Anti-PTK7 chimeric antibody and Anti-PTK7 humanized antibody, and uses thereof

Abstract

The present disclosure relates to an anti-PTK7 chimeric antibody, an anti-PTK7 humanized antibody, and uses thereof. According to the present invention, it was confirmed that the anti-PTK7 chimeric antibody and anti-PTK7 humanized antibody, which specifically bind to PTK7, suppress oncogenicity such as wound regeneration, migration, and invasion of cells derived from PTK7-expressing cancers, such as esophageal squamous cell carcinoma, triple-negative breast cancer, and the like, and inhibit tumor growth and in vivo angiogenesis in xenograft animal models of these cancers. Therefore, the antibodies can be used as anticancer therapeutic agents for various PTK7-positive cancers and therapeutic agents for angiogenic diseases. In addition, these antibodies can be used alone, in combination with drugs such as existing anticancer drugs with known efficacy, or in the form of antibody-drug conjugates for anticancer and anti-angiogenic therapy.

Description

Anti-PTK7 chimeric antibodies and anti-PTK7 humanized antibodies and uses thereof

[0001] One example of the present invention relates to an anti-PTK7 chimeric or humanized antibody that specifically binds to PTK7 (protein tyrosine kinase 7), a functional fragment thereof, and uses of the antibody.

[0002] A total of 58 types of receptor protein tyrosine kinases (RPTKs) are known in humans, and are composed of an extracellular domain that binds ligands, a transmembrane domain, and an intracellular tyrosine kinase domain. Generally, when ligand binds, RPTKs dimerize, and the cytoplasmic domain is phosphorylated and activated, inducing signal transduction.

[0003] Defective RPTKs are a subgroup of RPTKs in which the tyrosine kinase that catalyzes phosphorylation is inactive. In humans, defective RPTKs such as ErbB3, PTK7, EphA10, EphB6, and RYK have been reported. Despite their inactivity, these defective RPTKs have been suggested to have physiological functions, such as carcinogenesis. For example, ErbB3 has been identified to bind to other ErbB family members and induce oncogenic signaling cascades, and ErbB3-neutralizing humanized antibodies have been developed as non-resistant targeted anticancer therapeutics and are in clinical trials. Furthermore, ErbB3-neutralizing humanized antibodies have shown excellent antitumor effects when used in combination with other anticancer antibodies or anticancer drugs.

[0004] PTK7 comprises an extracellular domain containing seven immunoglobulin (Ig)-like loops, a transmembrane domain, and a cytoplasmic region containing an inactive tyrosine kinase catalytic domain. PTK7 expression, which is upregulated in various malignancies, negatively correlates with disease-free and / or overall survival in cancer patients. PTK7 functions as a co-receptor for active RPTKs, such as FGFR1, thereby enhancing oncogenic signaling. Furthermore, PTK7 expression has been found to be upregulated in endothelial cells, particularly during tube formation, suggesting a crucial role for PTK7 in angiogenesis. Increased expression of PTK7 has been observed in several cancer types, including colon cancer, and its involvement in carcinogenesis and metastasis has been elucidated. However, since the active site of PTK7 tyrosine kinase is mutated, it is not easy to develop an active inhibitor. Therefore, we developed an anti-PTK7 mouse antibody to inhibit PTK7 function and found that it can control carcinogenesis, cancer metastasis, and angiogenesis. The present inventors developed an anti-PTK7 chimeric antibody and an anti-PTK7 humanized antibody from the anti-PTK7 mouse antibody that neutralizes PTK7 function and confirmed the inhibitory efficacy against carcinogenesis and angiogenesis. In addition, we showed that the anti-PTK7 antibodies were efficiently endocytosed and degraded in the lysosome after binding to PTK7-expressing cells, suggesting that they can be utilized in antibody-drug conjugates.

[0005] One aspect is an anti-PTK7 antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7), wherein the anti-PTK7 antibody provides an anti-PTK7 chimeric antibody or an anti-PTK7 humanized antibody.

[0006] Another aspect is to provide a polynucleotide encoding the antibody or a functional fragment thereof.

[0007] Another aspect is to provide a vector comprising the polynucleotide.

[0008] Another aspect is to provide cells transformed with the above vector.

[0009] Another aspect comprises a step of culturing the cells to produce a polypeptide comprising light and heavy chain variable regions; and

[0010] The present invention provides a method for producing an antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7), which comprises a step of recovering the polypeptide from the cell or a culture medium in which the cell is cultured.

[0011] Another aspect is to provide an angiogenesis inhibitor comprising the anti-PTK7 antibody or a functional fragment thereof.

[0012] Another aspect is to provide a pharmaceutical composition for preventing or treating angiogenesis-related diseases, comprising the above angiogenesis inhibitor.

[0013] Another aspect is to provide a growth, migration or invasion inhibitor of tumor cells comprising the anti-PTK7 antibody or a functional fragment thereof.

[0014] Another aspect is to provide a pharmaceutical composition for preventing or treating cancer, comprising an inhibitor of growth, migration or invasion of the tumor cells.

[0015] Another aspect provides a method for preventing or treating an angiogenesis-related disease, comprising administering to a subject in need thereof an anti-PTK7 antibody or a functional fragment thereof.

[0016] Another aspect provides the use of an anti-PTK7 antibody or a functional fragment thereof for the manufacture of a medicament for the prevention or treatment of angiogenesis-related diseases.

[0017] Another aspect provides a method for preventing or treating cancer comprising administering to a subject in need thereof an anti-PTK7 antibody or a functional fragment thereof.

[0018] Another aspect provides the use of an anti-PTK7 antibody or a functional fragment thereof for the manufacture of a medicament for the prevention or treatment of cancer.

[0019] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0020] Under the above background, the present inventors have conducted research efforts to develop PTK7-neutralizing chimeric antibodies and humanized antibodies that can be used to inhibit angiogenesis and treat various cancers by inhibiting the function of PTK7. As a result, they have confirmed that PTK7 activity is inhibited by specifically binding to the extracellular region of PTK7, thereby inhibiting growth, migration, invasion, and angiogenesis of cancer cells, thereby completing the present invention.

[0021] In order to achieve the above-described purpose of the present invention, the present invention provides an anti-PTK7 antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7),

[0022] The above anti-PTK7 antibody is an anti-PTK7 chimeric antibody or an anti-PTK7 humanized antibody,

[0023] The above anti-PTK7 chimeric antibody or functional fragment thereof

[0024] i) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 3; and

[0025] A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 5, a CDR2-VL having an amino acid sequence of SEQ ID NO: 6, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 7;

[0026] ii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 9, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 10, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 11; and

[0027] A light chain variable region comprising a CDR1-VL comprising the amino acid sequence of SEQ ID NO: 13, a CDR2-VL comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3-VL comprising the amino acid sequence of SEQ ID NO: 15; or

[0028] iii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and

[0029] A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 21, a CDR2-VL having an amino acid sequence of SEQ ID NO: 22, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 23;

[0030] The above anti-PTK7 humanized antibody or functional fragment thereof

[0031] iv) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and

[0032] Provided is an anti-PTK7 antibody or a functional fragment thereof, characterized in that it comprises a light chain variable region comprising CDR1-VL comprising an amino acid sequence of SEQ ID NO: 21, CDR2-VL comprising an amino acid sequence of SEQ ID NO: 22, and CDR3-VL comprising an amino acid sequence of SEQ ID NO: 23. [Table 1, Table 2, Table 3].

[0033] As an embodiment of the present invention, under the background of the anti-PTK7 chimeric antibody, the present inventors have made efforts to develop a PTK7 neutralizing chimeric antibody and humanized antibody that can be used to inhibit angiogenesis and treat various cancers by inhibiting the function of PTK7, and as a result, they have confirmed that the activity of PTK7 is inhibited by specifically binding to the extracellular region of PTK7, thereby inhibiting the growth, migration, invasion, and angiogenesis of cancer cells, thereby completing the present invention.

[0034] In order to achieve the above-described purpose of the present invention, the present invention provides an anti-PTK7 antibody or a functional fragment thereof that specifically binds to PTK7,

[0035] The above anti-PTK7 antibody is an anti-PTK7 chimeric antibody or an anti-PTK7 humanized antibody,

[0036] The above anti-PTK7 chimeric antibody or functional fragment thereof

[0037] i) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 3; and

[0038] A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 5, a CDR2-VL having an amino acid sequence of SEQ ID NO: 6, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 7;

[0039] ii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 9, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 10, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 11; and

[0040] A light chain variable region comprising a CDR1-VL comprising the amino acid sequence of SEQ ID NO: 13, a CDR2-VL comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3-VL comprising the amino acid sequence of SEQ ID NO: 15; or

[0041] iii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and

[0042] A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 21, a CDR2-VL having an amino acid sequence of SEQ ID NO: 22, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 23;

[0043] The above anti-PTK7 humanized antibody or functional fragment thereof

[0044] iv) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and

[0045] Provided is an anti-PTK7 antibody or a functional fragment thereof, characterized in that it comprises a light chain variable region comprising CDR1-VL consisting of the amino acid sequence of SEQ ID NO: 21, CDR2-VL consisting of the amino acid sequence of SEQ ID NO: 22, and CDR3-VL consisting of the amino acid sequence of SEQ ID NO: 23.

[0046] In one embodiment of the present invention, the anti-PTK7 chimeric antibody or functional fragment thereof may comprise a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 4, 12, or 20, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 8, 16, or 24.

[0047] In another embodiment of the present invention, the anti-PTK7 humanized antibody or functional fragment thereof may comprise a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 25, 26 or 27, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 28, 29, 30 or 31.

[0048] In another embodiment of the present invention, the antibody may be at least one selected from the group consisting of IgG, IgA, IgM, IgE and IgD, and the functional fragment may be at least one antibody selected from the group consisting of diabody, Fab, F(ab'), F(ab')2, Fv, dsFv and scFv.

[0049] In another embodiment of the present invention, the antibody or functional fragment thereof may inhibit one or more selected from the group consisting of wound healing, chemotactic migration and invasion.

[0050] In another embodiment of the present invention, the antibody or functional fragment thereof may reduce the level of hemoglobin (Hb) in a tissue.

[0051] In another embodiment of the present invention, the antibody or functional fragment thereof may inhibit the interaction of protein tyrosine kinase 7 (PTK7) and a receptor that binds to PTK7.

[0052] In another embodiment of the present invention, the antibody or functional fragment thereof may inhibit cancer growth.

[0053] In addition, the present invention provides a polynucleotide encoding the chimeric antibody and humanized antibody or functional fragment thereof.

[0054] Additionally, the present invention provides a vector comprising a polynucleotide.

[0055] Additionally, the present invention provides a cell transformed with the above vector.

[0056] In addition, the present invention provides a method for producing an antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7), comprising the steps of culturing the cell to produce a polypeptide comprising light chain and heavy chain variable regions; and recovering the polypeptide from the cell or the culture medium in which the cell is cultured.

[0057] In addition, the present invention provides an angiogenesis inhibitor comprising the antibody or a functional fragment thereof as an active ingredient.

[0058] In addition, the present invention provides a pharmaceutical composition for preventing or treating angiogenesis-related diseases, which comprises the angiogenesis inhibitor as an active ingredient.

[0059] In one embodiment of the present invention, the angiogenesis-related disease may be at least one selected from the group consisting of cancer, endometriosis, obesity, arthritis, arteriosclerosis, hemangioma, angiofibroma, vascular malformation, vascular adhesion, edematous sclerosis, diabetic retinopathy, macular degeneration, angiogenic glaucoma, corneal disease caused by angiogenesis, psoriasis, telangiectasia, pyogenic granuloma, seborrheic dermatitis, and Alzheimer's disease.

[0060] In addition, the present invention provides a tumor cell growth, migration or invasion inhibitor comprising the antibody or a functional fragment thereof as an active ingredient.

[0061] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an inhibitor of growth, migration or invasion of the tumor cells as an active ingredient.

[0062] In one embodiment of the present invention, the cancer may be at least one selected from the group consisting of glioblastoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, renal cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer.

[0063] Additionally, the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need thereof an anti-PTK7 chimeric antibody and an anti-PTK7 humanized antibody or a functional fragment thereof.

[0064] The present invention also provides the use of anti-PTK7 chimeric antibodies and anti-PTK7 humanized antibodies or functional fragments thereof for the manufacture of medicaments for the prevention or treatment of cancer.

[0065] Additionally, the present invention provides a method for preventing or treating an angiogenesis-related disease, comprising administering an anti-PTK7 humanized antibody or a functional fragment thereof to a subject in need thereof.

[0066] The present invention also provides the use of an anti-PTK7 chimeric antibody and an anti-PTK7 humanized antibody or a functional fragment thereof for the manufacture of a medicament for the prevention or treatment of angiogenesis-related diseases.

[0067] The anti-PTK7 chimeric antibody and anti-PTK7 humanized antibody according to the present invention showed the effect of inhibiting wound regeneration, migration, and invasion of cells derived from esophageal squamous cell carcinoma and triple-negative breast cancer, and showed a tumor suppression effect in mouse models of esophageal squamous cell carcinoma and triple-negative breast cancer, and inhibited angiogenesis in mice. Therefore, it is expected that it can be used for the treatment of various PTK7-positive cancers and angiogenic diseases, and can be used as a key global therapeutic agent for such diseases. In addition, it can be used as an essential material for developing a new antibody-drug conjugate (ADC) drug that can be used in clinical practice by producing a form in which a drug is conjugated to a chimeric antibody or humanized antibody, and it can be utilized to maximize the effect of anticancer treatment not only alone but also in combination with drugs such as existing anticancer drugs whose effects have been identified.

[0068] Figure 1 shows the results of molecular weight analysis by SDS-PAGE and physical property analysis by SE-HPLC of purified products after transient expression of anti-PTK7 chimeric antibodies 32-chi, 43-chi, and 52-chi. NR: non-reduced, R: reduced.

[0069] Figure 2 shows the results of binding affinity measurements using SPR of mouse antibodies 32-m, 43-m, 52-m and chimeric antibodies 32-chi, 43-chi, 52-chi for human PTK7 protein (His capture method).

[0070] Figure 3 shows the results of binding affinity measurements using SPR of mouse antibodies 32-m, 43-m, 52-m and chimeric antibodies 32-chi, 43-chi, 52-chi for human PTK7 protein (Fc capture method).

[0071] Figure 4 shows the results of analyzing the inhibitory effects on wound healing, migration, and invasion by treatment with anti-PTK7 mouse monoclonal antibodies 32-m, 43-m, and 52-m and anti-PTK7 chimeric antibodies 32-chi, 43-chi, and 52-chi in KYSE-30 cells.

[0072] Figure 5 shows the results of analyzing the inhibitory effects on wound healing, migration, and invasion by treatment with anti-PTK7 mouse monoclonal antibodies 32-m, 43-m, and 52-m and anti-PTK7 chimeric antibodies 32-chi, 43-chi, and 52-chi in MDA-MB-231 cells.

[0073] Figure 6 shows the results of molecular weight analysis by SDS-PAGE and physical property analysis by SE-HPLC of purified products after transient expression of anti-PTK7 humanized antibodies 52-hu-10, 52-hu-22, 52-hu-44, 52-hu-46, and 52-hu-59. NR: non-reduced, R: reduced.

[0074] Figure 7 shows the results of comparing the binding affinity of humanized antibodies 52-hu-10, 52-hu-22, 52-hu-44, 52-hu-46, and 52-hu-59 to human PTK7 protein with that of 52-chi using SPR (His capture method).

[0075] Figure 8 shows the results of comparing the binding affinity of humanized antibodies 52-hu-10, 52-hu-22, 52-hu-44, 52-hu-46, and 52-hu-59 to human PTK7 protein with that of 52-chi using SPR (Fc capture method).

[0076] Figure 9 shows the results of analyzing the inhibitory effects on wound healing, migration, and invasion by treatment with anti-PTK7 mouse monoclonal antibody 52-m, anti-PTK7 humanized antibodies 52-hu-10, 52-hu-22, 52-hu-44, 52-hu-46, and 52-hu-59 in KYSE-30 cells.

[0077] Figure 10 shows the results of analyzing the inhibitory effects on wound healing, migration, and invasion by treatment with anti-PTK7 mouse monoclonal antibody 52-m, anti-PTK7 humanized antibodies 52-hu-10, 52-hu-22, 52-hu-44, 52-hu-46, and 52-hu-59 in MDA-MB-231 cells.

[0078] Figure 11 is a diagram showing the results of confirming the cross-species reactivity of humanized antibodies 52-hu-22 and 52-hu-44 and Cofetuzumab against Human PTK7 (sPTK7-h) and Cynomolgus PTK7 (sPTK7-c).

[0079] Figure 12 is a diagram showing the results of confirming the interspecies cross-reactivity of humanized antibodies 52-hu-22 and 52-hu-44 and Cofetuzumab against Mouse PTK7 (sPTK7-m).

[0080] Figure 13 shows the results of confirming that the epitopes of anti-PTK7 humanized antibodies 52-hu-22, 52-hu-44, 32-chi, and Cofetuzumab are different from each other using the epitope binning method.

[0081] Figure 14 shows the results of comparative analysis of anticancer activity by measuring the weight and volume of tumors and staining Ki-67 positive cells through IHC staining after administering anti-PTK7 humanized antibodies 52-hu-22 and 52-hu-44 to an ESCC mouse model created by injecting KYSE-30 cells into mice.

[0082] Figure 15 shows the results of comparative analysis of anticancer activity by measuring the weight and volume of tumors and staining Ki-67 positive cells through IHC staining after administering anti-PTK7 humanized antibodies 52-hu-22 and 52-hu-44 to a TNBC mouse model created by injecting MDA-MB-231 cells into mice.

[0083] Figure 16 shows the results of a comparative analysis of the degree of angiogenesis according to the inclusion or absence of anti-PTK7 humanized antibodies 52-hu-22 and 52-hu-44 in Matrigel at different concentrations when in vivo angiogenesis was induced by injecting Matrigel containing VEGF into mice, observing the Matrigel plugs recovered, and measuring the hemoglobin content.

[0084] Figure 17 shows the results of measuring the change in body weight over 14 days after single-dose intravenous administration of human IgG or anti-PTK7 humanized antibodies 52-hu-22, 52-hu-44 at different concentrations (0, 5, 10, 50 mg / kg) to mice.

[0085] Figure 18 shows the results of analyzing the weights of major organs (brain, heart, lungs, kidneys, spleen, ovaries, liver) 14 days after single-dose intravenous administration of human IgG or anti-PTK7 humanized antibodies 52-hu-22, 52-hu-44 at different concentrations (0, 5, 10, 50 mg / kg) into mice.

[0086] Figure 19 shows the results of measuring pharmacokinetic indices such as peak blood concentration and area under the curve by collecting blood over 42 days after administering a single dose of 10 mg / kg each of anti-PTK7 humanized antibodies 52-hu-22-IgG and 52-hu-44-IgG to a mouse intravenously.

[0087] Figure 20 shows the results of measuring the level of antibody remaining in the cells by performing western blotting after treating MDA-MB-468 cells with anti-PTK7 mouse monoclonal antibody 52-m and humanized antibodies 52-hu-22, 52-hu-44 and washing the cells hourly.

[0088] Figure 21 shows the results of analyzing the level of endocytosed intracellular antibodies by taking fluorescent photographs 0, 1, 2, and 4 hours after treatment of MDA-MB-468 cells with anti-PTK7 mouse monoclonal antibody 52-m and humanized antibodies 52-hu-22, 52-hu-44.

[0089] Figure 22 shows the results of analyzing the location of endocytosed antibodies by taking fluorescent photographs 0 and 2 hours after staining lysosomes with lysotracker in MDA-MB-468 cells and treating them with anti-PTK7 monoclonal antibody 52-m and humanized antibodies 52-hu-22 and 52-hu-44.

[0090] Figure 23 shows the results of analyzing the location of endocytosed antibodies by taking fluorescent photographs 0 and 2 hours after staining lysosomes with lysotracker in KYSE-30 cells and treating them with anti-PTK7 monoclonal antibody 52-m and humanized antibodies 52-hu-22 and 52-hu-44.

[0091] Hereinafter, the present invention will be described in detail.

[0092] The present invention relates to an anti-PTK7 antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7),

[0093] The above anti-PTK7 antibody is an anti-PTK7 chimeric antibody or an anti-PTK7 humanized antibody,

[0094] The above anti-PTK7 chimeric antibody or functional fragment thereof

[0095] i) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 3; and

[0096] A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 5, a CDR2-VL having an amino acid sequence of SEQ ID NO: 6, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 7;

[0097] ii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 9, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 10, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 11; and

[0098] A light chain variable region comprising a CDR1-VL comprising the amino acid sequence of SEQ ID NO: 13, a CDR2-VL comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3-VL comprising the amino acid sequence of SEQ ID NO: 15; or

[0099] iii) CDR1-VH consisting of an amino acid sequence of sequence number 17, amino acid sequence of sequence number 18, the present invention is described in detail.

[0100] The present invention relates to an anti-PTK7 antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7),

[0101] The above anti-PTK7 antibody is an anti-PTK7 chimeric antibody or an anti-PTK7 humanized antibody,

[0102] The above anti-PTK7 chimeric antibody or functional fragment thereof

[0103] i) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 3; and

[0104] A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 5, a CDR2-VL having an amino acid sequence of SEQ ID NO: 6, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 7;

[0105] ii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 9, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 10, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 11; and

[0106] A light chain variable region comprising a CDR1-VL comprising the amino acid sequence of SEQ ID NO: 13, a CDR2-VL comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3-VL comprising the amino acid sequence of SEQ ID NO: 15; or

[0107] iii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and

[0108] A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 21, a CDR2-VL having an amino acid sequence of SEQ ID NO: 22, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 23;

[0109] The above anti-PTK7 humanized antibody or functional fragment thereof

[0110] iv) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and

[0111] Provided is an anti-PTK7 antibody or a functional fragment thereof, characterized in that it comprises a light chain variable region comprising CDR1-VL consisting of the amino acid sequence of SEQ ID NO: 21, CDR2-VL consisting of the amino acid sequence of SEQ ID NO: 22, and CDR3-VL consisting of the amino acid sequence of SEQ ID NO: 23.

[0112] In one embodiment of the present invention, the anti-PTK7 chimeric antibody or functional fragment thereof may comprise a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 4, 12, or 20, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 8, 16, or 24.

[0113] In another embodiment of the present invention, the anti-PTK7 humanized antibody or functional fragment thereof may comprise a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 25, 26 or 27, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 28, 29, 30 or 31.

[0114] In another embodiment of the present invention, the antibody may be at least one selected from the group consisting of IgG, IgA, IgM, IgE and IgD, and the functional fragment may be at least one antibody selected from the group consisting of diabody, Fab, F(ab'), F(ab')2, Fv, dsFv and scFv.

[0115] In another embodiment of the present invention, the antibody or functional fragment thereof may inhibit one or more selected from the group consisting of wound healing, chemotactic migration and invasion.

[0116] In another embodiment of the present invention, the antibody or functional fragment thereof may reduce the level of hemoglobin (Hb) in a tissue.

[0117] In another embodiment of the present invention, the antibody or functional fragment thereof may inhibit the interaction of protein tyrosine kinase 7 (PTK7) and a receptor that binds to PTK7.

[0118] In another embodiment of the present invention, the antibody or functional fragment thereof may inhibit cancer growth.

[0119] The term "antibody" as used herein includes an immunoglobulin molecule that immunologically reactives with a specific antigen, and includes both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies), humanized antibodies, heterozygous antibodies (e.g., bispecific antibodies), and bispecific antibodies. In the present invention, the antibody is, for example, a monoclonal antibody.

[0120] The “anti-PTK7 chimeric antibody” and “anti-PTK7 humanized antibody” of the present invention are used in the broadest sense in the present invention, and specifically include a binding site that specifically binds to PTK7.

[0121] The anti-PTK7 antibody or functional fragment thereof according to the present invention specifically binds to PTK7, and in particular, binds specifically to the extracellular domain of PTK7 with very high affinity.

[0122] The above PTK7 is not particularly limited in its specific biological origin as long as it is known in the art as PTK7, and may be derived from mammals including, for example, a mouse, a human, a rat, a chicken, a dog or a monkey, and may mean derived from a human.

[0123] Typically, antibodies have heavy chains and light chains, each of which contains a constant region and a variable region (also known as a "domain"). The variable regions of the light and heavy chains are each composed of one domain, the heavy chain variable region (VH) or the light chain variable region (VL). The light and heavy chains are linked by a single covalent disulfide bond, with their variable and constant regions aligned side by side, and the heavy chains of the two molecules bound to the light chain are linked by two covalent disulfide bonds to form the whole antibody. The whole antibody specifically binds to an antigen through the variable regions of the heavy and light chains, and since the whole antibody is composed of two pairs of heavy and light chains (HC / LC), one whole antibody molecule has a bivalent monospecificity, binding to the same two antigens through its two variable regions.

[0124] The heavy chain typically consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), which defines the immunoglobulin's isotype. The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. The heavy chain constant region may additionally include a hinge region.

[0125] As with the heavy chain, each light chain typically consists of several regions: a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region typically consists of one domain, the CL.

[0126] The variable region, which includes the site where the antibody binds to the antigen, includes three variable regions called "complementarity-determining regions" (hereinafter, "CDRs") and four "framework regions." The CDRs primarily play a role in binding to epitopes on the antigen. The CDRs of each chain are typically arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences can be determined using the method provided by IMGT (the international ImMunoGeneTics information system).

[0127] Additionally, antibodies are identified by the chains they contain, which contain specific CDRs. However, not all CDR residues are necessarily directly involved in antigen binding.

[0128] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., derived from a rodent) and the constant region is derived from a different species, such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a term commonly used for various types of antibody modifications and is well known to those skilled in the art.

[0129] The term "humanized antibody," as used herein, refers to a genetically engineered non-human antibody comprising a non-human variable domain modified to have a high degree of sequence homology to human antibody constant domains and human variable domains. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs), which together form the antigen-binding site, onto homologous human acceptor framework regions (FRs). To fully reconstitute the binding affinity and specificity of the parent antibody, substitution (backmutation) of framework residues from the parent antibody (i.e., non-human antibody) into human framework regions may be necessary. Structural homology modeling can help identify amino acid residues within the framework regions that are critical to the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, a human framework region, optionally including one or more amino acid backmutations primarily to non-human amino acid sequences, and an entirely human constant region. Optionally, additional amino acid modifications, which are not necessarily back mutations, may be applied to obtain humanized antibodies with desirable characteristics, such as affinity and biochemical properties.

[0130] The amino acid sequence of antibodies of non-human origin is distinct from that of antibodies of human origin, and therefore, non-human antibodies are potentially immunogenic when administered to human patients. However, despite the non-human origin of an antibody, its CDR segments are responsible for the antibody's ability to bind to its target antigen, and humanization aims to maintain the antibody's specificity and binding affinity. Therefore, humanization of a non-human therapeutic antibody is performed to minimize its immunogenicity in humans while simultaneously maintaining the specificity and binding affinity of the non-human antibody.

[0131] In the present invention, the antibody may be at least one selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, and may be, for example, IgG. The IgG type antibody includes all of the IgG1, IgG2, IgG3, or IgG4 subtype forms.

[0132] The functional fragment of the present invention refers to a fragment of an antibody that maintains the antigen-specific binding affinity of the whole antibody, and the fragment has at least 20%, 50%, 70%, 80%, preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% or more of the PTK7 affinity of the parent antibody. Specifically, the fragment may be one or more selected from the group consisting of diabody, Fab, F(ab'), F(ab')2, Fv, dsFv and scFv, but is not limited thereto.

[0133] The term "tissue" used in the present invention means a tissue in which blood vessels can be formed, and the humanized antibody or functional fragment thereof may reduce blood vessel formation in the tissue, thereby reducing hemoglobin in the tissue.

[0134] The tissue may be one or more selected from the group consisting of, for example, liver, pancreas, heart, blood vessels, kidney, skin, lung, brain, stomach, large intestine, small intestine, duodenum, rectum, ovary, breast, lymph node, biliary tract, pancreatic islet, cornea, uterus, esophagus, prostate, penis, and anus.

[0135]

[0136] In addition, the present invention provides a polynucleotide encoding the chimeric antibody and humanized antibody or functional fragment thereof.

[0137] The term "polynucleotide" as used in the present invention may be described as an oligonucleotide or a nucleic acid, and includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The polynucleotide may be single-stranded or double-stranded.

[0138] The polynucleotide of the present invention is not particularly limited in its sequence as long as it encodes the antibody of the present invention or a fragment thereof.

[0139] A polynucleotide encoding an antibody or fragment thereof of the present invention can be obtained by methods well known in the art. For example, based on a DNA sequence encoding part or all of the heavy and light chains of the antibody or the corresponding amino acid sequence, the polynucleotide can be synthesized using an oligonucleotide synthesis technique well known in the art, such as the polymerase chain reaction (PCR) method.

[0140]

[0141] Additionally, the present invention provides a vector comprising a polynucleotide.

[0142] The term "vector" used in the present invention is used for the purpose of replication or expression of the polynucleotide of the present invention for the recombinant production of the antibody of the present invention or a fragment thereof, and generally includes at least one of a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The vector of the present invention may preferably be an expression vector, and more preferably may be a vector including the polynucleotide of the present invention operably linked to a regulatory sequence, for example, a promoter.

[0143]

[0144]

[0145] Additionally, the present invention provides a cell transformed with the above vector.

[0146] The type of cell of the present invention is not particularly limited as long as it can be used to express a polynucleotide encoding an antibody or a fragment thereof included in the expression vector of the present invention. The cell (host cell) transformed with the expression vector according to the present invention may be a prokaryote (e.g., Escherichia coli), a eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, insect cell, or a hybridoma derived therefrom), but preferably, it may be a cell derived from a mammal including a human.

[0147] The term "transformation" as used herein refers to a change in the genotype of a host cell due to the introduction of a foreign polynucleotide, and refers to the introduction of the foreign polynucleotide into the host cell, regardless of the method used for the transformation. The foreign polynucleotide introduced into the host cell may be maintained by integration into the genome of the host cell, or may be maintained without integration, and the present invention encompasses both.

[0148] The recombinant expression vector capable of expressing the anti-PTK7 antibody or functional fragment thereof according to the present invention can be introduced into cells for producing the antibody or fragment thereof and transformed by methods known in the art, such as transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and any known method for introducing nucleic acids into cells, but the transformation method is not limited thereto.

[0149]

[0150] In addition, the present invention provides a method for producing an antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7), comprising the steps of culturing the cell to produce a polypeptide comprising light chain and heavy chain variable regions; and recovering the polypeptide from the cell or the culture medium in which the cell is cultured.

[0151] The above cell culture may have different medium compositions and culture conditions depending on the type of cell, and these can be appropriately selected and controlled by a person skilled in the art.

[0152] The antibody molecule may be accumulated within the cytoplasm of the cell, secreted from the cell, or targeted to the periplasm or extracellular medium (supernatant) by an appropriate signal sequence. Furthermore, it is preferable to refold the produced antibody molecule into a functional conformation using a method well known to those skilled in the art. The recovery of the polypeptide may vary depending on the characteristics of the produced polypeptide and the characteristics of the cell, and this can be appropriately selected and controlled by those skilled in the art.

[0153]

[0154] In addition, the present invention provides an angiogenesis inhibitor comprising the antibody or a functional fragment thereof as an active ingredient.

[0155] In addition, the present invention provides a pharmaceutical composition for preventing or treating angiogenesis-related diseases, which comprises the angiogenesis inhibitor as an active ingredient.

[0156] In one embodiment of the present invention, the "angiogenesis-related disease" is a disease that can be induced by continuous abnormal or excessive angiogenesis, and specifically, may be selected from the group consisting of cancer, endometriosis, obesity, arthritis, arteriosclerosis, hemangioma, angiofibroma, vascular malformation, vascular adhesion, edematous sclerosis, diabetic retinopathy, macular degeneration, angiogenic glaucoma, corneal disease caused by angiogenesis, psoriasis, telangiectasia, pyogenic granuloma, seborrheic dermatitis, and Alzheimer's disease, but is not limited thereto.

[0157]

[0158] In addition, the present invention provides a tumor cell growth, migration or invasion inhibitor comprising the antibody or a functional fragment thereof as an active ingredient.

[0159] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an inhibitor of growth, migration or invasion of the tumor cells as an active ingredient.

[0160] In one embodiment of the present invention, the cancer is preferably one in which the expression or activity of PTK7 is increased, and specifically, it may be any one selected from the group consisting of bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, anal cancer, central nervous system tumor, liver cancer, and colon cancer, but is not limited thereto.

[0161]

[0162] The pharmaceutical composition according to the present invention comprises an angiogenesis inhibitor or a tumor cell growth, migration or invasion inhibitor comprising an anti-PTK7 antibody or a functional fragment thereof as an active ingredient, and may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in formulations, and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further comprise other conventional additives such as antioxidants and buffers as needed. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, emulsion, pills, capsules, granules or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, each ingredient can be preferably formulated using the method disclosed in the literature of Remington. The pharmaceutical composition of the present invention has no particular limitation on the formulation, but can be formulated as an injection, an inhalant, an external skin preparation, etc.

[0163] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0164] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0165] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitantly administered drugs, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0166]

[0167] Additionally, the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need thereof an anti-PTK7 chimeric antibody and an anti-PTK7 humanized antibody or a functional fragment thereof.

[0168] The present invention also provides the use of anti-PTK7 chimeric antibodies and anti-PTK7 humanized antibodies or functional fragments thereof for the manufacture of medicaments for the prevention or treatment of cancer.

[0169] Additionally, the present invention provides a method for preventing or treating an angiogenesis-related disease, comprising administering an anti-PTK7 humanized antibody or a functional fragment thereof to a subject in need thereof.

[0170] The present invention also provides the use of an anti-PTK7 chimeric antibody and an anti-PTK7 humanized antibody or a functional fragment thereof for the manufacture of a medicament for the prevention or treatment of angiogenesis-related diseases.

[0171]

[0172] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0173]

[0174] Example

[0175] 1. Experimental materials and methods

[0176] (1) Production of chimeric antibodies

[0177] To produce chimeric antibodies, the VH and VL regions of mouse antibodies 32-m, 43-m, and 52-m (Tables 1, 2, and 3) were amplified by PCR using a pcDNA3.4-based vector (Invitrogen), and the constant region (CL) was linked to IGHG1 (immunoglobulin heavy constant gamma 1; P01857) and IGKC (immunoglobulin kappa constant; P01834) (Tables 4, 5) to create an animal cell expression vector.

[0178] Clone name Amino acid sequence of variable region (underlined: CDR sequence) SEQ ID NO: 32-VH-CDR1GFDFSRYW SEQ ID NO: 132-VH-CDR2INPDSSTI SEQ ID NO: 232-VH-CDR3ARAYYIYYFDY SEQ ID NO: 332-chi-VHEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARAYYIYYFDYWGQGTTLTVSS SEQ ID NO: 432-VL-CDR1QSLLYSSNQKNY SEQ ID NO: 532-VL-CDR2WAS SEQ ID NO: 632-VL-CDR3QQYYSYPWT SEQ ID NO: 732-chi-VLDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASIRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPWTFGGGTKLEIKSeq ID 843-VH-CDR1GFNIKDTYSEQ ID 943-VH-CDR2IDPANGNTSEQ ID 1043-VH-CDR3ARGDANYGAYSEQ ID 1143-chi-VHEVLLQQSGADLVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQFSSLTSEDTAVYYCARGDANYGAYWGQGTLVTVSASEq ID 1243-VL-CDR1ESVDNYGISFSEQ ID NO: 1343-VL-CDR2AASSEQ ID NO: 1443-VL-CDR3QQSKEVPLTSEQ ID NO: 1543-chi-VLDIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQRSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPLTFGAGTKLELKSEQ ID NO: 1652-VH-CDR1GFNIEDTYSEQ ID NO: 1752-VH-CDR2IDPANGNDSEQ ID NO: 1852-VH-CDR3ARGDANYGSYSEQ ID NO:1952-chi-VHEVQLQQSGAELVKPGASVKLSCTASGFNIEDTYIHWVKQRPEQGLEWIGRIDPANGNDKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGDANYGSYWGQGTLVTVSASEQ ID NO: 2052-VL-CDR1ESVDHFGVSFSEQ ID NO: 2152-VL-CDR2AASEQ ID NO: 2252-VL-CDR3QQSKEVPLTSEQ ID NO: 2352-chi-VLDIVLTQSPASLAVSLGQRATISCRASESVDHFGVSFMNWFQQKPGQPPKLLIYAASNQRSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPLTFGAGTKLELKSEQ ID NO: 2452-hu-10-VH52-hu-22-VH52-hu-46-VHEVQLVQSGAEVKKPGATVKISCKVSGFNIEDTYMHWVQQAPGKGLEWMGRIDPANGNDKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARGDANYGSYWGQGTLVTVSSSEQ ID NO: 2552-hu-44-VHEVQLVQSGAEVKKPGESLKISCKGSGFNIEDTYIHWVHQMPGKGLEWMGRIDPANGNDKYDPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGDANYGSYWGQGTLVTVSSSEQ ID NO: 2652-hu-59-VHQVQLVQSGSELKKPGASVKVSCKASGFNIEDTYMHWVRQAPGQGLEWMGRIDPANGNDKYDPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGDANYGSYWGQGTLVTVSSSEQ ID NO: 2752-hu-10-VLDIVMTQSPDSLAVSLGERATINCRASESVDHFGVSFMNWFQQKPGQPPKLLIYAASNQRSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPLTFGGGTKVEIKSequence number2852-hu-22-VLDIQMTQSPSSLSASVGDRVTITCRASESVDHFGVSFMNWFQQKPGKAPKLLIYAASNQRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSKEVPLTFGGGTKVEIKSequence number 2952-hu-44-VL52-hu-46-VLDIVMTQSPDSLAVSLGERATINCRASESVDHFGVSFMNWYQQKPGQPPKLLIYAASNQRSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPLTFGGGTKVEIKSequence number 3052-hu-59-VLDIQMTQSPSSLSASVGDRVTITCRASESVDHFGVSFMNWYQQKPGKAPKLLIYAASNQRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSKEVPLTFGGGTKVEIKSequence number 31

[0179]

[0180] Clone name Sequence number Base sequence of variable region (underlined: CDR sequence) 32-VH-CDR1 Sequence number 36 GGATTCGATTTTAGTAGATACTGG 32-VH-CDR2 Sequence number 37 ATTAATCCAGAGAGCAGTACGATA 32-VH-CDR3 Sequence number 38 GCAAGAGCTACTATATATACTACTTTGACTAC 32-chi-VH Sequence number 39GAGGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAATTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGAGCCTACTATATATACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA32-VL-CDR1SEQ ID NO: 40CAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC32-VL-CDR2SEQ ID NO: 41TGGGCATCC32-VL-CDR3SEQ ID NO: 42CAGCAATATTATAGCTATCCGTGGACG32-chi-VL sequence number43GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGTTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCC ATTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA43-VH-CDR1 sequence Number 44GGCTTCAACATTAAAGACACCTAT43-VH-CDR2 SEQ ID NO. 45ATTGATCCTGCGAATGGTAATACT43-VH-CDR3 SEQ ID NO. 46GCTAGAGGGGATGCTAACTACGGTGCTTAC43-chi-VHSEQ ID NO: 47GAGGTTCTTCTGCAGCAGTCTGGGGCAGACCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATACACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATAT GACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGTTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAGGGGATGCTAACTACGGTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA43-VL-CDR1 sequence number 48GAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTC43-VL-CDR2 SEQ ID NO: 49GCTGCATCC43-VL-CDR3 SEQ ID NO: 50CAGCAAAGTAAGGAGGTTCCGCTCACG43-chi-VL SEQ ID NO:51GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAAGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0181]

[0182] Clone name Sequence number Base sequence of variable region (underlined: CDR sequence) 52-VH-CDR1 Sequence number 52GGCTTCAACATTGAAGACACCTAT 52-VH-CDR2 Sequence number 53ATTGATCCTGCGAATGGTAATGAT 52-VH-CDR3GCTAGAGGGGATGCTAACTACGGTTCTTAC 52-chi-VH Sequence number 54GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTGAAGACACCTATATACACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATGATAAATATG ACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACTTCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAGGGGATGCTAACTACGGTTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG52-VL-CDR1 sequence Number 55GAAAGTGTTGATCATTTTGGCGTTAGTTTTATGAAC52-VL-CDR2 SEQ ID NO. 56GCTGCATCC52-VL-CDR3 SEQ ID NO. 57CAGCAAAGTAAGGAGGTTCCGCTCACG52-chi-VL sequence number58GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATCATTTTGGCGTTAGTTTTATGAACTGGTTCCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAAGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA52-hu-10-VH52-hu-22-VH52-hu-46-VH서열 번호 59GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTTCAACATTGAAGACACCTATATGCACTGGGTGCAACAGGCCCCTGGAAAAGGGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTAATGATAAATATGACCCGAAGTTCCAGGGCAGAGTCACCATAACCGCGGACACGTCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCTAGAGGGGATGCTAACTACGGTTCTTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA52-hu-44-VH서열 번호60GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGCTTCAACATTGAAGACACCTATATACACTGGGTGCACCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGAAGGAATTGATCCTGCGAATGGTAATGATAAATATGACCCGAAGTTCCAGGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCTAGAGGGGATGCTAACTACGGTTCTTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA52-hu-59-VH서열 번호 61CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGCTTCAACATTGAAGACACCTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTAATGATAAATATGACCCGAAGTTCCAGGGCCGGTTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAGCCTAAAGGCTGAGGACACTGCCGTGTATTACTGTGCTAGAGGGGATGCTAACTACGGTTCTTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCA52-hu-10-VL서열 번호62GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGAGCCAGCGAAAGTGTTGATCATTTTGGCGTTAGTTTTATGAACTGGTTCCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAAGATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAAAGTAAGGAGGTTCCGCTCACGTTCGGCGGAGGGACCAAGGTGGAGATCAAA52-hu-22-VL서열 번호 63GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGAGCCAGCGAAAGTGTTGATCATTTTGGCGTTAGTTTTATGAACATGAACTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAACCAAAGATCCGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAAAGTAAGGAGGTTCCGCTCACGTTCGGCGGAGGGACCAAGGTGGAGATCAAA52-hu-44-VL52-hu-46-VL서열 번호64GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGAGCCAGCGAAAGTGTTGATCATTTTGGCGTTAGTTTTATGAACTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAAGATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAAAGTAAGGAGGTTCCGCTCACGTTCGGCGGAGGGACCAAGGTGGAGATCAAA52-hu-59-VL서열 번호 65GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGAGCCAGCGAAAGTGTTGATCATTTTGGCGTTAGTTTTATGAACATGAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAACCAAAGATCCGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAAAGTAAGGAGGTTCCGCTCACGTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0183]

[0184] 명칭서열 번호아미노산 서열P01857-IGHG1서열 번호 32ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKP01834-IGKC서열 번호 33RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0185]

[0186]

[0187] (2) Production of humanized antibodies

[0188] To produce a humanized antibody, the amino acid sequences of the heavy and light chain variable regions of mouse antibody 52-m were compared and analyzed with the human antibody amino acid sequences in the IMGT database, and the heavy chain genes IGHV1-69, IGHV5-51, IGHV7-4, IGHJ4 and the light chain genes IGKV4-1, IGKV1-39 and IGKJ4 of the human antibody variable regions with high similarity were selected. To humanize mouse antibody 52-m, the CDR of the mouse antibody was grafted onto a human antibody, and two amino acid residues (N35H, W50R) or four amino acid residues (L50R, I58K, A60D, E61P or G35H, I50R, I58K, A60D) of the humanized heavy chain FR and four amino acids (L33M, T53N, R54Q, E55R or L33M, S53N, L54Q, Q55R) of the humanized light chain were substituted with those of the mouse antibody. Additionally, A34N or Y36F was substituted with that of the mouse antibody. The heavy and light chains of the 52-hu-10 antibody designed as described above have amino acid sequences of SEQ ID NOS: 25 and 28, and the heavy and light chains of the 52-hu-22 antibody have amino acid sequences of SEQ ID NOS: 25 and 29. In addition, the heavy and light chains of the 52-hu-44 antibody have amino acid sequences of SEQ ID NOS: 26 and 30, and the heavy and light chains of the 52-hu-46 antibody have amino acid sequences of SEQ ID NOS: 25 and 30. The heavy and light chains of the 52-hu-59 antibody have amino acid sequences of SEQ ID NOS: 27 and 31. The 52-hu-10, 52-hu-22, and 52-hu-46 antibodies share the amino acid sequence of the heavy chain variable region (VH), and the 52-hu-44 and 52-hu-46 antibodies share the amino acid sequence of the light chain variable region (VL). The humanized antibodies were genetically recombined into a humanized antibody by recombining the variable region into an expression vector of a chimeric antibody.

[0189]

[0190] (3) Transient expression and purification of chimeric and humanized antibodies

[0191] Chimeric and humanized antibodies were transfected into Expi293 cells (Invitrogen) using Expifectamine 293 reagent with heavy and light chain expression vector plasmid DNA at a 1:1 ratio. Transfected cells were cultured in a shaking CO2 incubator at 37°C, 125 rpm, and 8% CO2 for 6 days. On the 6th day after transfection, the supernatant was centrifuged, and antibody proteins were purified using HiTrap MabSelect SuRe (Invitrogen).

[0192]

[0193] (4) Measurement of antigen binding affinity of chimeric antibodies and humanized antibodies

[0194] The binding affinity of antigens and chimeric and humanized antibodies was measured using SPR equipment. The His capture method was used for analysis, and the antigen (human PTK7 extracellular domain with His tag; human soluble PTK7-His; human sPTK7-His) was used as the ligand, and the chimeric and humanized antibodies were used as the analytes. First, 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were mixed at a 1:1 ratio (v / v) and flowed for 420 s to activate each flow cell. Afterwards, the antigen was flowed into each flow cell in immobilization buffer (pH 4.5 acetate buffer) at a flow rate of 10 μl / min to reach 200 RU. To inactivate the remaining carboxyl groups on the CM5 chip surface, 70 μL of 1 M ethanolamine-HCl (pH 8) was added. The analytes at each concentration, diluted 2-fold serially, were sequentially injected using a multi-cycle analysis method to measure the binding sensorgram. The binding kinetics were calculated using a 1:1 binding model evaluation model based on the BIAevaluation Software manual.

[0195]

[0196] (5) Analysis of anticancer efficacy of chimeric and humanized antibodies at the cellular level

[0197] The anticancer efficacy of three monoclonal mouse antibodies (32-m, 43-m, 52-m), three chimeric antibodies (32-chi, 43-chi, 52-chi) against each of these antibodies, and five humanized antibodies derived from 52-m (52-hu-10, 22, 44, 46, 59) was analyzed in KYSE-30 cells derived from esophageal squamous cell carcinoma and MDA-MB-231 cells derived from triple-negative breast cancer. The anticancer efficacy was measured by the degree of inhibition of wound healing, migration, and invasion of cancer cells. In the wound healing assay, a monolayer of cells starved for 24 h was scraped with a 1000p tip to create space. Next, KYSE-30 cells were treated with FBS media and antibodies (10 μg / mL) for 48 hours, and MDA-MB-231 cells were treated for 24 hours. Migration and invasion assays were performed by pre-incubating cells starved for 24 hours with antibodies for 30 minutes. For migration and invasion assays, gelatin (0.1%) was coated on the bottom of the Transwell, and for invasion assays, Matrigel (0.3 mg / mL) was additionally coated on the top of the Transwell. Antibody-treated cells were placed on the top of the Transwell, and the migration assay was performed for 24 hours and the invasion assay for 48 hours. After fixing the cells with 3.7% formaldehyde for 15 minutes, they were stained with 0.2% crystal violet for 5 minutes, and photographed under a microscope.

[0198]

[0199] (6) Analysis of heterologous cross-reactivity of humanized antibodies

[0200] The binding affinity of three different antigens to humanized antibodies was measured using SPR equipment. The His capture method was used for analysis, and the three antigens [human sPTK7-His (AcroBiosystem, Cat. No. PT7-H52H3), mouse sPTK7-His (AcroBiosystem, Cat. No. PT7-M52H3), cynomolgus sPTK7-His (AcroBiosystem, Cat. No. PT7-C52H3)] were used as ligands, and the humanized antibodies were used as analytes. The antigen-antibody binding was analyzed using the method described in (4) below.

[0201]

[0202] (7) Production, expression, and purification of cofetuzumab

[0203] The published heavy and light chain sequences of Cofetuzumab were digested with restriction enzymes EcoRI / BamHI and XhoI / AgeI, respectively, and then genetically recombined into the pcDNA3.4 vector digested with the same restriction enzymes. Transient expression and small-scale purification were performed in the same manner as in the transient expression and purification methods for chimeric and humanized antibodies described in the text (3), thereby securing highly pure antibodies.

[0204]

[0205] (8) Comparative analysis of epitopes of humanized antibodies and Cofetuzumab

[0206] We analyzed the epitopes of humanized antibodies and Cofetuzumab using SPR equipment. Analysis was performed using a tandem assay. After His-capture of the antigen, humanized antibodies, Cofetuzumab and humanized antibodies, or humanized antibodies and Cofetuzumab were sequentially injected, and binding sensorgrams were measured.

[0207]

[0208] (9) Analysis of anticancer efficacy of humanized antibodies in xenograft mouse models of esophageal squamous cell carcinoma and triple-negative breast cancer.

[0209] KYSE-30 and MDA-MB-231 cells were mixed 1:1 with Matrigel and PBS and injected subcutaneously into the backs of 5-week-old female BALB / c nu / nu mice. When tumor formation was confirmed 1-2 weeks after cell injection, 10 mg / kg of humanized antibodies 52-hu-22 and 52-hu-44 were injected intraperitoneally twice a week for 3 weeks. At this time, the same volume of PBS was injected to the antibody-naive control mice. Mice were sacrificed 1-2 weeks after antibody injection, and xenograft tumors were harvested. The tumor weight and volume were measured, fixed in 4% paraformaldehyde (PFA), and embedded in paraffin. The degree of proliferative cells was compared by immunohistochemical staining for Ki-67, a cell proliferation target protein, and hematoxylin / eosin staining in paraffin sections.

[0210]

[0211] (10) Analysis of anti-angiogenic efficacy of humanized antibodies through in vivo angiogenesis analysis

[0212] Five-week-old female BALB / c nu / nu mice were injected subcutaneously with growth factor-reduced Matrigel (0.5 mL) containing 32 U heparin and 250 ng mouse VEGF, plus humanized PTK7 antibodies 52-hu-22 and 52-hu-44 (3 and 10 μg / mL). After 12 days, the mice were sacrificed, and the Matrigel plugs were collected. To quantify the vascularization of the Matrigel plugs, the hemoglobin (Hb) content was measured using Drabkin's reagent kit 525 (Sigma-Aldrich).

[0213]

[0214] (11) Single-dose toxicity test of anti-PTK7 humanized antibody in mice

[0215] Six-week-old female mice (C57BL / 6) were administered a single dose of human IgG as a control group and anti-PTK7 humanized antibodies 52-hu-22 and 52-hu-44 as test groups at concentrations of 0, 5, 10, and 50 mg / kg, respectively, via the tail vein, and general symptoms were observed for 6 hours after administration and up to 14 days after administration. 20 mice per test substance, a total of 60 mice, were tested, and body weights were measured three times a week. On the final day of the test, the laparotomy was performed to visually observe the organs and measure the organ weights.

[0216]

[0217] (12) Pharmacodynamic analysis of anti-PTK7 humanized antibodies in mice

[0218] Mice (C57BL / 6) were administered a single intravenous dose of anti-PTK7 humanized antibodies 52-hu-22 and 52-hu-44 at a concentration of 10 mg / kg each, and blood samples were collected 19 times from before administration to 42 days after administration. 15 mice were collected per test substance, and a total of 30 mice were collected. Blood samples were analyzed for peak antibody concentration and kinetic parameters such as the area under the curve.

[0219]

[0220] (13) Analysis of the degradation level of humanized antibodies bound to PTK7-expressing cells

[0221] MDA-MB-468 triple-negative breast cancer cells with high PTK7 expression levels were treated with 10 μg / mL of PTK7 antibodies (52-m, 52-hu-22, and 52-hu-44) on ice for 30 min, washed twice with DMEM medium containing 10% fetal bovine serum, and then incubated in the same medium for 0, 1, 3, 9, and 27 h. After incubation, the level of intracellular antibodies was measured by Western blotting of cell lysates with mouse or human IgG antibodies and GAPDH antibodies to analyze the degree of antibody degradation.

[0222]

[0223] (14) Inclusion analysis of humanized antibodies bound to PTK7-expressing cells

[0224] MDA-MB-468 cells were stained with Lysotracker (50 nM) for 30 min, and then incubated with fluorescein-labeled anti-PTK7 monoclonal antibody 52-m, humanized antibodies 52-hu-22, and 52-hu-44 (10 μg / mL) on ice for 30 min. After washing twice with medium containing 10% fetal bovine serum, the cells were incubated with the same medium for 0, 1, 2, and 4 h. Endocytosis of Lysotracker and antibodies was observed using confocal fluorescence microscopy.

[0225] After staining MDA-MB-468 cells and KYSE-30 cells in the same manner as above, the degree of antibody co-localization with the Lysotracker staining site (lysosome) in cells cultured for 0 and 2 hours was observed.

[0226]

[0227] 2. Experimental Results

[0228] (1) Results of production and purification of anti-PTK7 chimeric antibodies

[0229] Light and heavy chain expression vectors for three chimeric antibodies (32-chi, 43-chi, 52-chi) against three anti-PTK7 monoclonal antibodies (32-m, 43-m, 52-m) were constructed, respectively, transiently expressed and cultured in Expi293 cells, and small amounts were purified using affinity chromatography. The non-reduced and reduced chimeric antibodies (32-chi, 43-chi, 52-chi) were analyzed by SDS-PAGE to confirm their molecular weight and basic properties (Figure 1, top). The chimeric antibodies were analyzed by FPLC and confirmed that all were purified with a purity of 98% or higher (Figure 1, bottom).

[0230]

[0231] (2) Results of antigen binding affinity measurement of anti-PTK7 chimeric antibodies

[0232] The antigen (sPTK7-His) binding affinity of three anti-PTK7 monoclonal mouse antibodies (32-m, 43-m, 52-m) and three chimeric antibodies (32-chi, 43-chi, 52-chi) was measured by the SPR method. When measured by the antigen immobilization (His capture) method, the antigen binding affinity was confirmed in the order 52-chi > 52-m > 43-chi > 43-m > 32-chi > 32-m (Fig. 2), and when measured by the antibody immobilization (antibody capture) method, the antigen binding affinity was confirmed in the order 52-m > 52-chi > 43-m > 43-chi > 32-chi > 32-m (Fig. 3). The binding affinity of the three chimeric antibodies was confirmed to be almost similar to that of the three monoclonal antibodies.

[0233]

[0234] (3) Results of anticancer activity analysis of anti-PTK7 chimeric antibodies at the cellular level

[0235] Wound healing, migration, and invasion assays were performed on mouse monoclonal antibodies (32-m, 43-m, 52-m) and chimeric antibodies (32-chi, 43-chi, 52-chi) selected from KYSE-30 esophageal squamous cell carcinoma cells and MDA-MB-231 triple-negative breast cancer cells. In the case of KYSE-30 cells, the order in wound healing assay was 32-m > 52-m > 52-chi > 43-m > 32-chi > 43-chi, in migration assay was 43-m > 52-m > 32-m > 43-chi > 52-chi > 32-chi, and in invasion assay was 43-m > 32-m > 52-m > 43-chi > 32-chi > 52-chi (Fig. 4). In the case of MDA-MB-231 cells, the order was 32-m > 52-m > 52-chi > 32-chi > 43-m > 43-chi in the wound healing assay, 32-m > 52-m > 43-m > 32-chi > 43-chi > 52-chi in the migration assay, and 52-m > 43-m > 32-m > 43-chi > 52-chi in the invasion assay (Fig. 5). In summary, both monoclonal and chimeric antibodies showed anticancer activity at the cellular level, and although the anticancer activity of the chimeric antibody was somewhat weaker than that of the monoclonal antibody, it was not statistically significant.

[0236] When the biochemical and cellular level analysis results of the chimeric antibody were combined, the antigen binding affinity and cellular level anticancer efficacy of the chimeric antibody 52-chi were the best, and therefore a humanized antibody was produced for it.

[0237]

[0238] (4) Results of production and purification of anti-PTK7 humanized antibodies

[0239] Various humanized antibodies against the anti-PTK7 chimeric antibody (52-chi) were produced (Figs. 5 to 7), and a primary screening was performed by confirming binding at the culture medium level, and a secondary screening was performed by analyzing affinity with the antigen after purifying a small amount. By comprehensively analyzing the physical properties and binding affinity of the humanized antibodies compared to the 52-chi antibody, five humanized antibodies (52-hu-10, 52-hu-22, 52-hu-44, 52-hu-46, 52-hu-59) were selected. The non-reduced and reduced humanized antibodies were analyzed by SDS-PAGE to confirm the molecular weight, and it was confirmed that all humanized antibodies were purified with excellent purity (Fig. 6, top). The humanized antibodies were analyzed by FPLC and it was confirmed that all were purified with a purity of 98% or more (Fig. 6, bottom).

[0240]

[0241] (5) Results of measurement of antigen binding affinity of humanized antibodies

[0242] The antigen (human sPTK7-His) binding affinity of chimeric antibodies (52-chi) and five selected 52-hu antibodies (52-hu-10, -22, -44, -46, -59) was measured using SPR. When the antigen binding affinity of antibodies was measured by the antigen immobilization method, the binding affinity was excellent in the order of 52-chi > 52-hu-10 > 52-hu-22 > 52-hu-46 > 52-hu-44 > 52-hu-59, and when measured by the antibody immobilization method, the binding affinity was excellent in the order of 52-chi > 52-hu-10 > 52-hu-46 > 52-hu-44 > 52-hu-22 > 52-hu-59 (Fig. 7, Fig. 8). Although there were some differences depending on the measurement method, four humanized antibodies (52-hu-10, 52-hu-22, 52-hu-44, and 52-hu-46) had antigen binding affinity (K D ) was excellent at the subnanomolar level or lower.

[0243]

[0244] (6) Results of measuring the anticancer activity of humanized antibodies at the cellular level

[0245] The anticancer efficacy of anti-PTK7 mouse monoclonal antibody 52-m and five humanized antibodies (52-hu-10, -22, -44, -46, -59) used as a control in KYSE-30 esophageal squamous cell carcinoma cells and MDA-MB-231 triple-negative breast cancer cells was analyzed through wound healing, migration, and invasion assays. For KYSE-30 cells, the order was 52-hu-22 > 52-hu-46 > 52-hu-44 > 52-hu-10 > 52-hu-59 in the wound healing assay, 52-hu-46 > 52-hu-44 > 52-hu-22 > 52-hu-10 > 52-hu-59 in the migration assay, and 52-hu-22 > 52-hu-44 > 52-hu-46 > 52-hu-10 > 52-hu-59 in the invasion assay (Fig. 9). In the case of MDA-MB-231 cells, the order was 52-hu-44 > 52-hu-22 > 52-hu-46 > 52-hu-59 > 52-hu-10 in the wound healing assay, 52-hu-22 > 52-hu-44 > 52-hu-46 > 52-hu-59 > 52-hu-10 in the migration assay, and 52-hu-22 > 52-hu-46 > 52-hu-59 > 52-hu-44 > 52-hu-10 in the invasion assay (Fig. 10). These results showed that all anti-PTK7 humanized antibodies selected at the cell level exhibited anticancer activity, and the three anti-PTK7 humanized antibodies with the highest inhibitory activity and the order were 52-hu-22 > 52-hu-44 ≥ 52-hu-46.

[0246] Based on the results of biochemical analysis, including antibody properties and antigen-antibody binding affinity, and the results of cell-level anticancer activity analysis, 52-hu-22 and 52-hu-44 were selected as anti-PTK7 humanized antibodies to be analyzed later.

[0247]

[0248] (7) Results of heterologous cross-reactivity analysis of humanized antibodies

[0249] The cross-species cross-reactivity of anti-PTK7 antibodies was analyzed by antigen binding using SPR. Human, cynomolgus monkey, and mouse sPTK7 were used as antigens, and two anti-PTK7 humanized antibodies (52-hu-22 and 52-hu-44) and Cofetuzumab, which is known to bind to human and cynomolgus monkey, were used as a control. The analysis results showed that anti-PTK7-humanized antibodies 52-hu-22 and 52-hu-44 bound to both human sPTK7 and cynomolgus monkey sPTK7, similar to Cofetuzumab, indicating cross-species cross-reactivity (Fig. 11). However, they did not bind to mouse sPTK7, confirming the absence of cross-species cross-reactivity (Fig. 12).

[0250]

[0251] (8) Results of antigen binding epitope analysis of humanized antibodies and Cofetuzumab

[0252] Using an epitope binning assay, the antigen (human sPTK7-His) binding sites of one anti-PTK7 chimeric antibody (32-chi), two anti-PTK7 humanized antibodies (52-hu-22, 52-hu-44), and Cofetuzumab antibody were analyzed. The epitopes of the two humanized antibodies (52-hu-22 and 52-hu-44) derived from the chimeric antibody 52-chi were confirmed to be identical, whereas the epitopes of the chimeric antibody 32-chi and the two humanized antibodies (52-hu-22, 52-hu-44) were confirmed to be different (Fig. 13). Furthermore, we confirmed that the epitopes of two humanized antibodies (52-hu-22, 52-hu-44) and Cofetuzumab (Pfizer, Binding domains 1–4) were different (Fig. 13). Therefore, it was found that the chimeric antibody 32-chi, two humanized antibodies (52-hu-22, 52-hu-44), and Cofetuzumab identified different epitopes of the PTK7 antigen.

[0253]

[0254] (9) Results of anticancer activity inhibition analysis by humanized antibodies in mice

[0255] The anticancer activity of two PTK7 humanized antibodies (52-hu-22, 52-hu-44) was compared in a mouse model of esophageal squamous cell carcinoma xenografted with KYSE-30 cells and a mouse model of triple-negative breast cancer xenografted with MDA-MB-231 cells. Tumor formation began to be observed after 7 days in the esophageal squamous cell carcinoma mouse model xenografted with KYSE-30 cells, and after 14 days in the triple-negative breast cancer mouse model xenografted with MDA-MB-231 cells. Mice in which tumor formation was observed were injected intraperitoneally with each of two humanized antibodies (52-hu-22, 52-hu-44) at a dose of 10 mg / kg for 3 weeks.

[0256] In the esophageal squamous cell carcinoma mouse model xenografted with KYSE-30 cells, inflammation was observed, and the xenograft tumor was recovered 1 week after antibody treatment. Compared with the control group administered PBS, the weight and volume of the xenograft tumor were significantly reduced in the humanized antibody-administered group, and the inhibitory effect of 52-hu-44 was somewhat superior to that of 52-hu-22 (Fig. 14). In addition, when IHC staining of the proliferation marker Ki-67 molecule in the xenograft tumor, the humanized PTK7 antibody significantly reduced the proportion of Ki-67-positive cells (Fig. 14).

[0257] Triple-negative breast cancer mouse models xenografted with MDA-MB-231 cells recovered xenograft tumors two weeks after antibody treatment. Compared with the PBS-treated control group, the humanized antibody-treated group showed a significant decrease in tumor weight and volume, and the degree of inhibition was similar between 52-hu-22 and 52-hu-44 (Fig. 15). As in the esophageal squamous cell carcinoma model, the humanized PTK7 antibody significantly reduced the proportion of Ki-67-positive cells in the xenograft tumors in the triple-negative breast cancer model (Fig. 15).

[0258]

[0259] (10) Analysis of inhibition of in vivo angiogenesis by humanized antibodies

[0260] To investigate the effect of humanized PTK7 antibodies on in vivo angiogenesis, a Matrigel plug assay was performed. Treatment with mouse VEGF resulted in deep red plugs, indicating angiogenesis induction. Co-treatment with 3 μg / mL of 52-hu-22 or 52-hu-44 and VEGF resulted in slightly lighter red or light red plugs, while co-treatment with 10 μg / mL of 52-hu-22 or 52-hu-44 and VEGF resulted in light orange plugs (Fig. 16). Furthermore, the degree of in vivo angiogenesis was quantified by measuring the hemoglobin (Hb) content within the plugs. As a result, the hemoglobin content in the plugs recovered from the VEGF-treated mice was 14.07 ± 2.22 g / dL, but co-treatment with 3 or 10 μg / mL 52-hu-22 and VEGF reduced the hemoglobin level to 7.01 ± 1.89 or 2.32 ± 0.21 g / dL, respectively. In addition, co-treatment with 3 or 10 μg / mL 52-hu-44 and VEGF reduced the hemoglobin level to 6.61 ± 0.71 or 2.08 ± 0.05 g / dL, respectively (Fig. 16). Therefore, it was confirmed that the two humanized antibodies (52-hu-22 and 52-hu-44) inhibited VEGF-induced angiogenesis in vivo in a dose-dependent manner, and the levels were similar.

[0261]

[0262] (11) Results of a single-dose toxicity test of anti-PTK7 humanized antibody in mice

[0263] Human IgG and anti-PTK7 humanized antibodies 52-hu-22 and 52-hu-44 were administered as a single dose to mice at concentrations of 0, 5, 10, and 50 mg / kg, respectively, and body weight changes and organ weights were measured for 14 days to analyze toxicity.

[0264] During the observation period, no deaths or general abnormalities were observed in any of the test substance-administered groups in mice. Furthermore, no significant changes in body weight were observed in the humanized antibody 52-hu-22 or 52-hu-44 groups compared to the control group (human IgG) (Figure 17). Post-necropsy organ analyses revealed no gross abnormalities in any of the control (human IgG) and humanized antibody 52-hu-22 or 52-hu-44 groups, and no significant changes in organ weight were observed compared to the control group (Figure 18).

[0265] In conclusion, no single-dose toxicity was observed with PTK7 humanized antibodies 52-hu-22 and 52-hu-44, and the lethal dose was determined to exceed 50 mg / kg of humanized antibody.

[0266]

[0267] (12) Pharmacodynamic analysis of anti-PTK7 humanized antibodies in mice

[0268] The indices of activity were analyzed over 6 weeks after a single intravenous administration of anti-PTK7 humanized antibodies 52-hu-22 and 52-hu-44 to mice.

[0269] Based on the mouse blood volume (1 ml per 20 g), the peak blood concentration was predicted to be 200 ug / ml when the antibody was administered at 10 mg / kg, but 52-hu-22 was observed at 1-1.6 times the predicted value, and 52-hu-44 was observed at 1.5-2.2 times the predicted value. In addition, 52-hu-44 showed a higher AUC (actually measured exposure) than 52-hu-22, and the area under the curve was measured higher in the 52-hu-44 administration group than in the 52-hu-22 administration group (Fig. 19).

[0270] In conclusion, when considering the pharmacokinetic parameters (peak blood concentration, area under the curve, etc.), the body exposure of all anti-PTK7 humanized antibodies was higher than expected, and 52-hu-44 was observed to have a higher body exposure than 52-hu-22.

[0271]

[0272] (13) Analysis of the degradation level of humanized antibodies bound to PTK7-expressing cells

[0273] After binding anti-PTK7 mouse antibody (52-m) and humanized antibodies (52-hu-22 and 52-hu-44) at 0°C in MDA-MB-468 triple-negative breast cancer cells that highly express PTK7, the levels of antibodies remaining in the cells at 0, 1, 3, 9, and 27 h at 37°C were quantified using the ImageJ program. When the level of antibody remaining immediately after antibody treatment (0 h) was set to 1.0, it decreased to 0.55, 0.48, and 0.45, respectively, after 9 h of treatment with PTK7 mouse antibody 52-m and humanized antibodies 52-hu-22 and 52-hu-44 (Fig. 20). Based on these results, 52-hu-22 and 52-hu-44 showed slightly higher levels of degradation than 52-m, and the intracellular degradation levels of the two humanized antibodies were similar.

[0274]

[0275] (14) Results of the internalization analysis of humanized antibodies bound to PTK7-expressing cells

[0276] After binding fluorescein-conjugated PTK7 monoclonal antibody 52-m or humanized antibodies 52-hu-22 and 52-hu-44 to MDA-MB-468 cells stained with Lysotracker at 0°C, changes in fluorescence were analyzed over time at 37°C. As a result, it was observed that the mouse antibody and humanized antibody bound to the cells gradually decreased as the incubation time at 37°C elapsed (Fig. 21).

[0277] After 2 hours of incubation at 37°C in MDA-MB-468 cells, antibody staining and Lysotracker staining clearly overlapped. Therefore, we analyzed the changes in fluorescence at 0 and 2 hours after binding the antibody together with Lysotracker staining in MDA-MB-468 triple-negative breast cancer cells and KYSE-30 esophageal squamous cell carcinoma cells. In particular, in MDA-MB-468 cells (Fig. 22) and KYSE-30 cells (Fig. 23) cultured for 2 hours, yellow staining indicating colocalization of fluorescein-conjugated monoclonal antibody 52-m, humanized antibodies 52-hu-22 and 52-hu-44 with Lysotracker was clearly observed, confirming that these antibodies were degraded in lysosomes after endocytosis.

Claims

1. An anti-PTK7 antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7), The above anti-PTK7 antibody is an anti-PTK7 chimeric antibody or an anti-PTK7 humanized antibody, The above anti-PTK7 chimeric antibody or functional fragment thereof i) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 3; and A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 5, a CDR2-VL having an amino acid sequence of SEQ ID NO: 6, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 7; ii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 9, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 10, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising a CDR1-VL comprising the amino acid sequence of SEQ ID NO: 13, a CDR2-VL comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3-VL comprising the amino acid sequence of SEQ ID NO: 15; or iii) An anti-PTK7 antibody or a functional fragment thereof that specifically binds to amino PTK7 (protein tyrosine kinase 7) of SEQ ID NO: 18, comprising a CDR1-VH having an amino acid sequence of SEQ ID NO: 17, The above anti-PTK7 antibody is an anti-PTK7 chimeric antibody or an anti-PTK7 humanized antibody, The above anti-PTK7 chimeric antibody or functional fragment thereof i) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 3; and A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 5, a CDR2-VL having an amino acid sequence of SEQ ID NO: 6, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 7; ii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 9, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 10, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 11; and A light chain variable region comprising a CDR1-VL comprising the amino acid sequence of SEQ ID NO: 13, a CDR2-VL comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3-VL comprising the amino acid sequence of SEQ ID NO: 15; or iii) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and A light chain variable region comprising a CDR1-VL having an amino acid sequence of SEQ ID NO: 21, a CDR2-VL having an amino acid sequence of SEQ ID NO: 22, and a CDR3-VL having an amino acid sequence of SEQ ID NO: 23; The above anti-PTK7 humanized antibody or functional fragment thereof iv) a heavy chain variable region comprising a CDR1-VH consisting of an amino acid sequence of SEQ ID NO: 17, a CDR2-VH consisting of an amino acid sequence of SEQ ID NO: 18, and a CDR3-VH consisting of an amino acid sequence of SEQ ID NO: 19; and A light chain variable region comprising a CDR1-VL comprising an amino acid sequence of SEQ ID NO: 21, a CDR2-VL comprising an amino acid sequence of SEQ ID NO: 22, and a CDR3-VL comprising an amino acid sequence of SEQ ID NO: 23; characterized in that it comprises; Anti-PTK7 antibody or a functional fragment thereof.

2. In claim 1, An anti-PTK7 antibody or functional fragment thereof, characterized in that the anti-PTK7 chimeric antibody or functional fragment thereof comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 4, 12, or 20, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 8, 16, or 24.

3. In claim 1, An anti-PTK7 antibody or functional fragment thereof, characterized in that the anti-PTK7 humanized antibody or functional fragment thereof comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 25, 26 or 27, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 28, 29, 30 or 31.

4. In claim 1, An anti-PTK7 antibody or a functional fragment thereof, characterized in that the antibody is at least one selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, and the functional fragment is at least one selected from the group consisting of diabody, Fab, F(ab'), F(ab')2, Fv, dsFv, and scFv.

5. In claim 1, An anti-PTK7 antibody or a functional fragment thereof, characterized in that the antibody or a functional fragment thereof inhibits at least one selected from the group consisting of wound healing, chemotactic migration, and invasion.

6. In claim 1, An anti-PTK7 antibody or a functional fragment thereof, characterized in that the antibody or a functional fragment thereof reduces the level of hemoglobin (Hb) in a tissue.

7. In claim 1, An anti-PTK7 antibody or functional fragment thereof, characterized in that the antibody or functional fragment thereof inhibits the interaction of PTK7 and a receptor that binds to PTK7.

8. In claim 1, An anti-PTK7 antibody or a functional fragment thereof, characterized in that the antibody or a functional fragment thereof inhibits cancer growth.

9. A polynucleotide encoding the antibody of claim 1 or a functional fragment thereof.

10. A vector comprising the polynucleotide of claim 9.

11. A cell transformed with the vector of claim 10.

12. A step of culturing the cell of claim 11 to produce a polypeptide comprising light chain and heavy chain variable regions; and A method for producing an antibody or a functional fragment thereof that specifically binds to PTK7 (protein tyrosine kinase 7), comprising a step of recovering the polypeptide from the cell or a culture medium in which the cell is cultured.

13. An angiogenesis inhibitor comprising the anti-PTK7 antibody of claim 1 or a functional fragment thereof.

14. A pharmaceutical composition for preventing or treating angiogenesis-related disease, comprising the angiogenesis inhibitor of claim 13.

15. In claim 14, A pharmaceutical composition characterized in that the above angiogenesis-related disease is at least one selected from the group consisting of cancer, endometriosis, obesity, arthritis, arteriosclerosis, hemangioma, angiofibroma, vascular malformation, vascular adhesion, edematous sclerosis, diabetic retinopathy, macular degeneration, angiogenic glaucoma, corneal disease caused by angiogenesis, psoriasis, telangiectasia, pyogenic granuloma, seborrheic dermatitis, and Alzheimer's disease.

16. An inhibitor of growth, migration or invasion of tumor cells comprising the anti-PTK7 antibody of claim 1 or a functional fragment thereof.

17. A pharmaceutical composition for preventing or treating cancer, comprising a growth, migration or invasion inhibitor of tumor cells according to claim 16.

18. In claim 17, A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer is at least one selected from the group consisting of glioblastoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, renal cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer.

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