growth factor receptors, or activation of signal transduction after growth factor stimulation is observed in the CDCP1-expressing cells.

[0068] The CDCP1-expressing cells in step (P) are not particularly limited as long as they express CDCP1 on the cell membrane, and cell lines, primary cultured cells, and the like can be used.

[0069] The test antibody in step (P) may be a polyclonal antibody or a monoclonal antibody obtained by immunizing an animal. The animal to be immunized is not particularly limited as long as it is an animal from which a hybridoma can be prepared, but preferably includes an animal with an autoimmune disease (for example, BXSB mouse in the case of a mouse). The immunization and preparation of a hybridoma can be carried out according to a known method (for example, Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 to 11.11).

[0070] The culture time in step (P) is 3 hours or more, more preferably 3 to 30 hours, even more preferably 3 to 20 hours, still more preferably 4 to 10 hours, and particularly preferably 5 to 10 hours.

[0071] In step (Q), the presence or absence of accumulation of the test antibody and / or CDCP1 on the cell membrane of a CDCP1-expressing cell is confirmed. The confirmation method is not particularly limited, and examples thereof include immunostaining of cells. When the test antibody is an antibody that binds to the N-terminal domain, the test antibody and / or CDCP1 is observed in the form of dots within the cell or is not observed. On the other hand, when the test antibody is a CDCP1 antibody of the present invention that binds to the C-terminal domain, the test antibody and / or CDCP1 is observed to be accumulated (accumulated in a linear form) on the cell membrane (plasma membrane). In the latter case, the CDCP1 antibody of the present invention can be obtained by selecting (screening) the test antibody as the CDCP1 antibody of the present invention.

[0072] Alternatively, in step (Q), enhancement or maintenance of CDCP1 in CDCP1-expressing cells, enhancement or maintenance of phosphorylated CDCP1, activation of the Src-PKC pathway, induction of accumulation or increase of growth factor receptors, or activation of signal transduction after growth factor stimulation are confirmed. The confirmation method is not particularly limited, and examples include the methods of Test Examples 7 and 8 described below. When at least one of these is confirmed, the test antibody is selected (screened) as the CDCP1 antibody of the present invention, thereby obtaining the CDCP1 antibody of the present invention.

[0073] 2-1. Creation of human antibodies When the CDCP1 antibody of the present invention is obtained as a human antibody, it can be produced based on the information below, and, if necessary, in accordance with or in accordance with other known information.

[0074] Immunoglobulin G (hereinafter simply referred to as "IgG") is composed of two light polypeptide chains (hereinafter referred to as "light chains") with a molecular weight of about 23,000 and two heavy polypeptide chains (hereinafter referred to as "heavy chains") with a molecular weight of about 50,000. Both the heavy and light chains have a repeating structure of a region with a conserved amino acid sequence consisting of about 110 residues, and these constitute the basic unit of the three-dimensional structure of IgG (hereinafter referred to as "domain"). The heavy and light chains are composed of four and two consecutive domains, respectively. In both the heavy and light chains, the amino terminal domain has a larger variation in amino acid sequence between each antibody molecule than the other domains, and this domain is called the variable domain (hereinafter referred to as the "V domain"). At the amino terminal of IgG, the V domains of the heavy and light chains associate in a complementary manner to form a variable region. In contrast, the remaining domains as a whole form a constant region. The constant region has a sequence characteristic of each animal species. For example, the constant region of mouse IgG is different from that of human IgG, so mouse IgG is recognized as a foreign substance by the human immune system, resulting in a human anti-mouse antibody (HAMA) response (Schroff RW. et al Cancer Res. (1985) 45, 879-85). Therefore, mouse antibodies cannot be administered repeatedly to humans. In order to administer such antibodies to humans, it is necessary to modify the antibody molecule so as not to cause a HAMA response while retaining the specificity of the antibody.

[0075] According to the results of X-ray crystal structure analysis, such domains generally have a long cylindrical structure in which two layers of antiparallel β sheets consisting of three to five β strands are stacked. In the variable region, three loops are assembled for each of the V domains of the heavy and light chains to form an antigen-binding site. Each of these loops is called a complementarity determining region (hereinafter referred to as "CDR"), and the amino acid sequence variation is the most significant. The part of the variable region other than the CDR generally plays a role in maintaining the structure of the CDR and is called a "framework". Kabatt et al. collected a large number of primary sequences of the variable regions of the heavy and light chains and created a table in which each primary sequence is classified into CDR and framework based on the conservation of the sequence (Kabatt et al. SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No.91-3242, EA).

[0076] Moreover, each framework was classified into several subgroups with common amino acid sequence characteristics. Furthermore, it was found that there are corresponding frameworks between humans and mice. From such research on the structural characteristics of IgG, the following method for producing humanized antibodies was devised. In the early stage of research, chimeric antibodies in which the variable region of a mouse-derived antibody was joined to a constant region of human origin were proposed (Morrison SL. et al Proc Natl Acad Sci US A. (1984)81,6851-5). However, such chimeric antibodies still contain many non-human amino acid residues and may induce HAMA responses, especially when administered for a long period of time (Begent et al., Br. J. Cancer, (1990)62, 487).

[0077] As a method for further reducing the number of amino acid residues derived from non-human mammals that may induce HAMA responses in humans, a method of incorporating only the CDR portion into an antibody derived from a human has been proposed (Peter T et al. Nature, (1986) 321, 522-5); however, generally, grafting only the CDR is insufficient to maintain immunoglobulin activity against an antigen. Meanwhile, Chothia et al., using X-ray crystal structure analysis data in 1987, found that (a) in the amino acid sequence of a CDR, there are sites that directly bind to antigens and sites that maintain the structure of the CDR itself, and that the three-dimensional structures that CDRs can take are classified into several typical patterns (canonical structures), and (b) the class of the canonical structure is determined by the type of amino acid at a specific position in not only the CDR but also the framework portion (Chothia C. et al. J. Mol. Biol. (1987)196, 901-17). Based on this finding, it was suggested that when using the CDR grafting method, it would be necessary to graft some framework amino acid residues as well as the CDR sequences onto a human antibody (Patent Publication No. 4-502408).

[0078] In general, an antibody derived from a non-human mammal having a CDR to be grafted is defined as a "donor," and a human antibody to which the CDR is grafted is defined as an "acceptor." When carrying out the CDR grafting method, it is important to consider how to preserve the CDR structure as much as possible and how to maintain the activity of the immunoglobulin molecule. To achieve this goal, it is necessary to consider two points: (a) which subgroup of acceptor should be selected, and (b) which amino acid residue should be selected from the donor framework.

[0079] Queen et al. proposed a design method in which amino acid residues in the framework of a donor are grafted onto an acceptor together with CDR sequences if they meet at least one of the following criteria (JP Patent Publication No. 4-502408): (a) an amino acid in the framework region of the acceptor is rare at that position and the corresponding amino acid in the donor is common at that position in the acceptor; (b) the amino acid is in close proximity to one of the CDRs; (c) the amino acid has a side chain atom within about 3 Å (angstroms) of a CDR in a three-dimensional immunoglobulin model and is predicted to be able to interact with an antigen or with a CDR of a humanized antibody.

[0080] 2-2. Generation of human antibodies When obtaining the CDCP1 antibody of the present invention as a human antibody, it can be produced based on the information below, and, if necessary, in accordance with or in accordance with other known information.

[0081] In the present invention, a "human antibody" or "human immunoglobulin" refers to an immunoglobulin in which all regions constituting the immunoglobulin, including the H chain variable region (VH) and H chain constant region (CH) and the L chain variable region (VL) and L chain constant region (CL), are derived from genes encoding human immunoglobulin. In other words, it refers to an antibody in which the H chain is derived from a human immunoglobulin heavy chain gene and the light chain is derived from a human immunoglobulin light chain gene.

[0082] Human antibodies can be produced in the same manner as monoclonal antibodies, for example by immunizing a transgenic animal, which is produced by incorporating at least a human immunoglobulin gene into the gene locus of a non-human mammal such as a mouse, with an antigen. For example, a transgenic mouse producing human antibodies can be produced according to the methods previously reported (Mendez MJ et al. Nature Genetics (1997) 15, 146-56, Green LL et al. Nature Genetics (1994) 7, 13-21, Table 4-504365; International Patent Publication WO94 / 25585; Nikkei Science, June issue, pp. 40-50, 1995; Nils Lonberg et al. Nature (1994) 368, 856-9, and JP-T-6-500233).

[0083] 3.Cells In one aspect, the present invention relates to a cell containing the polynucleotide of the present invention.

[0084] Such cells include, for example, cells transfected with a polynucleotide of the invention.

[0085] The cells are preferably isolated cells. The cells may be cultured cells, and may be primary cultured cells or subcultured cells. The cells may be established cell lines. The cells may be iPS cells.

[0086] Examples of the cells include Escherichia coli K12 and other Escherichia coli, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, Spodoptera frugiperda-derived Sf cell line or Trichoplusia ni-derived HighFive cell line, insect cells such as olfactory nerve cells, animal cells, etc. Preferred examples of the animal cells include cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, etc.

[0087] 4.Applications In one aspect, the present invention relates to a pharmaceutical composition, a reagent, etc., containing at least one selected from the group consisting of the CDCP1 antibody of the present invention, the polynucleotide of the present invention, and the cell of the present invention (hereinafter, sometimes referred to as an "active ingredient").

[0088] The CDCP1 antibody of the present invention can sustainably activate the Src-PKC pathway, induce accumulation of growth factor receptors, and enhance activation of signal transduction after growth factor stimulation. Therefore, the CDCP1 antibody of the present invention can be used, for example, for promoting cell proliferation, promoting repair of damaged tissue, or promoting tissue regeneration. Specific examples of target tissues include bone tissue, muscle tissue, adipose tissue, endothelial tissue, epithelial tissue, and the like.

[0089] The pharmaceutical composition and reagent of the present invention include Further comprising at least one selected from the group consisting of a growth factor and a polynucleotide comprising a coding sequence for a growth factor, or For use in combination with at least one selected from the group consisting of growth factors and polynucleotides comprising coding sequences for growth factors. This makes it more suitable for the above-mentioned applications.

[0090] The growth factor is not particularly limited as long as it is a protein that can promote cell growth and / or differentiation. Examples of growth factors include FGF (preferably FGF2), PDGF (preferably PDGF-BB), BMP (preferably BMP2), EGF, IGF, TGF, NGF, BDNF, VEGF, G-CSF, GM-CSF, EPO, TPO, HGF, NT3, etc. When a growth factor is contained, the content can be appropriately adjusted depending on the size of the damaged site, etc. The content is, for example, about 0.01 to 100 μg, preferably 0.1 to 10 μg, more preferably about 0.3 to 5 μg.

[0091] The target organisms for the CDCP1 antibody of the present invention are not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

[0092] The content of the active ingredient in the pharmaceutical composition of the present invention can be appropriately set in consideration of the type of target disease, the intended therapeutic effect, the administration method, the treatment period, the age and body weight of the patient, etc. For example, the content of the active ingredient in the pharmaceutical composition of the present invention can be about 0.0001 to 100 parts by weight, assuming that the entire pharmaceutical composition of the present invention is 100 parts by weight.

[0093] The administration form of the pharmaceutical composition of the present invention is not particularly limited as long as the desired effect can be obtained, and it can be administered to mammals including humans by any of the administration routes of oral administration and parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, and topical administration). The preferred administration form is parenteral administration, and more preferably intravenous injection. Dosage forms for oral administration and parenteral administration and their manufacturing methods are well known to those skilled in the art, and can be manufactured according to conventional methods by mixing the active ingredient with a pharma- ceutically acceptable carrier, etc.

[0094] Dosage forms for parenteral administration include injection preparations (e.g., drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, and the like. For example, injection preparations are prepared by dissolving antibodies or cells in distilled water for injection, and solubilizing agents, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as necessary. The pharmaceutical agent can also be a freeze-dried preparation for preparation when needed.

[0095] The pharmaceutical composition of the present invention may further contain other drugs effective for treating or preventing diseases. The pharmaceutical composition of the present invention may also contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids, as necessary.

[0096] Carriers used in formulating the pharmaceutical composition of the present invention include excipients, binders, disintegrants, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizing agents, soothing agents, and the like that are commonly used in the art.

[0097] The dosage of the pharmaceutical composition of the present invention can be determined by a clinician based on various factors, such as the route of administration, the type of disease, the degree of symptoms, the age, sex, and body weight of the patient, the severity of the disease, pharmacological findings such as pharmacokinetics and toxicological characteristics, the presence or absence of a drug delivery system, and whether the composition is administered as part of a combination of other drugs. The dosage of the pharmaceutical composition of the present invention can be, for example, about 1 μg / kg (body weight) to 10 g / kg (body weight) per day. The administration schedule of the pharmaceutical composition of the present invention can also be determined taking into account the same factors as the dosage. For example, the above daily dosage can be administered once a day to once a month.

[0098] The reagent of the present invention may be in the form of a composition containing an active ingredient. The composition may contain other ingredients as necessary. Examples of other ingredients include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0099] The reagent of the present invention may be in the form of a kit containing an active ingredient. The kit may also contain instruments, reagents, etc. Examples of such instruments and reagents include test tubes, microtiter plates, purification columns, labeled antibodies, standard samples (positive controls, negative controls), etc. EXAMPLES

[0100] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0101] The list of antibodies used in the following test examples is shown in Table 1.

[0102] [Table 1]

[0103] Test Example 1. Structural analysis of the extracellular domain of human CDCP1 The official name of CDCP1 is CUB domain-containing protein1, and its extracellular domain was predicted to have a structure of three CUB domains aligned based on amino acid sequence information. However, our analysis of the extracellular domain of human CDCP1 confirmed that the structure is significantly different from the previously predicted structure, and that it has two new structural domains aligned (Fig. 1). Specifically, the determined structure was two domains containing a large amount of β-sheets, from amino acid residues 30 to 336 and from amino acid residues 344 to 664, and a linker with no secondary structure, from amino acid residues 337 to 343, connecting these two domains. These domains are called new domains, the N-terminal side (30-336) is called the N-terminal domain (NTD) and the C-terminal side (344-664) is called the C-terminal domain (CTD).

[0104] The above analysis was carried out as follows. Diffraction data was collected from crystals of the extracellular domain of CDCP1 at SPring-8 BL44XU, and the diffraction data was processed using the program autoPROC. The initial phase was determined using the program Phenix, and the initial structure was obtained from the resulting electron density map. The initial structure was refined using the programs Phenix and Coot to obtain the final structure.

[0105] Test Example 2. Preparation of monoclonal antibodies against CDCP1 First, a human CDCP1 antigen (an antigen consisting of a region from the N-terminus of SEQ ID NO:27 to the 30th amino acid to the 667th amino acid) was prepared.

[0106] SEQ ID NO:27 is the full-length amino acid sequence of the human CDCP1 protein, the sequence of which is as follows: MAGLNCGVSIALLGVLLLGAARLPRGAEAFEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKMQEGVKMALHLPWFHPRNVSGFSIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSLQGCDQDAQSPGILRLQFQVLVQHPQNESNKIYVVDLSNERAMSLTIEPRPVKQSRKFVPGCFVCLESRTCSSNLTLTSGSKHKISFLCDDLTRLWMNVEKTISCTDHRYCQRKSYSLQVPSDILHLPVELHDFSWKLLVPKDRLSLVLVPAQKLQQHTHEKPCNTSFSYLVASAIPSQDLYFGSFCPGGSIKQIQVKQNISVTLRTFAPSFQQEASRQGLTVSFIPYFKEEGVFTVTPDTKSKVYLRTPNWDRGLPSLTSVSWNISVPRDQVACLTFFKERSGVVCQTGRAFMIIQEQRTRAEEIFSLDEDVLPKPSFHHHSFWVNISNCSPTSGKQLDLLFSVTLTPRTVDLTVILIAAVGGGVLLLSALGLIICCVKKKKKKTNKGPAVGIYNDNINTEMPRQPKKFQKGRKDNDSHVYAVIEDTMVYGHLLQDSSGSFLQPEVDTYRPFQGTMGVCPPSPPTICSRAPTAKLATEEPPPRSPPESESEPYTFSHPNNGDVSSKDTDIPLLNTQEPMEPAE。

[0107] The human CDCP1 antigen for producing monoclonal antibodies was a functionally active "cleaved" form. First, the extracellular domain of the uncleaved CDCP1 protein was expressed and adjusted, and then the CDCP1 protein was cleaved using a specific protease, matriptase. The CDCP1 protein used in this experiment had a Tobacco Etch Virus (TEV) protease recognition sequence and a histidine tag (6xHIS) added in tandem to the C-terminus of the CDCP1 extracellular domain (30-667 amino acids) (ECD-TEV-6xHIS). After purifying the protein with the 6xHIS tag, the tag can be removed by treatment with TEV protease.

[0108] The experimental procedure is as follows. ECD-TEV-6xHIS plasmid was transfected into wild-type Expi293F cells (Thermo Fisher Scientific). HE400AZ medium (HE400AZ, GMEP) was used for culture, and Gxpress 293 Transfection Kit (GX293-RK, GMEP) and Gxpress 293 Enhancer (GX293-EN, GMEP) were used for transfection. After 3 days of culture, the cell culture medium was collected and subjected to immobilized metal affinity chromatography (resin: TALON metal affinity resin (Z5504N, Takara)). The fraction eluted with imidazole was concentrated, and Recombinant Human Matriptase / ST14 Catalytic Domain (3946-SE, R&D) was added under 0.2 mM DTT conditions to cleave the protease recognition site. The digested sample was applied to Superdex 200 increase 10 / 300 GL (Cytiva) to remove the enzyme and replace the buffer. It was then treated with TEV protease under 0.2 mM DTT conditions, and the reaction solution was again applied to TALON metal affinity resin to recover the fraction containing CDCP1 protein. Finally, the buffer was replaced with PBS using a PD-10 column (Cytiva).

[0109] Using the obtained human CDCP1 antigen, rats were immunized, myeloma cells were prepared, and cells were fused according to standard methods. Furthermore, ELISA screening using CDCP1 peptides was performed to obtain several hybridomas producing CDCP1 antibodies.

[0110] The obtained hybridomas were further subjected to the following screening. 1. Cells (A498, ACHN) were seeded onto a 24-well plate and cultured in DMEM medium containing 10% FBS for 16 hours (37°C, in a 5% CO2 incubator). 2. When the cell density reached about 50%, the medium was replaced with serum-free medium and cultured for 24 hours (37°C, 5% CO2 incubator). 3. The hybridoma culture supernatant was replaced with serum-free medium containing 5% serum, and the cells were cultured for 5 hours (37°C, in a 5% CO2 incubator). 4. The cells were fixed with 4% PFA (room temperature, 15 minutes). 5. Wash with PBS buffer (10 min, total 3 sets). 6. Treat with PBS containing 0.03% TX100 for 30 minutes (room temperature). 7. The sections were treated with blocking solution (Blocking One, Nacalai Tesque) for 60 minutes (room temperature). 8. A blocking solution containing anti-CDCP1 primary antibody (CST #4115, diluted 1000 times) was added and reacted for 16 hours (4th cycle). 9. Wash with PBS buffer (10 minutes, 3 sets in total). 10. A PBS solution containing anti-rabbit secondary antibody (Alexa594-conjugated, Thermo Fisher A-11012, diluted 2000 times) and anti-rat secondary antibody (Alexa488, Thermo Fisher A-11006, diluted 2000 times) was added and reacted for 90 minutes (room temperature). 11. Wash with PBS buffer (10 minutes, total 3 sets). 12. Fixed cell samples were prepared using anti-fade mounting medium (Prolong gold, Thermo Fisher P36941). 13. Fixed cells were observed using a confocal laser fluorescence microscope. 14. One hybridoma (#14) was selected when endogenous CDCP1 was observed in the form of dots within the cells, while four hybridomas (#25, #26, #29, and #43) were selected when endogenous CDCP1 was observed in the form of lines on the cell membrane (plasma membrane).

[0111] The antibodies obtained from each hybridoma were purified according to standard methods and used in the following test examples.

[0112] Test Example 3. Identification of the amino acid sequence of a monoclonal antibody against CDCP1 The amino acid sequences of antibodies #25, #26, #29, and #43 were determined from the genomic information of the hybridomas (#25, #26, #29, and #43) cloned in Test Example 1. The amino acid sequences of the heavy chain variable region and light chain variable region of antibodies #25, #26, #29, and #43, as well as the CDR sequences (underlined portions), are as follows (the sequence numbers are shown in parentheses at the end of the sequences):

[0113] CDR was determined based on the following papers. A new clustering of antibody CDR loop conformations. Benjamin North, Andreas Lehmann, Ronal L. Dunbrack Jr. Journal of Molecular Biology, 406 (2): 228-256 (2011) DOI: 10.1016 / j.jmb.2010.10.030.

[0114] < #25 > (Heavy chain variable region) EVQLVESGGGLVQPGRSLKLSC AASGFTFRDYYMA WVRQAPKKGLEWVA FISYEGSSTYYGDSVKGRFTISRDNAKSTLYLQMNSLRSEDTATYYC ARGMYTTDYYSH WGQGTLVTVSS (SEQ ID NO: 23) (Heavy chain CDR1) AASGFTFRDYYMA (SEQ ID NO:7) (Heavy chain CDR2) FISYEGSSTY (SEQ ID NO:8) (Heavy Chain CDR3) ARGMYTTDYYSH (SEQ ID NO:9) (Light chain variable region) DIQMTQSPASLSASLEEIVTITC QASQDIGNWLA WYQQKPGKSPQLLI YGSTSLAD GVPSRFSGSRSGTQYSLKISRLQVEDIGIYYC LQAYSAPFT FGSGTKLEIK (SEQ ID NO:24) (Light Chain CDR1) QASQDIGNWLA (SEQ ID NO:10) (Light Chain CDR2) YGSTSLAD (SEQ ID NO:11) (Light Chain CDR3) LQAYSAPFT (SEQ ID NO:12) < #26 > (Heavy chain variable region) EVKLVESGGGLVQPGRSLKLSC AASGFNFNDYWMG WVRQAPGKGLEWIG EINKDSSSIN YTPSLKDKFSISRDNAQNTLSLQMSKLGSEDTAIYYC ARAGGYHNYWFAY WGQGTLVTVSS (SEQ ID NO: 25) (Heavy chain CDR1) AASGFNFNDYWMG (SEQ ID NO:13) (Heavy chain CDR2) EINKDSSSIN (SEQ ID NO:14) (Heavy Chain CDR3) ARAGGYHNYWFAY (SEQ ID NO:15) (Light chain variable region) DIQMTQSPPSLSASLGDKVTITC QASQNIGKYIA WYQQKPGKAPRLLI RYTSTLES GTPSRFSGGSGRDYSFSISNVESEDIASYYC LQYVNLPLT FGSGTKLEIK (SEQ ID NO:26) (Light Chain CDR1) QASQNIGKYIA (SEQ ID NO:16) (Light Chain CDR2) RYTSTLES (SEQ ID NO:17) (Light Chain CDR3) LQYVNLPLT (SEQ ID NO:18) < #29 > (Heavy chain variable region) EVKLVESGGGLVQPGRSLKLSC AASGFNFNDYWMG WVRQAPGKGLEWIG DINKDSSTIN YIPSLKDKFTFSRDNAQNTLYLQMSKLGSEDTAIYYC ARASYNRYYFDY WGQGVMVTVSS (SEQ ID NO: 20) (Heavy chain CDR1) AASGFNFNDYWMG (SEQ ID NO:1) (Heavy chain CDR2) DINKDSSTIN (SEQ ID NO:2) (Heavy Chain CDR3) ARASYNRYYFDY (SEQ ID NO:3) (Light chain variable region) DTVLTQSPALPVSPGERVTISC RASESVSTLMH WYQQKPGQQPKLLI YLASHLES GVPARFSGIGSGTDFTLTIDPVEADDTATYYC QQSWNDPCT FGGGTKLELR (SEQ ID NO: 22) (Light Chain CDR1) RASESVSTLMH (SEQ ID NO:4) (Light Chain CDR2) YLASHLES (SEQ ID NO:5) (Light Chain CDR3) QQSWNDPCT (SEQ ID NO:6) < #43 > (Heavy chain variable region) EVKLVESGGGLVQPGRSLKLSC AASGFNFNDYWMG WVRQAPGKGLEWIG DINKDSSTIN YIPSLKDKFTISRDNAQNTLYLQMSKLGSEDTAIYYC ARASYNRYYFDY WGQGVMVTVSS (SEQ ID NO: 19) (Heavy chain CDR1) AASGFNFNDYWMG (SEQ ID NO:1) (Heavy chain CDR2) DINKDSSTIN (SEQ ID NO:2) (Heavy Chain CDR3) ARASYNRYYFDY (SEQ ID NO:3) (Light chain variable region) DTVLTQSPALPVSPGERITISC RASESVSTLMH WYQQKPGQQPKLLI YLASHLES GVPARFSGIGSGTDFTLTIDPVEADDTATYYC QQSWNDPCT FGGGTKLELR (SEQ ID NO:21) (Light Chain CDR1) RASESVSTLMH (SEQ ID NO:4) (Light Chain CDR2) YLASHLES (SEQ ID NO:5) (Light Chain CDR3) QQSWNDPCT (SEQ ID NO:6)

[0115] Test Example 4. Determination of epitopes of monoclonal antibodies against CDCP1 <Candidate Selection Method> Structural information was obtained by performing crystal structure analysis of the complex between CDCP1-ECD (Extra Cellular Domain) (hereinafter ECD) and Fab antibodies (#14, #25, #26, #29, and #43). The obtained information was used to analyze pairs of amino acids that are close to each other using PISA (Protein, Interfaces, Structures and Assemblies: https: / / www.ebi.ac.uk / pdbe / pisa / ) of PDBe (Protein Data Bank in Europe). Amino acids in the ECD that are close to the antibody at a certain distance (3.5 to 4 Å) were selected as candidates for epitopes.

[0116] <Analysis method> First, a plasmid in which all of the candidate amino acids on the ECD side were replaced with alanine was prepared by mutagenesis PCR using primers containing the DNA sequence after the target amino acid mutation. 1. Expi293F cells were seeded in HE400 medium and rotated for 16 hours (37°C, 8% CO2 incubator). 2. The mutant ECD plasmid (with 6xHis tag) was transfected into Expi293F cells using PEI-MAX, and then the cells were rotated and cultured for 72 hours (37 degrees, in an 8% CO2 incubator). 3. After collecting the culture supernatant, the mutant ECD protein was purified using Ni-NTA. 4. The mutant ECD protein and antibody were mixed (room temperature, 1 hour) and subjected to size exclusion chromatography using a Superdex 200 Increase 10 / 300GL column.

[0117] <Evaluation method> The ECD and antibody complex was analyzed by size exclusion chromatography, and the peak position was used to determine whether the two proteins were bound (Figure 2). If the peak position changed, it suggested that the mutated ECD and antibody bound, and it was determined that the mutated amino acid was not the antibody epitope (Figure 2, top). If the peak position did not change, it suggested that the mutated ECD and antibody did not bind, and it was determined that the mutated amino acid was the antibody epitope (Figure 2, bottom).

[0118] <Result> Crystal structure analysis revealed that antibodies #25 and #43 bind to different sites on the CTD (Figure 3). Crystal structure analysis revealed that antibodies #26 and #43 bind to the same site on the CTD (Figure 4). Since antibody #29 has the same CDR sequence as antibody #43 (with slight differences in amino acids outside of the CDRs), the binding site of antibody #29 was predicted to be similar to that of antibody #43. On the other hand, crystal structure analysis revealed that antibody #14 binds to the NTD.

[0119] The amino acids (epitopes) on the CDCP1 side important for the binding of antibodies #14, #25, and #43 are shown below. Each number indicates the amino acid number counted from the N-terminus of the amino acid sequence shown in SEQ ID NO:27.

[0120] <#14> R63-S69-K71-E74-R75 <#25> D565-R566-E593-R594-V598-Q600-T646-S647-K649-Q650 <#43> R603-F605-I607-E616-E617-I618-E623-D624.

[0121] Test Example 5. Analysis of changes in intracellular localization The changes in intracellular localization of antibodies #14, #25, and #43 were analyzed. 1. ACHN cells were seeded onto a 24-well plate and cultured in DMEM medium containing 10% FBS for 16 hours (37°C, in a 5% CO2 incubator). 2. When the cell density reached about 50%, the medium was replaced with serum-free medium and cultured for 4 hours (37°C, in a 5% CO2 incubator). 3. Antibody (final concentration 1.0 mg / ml) was added and cultured for 0 to 24 hours (in a 37°C, 5% CO2 incubator). 4. The cells were fixed with 4% PFA (room temperature, 15 minutes). 5. Wash with PBS buffer (10 min, total 3 sets). 6. Treat with PBS containing 0.03% Triton X-100 for 30 minutes (room temperature). 7. The sections were treated with blocking solution (Blocking One, Nacalai Tesque) for 60 minutes (room temperature). 8. A blocking solution containing anti-CDCP1 primary antibody (1000-fold dilution) was added and reacted for 16 hours (4th cycle). 9. Wash with PBS buffer (10 minutes, 3 sets in total). 10. A PBS solution containing Alexa594-conjugated anti-rabbit secondary antibody (A-11012, Thermo Fisher) (2000-fold dilution) and Alexa488-conjugated anti-rat secondary antibody (A-11006, Thermo Fisher) (2000-fold dilution) was added and reacted for 90 minutes (room temperature). 11. Wash with PBS buffer (10 minutes, total 3 sets). 12. Fixed cell samples were prepared using anti-fade mounting medium Prolong gold (P36941, Thermo Fisher). 13. Fixed cells were observed using a confocal laser fluorescence microscope.

[0122] The results are shown in Figure 5. When antibody #14 was used, endogenous CDCP1 was observed in the form of dots within the cells, whereas when antibody #25 / 43 was used, it was observed in the form of lines on the cell membrane (plasma membrane).

[0123] Test Example 6. Analysis of dimer formation Antibodies #14, #25, and #43 were analyzed using the NanoBiT Protein:Protein interaction system (Promega) to determine whether they promote dimerization of CDCP1. In this system, LgBiT and SmBiT, which are fragments of the luminescent enzyme luciferase, are attached to the C-terminus of CDCP1, respectively, and only when CDCP1-LgBiT and CDCP1-SmBiT form dimers, do they have enzymatic activity. The analysis was performed by adding the substrate luciferin and detecting luminescence by luciferase. When dimerization was promoted by anti-CDCP1 antibodies, the amount of luminescence was higher than that of the control. As a control, we used CUB1 antibody (324002, Biolegend), which internalizes CDCP1. In order to evaluate the properties of antibodies that bind to two antigens, we also used Fabs (which can only bind to one antigen) treated with papain. 1. HEK293 cells were seeded in a 96-well cell culture plate (optical bottom, 165306, Thermo Fisher) and cultured in 100 uL of DMEM medium (without penicillin-streptomycin) containing 10% FBS for 16 hours (37°C, in a 5% CO2 incubator). 2. 100 ng each of the two plasmids (pBiT1.1-CDCP1-LgBiT and pBiT2.1-CDCP1-SmBiT) and 0.6 ug of PEI-MAX (24765-1, Polysciences) were added to OPTI-MEM, and the total volume was adjusted to 10 uL. The mixture was then incubated at 37°C for 15 minutes. 3. The mixed solution from step 2 above was added to the cultured cells and cultured for 23 hours (37°C, in a 5% CO2 incubator). 4. The medium was removed and replaced with 50 uL of OPTI-MEM medium, and then cultured for 1 hour (37°C, in a 5% CO2 incubator). 5. 50 uL of OPTI-MEM medium containing 2.0 mg / ml antibody was added, and immediately added 25 uL of Nano-Glo Live Cell Reagent (N2011, Promega). 6. The luminescence value was measured using a GloMAX Discover Microplate Reader (37 degrees, 1 hour).

[0124] The results are shown in Figure 6. When antibody #14 was used, CDCP1 dimer formation was promoted, as in the case of using existing antibodies. On the other hand, when antibody #25 / 43 was used, CDCP1 dimer formation was suppressed.

[0125] Test Example 7. Analysis of antibody function 1 The effects of antibodies #14, #25, and #43 on intracellular signaling were examined. 1. ACHN cells were seeded onto a 6-well plate and cultured in DMEM medium containing 10% FBS for 16 hours (37°C, in a 5% CO2 incubator). 2. The medium was replaced with serum-free medium and cultured for 4 hours (37°C, in a 5% CO2 incubator). 3. Antibody (final concentration 1.0 mg / ml) was added and cultured for 0 to 24 hours (in a 37°C, 5% CO2 incubator). 4. The cells were washed twice with cold PBS buffer, and ODG buffer (composition: 20 mM Tris-HCl pH 7.4, 1 mM EDTA pH 7.4, 150 mM NaCl, 5% glycerol, 1% NP40, 2% 1-On-octyl-bD-glucopyranoside (ODG), 1 mM Na3VO4, 20 mM NaF, protease inhibitor cocktail (25535-24, Nacalai Tesque)) was added, and the cells were collected with a scraper. 5. The cell lysate was transferred to a tube, pipetted, and centrifuged (15,000 rpm, 4 degrees, 15 minutes). 6. The supernatant was transferred to a new tube, being careful not to aspirate the pellet. 7. Protein concentration was quantified (BCA method) and adjusted. 8. An appropriate amount of sample buffer (containing BPB) was added and boiled (95°C, 5 minutes). 9. The required amount of sample was applied to an SDS-PAGE gel (any gel concentration) and electrophoresed. 10. After electrophoresis, the gel was transferred onto a PVDF membrane using a transfer device. 11. After transfer, the membrane was washed with distilled water and blocked with Blocking One (Nacalai Tesque). 12. The membrane was immersed in a diluted solution of primary antibody and reacted (4 times, overnight). 13. After washing the membrane with TTBS buffer, it was immersed in a diluted solution of secondary antibody and reacted at room temperature (30 minutes). 14. After washing the membrane with TTBS buffer, it was reacted with ECL solution and developed.

[0126] The results are shown in Figure 7. When antibody #14 was used, CDCP1 and its phosphorylation were decreased, and the Src-PKC pathway was activated for a short period of time but then suppressed. On the other hand, when antibody #25 / 43 was used, CDCP1 and its phosphorylation were increased or maintained, and the Src-PKC pathway was continuously activated.

[0127] Test Example 8. Analysis of antibody function 2 The effect of antibody #43 on intracellular signaling was examined. 1. ACHN cells were seeded onto a 6-well plate and cultured in DMEM medium containing 10% FBS for 16 hours (37°C, in a 5% CO2 incubator). 2. The medium was replaced with serum-free medium and cultured for 4 hours (37°C, in a 5% CO2 incubator). 3. Antibody (final concentration 1.0 mg / ml) was added and cultured for 24 hours (37°C, in a 5% CO2 incubator). 4. HGF (100-39H, PeproTech), EGF (AF-100-15, PeproTech), or FGF2 (100-18B, PeproTech) (final concentration 50 ng / ml) was added and the cells were cultured for 0-60 minutes (37°C, in a 5% CO2 incubator). 5. The cells were washed twice with cold PBS buffer, and ODG buffer (composition: 20 mM Tris-HCl pH 7.4, 1 mM EDTA pH 7.4, 150 mM NaCl, 5% glycerol, 1% NP40, 2% 1-On-octyl-bD-glucopyranoside (ODG), 1 mM Na3VO4, 20 mM NaF, protease inhibitor cocktail (25535-24, Nacalai Tesque)) was added, and the cells were collected with a scraper. 6. The cell lysate was transferred to a tube, pipetted, and centrifuged (15,000 rpm, 4 degrees, 15 minutes). 7. The supernatant was transferred to a new tube, being careful not to aspirate the pellet. 8. Protein concentration was quantified (BCA method) and the concentration was adjusted. 9. An appropriate amount of sample buffer (containing BPB) was added and boiled (95°C, 5 minutes). 10. The required amount of sample was applied to an SDS-PAGE gel (any gel concentration) and electrophoresed. 11. After electrophoresis, the gel was transferred to a PVDF membrane using a transfer device. 12. After transfer, the membrane was washed with distilled water and blocked with Blocking One (Nacalai Tesque). 13. The membrane was immersed in a diluted solution of primary antibody and reacted (4 times, overnight). 14. After washing the membrane with TTBS buffer, it was immersed in a diluted solution of secondary antibody and reacted at room temperature (30 minutes). 15. After washing the membrane with TTBS buffer, it was reacted with ECL solution and developed.

[0128] The results are shown in Figure 8. Antibody #43 not only induced the accumulation of CDCP1 on the cell membrane surface, but also the accumulation of receptor proteins that interact with CDCP1. In addition to MET and EGFR, which have previously been reported to interact with CDCP1, FGFR1 was also found to accumulate. When growth factors (HGF, EGF, and FGF2, respectively) were added to the cells, the phosphorylation levels of each receptor increased compared to samples not treated with the antibody. Furthermore, the phosphorylation levels of ERK in the intracellular signaling MAPK pathway and S6 in the mTOR pathway were also increased.

[0129] Summary of Test Examples 1-8 Conventional anti-CDCP1 antibodies induce the internalization and degradation of CDCP1. This effect can suppress the proliferation of cancer cells. Furthermore, the addition of an anticancer drug has been reported to have an antitumor effect. In contrast, the CDCP1 antibody of the present invention (an antibody that binds to the C-terminal domain of the extracellular region of the CDCP1 protein) is different from the conventional concept in that it induces an increase in the expression level of CDCP1 and an associated increase in activated (phosphorylated) CDCP1. Furthermore, the CDCP1 antibody of the present invention can induce the accumulation of growth factor receptors and can enhance the activation of signal transduction after stimulation with growth factors. Therefore, when growth factors are required for tissue repair, regeneration, etc., the novel antibody of the present invention can enhance the effect.

Claims

1. An antibody or an antigen-binding fragment thereof that has binding affinity to region A in the C-terminal domain of the extracellular region of CDCP1 protein.

2. The C-terminal domain is A region C1 from the 344th amino acid to the 664th amino acid from the N-terminus of the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region C2 corresponding to the region C1 in the CDCP1 protein of an organism other than human; The antibody or antigen-binding fragment thereof according to claim 1 .

3. The region A is A region A1 from the 450th amino acid to the 664th amino acid from the N-terminus of the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region A2 corresponding to the region A1 in the CDCP1 protein of an organism other than human; The antibody or antigen-binding fragment thereof according to claim 1 .

4. The region A is A region A11 from the 500th amino acid to the 664th amino acid from the N-terminus of the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region B12 corresponding to the region A11 in the CDCP1 protein of an organism other than human; The antibody or antigen-binding fragment thereof according to claim 1 .

5. The region A is A region A111 from the N-terminus of the 550th amino acid to the 664th amino acid in the human CDCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 27, or A region B112 corresponding to the region A111 in the CDCP1 protein of an organism other than human; The antibody or antigen-binding fragment thereof according to claim 1 .

6. （a） A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:1; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:3; and / or A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:4; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; A light chain variable region comprising an antibody a; （b） A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; and / or A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12; A light chain variable region comprising or （c） A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13; A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and / or A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18; A light chain variable region comprising an antibody c comprising: The antibody or antigen-binding fragment thereof according to claim 1 .

7. The antibody a comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 19 or 20, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 21 or 22, The antibody b comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 23, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 24, or The antibody c comprises a heavy chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 25, and / or a light chain variable region comprising an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 26, The antibody or antigen-binding fragment thereof described in claim 6.

8. A polynucleotide comprising a coding sequence for the antibody or antigen-binding fragment thereof of claim 1.

9. A cell containing the polynucleotide of claim 8.

10. A pharmaceutical composition comprising at least one selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the polynucleotide according to claim 8, and the cell according to claim 9.

11. The pharmaceutical composition according to claim 10, which is for promoting cell proliferation, promoting tissue damage repair, or promoting tissue regeneration.

12. Further comprising at least one selected from the group consisting of a growth factor and a polynucleotide comprising a coding sequence for a growth factor, or For use in combination with at least one selected from the group consisting of a growth factor and a polynucleotide comprising a coding sequence for the growth factor; The pharmaceutical composition according to claim 10.

13. A reagent comprising at least one selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the polynucleotide according to claim 8, and the cell according to claim 9.

14. (P) culturing CDCP1-expressing cells in a state in which the cells can be contacted with a test antibody for 3 hours or more; and (Q) A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a step of selecting a test antibody when a test antibody and / or CDCP1 is accumulated on the cell membrane of the CDCP1-expressing cells after culture, or when enhancement or maintenance of CDCP1, enhancement or maintenance of phosphorylated CDCP1, activation of the Src-PKC pathway, induction of accumulation or increase of growth factor receptors, or activation of signal transduction after stimulation with a growth factor is observed in the CDCP1-expressing cells.

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