Humanized anti-GPC-1 antibody

Abstract

The present disclosure provides a humanized antibody specifically binding to glypican-1 (GPC-1) and a technique, a method, a drug, etc., each related thereto. In one aspect, the present disclosure provides a humanized anti-GPC-1 monoclonal antibody that has high affinity to GPC-1 and exhibits high internalization activity in GPC-1 positive cells. In another aspect, the present disclosure provides a composition for preventing or treating glypican-1-positive cancer, said composition comprising a complex of the humanized anti-GPC-1 monoclonal antibody with a drug having cytotoxic activity.

Description

[Technical field]

[0001] The present disclosure relates to humanized antibodies that specifically bind to Glypican-1 (GPC-1), as well as related techniques, methods, agents, and the like. [Background technology]

[0002] It has been found that the Glypican-1 molecule is expressed significantly more strongly in esophageal cancer cells than in normal cells, and can be used as a tumor marker (Patent Document 1). In addition, an antibody that specifically binds to Glypican-1 has also been isolated (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2015 / 098112 Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the present disclosure provides a humanized anti-GPC-1 monoclonal antibody that has high affinity to GPC-1 and has high internalization activity in GPC-1 positive cells.Humanized anti-GPC-1 antibodies have not been developed so far, and it was not expected that humanized anti-GPC-1 antibodies would have high activity.The antibody of the present disclosure can be used for various therapeutic applications.

[0005] Therefore, in another aspect, the present disclosure also provides a composition for preventing or treating Glypican-1 positive cancer, comprising a conjugate of a humanized anti-GPC-1 monoclonal antibody and a drug having cytotoxic activity.

[0006] Such antibodies or fragments thereof can be used as companion diagnostic agents and targeted therapies or personalized medicines in combination with companion diagnostic agents.

[0007] Based on the above, the present disclosure provides, for example, the following items. (Item 1) A humanized anti-GPC-1 antibody or an antigen-binding fragment thereof, which is humanized based on an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:6, and which has a higher affinity for GPC-1 than an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:6. (Item 2) the humanized antibody or antigen-binding fragment thereof comprising: (a) comprising heavy chain CDRs 1 to 3 having the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively; or (b) (a) has three or less amino acid substitutions, deletions and / or additions in the heavy chain CDRs 1 to 3; Item 1. The composition according to item 1. (Item 3) the humanized antibody or antigen-binding fragment thereof comprising: (a) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; or (b) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively; or (c) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; or (d) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 40 to 42, respectively; or (e) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 43 to 45, respectively; or (f) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 46 to 48, respectively; or (g) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 49 to 51, respectively; or (h) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 52 to 54, respectively; or (i) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 55 to 57, respectively; or (j) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively; or (k) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 61 to 63, respectively; or (l) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 64 to 66, respectively; or (m) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 67 to 69, respectively; or (n) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 70 to 72, respectively; or (o) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 73 to 75, respectively; or (p) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 76 to 78, respectively; or (q) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 79 to 81, respectively; or (r) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 82 to 84, respectively; or (s) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 85 to 87, respectively; or (t) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 88 to 90, respectively; or (u) having three or less amino acid substitutions, deletions and / or additions in light chain CDR1 to CDR3 in any of (a) to (t); 3. The humanized antibody or antigen-binding fragment thereof according to item 1 or 2. (Item 4) 4. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 3, comprising a heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7. (Item 5) 5. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 4, comprising a light chain variable region having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8 to 27. (Item 6) 6. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 5, comprising a light chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8 to 27. (Item 7) 7. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 6, comprising a light chain variable region having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8 to 17. (Item 8) 8. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 7, comprising a light chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8 to 17. (Item 9) 9. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein the epitope of the antibody comprises positions 339 to 358 and / or positions 388 to 421 of SEQ ID NO: 2. (Item 10) The antibody has a molecular weight of 1.02E based on analysis by surface plasmon resonance. -8 The following K D 10. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 9, which binds to Glypican-1 at (Item 11) 11. A pharmaceutical composition for intracellular internalization of an active ingredient, comprising the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 10. (Item 12) 11. A conjugate of the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 10 and a drug having cytotoxic activity. (Item 13) The conjugate according to item 12, wherein the humanized antibody or antigen-binding fragment thereof is operably linked to the agent having cytotoxic activity via a linker. (Item 14) 14. The conjugate according to item 12 or 13, wherein the agent having cytotoxic activity is selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), DM1, DM4, calicheamicin, duocarmycin, pyrrolobenzodiazepines (PBDs), and topoisomerase inhibitors. (Item 15) 15. The conjugate according to item 13 or 14, wherein the agent having cytotoxic activity is cell membrane permeable. (Item 16) The conjugate according to any one of items 13 to 15, wherein the drug having cytotoxic activity is selected from MMAE, PBD, Eribulin, SN-38, Dxd, and DM4. (Item 17) 17. The conjugate according to any one of items 13 to 16, wherein the linker is selected from the group consisting of an enzyme-cleavable linker, an acid-labile linker, and a disulfide linker. (Item 18) 18. The conjugate according to any one of items 13 to 17, wherein the linker is a cleavable linker that is cleaved by cathepsin B. (Item 19) The complex has an IC of about 0.1 nM or less in Glypican-1 positive cells. 50 19. The complex according to any one of items 12 to 18, wherein (Item 20) A composition for preventing or treating Glypican-1 positive cancer, comprising the complex according to any one of items 12 to 19. (Item 21) 21. The composition of item 20, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 22) 22. The composition according to item 20 or 21, wherein the Glypican-1 positive cancer is esophageal cancer or pancreatic cancer. (Item 23) 23. The composition according to any one of items 20 to 22, wherein the cancer has cancer-associated fibroblasts (CAFs). (Item 24) 24. The composition according to any one of items 20 to 23, characterized in that the composition is administered together with an immune checkpoint inhibitor. (Item 25) 25. The composition of claim 24, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. (Item 26) 20. A composition for preventing or treating metastatic cancer of a Glypican-1 positive cancer, comprising the complex according to any one of items 12 to 19. (Item 27) 27. The composition of item 26, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 28) 28. The composition of claim 26 or 27, wherein the metastatic cancer is a metastatic cancer of pancreatic cancer. (Item 29) 29. The composition according to any one of items 26 to 28, wherein the metastatic cancer is a metastatic cancer that has metastasized to the liver, esophagus, bile duct, cervix, lung, head and neck, breast, uterine smooth muscle, prostate, oral squamous epithelium, brain, bone, peritoneum, or adrenal gland. (Item 1A) A method for intracellular internalization of an active ingredient, the method comprising the step of administering to a cell the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 10. (Item 2A) 20. A method for preventing or treating Glypican-1 positive cancer in a subject, the method comprising administering to the subject the complex according to any one of items 12 to 19. (Item 3A) The method according to item 2A, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 4A) The method according to item 2A or 3A, wherein the Glypican-1 positive cancer is esophageal cancer or pancreatic cancer. (Item 5A) The method according to any one of items 2A to 4A, wherein the cancer has cancer-associated fibroblasts (CAFs). (Item 6A) The method according to any one of items 2A to 5A, further comprising administering an immune checkpoint inhibitor. (Item 7A) The method of item 6A, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. (Item 8A) 20. A method for preventing or treating metastatic cancer of a Glypican-1 positive cancer in a subject, the method comprising a step of administering to the subject a complex according to any one of items 12 to 19. (Item 9A) The method of item 8A, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 10A) The method according to item 8A or 9A, wherein the metastatic cancer is a metastatic cancer of a pancreatic cancer. (Item 11A) The method according to any one of items 8A to 10A, wherein the metastatic cancer is a metastatic cancer that has metastasized to the liver, esophagus, bile duct, cervix, lung, head and neck, breast, uterine smooth muscle, prostate, oral squamous epithelium, brain, bone, peritoneum, or adrenal gland. (Item 1B) 11. Use of the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 10 in the manufacture of a medicament for intracellular internalization of an active ingredient. (Item 2B) 20. Use of the conjugate according to any one of items 12 to 19 in the manufacture of a medicament for preventing or treating Glypican-1 positive cancer in a subject. (Item 3B) The use according to item 2B, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 4B) The use according to item 2B or 3B, wherein the Glypican-1 positive cancer is esophageal cancer or pancreatic cancer. (Item 5B) The use according to any one of items 2B to 4B, wherein the cancer has cancer-associated fibroblasts (CAFs). (Item 6B) The use according to any one of items 2B to 5B, characterized in that the complex is administered together with an immune checkpoint inhibitor. (Item 7B) The use according to item 6B, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. (Item 8B) 20. Use of the conjugate according to any one of items 12 to 19 in the manufacture of a medicament for preventing or treating metastatic cancer of a Glypican-1 positive cancer in a subject. (Item 9B) The use according to item 8B, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 10B) The use according to item 8B or 9B, wherein the metastatic cancer is a metastatic cancer of a pancreatic cancer. (Item 11B) The use according to any one of items 8B to 10B, wherein the metastatic cancer is a metastatic cancer that has metastasized to the liver, esophagus, bile duct, cervix, lung, head and neck, breast, uterine smooth muscle, prostate, oral squamous epithelium, brain, bone, peritoneum, or adrenal gland. (Item 1C) 11. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 10, for intracellular internalization of an active ingredient. (Item 2C) The conjugate according to any one of items 12 to 19, for preventing or treating Glypican-1 positive cancer in a subject. (Item 3C) The conjugate according to item 2C, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 4C) The conjugate according to item 2C or 3C, wherein the Glypican-1 positive cancer is esophageal cancer or pancreatic cancer. (Item 5C) The complex according to any one of items 2C to 4C, wherein the cancer has cancer-associated fibroblasts (CAFs). (Item 6C) The conjugate according to any one of items 2C to 5C, wherein the conjugate is administered together with an immune checkpoint inhibitor. (Item 7C) The conjugate according to item 6C, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. (Item 8C) The conjugate according to any one of items 12 to 19, for preventing or treating metastatic cancer of a Glypican-1 positive cancer in a subject. (Item 9C) The conjugate according to item 8C, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. (Item 10C) The conjugate according to item 8C or 9C, wherein the metastatic cancer is a metastatic cancer of pancreatic cancer. (Item 11C) The conjugate according to any one of items 8C to 10C, wherein the metastatic cancer is a metastatic cancer that has metastasized to the liver, esophagus, bile duct, cervix, lung, head and neck, breast, uterine smooth muscle, prostate, oral squamous epithelium, brain, bone, peritoneum, or adrenal gland.

[0008] It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. Effect of the Invention

[0009] According to the present disclosure, a clinically applicable humanized anti-Glypican-1 antibody is provided, which can be used as a detection agent for the diagnosis and companion therapy of Glypican-1 positive cancers, and further, a conjugate of the antibody with a drug is highly effective in the treatment of Glypican-1 positive cancers. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the equilibrium dissociation constants (KD) of 20 clones measured by Biacore®. [Diagram 2] FIG. 2 shows a schematic diagram of the ADC assay of Example 2. [Diagram 3] FIG. 3 shows the results of the cytotoxic activity (IC50) of 20 clones in the TE14 cell line. [Figure 4] FIG. 4 shows the results of antigen affinity analysis by FACS of the anti-GPC-1 chimeric antibody (01a033) and clone T2. [Diagram 5] FIG. 5 shows a schematic diagram of the structure of an exemplary ADC. [Figure 6]FIG. 6 shows a schematic diagram of the Drug-to-Antibody Ratio (DAR) setting. [Figure 7] FIG. 7 shows the results of antigen affinity analysis by FACS of humanized GPC1-ADC and unlabeled antibody. [Figure 8] Figure 8 shows the results of intracellular internalization assay of humanized anti-GPC-1 antibody (clone T2) and humanized GPC1-ADC (MMAE). Green indicates staining of GPC1, red indicates staining of CD107a, and blue indicates staining of DAPI. Scale indicates 10 μm. The left bar of each group in the graph indicates 4 ° C, and the right bar indicates 37 ° C. [Figure 9] FIG. 9 shows the results of GPC1 expression analysis by FACS in various cell lines. [Figure 10] FIG. 10 shows the results of an in vitro ADC assay using humanized GPC1-ADC(MMAE). [Figure 11] FIG. 11 shows the results of GPC1 expression analysis and ADC assay in HeLa, Detroit562, and FaDu cell lines. [Figure 12] FIG. 12 shows the results of GPC1 expression analysis and ADC assay in Ho-1-u-1, KOSC2 cl3-43, and KON cell lines. [Figure 13] Figure 13 shows the results of an in vivo efficacy test of humanized GPC1-ADC (MMAE) using pancreatic cancer PDX (PK565) with heterogeneous GPC1 expression. There was no significant change in body weight change in any group. Tumor volume was almost unchanged in humanized GPC1-ADC (10 mg / kg), slightly increased in humanized GPC1-ADC (3 mg / kg), then increased in humanized GPC-ADC (1 mg / kg) and Control-ADC (10 mg / kg), with almost the same magnitude, and increased most in the vehicle control. [Figure 14]Figure 14 shows the results of an in vivo efficacy test of humanized GPC1-ADC (MMAE) using pancreatic cancer PDX (PK175) in which GPC1 is expressed in both cancer cells and CAFs. Body weight increased slightly in humanized GPC1-ADC (10 mg / kg) and humanized GPC1-ADC (3 mg / kg). There was no significant change in humanized GPC-ADC (1 mg / kg) and vehicle control. Tumor volume decreased in humanized GPC1-ADC (10 mg / kg), being the smallest, increased slightly in humanized GPC1-ADC (3 mg / kg), then gradually increased in humanized GPC-ADC (1 mg / kg) and vehicle control, with vehicle control being the largest. [Figure 15] Figure 15 shows the results of an in vivo efficacy test of humanized GPC1-ADC (MMAE) using esophageal cancer PDX (ESCC14) in which GPC1 is uniformly and highly expressed. There was no significant change in body weight in any group. Tumor volume was similar and smallest in humanized GPC1-ADC (10 mg / kg) and humanized GPC1-ADC (3 mg / kg), followed by humanized GPC-ADC (1 mg / kg), which was almost flat, and gradually increased in the vehicle control. [Figure 16] FIG. 16 shows the results of GPC1 expression analysis and ADC assay in a mouse colon cancer cell line (mGPC1-MC38) overexpressing mGPC1. [Figure 17] FIG. 17 shows the results of quantification of HMGB-1 in culture supernatants by ELISA. [Figure 18] Figure 18 shows the results of the antitumor effect of the combined use of humanized GPC1-ADC (MMAE) and anti-PD1 antibody using a colorectal cancer syngenic model mouse. There was no significant change in body weight in any of the groups. The tumor volume was smallest in the humanized GPC1-ADC (10 mg / kg) + anti-PD1 Ab (3 mg / kg) group, followed by anti-PD1 Ab (3 mg / kg), humanized GPC1-ADC (10 mg / kg), and vehicle control. [Figure 19]Figure 19 shows the results of measuring the G2 / M phase cell cycle marker (phospho-Histone H3 (Ser10)) in pancreatic cancer PDX (PK565). The scale bar indicates 100 μm. One-way ANOVA after Dunnett's post hoc test, **P<0.01. [Figure 20] Figure 20 shows the results of immunohistochemical staining of GPC1 expression in pancreatic cancer distant metastasis tissues. The scale bar indicates 100 μm. [Figure 21] Figure 21 shows an overview of the creation of a pancreatic cancer liver metastasis model and the efficacy test using humanized GPC1-ADC. IV administration was performed on days 0, 3, 7, and 10. IVIS measurements were performed on days 0, 7, 14, 21, 28, 35, 42, and 49. [Figure 22] Figure 22 shows the results of a drug efficacy test using humanized GPC1-ADC in a pancreatic cancer liver metastasis model. PBS administration group: n=13, GPC1-ADC 10 mg / kg administration group: n=10, *: p<0.05, **: p<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure will be described below. Throughout this specification, the singular expression should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence. "About" means ±10% of the indicated value.

[0012] (definition) First, terms and general techniques used in this disclosure are explained.

[0013] As used herein, "Glypican-1", "GPC-1" or "GPC1" are terms used interchangeably and are glycosylphosphatidylinositol (GPI)-anchored cell surface proteoglycans that contain heparan sulfate. They are believed to be involved in cell adhesion, migration, lipoprotein metabolism, growth factor activity regulation and blood coagulation inhibition. They are said to bind to several fibroblast growth factors (FGFs), such as FGF-1, FGF-2 and FGF-7. Glypican-1 is believed to function as an extracellular chaperone for VEGF165, helping to restore receptor binding ability after oxidation. Six types of glypican are currently known, Glypican-1 to Glypican-6, but in terms of their relationship to cancer, even if they are members of the Glypican family, they are not necessarily recognized as cancer markers, and the members appear to be unrelated to each other. Glypican-1 is registered in UniProt under the accession number P35052 (see http: / / www.uniprot.org / uniprot / P35052), and is also registered in NCBI under NP_002072.2 (precursor amino acid sequence), NM_002081.2 (mRNA), and in EMBL, GenBank and DDBJ under X54232.1 (mRNA), BC051279.1 (mRNA), and AC110619.3 (genomic). All of these are information that can be used in the present specification, and the information is incorporated herein by reference. For Glypican-1, see David G et al., J Cell Biol. 1990 Dec;111(6 Pt 2):3165-76; Haecker U et al., Nat Rev Mol Cell Biol. 2005 Jul;6(7):530-41; Aikawa T et al., J Clin Invest. 2008 Jan;118(1):89-99.; Matsuda K, et al., Cancer Res. 2001 Jul 15;61(14):5562-9. etc. A representative example of the nucleic acid sequence (full length) of human Glypican-1 is SEQ ID NO:1, and a representative example of the amino acid sequence is SEQ ID NO:2.Furthermore, a representative example of the nucleic acid sequence (full length) of mouse Glypican-1 is SEQ ID NO: 3, and a representative example of the amino acid sequence is SEQ ID NO: 4. As used for purposes of this specification, "Glypican-1", "GPC-1" or "GPC1" refers not only to a protein having an amino acid sequence set forth in a particular SEQ ID NO or accession number (or a nucleic acid encoding the same), but also to a functionally active analog or derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a mutant encoded by a nucleic acid that hybridizes to a nucleic acid encoding the protein under high or low stringency conditions, as long as it meets the specific purpose of this disclosure.

[0014] As used herein, "derivative", "analog" or "variant" preferably includes, but is not limited to, molecules that contain a region substantially homologous to the protein of interest (e.g., Glypican-1, antibodies), which in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical over the same size amino acid sequence or when compared to sequences aligned by computer homology programs known in the art, or the nucleic acid encoding such a molecule is hybridizable to sequences encoding the component protein under (highly) stringent, moderately stringent or non-stringent conditions. This refers to proteins that are the product of protein modification by amino acid substitutions, deletions and additions, respectively, and whose derivatives still exhibit the biological functions of the original protein, although not necessarily to the same degree. For example, the biological function of such proteins can be examined by suitable available in vitro assays described herein or known in the art. "Functionally active" as used herein refers to a polypeptide, i.e., a fragment or derivative, that has a structural, regulatory, or biochemical function of a protein, such as a biological activity, according to the embodiment to which the polypeptide, i.e., fragment or derivative of the present disclosure relates.

[0015] In this disclosure, Glypican-1 is mainly discussed in humans, but since many animals other than humans, such as chimpanzees (Pantroglodytes) (K7B6W5), rhesus monkeys (Macaca mulatta) (F6VPW9), mice (Mus musculus) (Q9QZF2), rats (Rattus norvegicus) (P35053), and chickens (Gallus gallus) (F1P150), are known to express Glypican-1 protein, it is understood that these animals, particularly mammals, are also within the scope of this disclosure. Preferably, functional domains of Glypican-1, such as the extracellular domain (approximately 500 amino acids and including 12 cysteine ​​residues) and the C-terminal hydrophobic region (GPI-anchor domain), are conserved.

[0016] As used herein, "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety). The definition also includes, for example, polypeptides containing one or more analogs of amino acids (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" is a collective term for an organic compound having an amino group and a carboxyl group. When an antibody according to an embodiment of the present disclosure includes a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Any of the amino acids in the amino acid sequence may form a salt or a solvate. Any of the amino acids in the amino acid sequence may be L-type or D-type. In such cases, the protein according to the embodiment of the present disclosure may contain the above-mentioned "specific amino acid sequence". Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, sugar chain addition, etc.). The amino acids may be natural or unnatural as long as they satisfy the purpose of the present disclosure.

[0017] As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. This term also includes "oligonucleotide derivative" or "polynucleotide derivative". "Oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that contains a derivative of nucleotides or has a bond between nucleotides that is different from the usual one, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in an oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in an oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in an oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which the uracil in an oligonucleotide has been replaced with C-5 propynyl uracil, oligonucleotide derivatives in which the uracil in an oligonucleotide has been replaced with C-5 thiazole uracil, oligonucleotide derivatives in which the cytosine in an oligonucleotide has been replaced with C-5 propynyl cytosine, oligonucleotide derivatives in which the cytosine in an oligonucleotide has been replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA has been replaced with 2'-O-propyl ribose, and oligonucleotide derivatives in which the ribose in an oligonucleotide has been replaced with 2'-methoxyethoxy ribose. Unless otherwise indicated, a particular nucleic acid sequence is also intended to encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" may be natural or non-natural.

[0018] As used herein, the term "gene" refers to a factor that determines a genetic trait, and may refer to a "polynucleotide," an "oligonucleotide," and a "nucleic acid."

[0019] As used herein, the term "homology" of a gene refers to the degree of identity between two or more gene sequences, and generally, "homology" refers to a high degree of identity or similarity. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes have homology can be determined by direct comparison of sequences, or, in the case of nucleic acids, by hybridization methods under stringent conditions. When two gene sequences are compared directly, the genes have homology if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical. Thus, as used herein, "homolog" or "homologous gene product" refers to a protein in another species, preferably a mammal, that exerts the same biological function as a protein component of a complex as further described herein. Such a homolog may also be referred to as an "orthologous gene product." It is understood that such homologs, homologous gene products, orthologous gene products, etc. can also be used so long as they are consistent with the purposes of the present disclosure.

[0020] Amino acids may be referred to herein by either their commonly known three letter symbols or the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one letter codes. In this specification, the comparison of similarity, identity and homology of amino acid and base sequences is calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST2.2.28 (published 2013.4.2). The identity value in this specification usually refers to the value when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value is taken as the identity value. When identity is evaluated in multiple regions, the highest value among them is taken as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.

[0021] In one embodiment of the present disclosure, "90% or more" may be, for example, 90, 95, 96, 97, 98, 99, or 100% or more, or may be within the range of any two of these values. The above-mentioned "homology" may be calculated by calculating the ratio of the number of homologous amino acids in two or more amino acid sequences according to a method known in the art. Before calculating the ratio, the amino acid sequences of the amino acid sequences to be compared are aligned, and gaps are introduced into a part of the amino acid sequence if necessary to maximize the ratio of identical amino acids. Methods for alignment, methods for calculating the ratio, methods for comparison, and computer programs related thereto are conventionally well known in the art (e.g., BLAST, GENETYX, etc.). In this specification, "homology" can be expressed as a value measured by NCBI's BLAST unless otherwise specified. For the algorithm when comparing amino acid sequences with BLAST, Blastp can be used with default settings. The measurement results are quantified as Positives or Identities.

[0022] As used herein, the term "functional equivalent" refers to any entity that has the same intended function but a different structure compared to the original entity of interest. Thus, it is understood that the functional equivalent of "Glypican-1" or its antibody is not Glypican-1 or its antibody itself, but includes mutants or variants of Glypican-1 or its antibody (e.g., amino acid sequence variants, etc.) that have the biological action of Glypican-1 or its antibody, as well as those that can be changed into Glypican-1 or its antibody itself or mutants or variants of this Glypican-1 or its antibody at the time of acting (e.g., nucleic acid encoding Glypican-1 or its antibody itself or mutants or variants of Glypican-1 or its antibody, and vectors, cells, etc. containing the nucleic acid). In the present disclosure, it is understood that the functional equivalent of Glypican-1 or its antibody can be used in the same way as Glypican-1 or its antibody, even if not specifically mentioned. Functional equivalents can be found by searching databases, etc. As used herein, the term "search" refers to the use of a nucleic acid sequence to find other nucleic acid sequences with specific functions and / or properties, either electronically or by biological or other methods. Electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)).Biological searches include, but are not limited to, stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays in which genomic DNA is attached to a glass plate (microarray assay), PCR, in situ hybridization, etc. In this specification, it is intended that the gene used in this disclosure should also include corresponding genes identified by such electronic searches and biological searches.

[0023] As the functional equivalent of the present disclosure, an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both ends thereof can be used. In the present specification, "an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both ends thereof" means that the amino acid sequence has been modified by a well-known technical method such as site-directed mutagenesis, or by natural mutation, by substitution of a number of amino acids to the extent that would occur naturally. The modified amino acid sequence may be, for example, one in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted or deleted, or added to one or both ends thereof. The modified amino acid sequence may preferably be an amino acid sequence having one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of Glypican-1. Here, "conservative substitution" means replacing one or more amino acid residues with other chemically similar amino acid residues so as not to substantially alter the function of the protein. For example, a hydrophobic residue may be replaced with another hydrophobic residue, a polar residue may be replaced with another polar residue having the same charge, and the like. Functionally similar amino acids that can be used for such substitutions are known in the art for each amino acid. Specific examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0024] In one embodiment of the present disclosure, "anti-Glypican-1 antibody" includes an antibody that has binding ability to Glypican-1. The method for producing this anti-Glypican-1 antibody is not particularly limited, and for example, the antibody may be produced by immunizing a mammal or bird with Glypican-1. An "antigen-binding fragment" of an anti-Glypican-1 antibody refers to a fragment of the antibody that retains the ability to bind to Glypican-1. Examples of such antigen-binding fragments include, but are not limited to, single-chain antibodies, scFv, Fab fragments, or F(ab') 2 Fragments, etc.

[0025] The antibody class of the anti-Glypican-1 antibody according to one embodiment of the present disclosure is not particularly limited, and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.

[0026] The anti-Glypican-1 antibody according to one embodiment of the present disclosure may be an antibody fragment having antigen-binding activity (hereinafter, also referred to as an "antigen-binding fragment"). In this case, there are effects such as increased stability or antibody production efficiency.

[0027] The anti-Glypican-1 antibody according to one embodiment of the present disclosure may be a fusion protein. This fusion protein may comprise an anti-Glypican-1 antibody bound to a polypeptide or oligopeptide at its N- or C-terminus. The oligopeptide may be a His tag.

[0028] The anti-Glypican-1 antibody according to one embodiment of the present disclosure may be, for example, an antibody obtained by a process of immunizing an organism with purified Glypican-1, Glypican-1-expressing cells, or a lipid membrane containing Glypican-1. From the viewpoint of enhancing the therapeutic effect against Glypican-1-positive cancer, it is preferable to use Glypican-1-expressing cells for immunization.

[0029] The anti-Glypican-1 antibody according to one embodiment of the present disclosure may be an antibody having a CDR set of an antibody obtained by a process of immunizing an organism with purified Glypican-1, Glypican-1-expressing cells, or a lipid membrane containing Glypican-1. From the viewpoint of enhancing the therapeutic effect against Glypican-1-positive cancer, it is preferable to use Glypican-1-expressing cells for immunization. The CDR set is a set of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3.

[0030] In one embodiment of the present disclosure, the "Glypican-1 expressing cells" may be obtained, for example, by introducing a polynucleotide encoding Glypican-1 into a cell and then expressing Glypican-1. Here, Glypican-1 includes a Glypican-1 fragment. In addition, in one embodiment of the present disclosure, the "Glypican-1-containing lipid membrane" may be obtained, for example, by mixing Glypican-1 with a lipid bilayer membrane. Here, Glypican-1 includes a Glypican-1 fragment. In addition, from the viewpoint of enhancing the therapeutic effect against Glypican-1-positive cancer, the anti-Glypican-1 antibody according to one embodiment of the present disclosure is preferably an antibody obtained via a process of immunizing chickens with an antigen, or an antibody having a CDR set of such an antibody.

[0031] The anti-Glypican-1 antibody of the present disclosure may have any binding strength as long as the objective is achieved. For example, even if the anti-Glypican-1 antibody of the present disclosure has low affinity for Glypican-1, it is sufficient as long as it has internalization activity and / or ADC activity. However, for diagnostic or companion reagent purposes, strong binding strength is preferable. For example, a KD value (kd / ka) of 1.0×10 -7 (M) or less, 1.0×10 -8 (M) or less, 1.0×10 -9 (M) or less or 1.0×10 -10(M) or less. Preferably, the binding affinity of the anti-Glypican-1 antibody for diagnostic or companion reagent purposes is less than or equal to 1.0×10 in terms of KD value (kd / ka). -8 (M) or less.

[0032] The anti-Glypican-1 antibody according to one embodiment of the present disclosure may be an antibody that binds to wild-type or mutant Glypican-1. Mutant types include those resulting from differences in DNA sequences between individuals. The amino acid sequence of wild-type or mutant Glypican-1 has a homology of preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more to the amino acid sequence shown in SEQ ID NO:2.

[0033] As used herein, the term "antibody" includes a molecule or a group thereof that can specifically bind to a specific epitope on an antigen. Antibodies can exist in various forms, and may be in one or more forms selected from the group consisting of full-length antibodies (antibodies having a Fab region and an Fc region), Fv antibodies, Fab antibodies, F(ab')2 antibodies, Fab' antibodies, diabodies, single-chain (single-chain) antibodies (e.g., scFv), sc(Fv)2 (single chain (Fv)2), scFv-Fc, dsFv, multivalent specific antibodies (e.g., oligospecific antibodies, bivalent specific antibodies), diabodies, peptides or polypeptides having antigen-binding properties, chimeric antibodies (e.g., mouse-human chimeric antibodies, chicken-human chimeric antibodies, etc.), mouse antibodies, chicken antibodies, humanized antibodies, human antibodies, or equivalents thereof (or equivalents). Antibodies also include modified or unmodified antibodies. Modified antibodies may be antibodies bound to various molecules, such as polyethylene glycol. The modified antibody can be obtained by chemically modifying the antibody using a known method. Furthermore, such an antibody may be covalently bound or recombinantly fused to an enzyme, such as alkaline phosphatase, horseradish peroxidase, α-galactosidase, etc. The anti-Glypican-1 antibody used in the present disclosure may be of any origin, type, shape, etc., as long as it binds to the Glypican-1 protein. Specifically, known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. The antibody is preferably specifically bound to the Glypican-1 protein. The antibody may be a modified or unmodified antibody. The modified antibody may be bound to various molecules, such as polyethylene glycol. The modified antibody can be obtained by chemically modifying the antibody using a known method.

[0034] In one embodiment of the present disclosure, a "monoclonal antibody" includes a population of antibodies in which each antibody corresponds to a substantially single epitope, except for antibodies with a small amount of naturally occurring mutations. Alternatively, each antibody in the population may be substantially identical, except for antibodies with a small amount of naturally occurring mutations. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain different antibodies corresponding to different epitopes. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized from a hybridoma culture that is not contaminated by other immunoglobulins. The term "monoclonal" may indicate the characteristic of being obtained from a substantially homogeneous antibody population, but does not imply that the antibody must be produced by any particular method. For example, monoclonal antibodies may be produced by methods similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug 7;256(5517):495-497." Alternatively, the monoclonal antibodies may be produced by recombinant methods similar to those described in U.S. Patent No. 4,816,567. Alternatively, the monoclonal antibodies may be isolated from phage antibody libraries using techniques similar to those described in "Clackson et al., Nature. 1991 Aug 15;352(6336):624-628," or "Marks et al., J MolBiol. 1991 Dec 5;222(3):581-597." Alternatively, the monoclonal antibodies may be produced by the methods described in "Protein Experiment Handbook, Yodosha (2003):92-96."

[0035] Any method known in the art can be used for the mass production of antibodies, but the following can be exemplified as a representative example of the construction of a mass production system for antibodies and the production of antibodies. That is, an H-chain antibody expression vector and an L-chain antibody expression vector are transfected into CHO cells, cultured using the selection reagents G418 and Zeocin, and cloned by limiting dilution. After cloning, clones that stably express the antibody are selected by ELISA. The selected CHO cells are expanded and the culture supernatant containing the antibody is collected. The antibody can be purified from the collected culture supernatant by Protein A or Protein G purification. The humanized antibody of the present disclosure can be obtained as a polyclonal, fully human antibody by immunizing a human antibody-producing mouse with the desired antigen, and can be used in the present disclosure after being converted to a monoclonal state.

[0036] In one embodiment of the present disclosure, a "humanized antibody" is an antibody that has, for example, one or more CDRs from a non-human species, a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin, and binds to a desired antigen. Antibody humanization can be performed using various techniques known in the art (Almagro et al., Front Biosci. 2008 Jan 1;13:1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1; 93(11): 3922-3930.), Re-surfacing (Roguska et al., Proc Natl Acad Sci US A. 1994 Feb 1; 91(3): 969-973.), or FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr; 44(11): 3049-3060. Epub 2007 Jan 22.) can be used. To alter (preferably improve) antigen binding, amino acid residues in the human FR region may be replaced with corresponding residues from the CDR donor antibody. This FR replacement can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24; 332(6162): 323-327.). For example, FR residues important for antigen binding may be identified by modeling the interaction of CDR and FR residues. Alternatively, unusual FR residues at specific positions may be identified by sequence comparison. In humanization, amino acids in the CDRs may be left unchanged or may be changed. In addition to the above, humanized antibodies may be produced as described in the production examples of the present disclosure. Specifically, screening may be performed from a heavy chain-fixed Vκ library in which the CDRs of non-human derived antibodies are grafted onto a human heavy chain framework. In this humanization method, the heavy chain CDRs are not changed, but the light chain CDRs may be changed.

[0037] In one embodiment of the present disclosure, the "heavy chain" is typically the main component of a full-length antibody. The heavy chain is usually bound to the light chain by disulfide bonds and non-covalent bonds. The N-terminal domain of the heavy chain contains a region called a variable region (VH), whose amino acid sequence is not constant even in the same class of antibody of the same species, and it is generally known that the VH contributes greatly to the specificity and affinity for the antigen. For example, "Reiter et al., J Mol Biol. 1999 Jul 16;290(3):685-98" describes that when a molecule consisting of only VH was prepared, it bound specifically to the antigen with high affinity. Furthermore, "Wolfson W,Chem Biol. 2006 Dec;13(12):1243-1244" describes that there are antibodies consisting of only heavy chains and no light chains among camel antibodies.

[0038] In one embodiment of the present disclosure, the "CDR (complementarity determining region)" is a region of an antibody that actually contacts an antigen to form a binding site. Generally, CDR is located on the Fv (variable region: including a heavy chain variable region (VH) and a light chain variable region (VL)) of an antibody. Generally, CDRs include CDR1, CDR2, and CDR3, each of which is composed of about 5 to 30 amino acid residues. It is known that the CDR of the heavy chain in particular contributes to the binding of the antibody to the antigen. It is also known that, among the CDRs, CDR3 contributes most to the binding of the antibody to the antigen. For example, "Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128" describes that the binding ability of an antibody is increased by modifying the heavy chain CDR3. The Fv region other than the CDRs is called the framework region (FR), which consists of FR1, FR2, FR3, and FR4, and is relatively well conserved among antibodies (Kabat et al., "Sequence of Proteins of Immunological Interest," US Dept. Health and Human Services, 1983.). In other words, it can be said that the factors that characterize the reactivity of an antibody are the CDRs, and particularly the heavy chain CDRs.

[0039] There are several reported methods for defining CDRs and determining their positions. For example, the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) or Chothia's definition (Chothia et al., J. Mol.Biol.,1987;196:901-917) may be adopted. In one embodiment of the present disclosure, the Kabat definition is adopted as a preferred example, but is not necessarily limited thereto. In some cases, the definition may be determined taking into consideration both the Kabat definition and the Chothia definition. For example, the overlapping portion of CDRs according to each definition, or a portion including both CDRs according to each definition, may be regarded as a CDR. A specific example of such a method is the method of Martin et al. (Proc. Natl. Acad. Sci. USA, 1989; 86: 9268-9272) using Oxford Molecular's AbM antibody modeling software, which is a compromise between the Kabat and Chothia definitions. Such CDR information can be used to produce variants that can be used in the present disclosure. Such antibody variants can be produced that contain one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions, additions, or deletions in the framework of the original antibody, but do not contain mutations in the CDRs.

[0040] As used herein, "antigen" refers to any substance that can be specifically bound by an antibody molecule. As used herein, "immunogen" refers to an antigen that can initiate lymphocyte activation resulting in an antigen-specific immune response. As used herein, "epitope" or "antigenic determinant" refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well-known and conventional techniques when provided with the primary sequence of nucleic acid or amino acid. It is understood that the antibodies of the present disclosure can be similarly utilized with antibodies having other sequences, as long as the epitopes are the same.

[0041] It is understood that the antibody used herein may have any specificity as long as false positives are reduced.

[0042] The "cancer" covered by the present disclosure includes one or more selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, ovarian cancer, bile duct cancer, uterine cancer, bladder cancer, prostate cancer, ureter cancer, renal pelvis cancer, ureter cancer, penile cancer, testicular cancer, brain tumor, central nervous system cancer, peripheral nervous system cancer, head and neck cancer, glioma, glioblastoma multiforme, skin cancer, melanoma, thyroid cancer, salivary gland cancer, malignant lymphoma, carcinoma, sarcoma, leukemia, and hematological malignancies. "Cancer" and "tumor" are used interchangeably. Here, ovarian cancer includes, for example, ovarian serous adenocarcinoma or ovarian clear cell duct cancer. Uterine cancer includes, for example, endometrial cancer or cervical cancer. Head and neck cancers include, for example, oral cavity cancer, pharyngeal cancer, laryngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, or thyroid cancer. Lung cancers include, for example, non-small cell lung cancer, or small cell lung cancer. The malignant tumor may also be PD-L1 positive.

[0043] In this specification, the term "esophageal cancer" is used in the usual sense and in a broad sense including cancer in the esophagus. Esophageal cancer includes, but is not limited to, squamous cell carcinoma, adenocarcinoma, and lymph node metastasis. It is believed that about half of esophageal cancer cases in Japanese people develop near the center of the esophagus in the chest, and then one-quarter develop in the lower part of the esophagus, and it is understood that the present disclosure covers both. Without wishing to be bound by theory, it is expected that the present disclosure can be used as an indicator of esophageal cancer as a whole, including squamous cell carcinoma, adenocarcinoma, and lymph node metastasis.

[0044] As used herein, "pancreatic cancer" refers to a malignant tumor originating in the pancreas, generally refers to pancreatic ductal carcinoma, and also includes tumors in other parts of the pancreas.

[0045] As used herein, "cervical cancer" refers to a type of uterine cancer, which is divided into cervical cancer and uterine body cancer. Cervical cancer develops in the part of the uterus called the cervix, which is the entrance to the uterus.

[0046] As used herein, "lung cancer" refers to a malignant tumor derived from epithelial cells that occurs in the lungs, and includes small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

[0047] As used herein, "head and neck cancer" refers to a tumor in the head and neck, particularly in the area from the face to the neck, such as the nose, mouth, throat, upper jaw, lower jaw, and ears.

[0048] As used herein, "breast cancer" refers to a tumor that occurs in the breast, and includes ductal carcinoma, lobular carcinoma, and the like.

[0049] As used herein, the term "uterine leiomyosarcoma" refers to uterine sarcoma that occurs in the smooth muscle of the uterus. Uterine leiomyosarcoma can be histologically diagnosed based on cell density, cell division rate, cell atypia, and the presence or absence of coagulation necrosis of tumor cells.

[0050] As used herein, "prostate cancer" refers to a tumor that develops in the prostate.

[0051] As used herein, "oral squamous cell carcinoma" refers to a type of oral cancer that is a tumor occurring in the epithelium of the oral mucosa.

[0052] As used herein, "cholangiocarcinoma" refers to a tumor occurring in the bile duct, and is classified into intrahepatic cholangiocarcinoma occurring in the bile duct within the liver, perihilar cholangiocarcinoma occurring in the hilar area outside the liver, and distal cholangiocarcinoma occurring in the distal area outside the liver.

[0053] As used herein, "cancer-associated fibroblasts (CAFs)" refers to fibroblasts that make up the stroma of cancer. CAFs produce many extracellular matrices, such as collagen, which make the stroma of cancer very hard, and as a result, blood vessels are crushed (collapsed), preventing the efficient penetration of anticancer drugs. Crushing of blood vessels also induces a hypoxic state in cancer tissues, which further increases the malignancy of cancer cells. In addition, an increase in the hardness of the stroma itself is one of the factors that increases the proliferation and invasive ability (ability to spread to surrounding tissues) of cancer cells. In addition, CAFs produce many growth factors that act on cancer cells to promote their proliferation and invasion, and suppress antitumor immune responses.

[0054] As used herein, the term "stroma" of cancer refers to tissue that surrounds cancer cells in cancer tissue, and contains CAFs as its main component, as well as other components such as immune cells, bone marrow-derived cells, tumor blood vessels, lymphatic vessels, and extracellular matrix produced by CAFs.

[0055] As used herein, the term "subject" refers to a subject that is the target of diagnosis, detection, or treatment, etc., of the present disclosure (for example, an organism such as a human, or cells, blood, serum, etc. extracted from an organism).

[0056] As used herein, the term "sample" refers to any substance obtained from a subject, etc., and includes, for example, serum, etc. Those skilled in the art can appropriately select a preferred sample based on the description of the present specification.

[0057] In the present specification, the terms "drug", "agent" or "factor" (each of which corresponds to the English term agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof. Substances that bind to Glypican-1 may also be such drugs. Representative examples of factors specific to a polynucleotide include, but are not limited to, polynucleotides that have a certain sequence homology (e.g., 70% or more sequence identity) with respect to the sequence of the polynucleotide and polypeptides such as transcription factors that bind to promoter regions. Representative examples of factors specific to a polypeptide include, but are not limited to, an antibody or a derivative or analog thereof (e.g., a single-chain antibody) specifically directed against the polypeptide, a specific ligand or receptor when the polypeptide is a receptor or ligand, and a substrate when the polypeptide is an enzyme.

[0058] As used herein, "diagnosis" refers to identifying various parameters related to a disease, disorder, condition (e.g., esophageal cancer) in a subject and determining the current or future state of such a disease, disorder, or condition. By using the method, device, and system of the present disclosure, the internal state can be examined, and such information can be used to select various parameters of the disease, disorder, condition, treatment or prevention formulation or method to be administered in the subject. In the narrow sense of the present specification, "diagnosis" refers to diagnosing the current state, but in the broad sense, it includes "early diagnosis," "predictive diagnosis," "pre-diagnosis," and the like. The diagnostic method of the present disclosure is, in principle, industrially useful because it can utilize what comes out of the body and can be performed without the hands of medical professionals such as doctors. In the present specification, in order to clarify that it can be performed without the hands of medical professionals such as doctors, it is sometimes referred to as "assisting" "predictive diagnosis, pre-diagnosis, or diagnosis."

[0059] As used herein, the term "detection agent" or "test agent" refers broadly to any agent capable of detecting or testing an object of interest.

[0060] As used herein, the term "diagnostic agent" refers in a broad sense to any agent capable of diagnosing a target condition (eg, a disease such as esophageal cancer).

[0061] As used herein, "treatment" refers to, in the case of a certain disease or disorder (e.g., esophageal cancer), preventing the worsening of such a disease or disorder when such a state occurs, preferably maintaining the current state, more preferably alleviating, and even more preferably causing the disease or disorder to disappear, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is called "companion treatment," and diagnostic agents for this purpose are sometimes called "companion diagnostic agents."

[0062] In the present specification, the term "therapeutic agent (agent)" refers, in a broad sense, to any drug capable of treating a target condition (e.g., a disease such as esophageal cancer). In one embodiment of the present disclosure, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition may be produced, for example, by mixing the active ingredient with the above-mentioned carrier and using any method known in the technical field of pharmaceutical formulations. The therapeutic agent may be in any form as long as it is used for treatment, and may be an active ingredient alone or a mixture of the active ingredient and any ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The therapeutic agent for esophageal cancer includes a drug used for preventing esophageal cancer (prophylactic drug) or an inhibitor of the proliferation of esophageal cancer cells.

[0063] As used herein, "prevention" refers to preventing a disease or disorder (e.g., esophageal cancer) from becoming a certain condition before it occurs. The drug of the present disclosure can be used to perform diagnosis, and if necessary, the drug of the present disclosure can be used to prevent, for example, esophageal cancer, or measures for prevention can be taken.

[0064] As used herein, the term "prophylactic drug (agent)" refers in a broad sense to any drug that can prevent a target condition (eg, a disease such as esophageal cancer).

[0065] As used herein, the term "interaction" refers to the mutual exertion of force (e.g., intermolecular forces (van der Waals forces), hydrogen bonds, hydrophobic interactions, etc.) between one substance and the other. Usually, two substances that have interacted are in an associated or bonded state. The detection, testing, and diagnosis of the present disclosure can be achieved by utilizing such interactions.

[0066] As used herein, "detection" or "quantification" of polynucleotide or polypeptide expression can be achieved using suitable methods, including, for example, binding or interaction with detection agents, testing agents or diagnostic agents (including applications as companion reagents), including measurement of mRNA and immunological measurement methods. Examples of molecular biological measurement methods include, for example, Northern blot, dot blot, or PCR. Examples of immunological measurement methods include, for example, ELISA using microtiter plates, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion (SRID), immunoturbidimetric (TIA), Western blot, and immunohistochemical staining. Examples of quantification methods include ELISA or RIA. Genetic analysis can also be performed using arrays (e.g., DNA arrays, protein arrays). DNA arrays are broadly reviewed in "DNA Microarrays and the Latest PCR Methods," a special edition of Cell Engineering, edited by Shujunsha). Protein arrays are described in detail in Nat Genet. 2002 Dec;32 Suppl:526-532. In addition to the above, gene expression analysis methods include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like. Such further analysis methods are described, for example, in Genome Analysis Experimental Methods, Nakamura Yusuke Lab Manual, edited by Nakamura Yusuke Yodosha (2002), and all of the descriptions therein are incorporated by reference herein.

[0067] As used herein, "reduction" or "suppression" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to a decrease in the quantity, quality, or effect of a particular activity, transcript, or protein, or an activity that is reduced. When "elimination" is used as a reduction, it refers to an activity, expression product, etc. falling below the detection limit, and is sometimes specifically referred to as "elimination." As used herein, "elimination" is encompassed by "reduction" or "suppression."

[0068] As used herein, the term "label" refers to an entity (e.g., a substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include the RI (radioisotope) method, the fluorescence method, the biotin method, and the chemiluminescence method. When labeling a plurality of markers of the present disclosure or factors or means for capturing them by the fluorescence method, labeling is performed with fluorescent substances having mutually different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. When labeling a ligand, any substance that does not affect the function can be used, but examples of fluorescent substances include Alexa TM Fluor is preferred. Alexa TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. It is very stable, bright, and has low pH sensitivity compared to other fluorescent dyes of the corresponding wavelengths. Alexa TM 555 and Alexa TM 633 combinations, Alexa TM 488 and Alexa TM 555 combination, etc. When labeling a nucleic acid, any material that can bind to the base portion can be used, but it is preferable to use cyanine dyes (e.g., Cy3, Cy5, etc. of the CyDyeTM series), rhodamine 6G reagent, 2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), etc. Examples of fluorescent substances with a difference in maximum fluorescence emission wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein. In the present disclosure, such labels can be used to modify the target object so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods appropriately depending on the label and the target object.

[0069] As used herein, "in vivo" refers to the inside of a living organism. In certain contexts, "in vivo" refers to the location where a substance of interest is to be placed.

[0070] As used herein, the term "in vitro" refers to a state in which a part of a living body is isolated or separated "outside of a living body" (e.g., in a test tube) for various research purposes. This term is in contrast to in vivo.

[0071] As used herein, "ex vivo" refers to a series of operations that are performed outside the body and are intended to be returned to the body afterwards. In the present disclosure, an embodiment can be envisioned in which cells in a living body are treated with the agent of the present disclosure and then returned to the patient.

[0072] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually separated into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed and used immediately before use. Such a kit is advantageously provided with instructions or instructions that preferably describe how to use the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, etc.) or how to handle the reagents. When the kit is used as a reagent kit in the present specification, the kit usually includes instructions that describe how to use the test agents, diagnostic agents, therapeutic agents, antibodies, etc.

[0073] In this specification, the term "instructions" refers to a description of a method for using the present disclosure to a physician or other user. The instructions include a description of the detection method of the present disclosure, a description of how to use a diagnostic agent, or a description of how to administer a medicine or the like. The instructions may also include a description of the administration site, such as oral or esophageal administration (e.g., by injection). The instructions are prepared in accordance with a format prescribed by a supervisory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and it is clearly stated that the instructions have been approved by the supervisory agency. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, e-mail).

[0074] As used herein, "internalization" refers to the uptake of a substance bound to an antigen by a cell through endocytosis or phagocytosis, mediated by the substance bound to the antigen on the cell surface. A substance that binds to Glypian-1 having such activity (e.g., an anti-Glypican-1 antibody) can internalize a target active ingredient into a cell expressing Glypian-1 on the cell surface, and produce a desired effect of the target active ingredient in the Glypian-1-expressing cell. Examples of the target active ingredient include, but are not limited to, drugs with cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, etc.

[0075] As used herein, the term "complex" refers to any construct containing two or more parts. For example, when one part is a polypeptide, the other part may be a polypeptide or other substances (e.g., sugars, lipids, nucleic acids, other carbohydrates, low molecular weight compounds, etc.). As used herein, the two or more parts constituting a complex may be bonded by a covalent bond or other bonds (e.g., hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waals forces, etc.). Thus, as used herein, the term "complex" includes molecules formed by linking multiple types of molecules such as polypeptides, nucleic acids, lipids, sugars, low molecular weight compounds, etc.

[0076] As used herein, the term "linker" refers to a molecule for linking and spatially separating at least two components. Examples of linkers include, but are not limited to, enzyme-cleavable linkers, acid-labile linkers, and disulfide linkers. The term "enzyme-cleavable linker" refers to a linker that is cleaved by an enzyme present in a cell. The term "acid-labile linker" refers to a linker that is cleaved under the production conditions of endosomes or lysosomes in a cell. The term "disulfide linker" refers to a linker that is cleaved under a reducing environment in a cell.

[0077] As used herein, the term "antibody drug conjugate (ADC)" refers to an antibody or antigen-binding fragment thereof chemically linked to one or more active ingredients of interest. In a preferred embodiment, the ADC is operably linked via a linker. As used herein, "operably linked" refers to a relationship in which the linked components are capable of operating to perform their functions. Examples of active ingredients of interest that may be included in an ADC include, but are not limited to, drugs having cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, and the like. The linker may be a cleavable linker or a non-cleavable linker. Examples of cleavable linkers include, but are not limited to, linkers having a sequence cleaved by a protease, acid-labile linkers, disulfide linkers, and the like. Examples of non-cleavable linkers include, but are not limited to, MCC linkers, and the like.

[0078] As used herein, the term "cytotoxic activity" refers to, for example, causing pathological changes in cells, not only direct trauma, but also directly or indirectly blocking cell function through any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division system, reduction in the activity of various enzymes, etc. Thus, examples of "drugs having cytotoxic activity" include, but are not limited to, alkylating agents, tumor necrosis factor inhibitors, intercalators, microtubule inhibitors, kinase inhibitors, proteasome inhibitors, and topoisomerase inhibitors.

[0079] As used herein, "50% inhibitory concentration (IC 50 "IC" refers to the concentration of a compound required to kill 50% of cells. As used herein, IC 50 is measured.

[0080] As used herein, "affinity" refers to the equilibrium constant of the reversible binding of two substances (e.g., an antibody and an antigen), and is expressed as the equilibrium dissociation constant (K DThe equilibrium dissociation constant can be measured by a surface plasmon resonance (SPR) method, for example, Biacore (registered trademark). TM (BioNavis), OpenPlex (Horiba), and Smart SPR (RIBM). D Unless otherwise specified, values ​​refer to values ​​measured by surface plasmon resonance (SPR) method.

[0081] (Preferred embodiment) The preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0082] (antibody) In one aspect, the present disclosure provides a humanized anti-GPC-1 antibody or an antigen-binding fragment thereof, which is humanized based on an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 6. The present inventors humanized 01a033 (comprising the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6), an anti-GPC-1 mouse monoclonal antibody having high affinity for GPC-1 disclosed in International Publication WO 2018 / 199318 (incorporated herein by reference), and unexpectedly succeeded in obtaining a humanized antibody having a higher affinity than 01a033. Thus, the humanized antibody of the present disclosure may have a higher affinity for GPC-1 than an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 6.

[0083] In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure may comprise a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto.

[0084] In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure may comprise a light chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8-27, or an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In a preferred embodiment, an antibody or antigen-binding fragment thereof of the present disclosure may comprise a light chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8-17, or an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto.

[0085] In some embodiments, the antibody of the present disclosure may be of any class, for example, IgM, IgD, IgG, IgA, IgE, or IgY. In certain embodiments, the antibody of the present disclosure may be IgG. In further specific embodiments, the antibody of the present disclosure is of a subclass selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4, preferably human IgG4.

[0086] It is known that human IgG4 undergoes a reaction in which the heavy chain of human IgG4 exchanges with another human IgG4 (Fab-arm exchange) (https: / / www.nature.com / articles / nrneph.2015.95 / figures / 2). To prevent this, the S228P mutation can be introduced (J Biol Chem. 2015 Feb 27;290(9):5462-9.). For example, OPDIVOR (nivolumab) and KEYTRUDAR (pembrolizumab) have the S228P mutation introduced, and in dupilumab, the 233rd amino acid residue in the heavy chain is replaced with Pro. Since Fab-arm exchange does not occur in classes other than human IgG4, it is not necessary to introduce the amino acid mutation.

[0087] In some embodiments, an antibody of the disclosure may comprise a heavy chain constant region having an amino acid sequence set forth in SEQ ID NO: 91, or an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, an antibody of the disclosure may comprise a light chain constant region having an amino acid sequence set forth in SEQ ID NO: 92, or an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto.

[0088] In some embodiments, CDRs 1 to 3 of the heavy chain variable region may have the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively.

[0089] In some embodiments, CDRs 1 to 3 of the light chain variable region may have the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively, or SEQ ID NOs: 34 to 36, respectively, or SEQ ID NOs: 37 to 39, respectively, or SEQ ID NOs: 40 to 42, respectively, or SEQ ID NOs: 43 to 45, respectively, or SEQ ID NOs: 46 to 48, respectively, or SEQ ID NOs: 49 to 51, respectively, or SEQ ID NOs: 52 to 54, respectively, or SEQ ID NOs: 55 to 57, respectively, or SEQ ID NOs: 58 to 60, respectively, or SEQ ID NOs: 61 to 63, respectively, or SEQ ID NOs: 64 to 66, respectively, or SEQ ID NOs: 67 to 69, respectively, or SEQ ID NOs: 70 to 72, respectively, or SEQ ID NOs: 73 to 75, respectively, or 76 to 78, or 79 to 81, respectively, or 82 to 84, or 85 to 87, or SEQ ID NOs: 88 to 90, respectively. In a preferred embodiment, CDRs 1 to 3 of the light chain variable region may have the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively, or SEQ ID NOs: 34 to 36, respectively, or SEQ ID NOs: 37 to 39, respectively, or SEQ ID NOs: 40 to 42, respectively, or SEQ ID NOs: 43 to 45, respectively, or SEQ ID NOs: 46 to 48, respectively, or SEQ ID NOs: 49 to 51, respectively, or SEQ ID NOs: 52 to 54, respectively, or SEQ ID NOs: 55 to 57, respectively, or SEQ ID NOs: 58 to 60, respectively.

[0090] In some embodiments, the epitope of an antibody or antigen-binding fragment thereof of the present disclosure may include positions 339-358 and / or positions 388-421 of SEQ ID NO: 2 (full-length sequence of human GPC-1). In certain embodiments, the epitope of an antibody of the present disclosure may include positions 339-358 and positions 388-421 of SEQ ID NO: 2 (full-length sequence of human GPC-1).

[0091] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is about 5×10 -9 M, about 6 x 10 -9 M, about 7 x 10 -9 M, about 8 x 10 -9 M, about 9 x 10 -9 M, about 10 -8 M or less, approximately 2×10 -8 M, about 3 x 10 -8M, approx. 4 x 10 -8 M, about 5 x 10 -8 M, about 10 -7 M, or approximately 5 x 10 -7 Affinity of M (K D In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure may bind to Glypican-1 with a binding affinity of 3.16×10 -8 M or approx. 1.02 x 10 -8 Affinity of M (K D It can bind to Glypican-1 at a concentration of 0.15 mM.

[0092] In a further aspect, the present disclosure provides a pharmaceutical composition for intracellular internalization of an active ingredient, comprising the humanized antibody of the present disclosure or an antigen-binding fragment thereof. The antibody of the present disclosure unexpectedly has an internalization activity equivalent to or greater than that of 01a033.

[0093] (Complex) In a further aspect, the present disclosure provides a conjugate of the humanized antibody or antigen-binding fragment thereof of the present disclosure and an agent having cytotoxic activity. The agent having cytotoxic activity may be operably linked to the humanized antibody or antigen-binding fragment thereof via a linker.

[0094] Examples of drugs having cytotoxic activity include, but are not limited to, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1, DM4, calicheamicin, duocarmycin, pyrrolobenzodiazepine (PBD), and topoisomerase inhibitors. Although not intended to be limiting, drugs having cytotoxic activity are preferably those having a bystander effect. Bystander refers to an effect in which a drug released in a cancer cell permeates the cell membrane and is effective against surrounding cancer cells. Thus, in some embodiments, a drug having cytotoxic activity may be cell permeable. Examples of drugs having cytotoxic activity with a bystander effect include, but are not limited to, MMAE, PBD, Eribulin, SN-38, Dxd, DM4, etc.

[0095] In some embodiments, the linker may be any linker that is cleaved in cancer cells, such as an enzyme-cleavable linker, an acid-labile linker, a disulfide linker, etc. In certain embodiments, the linker may be a cleavable linker that is cleaved by cathepsin (e.g., cathepsin B), such as maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl.

[0096] In some embodiments, a complex of the disclosure has an IC of about 0.04 nM or less, about 0.05 nM or less, about 0.06 nM or less, about 0.07 nM or less, about 0.08 nM or less, about 0.09 nM or less, about 0.1 nM or less, about 0.11 nM or less, about 0.12 nM or less, about 0.13 nM or less, about 0.14 nM or less, or about 0.15 nM or less in Glypican-1 positive cells. 50 It can be shown that IC 50 may be the value measured in the ADC assay of Example 2. In a preferred embodiment, the IC 50 It can be shown that

[0097] In another embodiment, the linker used in the conjugate of the present disclosure may be an acid-labile linker. By utilizing the acidic pH in endosomes or lysosomes after intracellular internalization, a linker that is responsive to acidic and unstable pH can be used. Such linkers include, for example, hydrazones.

[0098] In yet another embodiment, the linker used in the conjugates of the present disclosure may be a disulfide linker, such linkers including N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), and the like.

[0099] The linker used in the conjugates of the present disclosure may be a non-cleavable linker, including, but not limited to, maleimidomethylcyclohexane-1-carboxylic acid (MCC linker).

[0100] Pharmaceutical Composition In a further aspect, the present disclosure provides a composition for preventing or treating Glypican-1 positive cancer, comprising the complex of the present disclosure.Can be selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma or any combination thereof.In certain embodiments, the Glypican-1 positive cancer can be esophageal cancer or pancreatic cancer.

[0101] Compositions for the Treatment of Metastatic Cancer In a further aspect, a composition for preventing or treating metastatic cancer of Glypican-1 positive cancer is provided, comprising the complex of the present disclosure. The present inventors have found that metastatic cancer of Glypican-1 positive cancer expresses Glypican-1, and that metastatic cancer of Glypican-1 positive cancer can be treated with the complex of the present disclosure. An antigen expressed at a primary site does not necessarily express the same antigen in a metastatic cancer. Therefore, it is necessary to confirm whether a cancer antigen whose expression has been confirmed at a primary site is expressed in a metastatic cancer. It has been confirmed that GPC1 is positively expressed in tumor tissues of metastases such as pancreatic cancer lymph node metastasis, pancreatic cancer liver metastasis, and esophageal cancer lymph node metastasis, and is significant as a therapeutic target.

[0102] In some embodiments, the Glypican-1 positive cancer may be selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof. In certain embodiments, the Glypican-1 positive cancer may be pancreatic cancer.

[0103] In some embodiments, the metastatic cancer can be a metastatic cancer that has metastasized to the liver, esophagus, bile duct, cervix, lung, head and neck, breast, uterine smooth muscle, prostate, oral squamous epithelium, brain, bone, peritoneum, adrenal gland, etc.

[0104] In some embodiments, the cancer may have cancer-associated fibroblasts (CAFs).

[0105] In further embodiments, the conjugates of the present disclosure may be administered together with immune checkpoint inhibitors, including but not limited to anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-VISTA antibodies, and the like.

[0106] (Creating variants) According to methods well known in the art, a person skilled in the art can produce a variant having an affinity for GPC-1 equivalent to or higher than that of 01a033 or the antibody of the present disclosure based on the sequence of the antibody in the present disclosure. For example, a person skilled in the art can analyze antibodies with slightly different CDR sequences using phage display method and screen for an antibody having an affinity equivalent to or higher than that of the antibody of interest. Other methods for producing variants include CDR-walking method, random mutagenesis method, etc. In some embodiments, the variant may have 3 or less amino acid substitutions, deletions and / or additions, preferably 2 or less amino acid substitutions, deletions and / or additions, more preferably 1 or less amino acid substitutions, deletions and / or additions in each CDR of the antibody or antigen-binding fragment thereof of the present disclosure. These variants may have an affinity for GPC-1 equivalent to or higher than that of 01a033 or the antibody of the present disclosure.

[0107] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range of two values," the range includes the two values ​​themselves.

[0108] All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0109] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0110] Examples are described below. When necessary, the handling of animals used in the following examples complied with the standards stipulated by the National Institute of Biomedical Innovation and was carried out in accordance with the Declaration of Helsinki. The reagents used were specifically the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldric, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

[0111] In this example, we humanized an anti-GPC1 monoclonal antibody (clone 01a033) that was discovered as an antibody with high internalization activity into cells, screened for higher internalization activity into cells than before humanization, and created a humanized GPC1-ADC by binding an anticancer drug to the humanized anti-GPC1 monoclonal antibody via a linker, and clarified the antitumor effect of the humanized GPC1-ADC in vitro and in vivo. In addition, since the payload MMAE has the property of inducing immunogenic cell death, we also aim to confirm that a synergistic antitumor effect can be obtained by combining the humanized GPC1-ADC with an immune checkpoint inhibitor.

[0112] (Production Example: Production of Humanized Antibody) The humanized antibodies of the present disclosure were commissioned to be produced by HD Biosciences (China) Co., Ltd.

[0113] In brief, humanization was performed by library construction, antigen panning and screening, binding ELISA, and affinity determination. A human Vκ library was constructed with mouse antibody CDR and human framework fixed heavy chain, followed by antigen panning and screening. From the Vκ library panning, a total of 30 humanized anti-glypican-1 antibodies were found and confirmed by phage ELISA. 20 of them were selected for antibody expression and purification, and then affinity was determined by Biacore® 8K.

[0114] (material and method) <Antibody library creation> Humanized H chain VH region created by CDR grafting of mouse antibody (01a033) The abYsis database was searched for alleles highly homologous to the VH of 01a033, and the framework of the human germline sequence VH1-46 was selected to create a gene having an amino acid sequence to which the CDR sequence of the VH of 01a033 was grafted.

[0115] The VL region was derived from a library of human VL gene pools (LC-Library: Library diversity 4.53 × 10 5 / Library size 1.06×10 13 The VL library gene + Ck gene + the above humanized VH gene + CH1 were inserted into the N-terminus of the phage GIII antigen.

[0116] A phage library (humanized antibody library) was created that displayed random human L chains (k) and humanized H chains (VH+CH1) having the CDR sequences of 01a033.

[0117] <Panning> Panning was performed to select phages expressing antibodies that actually bind to GPC-1 from a humanized antibody library.

[0118] The antigen GPC-1 was bound to a solid phase (test tube, particle), a phage population was added thereto, washed, and the bound phages were recovered.

[0119] <Isolation of phage clones by filter lifting and verification of antigen-binding ability> The phages that bound to the antigen in panning and were eluted were infected with Escherichia coli - seeded plates, and the resulting lytic plaques were transferred onto a filter to clone the phages.

[0120] The obtained phage clones were amplified, and the binding ability of each clone to the ELISA method (human GPC-1 antigen immobilized) was examined, and clones that obtained a binding signal above a certain value were selected.

[0121] <Expression and purification of IgG-converted antibody clones> IgG-converted antibodies were expressed from the genes of 20 selected phage clones in HEK293 cells (vectors: pFUSE2ss-CLIg-hkpFUSE2ss-CLIg-hk (light chain, Invivogen, catalog number: pfuse2ss-hclk) and pFUSEss-CHIg-hG1 (heavy chain, Invivogen, catalog number: pfusess-hchg1); cell line: FreeStyle TM 293F Cells (Invitrogen, catalog number: R790-07)). Antibodies were purified from the culture supernatant 4 days after transfection for quantification and confirmation by SDS-PAGE. Antibody production (presence and molecular weight of H chain and L chain) was confirmed for all 20 clones.

[0122] <Measurement of antigen-binding affinity of each clone by surface plasmon resonance method> The antigen-binding affinity of the antibody of 20 clones obtained by surface plasmon resonance (Biacore® 8K) was measured. Anti-human IgG antibody was immobilized on a chip, and each antibody was poured onto the chip to bind the antibody. After washing, human GPC-1 antigen was poured onto the chip to measure the binding constant (Ka) and dissociation constant (Kd). Seven clones showed higher affinity than the mouse antibody.

[0123] <Measurement of antigen-binding affinity of mouse antibody (01a033) using surface plasmon resonance> The binding affinity of mouse antibodies to human GPC-1 was measured by surface plasmon resonance (Biacore® 8K). Anti-mouse Fc antibodies were immobilized on a chip, and mouse antibodies (01a033) were poured onto the chip to bind to the antibodies. After washing, human GPC-1 was poured onto the chip to measure the binding constant (Ka) and dissociation constant (Kd).

[0124] <Creation of mouse anti-GPC-1 chimeric antibody> The VH / VL genes of 01a033 were linked to human Ck / CH genes to create a human antibody chimeric gene, which was expressed in HEK293F cells.

[0125] (Example 1: Affinity analysis of humanized anti-GPC1 monoclonal antibody) (material and method) Mouse anti-human GPC1 monoclonal antibody was humanized (subclass IgG4), and 20 clones (T1, T2, T3, T7, T8, T10, T11, T22, T26, T28, T29, T31, T32, T33, T34, T36, T43, T56, T57, T59) were prepared. The affinity of these clones with recombinant human GPC1 protein (R&D systems) was measured using a Human antibody capture kit (GE Healthcare) by immobilizing anti-human IgG (Fc) on a sCM5 sensor chip (GE Healthcare) and using a Biacore (registered trademark) 8K (GE Healthcare).

[0126] (result) We independently humanized anti-Glypican-1 (GPC-1) monoclonal antibody (01a033), and as a result, we obtained 20 candidate clones. The equilibrium dissociation constants (K D ) was measured (Figure 1). As a result, seven clones (clones T1, T2, T3, T8, T10, T36, and T56) were identified that showed stronger affinity than the chimeric mouse antibody (01a033), the antibody before humanization (Figure 1).

[0127] (Example 2: ADC assay combining humanized anti-GPC-1 antibody and MMAF-binding secondary antibody) (material and method) (Confirmation of anticancer drug sensitivity of TE14 cells) 2000 TE14 cells were added to a 96-well plate in 80 μl. Incubate overnight at 37°C, 5% CO 2 The cells were cultured in an incubator, and the next day, various clones of humanized anti-GPC1 antibodies, 10 times concentrated relative to the final concentration, were added as primary antibodies at 10 μl per well. Next, anti-human IgG antibodies (Fab) (MORADEC, model number AH-202AF-50) linked to an anticancer drug (MMAF) via a linker were added at 10 μl per well as secondary antibodies, for a total of 100 μl. The cells were cultured for 6 days in a 37°C, 5% CO2 incubator, and cell viability was measured by detecting the amount of ATP using the Cell Titer-Glo Luminescent Cell Viability Assay reagent. RPMI1640 + 10% FBS + 1% PS was used as the medium.

[0128] (ADC used in the assay) A schematic diagram of the ADC used in the assay is shown in Figure 2. Twenty types of clones were screened for clones with high intracellular internalization activity using the method shown in Figure 2. In this method, the humanized anti-GPC1 antibody of the clone obtained in the above manufacturing example is added as the primary antibody to the TE14 cell line, an esophageal cancer cell line that expresses GPC1, and an anti-human IgG antibody conjugated with an anti-cancer drug (MMAF) is added as the secondary antibody, allowing the anti-cancer drug to be taken up into the cancer cells as a complex of the primary and secondary antibodies, and cell death is induced by the action of the anti-cancer drug.

[0129] (result) Ten clones were identified that showed higher intracellular internalization activity than the chimeric mouse antibody (01a033) (clones T2, T7, T8, T1, T36, T57, T10, T34, T56, and T3) (Figure 3).

[0130] (Example 3: Antigen affinity analysis by FACS) As a result of Example 2, clone T2 showed the highest activity, and therefore clone T2 was selected as a humanized anti-GPC1 antibody with high intracellular internalization activity and analyzed.

[0131] (material and method) For FACS analysis using antibodies, human esophageal cancer cell line (TE8) cells were used as GPC1 positive cells, and TE8-GPC1 KO cells in which GPC1 was knocked out using CRISPR-Cas9 technology were used as GPC1 negative cells. Human / mouse chimeric anti-GPC1 antibody (clone 01a033) and humanized anti-GPC1 antibody (clone T2) were used as primary antibodies, and FITC-labeled anti-human IgG-FITC (Jackson Immuno research, model number: 109-095-098) was used as secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using BD FACSDiva software (BD).

[0132] (material and method) Human / mouse chimeric anti-GPC1 antibody (clone 01a033) and humanized anti-GPC1 antibody (clone T2) were used as primary antibodies at various concentrations, and FITC-labeled anti-human IgG-FITC (Jackson Immuno research, model number: 109-095-098) was used as secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using BD FACSDiva software (BD).

[0133] (result) FACS analysis was performed on TE8, a GPC1-positive esophageal cancer cell line, and TE8-GPC1-KO cells in which GPC1 was knocked out using the CRISPR / Cas9 system, using a chimeric mouse antibody (01a033) and a humanized anti-GPC1 antibody (T2). As a result, the humanized anti-GPC1 antibody (T2) was found to specifically bind to GPC1, since it showed no reactivity with TE8-GPC1-KO cells and only showed reactivity with TE8 cells (Figure 4). In addition, it was revealed that the humanized anti-GPC1 antibody (T2) showed a higher affinity with GPC1 on the TE8 cell surface than the chimeric mouse antibody (01a033) (Figure 4).

[0134] Example 4: Preparation of ADCs In this example, ADCs were prepared as follows.

[0135] The mAb was conjugated to a humanized anti-GPC1 antibody (clone T2) with maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E (MC-vc-PAB-MMAE). The conjugation was performed using a maleimide-cysteine-based method in which the interchain disulfide bonds of the mAb were first reduced with TCEP at 37°C, and then the maleimide moiety of the drug was attached to the reduced cysteine. The ADC was desalted with Amicon Ultra - 0.5 mL Centrifugal filters - 30 K (Millipore) to remove unreacted toxin, and then buffer exchanged into ADC formulation buffer (20 mM histidine, 7% sucrose, 0.02% (w / v) PS80, pH 5.5). The distribution of drug-antibody ratio (DAR) was analyzed by hydrophobic interaction chromatography (HIC), and the degree of aggregation was analyzed by size exclusion chromatography (SEC). The conjugation conditions were set so that the drug-antibody ratio (DAR) was 4.

[0136] (result) A schematic diagram of the structure of an exemplary ADC is shown in Figure 5. As shown in Figure 5, a payload with bystander effect, MMAE, was conjugated to a cysteine ​​residue of a humanized anti-GPC1 antibody (clone T2) via a cleavable linker that is cleaved by cathepsin B. The humanized GPC1-ADC showed a DAR of 4.0 (Figure 6).

[0137] (Example 5: Antigen affinity analysis of humanized GPC1-ADC and unlabeled antibody by FACS) (material and method) For FACS analysis using antibodies, human pancreatic cancer cell line (BxPC-3) cells were used as GPC1 positive cells. Humanized anti-GPC1 antibody (clone T2) and humanized GPC1-ADC (MMAE) were used as primary antibodies at various concentrations, and FITC-labeled anti-human IgG-FITC (Jackson Immuno research, model number: 109-095-098) was used as secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using BD FACSDiva software (BD).

[0138] (result) Regarding the effect of conjugating a payload to the humanized anti-GPC1 antibody (T2) on antigen affinity, antibody concentration-dependent binding ability was evaluated by FACS analysis using GPC1-expressing positive BxPC3 cells. As a result, the humanized anti-GPC1-ADC had the same K D The values ​​shown in the Table 1 confirmed that the payload conjugate did not significantly reduce the antigen affinity (Figure 7).

[0139] (Example 6: Intracellular internalization assay of humanized anti-GPC-1 antibody (clone T2) and humanized GPC1-ADC (MMAE)) In this example, the internalization activity of humanized GPC1-ADC (MMAE) was examined using FACS with the GPC1-positive BxPC-3 cell line.

[0140] (material and method) Cells treated with humanized anti-GPC1 antibody (clone T2) and humanized GPC1-ADC (MMAE) were detected with anti-GPC1 mAb (biotin-labeled 02b006) and PE-labeled streptavidin.

[0141] BxPC-3 cells were detached from a 10 cm plate using 0.02% EDTA and collected. The cells were cultured in RPMI1640 + 10% FBS + 1% GlutaMAX. TMI + 100U / ml penicillin + 100μg / ml streptomycin, and place in a 1.5ml tube, 2.0x10 6 The mixture was prepared at 0.9 ml per cell. To reduce internalization activity, the mixture was left on ice for 1 hour. 1 mg / ml of humanized anti-GPC1 antibody (clone T2) and humanized GPC1-ADC (MMAE) were added to ice-cold RPMI1640 + 10% FBS + 1% GlutaMAX TM The antibody was diluted to 24μg / ml (160nM) with I +100U / ml penicillin + 100μg / ml streptomycin. 100μl of the above antibody was added to the cell suspension, and the suspension was left on ice for 30 minutes to stain the antigens on the cell surface. After 30 minutes, 100μl of the cell suspension was dispensed into 6 tubes. The cell suspension was divided into 12 tubes, one for incubation at 4℃ and one for incubation at 37℃. The incubation times were 0h, 1h, 2h, 3h, 4h, and 6h. After the incubation time was reached, the cells were centrifuged at 1500rpm at 4℃ for 5 minutes to remove the supernatant. The cells were washed with 100μl of ice-cold PBS + 0.2% BSA and centrifuged to wash the cells. The washing operation was performed a total of 3 times. 50 μL of biotin-labeled anti-GPC1 antibody 02b006 (International Publication No. 2018 / 199318) at a concentration of 1 μg / ml was added and suspended. The antigen on the cell surface was stained on ice for 30 minutes (non-internalized GPC1 was stained). After the incubation time was reached, the cells were centrifuged at 1500 rpm and 4 ° C for 5 minutes to remove the supernatant. The cells were washed with 100 μl of ice-cold PBS + 0.2% BSA and centrifuged to wash the cells. The washing operation was performed a total of three times. Streptavidin-PE (PharMingen, #554061) was diluted 200 times with ice-cold PBS + 0.2% BSA, 50 μL was added to the cells, and the cells were suspended. The reaction was allowed to proceed on ice for 30 minutes in the dark. After the incubation time was reached, the cells were centrifuged at 1500 rpm and 4 ° C for 5 minutes to remove the supernatant. The cells were washed with 100 μl of ice-cold PBS + 0.2% BSA and centrifuged to wash the cells. The washing procedure was performed three times in total. 150 μl of ice-cold PBS + 0.2% BSA was added, and the cells were measured using FACS CantoII and analyzed using BD FACSDiva software (BD).

[0142] Furthermore, the intracellular localization of the humanized anti-GPC1 antibody (clone T2) and the humanized GPC1-ADC (MMAE) when internalized using the GPC1-positive BxPC-3 cell line was examined using a fluorescence microscope.

[0143] BxPC-3 cells were cultured at 7.5x10 in a 12-well plate (Corning: 3513) containing 18mm microcover glass (Matsunami). 4 Cells were seeded at 1 ml / well and incubated at 37°C, 5% CO 2 The cells were cultured overnight in an incubator.

[0144] The next day, remove the medium and add 1.0 ml of ice-cold RPMI1640 + 10% FBS + 1% GlutaMAX to the wells. TM I + 100 U / ml penicillin + 100 μg / ml streptomycin was added and incubated at 4° C. for 1 hour to reduce the internalization activity of the cells.

[0145] Next, the medium inside the wells was removed, and 10 μg / ml of primary antibody (ice-cold humanized anti-GPC1 antibody (clone T2) and humanized GPC1-ADC (MMAE)) was added at 500 μl / well and incubated at 4° C. for 15 minutes.

[0146] After 15 minutes, aspirate the antibody solution and add 1.0 ml of ice-cold RPMI1640 + 10% FBS + 1% GlutaMAX TM I + 100 U / ml penicillin + 100 μg / ml streptomycin was added and the cells were washed. A sample at this time point was collected as the 0-hour sample (0 h).

[0147] For samples to be internalized, aspirate the solution and add 1.0 ml of RPMI 1640 + 10% FBS + 1% GlutaMAX TM Add I + 100 U / ml penicillin + 100 μg / ml streptomycin (warmed at 37°C) and incubate at 37°C in 5% CO2 The cells were cultured in an incubator for 4 hours, and samples at this time point were collected as 4-hour samples (4h).

[0148] For samples at 0 and 4 hours, the medium in the wells was removed and the cells were washed with 1 ml ice-cold PBS.

[0149] Next, 1 ml of 100% (ice-cold) methanol was added, and the cells were fixed by incubating at -20°C for 15 minutes. After fixation, the cells were washed three times with 1 ml of PBS, and 1 ml of 1% BSA, 0.3% Triton X-100 / PBS(-) was added, followed by blocking treatment at room temperature for 1 hour. After blocking, the solution was removed, and 0.5 ml / well of a primary antibody (anti-CD107a monoclonal antibody (CST, model number #9091 LAMP1(D2D11)XP(registered trademark) Rabbit mAb)) diluted 100-fold with 1% BSA, 0.3% Triton X-100 / PBS(-) was added, and the reaction was allowed to proceed overnight at 4°C in the dark.

[0150] After overnight primary antibody reaction, the sections were washed three times with 1 ml of PBS and then resuspended in 1% BSA, 0.3% The adsorbed secondary antibodies goat anti-human IgG(H+L)-Alexa Fluor 488 (Life Technologies, A-11013, lot 2110842) diluted 200-fold with Triton X-100 / PBS(-) and donkey anti-rabbit IgG-Alexa647 (Life Technologies, A31573, lot 1626613) diluted 200-fold were added at 0.5 ml / well and reacted at room temperature in the dark for 1 hour.

[0151] After the secondary antibody reaction, the cells were washed three times with 1 ml of PBS, and the cover glass to which the cells had adhered was removed and placed on a slide glass, sealed with Vectashield (vectashield H1200), and observed under an all-in-one fluorescence microscope (Keyence, BZ-X800).

[0152] (result) The internalization activity into cells was evaluated. As a result of FACS analysis using BxPC3 cells, it was confirmed that after addition of both the humanized anti-GPC1 antibody (T2) and the humanized anti-GPC1-ADC to BxPC3 cells, 60 to 70% or more of GPC1 was rapidly internalized into the cells within 2 hours (Figure 8). In order for this ADC to exert its antitumor effect, the linker portion of the ADC must be cleaved by the lysosomal enzyme cathepsin B. Regarding intracellular localization, the localization of GPC1 with the lysosomal marker CD107a was evaluated by fluorescent double staining. As a result, both the humanized anti-GPC1 antibody (T2) and the humanized anti-GPC1-ADC colocalized with CD107a, confirming that the humanized anti-GPC1 antibody (T2) and the humanized anti-GPC1-ADC bind to GPC1 and then migrate to the lysosome (Figure 8).

[0153] Example 7: In vitro ADC assay using humanized GPC1-ADC (MMAE) (material and method) GPC1 expression analysis was performed by FACS.

[0154] The expression of GPC1 was analyzed by FACS analysis for esophageal cancer cell lines (TE8, TE14), pancreatic cancer cell lines (BxPC3, KP-2, PK-8), TE8-GPC1 KO, TE14-GPC1 KO cells, BxPC3-GPC1 KO cells, cervical cancer cell line (HeLa), head and neck tumor cell lines (Detroit562, FaDu), and oral squamous cell carcinoma cell lines (Ho-1-u-1, KOSC2 cl3-43, KON). Mouse anti-GPC1 antibody (clone 01a033) was used as the primary antibody, and FITC-labeled goat anti-mouse IgG (H+L chain specific) (southern biotech) was used as the secondary antibody. The measurement was performed using a FACS CantoII (BD). The measurement data was recorded using FlowJoint. TM The analysis was performed using software (Tree star).

[0155] An in vitro ADC assay using the humanized GPC1-ADC (MMAE) prepared in Example 4 was carried out as follows. (1) Cells were seeded (90 μL of cell suspension) onto a 96-well white plate (model number 136101) manufactured by Thermo Fisher Scientific. The medium used was RPMI1640 + 10% FBS + 100 U / ml penicillin + 100 μg / ml streptomycin (for BxPC-3 cells, RPMI1640 + 10% FBS + 1% GlutaMAX TM (I + 100U / ml penicillin + 100μg / ml streptomycin, Detroit562, FaDu, and KON were used with DMEM + 10% FBS + 100U / ml penicillin + 100μg / ml streptomycin, and Ho-1-u-1 was used with DMEM / F12 + 10% FBS + 100U / ml penicillin + 100μg / ml streptomycin). Cells were seeded in the 60 central wells of the plate, and 100μL of medium was added to the outer 36 wells. Incubated at 37℃, 5% CO 2 The cells were cultured in an incubator. (2) The next day, 10 μL of ADC was added to the cells (total volume 100 μL). (3) After culturing for 144 hours, 100 μL / well of CellTiter-Glo (registered trademark) Luminescence Cell Viability Assay reagent (Promega) was added and mixed. (4) Measured using a plate reader. (5)Analyzed using GraphPad Prism6.

[0156] The IC50 value was calculated using the following formula (Hossain MM, Hosono-Fukao T, Tang R, Sugaya N, van Kuppevelt TH, Jenniskens GJ, Kimata K, Rosen SD, Uchimura K (2010) Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulphate and their inhibition by PI-88. Glycobiology 20(2): 175-186.). IC50=10^(Log[A][B]×(50C) / (DC)+Log[B]) A: High concentration of 50% or more B: Low concentration between 50% C: Inhibition rate in B D: Inhibition rate in A.

[0157] (result) To evaluate the efficacy of the humanized GPC1-ADC, we prepared cell lines positive and negative for GPC1 expression. For the GPC1-positive esophageal cancer cell lines TE8 and TE14, and the pancreatic cancer cell line BxPC3, we created TE8-GPC1-KO cells, TE14-GPC1-KO cells, and BxPC3-GPC1-KO cells in which GPC1 was knocked out using the CRISPR / Cas9 system, and confirmed that GPC1 was knocked out by FACS analysis (Figure 9). As a result of in vitro ADC assay, the humanized GPC1-ADC was effective against GPC1-positive cancer cell lines, but not against GPC1-negative cancer cell lines, confirming that the humanized GPC1-ADC exhibits efficacy dependent on GPC1 expression (Figure 10). In addition to pancreatic cancer and esophageal cancer cell lines, humanized GPC1-ADC was also confirmed to exhibit GPC1 expression-dependent efficacy against cervical cancer, head and neck tumor, and oral squamous cell carcinoma cell lines (Figures 11 and 12).

[0158] (Example 9: In vivo efficacy test of humanized GPC1-ADC (MMAE) using pancreatic cancer PDX (PK565)) (material and method) GPC1 expression in pancreatic cancer PDX (PK565) was confirmed as follows.

[0159] Paraffin-embedded tissue was sliced, deparaffinized, and dehydrated with alcohol. Immunohistochemical staining for GPC1 was performed using anti-GPC-1 antibody (Genetex: GTX104557) and ChemMate Envision kit HRP 500T (Dako: K5007). GPC1 expression in PK565 was confirmed to be a heterogeneous tumor with a mixture of positive and negative pancreatic cancer cells.

[0160] Pancreatic cancer PDXs were subcutaneously implanted into 6-week-old NOG female mice, and the tumor size after implantation was approximately 100 mm 3 When this occurred, administration of PBS, control ADC (10 mg / kg), and humanized anti-GPC1-ADC (MMAE) (1 mg / kg), (3 mg / kg), and (10 mg / kg) prepared in Example 4 was started. The day administration started was set as day 0, and the mice were intravenously administered on days 0, 7, 14, and 21. Tumor volume and body weight were measured twice a week from day 0 until day 42.

[0161] (result) In an in vivo efficacy test, the humanized GPC1-ADC showed a high concentration-dependent antitumor effect when the drug was administered intravenously a total of four times every week to GPC1-positive pancreatic cancer PDX mice (PK565) (Figure 13). Since no weight loss was observed, the GPC1-ADC did not show characteristic toxicity compared to the control ADC. This means that in vivo, the use of GPC1-ADC can specifically deliver anticancer drugs to GPC1-positive cancer cells, achieving high efficacy and low toxicity compared to systemic administration of anticancer drugs, leading to the development of a groundbreaking therapeutic drug for GPC1-positive cancer.

[0162] (Example 10: In vivo efficacy test of humanized GPC1-ADC (MMAE) using pancreatic cancer PDX (PK175)) (material and method) GPC1 expression in pancreatic cancer PDX (PK175) was confirmed as follows.

[0163] Paraffin-embedded tissues were sliced, deparaffinized, and dehydrated with alcohol. Immunohistochemical staining for GPC1 was performed using anti-GPC-1 antibody (Genetex: GTX104557) and ChemMate Envision kit HRP 500T (Dako: K5007). GPC1 expression in PK175 was confirmed as a positive tumor in both pancreatic cancer cells and cancer-associated fibroblasts (CAFs).

[0164] Pancreatic cancer PDXs were subcutaneously implanted into 6-week-old NOG female mice, and the tumor size after implantation was approximately 100 mm 3 When this occurred, administration of PBS and humanized anti-GPC1-ADC (MMAE) (1 mg / kg), (3 mg / kg), and (10 mg / kg) was started. The day administration began was designated as day 0, and the drugs were administered intravenously on days 0, 7, 14, and 21. Tumor volume and body weight were measured twice a week from day 0 until day 42.

[0165] (result) In an in vivo efficacy test, the humanized GPC1-ADC exerted a high concentration-dependent antitumor effect when the drug was administered intravenously a total of four times every week to GPC1-positive pancreatic cancer PDX mice (PK175) (Figure 14). Since no weight loss was observed, the GPC1-ADC did not show characteristic toxicity compared to the control ADC. This means that in vivo, the use of GPC1-ADC can specifically deliver anticancer drugs to GPC1-positive cancer cells, achieving high efficacy and low toxicity compared to systemic administration of anticancer drugs, leading to the development of a groundbreaking therapeutic drug for GPC1-positive cancers.

[0166] (Example 11: In vivo efficacy test of GPC1-ADC using esophageal cancer PDX (ESCC14)) (material and method) GPC1 expression in esophageal cancer PDX (ESCC14) was confirmed as follows.

[0167] Paraffin-embedded tissues were thinly sectioned, deparaffinized, and dehydrated with alcohol. Immunohistochemical staining for GPC1 was performed using anti-GPC-1 antibody (Genetex: GTX104557) and ChemMate Envision kit HRP 500T (Dako: K5007). GPC1 expression in ESCC14 was confirmed to be homogeneous, with high expression uniformly in esophageal cancer cells.

[0168] Esophageal cancer PDXs were subcutaneously transplanted into 6-week-old NOG female mice, and the tumor size after transplantation was approximately 100 mm 3 When this occurred, administration of PBS as a control and humanized anti-GPC1-ADC (MMAE) (1 mg / kg), (3 mg / kg), and (10 mg / kg) prepared in Example 4 was started. The day administration started was set as day 0, and the tumors were intravenously administered on days 0, 7, 14, and 21. Tumor volume and body weight were measured twice a week from day 0 until day 42.

[0169] (result) In an in vivo efficacy test, the humanized GPC1-ADC showed a high concentration-dependent antitumor effect when the drug was administered intravenously a total of four times every week to GPC1-positive esophageal cancer PDX mice (ESCC14) (Figure 15). Since no weight loss was observed, the GPC1-ADC did not show characteristic toxicity compared to the control ADC. This means that in vivo, the use of GPC1-ADC can specifically deliver anticancer drugs to GPC1-positive cancer cells, achieving high efficacy and low toxicity compared to systemic administration of anticancer drugs, leading to the development of a groundbreaking therapeutic drug for GPC1-positive cancers.

[0170] (Example 12: GPC1 expression analysis by FACS analysis of mGPC1-expressing mouse colon cancer cell line (mGPC1-MC38)) (material and method) For mGPC1-MC38 cells, humanized anti-GPC1 antibody (clone T2) was used as the primary antibody, and FITC-labeled anti-human IgG-FITC (Jackson Immuno research, model number: 109-095-098) was used as the secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using BD FACSDiva software (BD).

[0171] Furthermore, an in vitro ADC assay was performed on mGPC1-MC38 cells in a manner similar to that described in Example 8.

[0172] (result) It is known that some types of payloads used in ADCs, such as MMAE, induce cell death accompanied by immunogenic cell death (ICD) in cancer cells. Nuclear proteins such as HMGB1, known as damage-associated molecular patterns (DAMPs), are released from cancer cells that have undergone ICD, and antigen-specific T cells that promote maturation and activation of dendritic cells via TLR-4 infiltrate into the tumor and exert an antitumor effect. Therefore, it is expected that a synergistic antitumor effect can be obtained by combining humanized GPC1-ADC with immune checkpoint inhibitors. Therefore, in vitro experiments were performed using MC38-mGPC1, a mouse colon cancer cell line in which mouse GPC1 was stably expressed, and in vivo experiments were performed using syngenic mice. Humanized anti-GPC1-ADC showed cell proliferation inhibitory activity against MC38-mGPC1 in vitro (Figure 16).

[0173] Example 13: Quantification of HMGB-1 in culture supernatant by ELISA (material and method) Control-ADC, humanized GPC1-ADC and MMAE were added to MC38-mGPC1 in vitro, and the culture supernatant was collected 48 hours later. The concentration of HMGB-1 in the culture supernatant was measured using HMGB1 ELISA KIT II - HMGB1 measurement reagent (Shinotestu Co., Ltd., model number SNO-326054329).

[0174] (result) The humanized anti-GPC1-ADC prepared in Example 4 was added to MC38-mGPC1, and the amount of HMGB1 in the culture supernatant was quantified by ELISA, confirming that the extracellular release of HMGB1 was induced (Figure 17). HMGB1 (high mobility group box-1) is a nuclear protein known as one of the DAMPs (damage-associated molecular patterns). Certain anticancer drugs release HMGB1 extracellularly when cancer cells undergo cell death, and therefore it is known as an indicator of immunogenic cell death. The extracellularly released HMGB1 binds to TLR4 on dendritic cells, promoting the activation and maturation of dendritic cells, which in turn promotes the infiltration of T cells into tumor tissues.

[0175] (Example 14: Confirmation of synergistic effect by combined use of humanized GPC1-ADC (MMAE) and anti-PD1 antibody using a colorectal cancer syngenic model mouse) (material and method) MC38-mGPC1 was subcutaneously implanted into 8-week-old C57BL / 6 female mice, and the tumor size after implantation was approximately 75 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into 4 groups, and administration of PBS, humanized anti-GPC1-ADC (MMAE) (10 mg / kg) prepared in Example 4, anti-mouse PD1 antibody (clone RMP1-14) (3 mg / kg), and humanized anti-GPC1-ADC (MMAE) (10 mg / kg) + anti-mouse PD1 antibody (clone RMP1-14) (3 mg / kg) was started. The day administration started was set as day 0, and ADC was administered intravenously on days 0, 3, 7, and 10, and anti-mouse PD1 antibody was administered intraperitoneally. Tumor volume and body weight were measured twice a week from day 0 until day 21.

[0176] (result) A syngenic model in which MC38-mGPC1 was subcutaneously transplanted into C57BL / 6 mice was divided into four groups: vehicle control, humanized anti-GPC1-ADC, anti-PD1 antibody, and humanized anti-GPC1-ADC + anti-PD1 antibody. The drugs were administered twice a week for a total of four times, and the anti-tumor effects were evaluated. As a result, a significant anti-tumor effect was observed in the combination group compared to the single-drug administration group of humanized GPC1-ADC and anti-PD1 antibody. On the other hand, no weight loss was observed with the combination administration of humanized GPC1-ADC and anti-PD1 antibody (Figure 18). These results suggest that humanized GPC1-ADC is expected to have a synergistic anti-tumor effect when used in combination with a checkpoint inhibitor.

[0177] (Example 15: Analysis of the in vivo mechanism of action of humanized GPC1-ADC (MMAE) using pancreatic cancer PDX (PK565))

[0178] (material and method) Pancreatic cancer PDX (PK565) was subcutaneously transplanted into 6-week-old NOG female mice, and the tumor size after transplantation was approximately 100 mm 3 When the tumor reached the threshold of 0.01 mg / kg, PBS, control ADC (10 mg / kg), and humanized anti-GPC1-ADC (MMAE) (1 mg / kg), (3 mg / kg), and (10 mg / kg) prepared in Example 4 were administered. The tumor was excised 24 hours after administration. The excised tissue was fixed in 10% neutral formalin buffer, and then paraffin-embedded tissue was prepared and thin sections were prepared. For each thin section, the expression of phospho-histone H3 (Ser10) was analyzed by immunohistochemical staining to evaluate cancer cells whose cell cycle was arrested in the G2 / M phase.

[0179] (result) As a result, it was confirmed that the proportion of cancer cells whose cell cycle was arrested at the G2 / M phase in a concentration-dependent manner was significantly increased in the humanized anti-GPC1-ADC (MMAE) group compared to the PBS and control ADC (10 mg / kg) groups (Figure 19). When the ADC payload acts on cancer cells to arrest the cell cycle at the G2 / M phase, the proliferation of cancer cells stops, which may contribute to extending the survival time of cancer patients.

[0180] (Example 16: Analysis of GPC1 expression in pancreatic cancer distant metastasis tissues by immunohistochemical staining) In pancreatic cancer, distant metastasis such as lymph node metastasis and liver metastasis is known to be a poor prognostic factor. Therefore, if GPC1 is expressed not only in the primary focus of pancreatic cancer but also in distant metastatic focus, it is considered to be highly useful as a therapeutic target. Therefore, the expression of GPC1 was evaluated in lymph node metastasis, liver metastasis, and adrenal metastasis tissues of pancreatic cancer by immunohistochemical staining. As a result, it was confirmed that the expression of GPC1 was positive in all tissues of lymph node metastasis, liver metastasis, and adrenal metastasis of pancreatic cancer (Figure 20). Therefore, it is suggested that the humanized GPC1-ADC (MMAE) of the present invention is effective in treating or preventing metastatic cancer of GPC1-positive cancer.

[0181] (Example 17: Creation of pancreatic cancer liver metastasis model and efficacy test of humanized GPC1-ADC)

[0182] In order to confirm the therapeutic or preventive effect of humanized GPC1-ADC (MMAE) on metastatic cancer as suggested in Example 16, a pancreatic cancer liver metastasis model was created.

[0183] (material and method) According to the procedure shown in FIG. 21, BxPC3-Luc cells were administered into the spleen of SCID mice, and the spleen was removed 1 to 2 weeks after administration to prepare a pancreatic cancer liver metastasis model. The proliferation of BxPC3-Luc cells metastasized to the liver was monitored by measuring luciferase activity using IVIS. The liver metastasis model mice were divided into two groups, a PBS administration group and a humanized anti-GPC1-ADC (MMAE) (10 mg / kg) administration group prepared in Example 4, and a drug efficacy test was performed using the same procedure as in Example 16.

[0184] (result) As a result, as shown on the left in Figure 22, the fermentation value measured by IVIS was significantly inhibited in the humanized anti-GPC1-ADC (MMAE) (10 mg / kg) group compared to the PBS-administered group. In addition, survival analysis confirmed that humanized anti-GPC1-ADC (MMAE) (10 mg / kg) significantly extended the survival time of the pancreatic cancer liver metastasis model.

[0185] (Example 18: Efficacy test of humanized GPC1-ADC in metastatic cancer) We will create a metastasis model in which the cancer metastasizes to the liver, lungs, brain, bone, peritoneum, adrenal gland, etc., and confirm the efficacy of humanized GPC1-ADC in the metastasis model. Humanized GPC1-ADC is expected to be effective against both primary and metastatic lesions, and to extend survival time.

[0186] Example 19: Formulation Example The humanized anti-GPC1-ADC (MMAE) of the present invention can be provided as the following intravenous injection formulation. Humanized anti-GPC1-ADC 100mg Polysorbate 80 5mg Sodium chloride 100mg Sodium citrate hydrate 80mg pH adjuster (appropriate amount) Total volume 10mL

[0187] As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the scope of the claims. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically described in this specification. This application claims the benefit of priority from Japanese Patent Application No. 2020-101856 filed on June 11, 2020, the contents of which are incorporated herein by reference. [Industrial Applicability]

[0188] A medicine for treating or preventing cancer is provided. Technology that can be used in industries (such as pharmaceuticals) based on such technology is provided. [Sequence List Free Text]

[0189] SEQ ID NO: 1: Nucleic acid sequence of human Glypican-1 SEQ ID NO: 2: Amino acid sequence of human Glypican-1 SEQ ID NO:3: Nucleic acid sequence of mouse Glypican-1 SEQ ID NO: 4: Amino acid sequence of mouse Glypican-1 SEQ ID NO: 5:01a033 Amino acid sequence of the heavy chain variable region SEQ ID NO:6:01a033 Amino acid sequence of the light chain variable region SEQ ID NO: 7: Amino acid sequence of the humanized heavy chain variable region SEQ ID NO: 8: Amino acid sequence of the humanized light chain variable region of clone T2 SEQ ID NO: 9: Amino acid sequence of the humanized light chain variable region of clone T7 SEQ ID NO: 10: Amino acid sequence of the humanized light chain variable region of clone T8 SEQ ID NO: 11: Amino acid sequence of the humanized light chain variable region of clone T1 SEQ ID NO: 12: Amino acid sequence of the humanized light chain variable region of clone T36 SEQ ID NO: 13: Amino acid sequence of the humanized light chain variable region of clone T57 SEQ ID NO: 14: Amino acid sequence of the humanized light chain variable region of clone T10 SEQ ID NO: 15: Amino acid sequence of the humanized light chain variable region of clone T34 SEQ ID NO: 16: Amino acid sequence of the humanized light chain variable region of clone T56 SEQ ID NO: 17: Amino acid sequence of the humanized light chain variable region of clone T3 SEQ ID NO: 18: Amino acid sequence of the humanized light chain variable region of clone T11 SEQ ID NO: 19: Amino acid sequence of the humanized light chain variable region of clone T28 SEQ ID NO: 20: Amino acid sequence of the humanized light chain variable region of clone T43 SEQ ID NO: 21: Amino acid sequence of the humanized light chain variable region of clone T59 SEQ ID NO: 22: Amino acid sequence of the humanized light chain variable region of clone T31 SEQ ID NO: 23: Amino acid sequence of the humanized light chain variable region of clone T22 SEQ ID NO: 24: Amino acid sequence of the humanized light chain variable region of clone T26 SEQ ID NO: 25: Amino acid sequence of the humanized light chain variable region of clone T32 SEQ ID NO: 26: Amino acid sequence of the humanized light chain variable region of clone T33 SEQ ID NO: 27: Amino acid sequence of the humanized light chain variable region of clone T29 SEQ ID NO: 28: Amino acid sequence of heavy chain CDR1 of the humanized heavy chain SEQ ID NO: 29: Amino acid sequence of the heavy chain CDR2 of the humanized heavy chain SEQ ID NO: 30: Amino acid sequence of the heavy chain CDR3 of the humanized heavy chain SEQ ID NO: 31: Amino acid sequence of the light chain CDR1 of clone T2 SEQ ID NO: 32: Amino acid sequence of the light chain CDR2 of clone T2 SEQ ID NO: 33: Amino acid sequence of the light chain CDR3 of clone T2 SEQ ID NO: 34: Amino acid sequence of the light chain CDR1 of clone T7 SEQ ID NO: 35: Amino acid sequence of the light chain CDR2 of clone T7 SEQ ID NO: 36: Amino acid sequence of the light chain CDR3 of clone T7 SEQ ID NO: 37: Amino acid sequence of the light chain CDR1 of clone T8 SEQ ID NO: 38: Amino acid sequence of the light chain CDR2 of clone T8 SEQ ID NO: 39: Amino acid sequence of the light chain CDR3 of clone T8 SEQ ID NO: 40: Amino acid sequence of the light chain CDR1 of clone T1 SEQ ID NO: 41: Amino acid sequence of the light chain CDR2 of clone T1 SEQ ID NO: 42: Amino acid sequence of the light chain CDR3 of clone T1 SEQ ID NO: 43: Amino acid sequence of the light chain CDR1 of clone T36 SEQ ID NO: 44: Amino acid sequence of the light chain CDR2 of clone T36 SEQ ID NO: 45: Amino acid sequence of the light chain CDR3 of clone T36 SEQ ID NO: 46: Amino acid sequence of the light chain CDR1 of clone T57 SEQ ID NO: 47: Amino acid sequence of the light chain CDR2 of clone T57 SEQ ID NO: 48: Amino acid sequence of the light chain CDR3 of clone T57 SEQ ID NO: 49: Amino acid sequence of the light chain CDR1 of clone T10 SEQ ID NO: 50: Amino acid sequence of the light chain CDR2 of clone T10 SEQ ID NO: 51: Amino acid sequence of the light chain CDR3 of clone T10 SEQ ID NO: 52: Amino acid sequence of the light chain CDR1 of clone T34 SEQ ID NO: 53: Amino acid sequence of the light chain CDR2 of clone T34 SEQ ID NO: 54: Amino acid sequence of the light chain CDR3 of clone T34 SEQ ID NO: 55: Amino acid sequence of the light chain CDR1 of clone T56 SEQ ID NO: 56: Amino acid sequence of the light chain CDR2 of clone T56 SEQ ID NO: 57: Amino acid sequence of the light chain CDR3 of clone T56 SEQ ID NO: 58: Amino acid sequence of the light chain CDR1 of clone T3 SEQ ID NO: 59: Amino acid sequence of the light chain CDR2 of clone T3 SEQ ID NO: 60: Amino acid sequence of the light chain CDR3 of clone T3 SEQ ID NO: 61: Amino acid sequence of the light chain CDR1 of clone T11 SEQ ID NO: 62: Amino acid sequence of the light chain CDR2 of clone T11 SEQ ID NO: 63: Amino acid sequence of the light chain CDR3 of clone T11 SEQ ID NO: 64: Amino acid sequence of the light chain CDR1 of clone T28 SEQ ID NO: 65: Amino acid sequence of the light chain CDR2 of clone T28 SEQ ID NO: 66: Amino acid sequence of the light chain CDR3 of clone T28 SEQ ID NO: 67: Amino acid sequence of the light chain CDR1 of clone T43 SEQ ID NO: 68: Amino acid sequence of the light chain CDR2 of clone T43 SEQ ID NO: 69: Amino acid sequence of the light chain CDR3 of clone T43 SEQ ID NO: 70: Amino acid sequence of the light chain CDR1 of clone T59 SEQ ID NO: 71: Amino acid sequence of the light chain CDR2 of clone T59 SEQ ID NO: 72: Amino acid sequence of the light chain CDR3 of clone T59 SEQ ID NO: 73: Amino acid sequence of the light chain CDR1 of clone T31 SEQ ID NO: 74: Amino acid sequence of the light chain CDR2 of clone T31 SEQ ID NO: 75: Amino acid sequence of the light chain CDR3 of clone T31 SEQ ID NO: 76: Amino acid sequence of the light chain CDR1 of clone T22 SEQ ID NO: 77: Amino acid sequence of the light chain CDR2 of clone T22 SEQ ID NO: 78: Amino acid sequence of the light chain CDR3 of clone T22 SEQ ID NO: 79: Amino acid sequence of the light chain CDR1 of clone T26 SEQ ID NO: 80: Amino acid sequence of the light chain CDR2 of clone T26 SEQ ID NO: 81: Amino acid sequence of the light chain CDR3 of clone T26 SEQ ID NO: 82: Amino acid sequence of the light chain CDR1 of clone T32 SEQ ID NO: 83: Amino acid sequence of the light chain CDR2 of clone T32 SEQ ID NO: 84: Amino acid sequence of the light chain CDR3 of clone T32 SEQ ID NO: 85: Amino acid sequence of the light chain CDR1 of clone T33 SEQ ID NO: 86: Amino acid sequence of the light chain CDR2 of clone T33 SEQ ID NO: 87: Amino acid sequence of the light chain CDR3 of clone T33 SEQ ID NO: 88: Amino acid sequence of the light chain CDR1 of clone T29 SEQ ID NO: 89: Amino acid sequence of the light chain CDR2 of clone T29 SEQ ID NO: 90: Amino acid sequence of the light chain CDR3 of clone T29 SEQ ID NO: 91: Amino acid sequences of the heavy chain constant regions of chimeric and humanized antibodies SEQ ID NO: 92: Amino acid sequences of the light chain constant region of chimeric and humanized antibodies

Claims

1. A humanized anti-GPC-1 antibody or antigen-binding fragment thereof, heavy chain CDR1-CDR3 having the amino acid sequences set forth in SEQ ID NOs: 28-30, respectively; and (a) comprising light chain CDR1-CDR3 having the amino acid sequences set forth in SEQ ID NOs: 31-33, respectively; or (b) light chain CDR1-CDR3 having the amino acid sequences set forth in SEQ ID NOs: 37-39, respectively; or (c) light chain CDR1-CDR3 having the amino acid sequences set forth in SEQ ID NOs: 40-42, respectively; or (d) light chain CDR1-CDR3 having the amino acid sequences set forth in SEQ ID NOs: 43-45, respectively; or (e) light chain CDR1-CDR3 having the amino acid sequences set forth in SEQ ID NOs: 49-51, respectively; or (f) light chain CDR1-CDR3 having the amino acid sequences set forth in SEQ ID NOs: 55-57, respectively; or (g) comprising light chain CDR1 to CDR3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively; A humanized anti-GPC-1 antibody or an antigen-binding fragment thereof.

2. 2. The humanized anti-GPC-1 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

7.

3. The humanized anti-GPC-1 antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a light chain variable region having an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8, 10, 11, 12, 14, 16, and 17.

4. The humanized anti-GPC-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a light chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 8, 10, 11, 12, 14, 16, and 17.

5. The humanized anti-GPC-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the epitope of the antibody comprises positions 339 to 358 and / or positions 388 to 421 of SEQ ID NO:

2.

6. The antibody has a molecular weight of 1.02E based on analysis by surface plasmon resonance. －８ The following K Ｄ The humanized anti-GPC-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to Glypican-1 at the agonistic level.

7. A pharmaceutical composition for intracellular internalization of an active ingredient, comprising the humanized anti-GPC-1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A conjugate of the humanized anti-GPC-1 antibody or its antigen-binding fragment according to any one of claims 1 to 6 and a drug having cytotoxic activity.

9. 9. The conjugate of claim 8, wherein the cytotoxic agent is selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), DM1, DM4, calicheamicin, duocarmycin, pyrrolobenzodiazepines (PBDs), and topoisomerase inhibitors.

10. The conjugate according to claim 8 or 9, wherein the drug having cytotoxic activity is cell membrane permeable.

11. The conjugate according to any one of claims 8 to 10, wherein the agent having cytotoxic activity is selected from MMAE, PBD, Eribulin, SN-38, Dxd, and DM4.

12. The conjugate according to any one of claims 8 to 11, wherein the humanized anti-GPC-1 antibody or antigen-binding fragment thereof is operably linked to the agent having cytotoxic activity via a linker.

13. 13. The conjugate of claim 12, wherein the linker is selected from the group consisting of an enzyme-cleavable linker, an acid-labile linker, and a disulfide linker.

14. The complex according to claim 12 or 13, wherein the linker is a cleavable linker that is cleaved by cathepsin B.

15. The complex has an IC of about 0.1 nM or less in Glypican-1 positive cells. ５０ The complex according to any one of claims 8 to 14, wherein

16. A composition for preventing or treating Glypican-1 positive cancer, comprising the complex according to any one of claims 8 to 15.

17. The composition of claim 16, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof.

18. The composition according to claim 16 or 17, wherein the Glypican-1 positive cancer is esophageal cancer or pancreatic cancer.

19. The composition of any one of claims 16 to 18, wherein the Glypican-1 positive cancer has cancer-associated fibroblasts (CAFs).

20. The composition according to any one of claims 16 to 19, characterized in that the composition is administered together with an immune checkpoint inhibitor.

21. The composition of claim 20, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

22. A composition for preventing or treating metastatic cancer of Glypican-1 positive cancer, comprising the complex according to any one of claims 8 to 15.

23. The composition of claim 22, wherein the Glypican-1 positive cancer is selected from esophageal cancer, pancreatic cancer, bile duct cancer, cervical cancer, lung cancer, head and neck cancer, breast cancer, uterine leiomyosarcoma, prostate cancer, oral squamous cell carcinoma, or any combination thereof.

24. The composition of claim 22 or 23, wherein the metastatic cancer is a metastatic cancer of a pancreatic cancer.

25. The composition of any one of claims 22 to 24, wherein the metastatic cancer is a metastatic cancer that has metastasized to the liver, esophagus, bile duct, cervix, lung, head and neck, breast, uterine smooth muscle, prostate, oral squamous epithelium, brain, bone, peritoneum, or adrenal gland.

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