Antibody-drug conjugate of Anti-EGFR / MUC1 bispecific antibody

Abstract

The present disclosure relates to: an antibody-drug conjugate of an anti-EGFR / MUC1 bispecific antibody; and a use thereof for tumor treatment.

Description

[0001]

Description of the Invention

[0002]

Title of Invention

[0003] Antibody-drug conjugate of anti-EGFR / MUC1 bispecific antibody

[0004]

Cross-reference to related applications

[0005] The present application claims priority to Korean patent application No. 10-2025-0004254, filed January 10, 2025; Korean patent application No. 10-2025-0142902, filed September 30, 2025; and Korean patent application No. 10-2025-0153530, filed October 22, 2025, the entire contents of each of these applications are incorporated by reference into the present disclosure.

[0006]

Technology Field

[0007] The technical field of the present disclosure relates to antibody-drug conjugates, the use thereof for treating tumors, and methods for treating tumors using the same.

[0008]

Background Skills

[0009] Since the development of Mylotarg® (gemtuzumab ozogamicin), the first Antibody-Drug Conjugate (ADC) approved by the US Food and Drug Administration (FDA) in 2000, research on ADCs has been actively underway. In particular, ADCs are referred to as "magic bullets" and "biological misses," attracting attention from academia and industry researching oncology. However, despite the research of numerous ADCs, only 14 ADCs have received market approval worldwide as of 2022. Although research on various antibodies and drugs for ADC development has been conducted for approximately 20 years, the development of effective ADCs remains a challenge.

[0010] Meanwhile, the epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is a 170 kDa type 1 transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. The epidermal growth factor receptor is a member of the ErbB receptor family, which includes four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), HER3 (ErbB-3), and Her4 (ErbB-4). In various cancers, mutations affecting the expression or activity of EGFR can lead to cancer. Since increased expression levels or kinase activity of EGFR are associated with various human cancers, EGFR is recognized as a promising target for therapeutic intervention. In non-small cell lung cancer, increases in both EGFR gene copy number and protein expression are associated with a good response to the EGFR tyrosine kinase inhibitor IRESSA™ (gef-it-inib). Representative examples of antibodies for cancer therapy targeting EGFR include,

[0011] Examples include cetuximab, panitumumab, and necitumumab. For a detailed review of EGFR, refer to the literature [Sabbah, DA, Haj jo, R., & Sweidan, K. (2020). Review on epidermal growth factor receptor (EGFR) structure, signaling pathways, interactions, and recent updates of EGFR inhibitors. Current topics in medicinal chemistry.] °].

[0012] Mucin 1 (MUCl) (also known as episialin, PEM, H23Ag, EMA, CA15-3, and MCA) is a single-pass type 1 transmembrane protein with a highly glycosylated extracellular domain extending approximately 200 to 500 nm from the cell surface. MUC1 is normally expressed in glandular or luminal epithelial cells of the breast, esophagus, stomach, duodenum, pancreas, uterus, prostate, and lungs, and to a lesser extent in hematopoietic cells. However, it is not expressed in skin epithelium and mesenchymal cells. Meanwhile, abnormally glycosylated MUC1 is overexpressed in most human cancers. MUC1 is attracting attention as an oncogeneic molecule and is one of the targets currently in the spotlight in the field of cancer treatment.

[0013] An anti-EGFR and anti-MUC1 bispecific antibody designed to target both EGFR and MUC1 is disclosed in PCT patent application publication number W02024 / 149195A1.

[0014]

Description of the Invention

[0015]

Technical Challenges

[0016] The problem to be solved by the present disclosure is to provide a bispecific antibody-drug conjugate (ADC) targeting EGFR and MUC1 that is usable for tumor treatment, and a method for treating a tumor using said antibody-drug conjugate.

[0017]

Technical Solution

[0018] The present disclosure provides novel antibody-drug conjugates based on bispecific antibodies targeting EGFR and MUC1 (e.g., anti-EGFR and anti-MUC1 bispecific antibodies). The antibody-drug conjugates of the present disclosure may be used for tumor treatment. For example, the antibody-drug conjugates of the present disclosure may be used to treat humans with tumors (e.g., tumors expressing at least one of EGFR and MUC1). Some embodiments of the present disclosure provide antibody-drug conjugates or pharmaceutically acceptable salts thereof.

[0019] Some embodiments of the present disclosure provide an antibody-drug conjugate having the structure of Formula 13 below or a pharmaceutically acceptable salt thereof:

[0020] [Equation 13]

[0021]

[0022] At this time,

[0023] AB is an antibody, wherein the antibody is an anti-EGFR / MUC1 bispecific antibody, and the anti-EGFR / MUC1 bispecific antibody comprises a first antigen-binding fragment targeting EGFR and a second antigen-binding fragment targeting MUC1, and

[0024] a is independently 0 or 1, and

[0025] y is 1 or 2.

[0026] In some embodiments, the first antigen-binding fragment may comprise HCDR1 having an amino acid sequence of sequence no. 1, HCDR2 having an amino acid sequence of sequence no. 2, HCDR3 having an amino acid sequence of sequence no. 3, LCDR1 having an amino acid sequence of sequence no. 4, LCDR2 having an amino acid sequence of sequence no. 5, and LCDR3 having an amino acid sequence of sequence no. 6, and the second antigen-binding fragment may comprise HCDR1 having an amino acid sequence of sequence no. 7, HCDR2 having an amino acid sequence of sequence no. 8, and HCDR3 having an amino acid sequence of sequence no. 9, LCDR1 having an amino acid sequence of sequence no. 4, LCDR2 having an amino acid sequence of sequence no. 5, and LCDR3 having an amino acid sequence of sequence no. 6.

[0027] In some embodiments, the first antigen-binding fragment may include a first heavy chain variable region of sequence number 10 and a light chain variable region of sequence number 15, and the second antigen-binding fragment may include a second heavy chain variable region of sequence number 22 and a light chain variable region of sequence number 15.

[0028] In some embodiments, the structure connected to AB in Formula 13 may be connected to an Asn residue located within the heavy chain constant region 2 (CH2) of the anti-EGFR / MUC1 bispecific antibody.

[0029] In some embodiments, the structure connected to AB in Formula 13 may be connected to Asn297 of the anti-EGFR / MUC1 bispecific antibody.

[0030] In some embodiments, the antibody-drug conjugate may have the structure of Formula 14 below:

[0031] [Equation 14]

[0032]

[0033] In this case, AB is the above-mentioned anti-EGFR / MUC1 bispecific antibody, and

[0034] a is independently 0 or 1, and

[0035] y is 1 or 2.

[0036] In some embodiments, y can be 2.

[0037] Some embodiments of the present disclosure provide a pharmaceutical composition for treating a target tumor, comprising an antibody-drug conjugate according to some embodiments of the present disclosure or a pharmaceutically acceptable salt thereof.

[0038] Some embodiments of the present disclosure provide a method for treating a tumor of a subject, comprising administering to the subject a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to some embodiments of the present disclosure.

[0039] Some embodiments of the present disclosure provide a use for manufacturing a therapeutic agent for treating a tumor targeting an antibody-drug conjugate or a salt thereof according to some embodiments of the present disclosure.

[0040] In some embodiments, the tumor may be a tumor expressing one or more of EGFR and MUC1. In some embodiments, the tumor may be an EGFR-positive and / or MUC1-positive tumor.

[0041] In some embodiments, the tumor may be a tumor expressing both EGFR and MUC1.

[0042] In some embodiments, the tumor may be an EGFR-positive and MUC1-positive tumor.

[0043] In some embodiments, the tumor is colon cancer, rectal cancer, renal cancer, renal cell carcinoma, urothelial cancer, breast cancer, triple-negative breast cancer, epithelial breast cancer, liver cancer, lung cancer, small cell lung cancer (SCLC), non-smal cell lung cancer (Sq-NSCLC), squamous non-smal cell lung cancer (Sq-NSCLC), non-squamous non-smal cell lung cancer (NSq-NSCLC), gastric cancer (Gastr ic cancer), colorectal cancer, colorectal adenocarcinoma, prostate cancer, small intestine cancer, esophageal cancer, hepatocellular carcinoma, melanoma, orthotopic cancer, bone cancer, ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, skin cancer, head and neck cancer,Head and neck squamous cell carcinoma, Head and neck adenocarcinoma, Neuroendocrine tumor (Carcinoid), Cutaneous or intraocular malignant melanoma, Uterine cancer, Ovarian cancer, Epithelial ovarian cancer, Rectal cancer, Cancer of the anal region, Stomach cancer, Testicular cancer, Carcinoma of the fallopian tubes, Endometrial cancer, Cervical cancer, Carcinoma of the vagina, Vulvar cancer (Carcinoma of the vulva), Hodgkin's Disease, non-Hodgkin's lymphoma, lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma, cholangiocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), esophageal adenocarcinoma,Salivary gland cancer, Sarcoma, Soft tissue sarcoma, Cancer of the urethra, Urothelial carcinoma, Cancer of the penis, Solid tumors of childhood, Bladder cancer, Bladder squamous cell carcinoma (bladder SCC), Cancer of the kidney or ureter, Carcinoma of the renal pelvis, Neurological cancer, Neoplasm of the central nervous system (CNS), Primary CNS lymphoma, Spinal axis tumor, Glioma It may be one or more selected from (G1 ioma), brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, and T-cell lymphoma.

[0044] Some embodiments of the present disclosure provide a method for manufacturing an antibody-drug conjugate comprising the following:

[0045] Engineered bispecific antibody having the structure of Formula 03 below

[0046] - [Essence 03]

[0047]

[0048] At this time,

[0049] AB is an antibody, wherein the antibody is an anti-EGFR / MUC1 bispecific antibody, and the anti-EGFR / MUC1 bispecific antibody comprises a first antigen-binding fragment targeting EGFR and a second antigen-binding fragment targeting MUC1, and

[0050] a is independently 0 or 1, and

[0051] x is 1 or 2 - ; and

[0052] Linker-payload compound having the structure of Formula 08 below

[0053] - [Essence 08]

[0054]

[0055] Contacting.

[0056] In some embodiments, the first antigen-binding fragment comprises HCDR1 having the amino acid sequence of sequence no. 1, HCDR2 having the amino acid sequence of sequence no. 2, HCDR3 having the amino acid sequence of sequence no. 3, LCDR1 having the amino acid sequence of sequence no. 4, LCDR2 having the amino acid sequence of sequence no. 5, and LCDR3 having the amino acid sequence of sequence no. 6, and the second antigen-binding fragment may comprise HCDR1 having the amino acid sequence of sequence no. 7, HCDR2 having the amino acid sequence of sequence no. 8, and HCDR3 having the amino acid sequence of sequence no. 9, LCDR1 having the amino acid sequence of sequence no. 4, LCDR2 having the amino acid sequence of sequence no. 5, and LCDR3 having the amino acid sequence of sequence no. 6.

[0057]

Effects of the Invention

[0058] The antibody-drug conjugate of the present disclosure has excellent anticancer activity and low toxicity. The antibody-drug conjugate of the present disclosure can be used to treat cancer in patients.

[0059]

Brief Description of Drawing

[0060] FIG. 02 relates to an example of a bispecific antibody structure of the present disclosure. FIG. 03 relates to an example of an antibody engineered through glyco-engineering. FIG. 04 relates to an example of a DAR4 antibody-drug conjugate.

[0061] FIG. 05 shows an example of a DAR2 antibody-drug conjugate, specifically, an example of a DAR2 antibody-drug conjugate made by conjugating one linker-payload compound to an engineered antibody having two azides.

[0062] FIG. 06 shows an example of a DAR2 antibody-drug conjugate, specifically, an example of a DAR2 antibody-drug conjugate made by conjugating a linker-payload compound to a engineered antibody having one azide.

[0063] Figure 07 shows an example of the antibody-drug conjugate of Equation 14 (when y=2).

[0064] Figure 08 shows the RP-UPLC chromatogram analysis results of the dual antibody ADC (AB0799).

[0065] Figure 09 relates to the results of the analysis of the cell binding affinity of antibodies, including dual antibodies. Specifically, Figure 09 shows the results of the analysis of the cell binding affinity of antibodies against NUGC-4 and HCC70 cell lines.

[0066] Figure 10 relates to the results of the analysis of the cell binding affinity of antibodies, including dual antibodies. Specifically, Figure 10 shows the results of the analysis of the cell binding affinity of antibodies against HCC1954, PANC02.03, and A431 cell lines.

[0067] Figure 11 shows the results of the cell binding affinity analysis of AB0799 to CFPAC-1 and NCI-H1975 cell lines.

[0068] Figure 12 relates to the results of the cellular internal isolation analysis of antibodies and ADCs. Specifically, Figure 12 shows the results of the cellular internal isolation analysis of ADCs and antibodies, including AB0799, on HCC70 and HSC4 cell lines.

[0069] Figure 13 relates to the results of the cellular internal isolation analysis of antibodies and ADCs. Specifically, Figure 13 shows the results of the cellular internal isolation analysis of ADCs and antibodies, including AB0799, on 647-V and MDA-MB-468 cell lines. Figure 14 relates to the results of the toxicity evaluation of AB0799 on HEKn (Human Epidermal Keratocytes, neonatal) cells.

[0070] Figure 15 relates to the results of evaluating the effect of AB0799 in inducing exposure to calret icul-in.

[0071] Figure 16 relates to the results of evaluating the effect of AB0799 inducing HMGB1 release.

[0072] Figure 17 shows the results of confirming the cell 1 cycle arrest-inducing effect of AB0799.

[0073] Figure 18 shows the results of confirming the caspase-3 / 7 activity-inducing effect of AB0799.

[0074] Figure 19 relates to the results confirming the apoptosis-inducing effect of AB0799. Figure 20 relates to the results confirming the bystander effect of AB0799. Figure 21 relates to the results confirming whether AB0799 inhibits EGF signaling effects, in which EGFR or phosphorylated EGFR was evaluated by Western blotting under each condition. Figure 22 relates to the results confirming whether AB0799 inhibits EGF signaling effects, in which the Western blotting results of Figure 21 were quantified using an image analysis program.

[0075] Figure 23 shows the results of the efficacy evaluation of AB0799 in a human-derived breast cancer cell line HCC1954 xenograft mouse model.

[0076] Figure 24 shows the results of the efficacy evaluation of AB0799 in a human-derived pancreatic cancer cell line CFPAC-1 xenograft mouse model.

[0077] Figure 25 relates to the results of a comparison of the unit price and efficacy of AB0799 against its divalent counterparts in a human-derived pancreatic cancer cell line CFPAC-1 xenograft mouse model.

[0078] Figure 26 shows the results of the efficacy comparison of AB0799O and DS-3939a in a human-derived pancreatic cancer cell line CFPAC-1 xenograft mouse model.

[0079] Figure 27 relates to the evaluation of the efficacy of AB0799 in a human-derived pancreatic cancer cell line xenograft mouse model, specifically showing the results for the AsPC-1 cell line.

[0080] Figure 28 shows the evaluation of the efficacy of AB0799 in a human-derived pancreatic cancer cell line xenograft mouse model, specifically showing the results for the HPAF-II cell line.

[0081] Figure 29 shows the evaluation of the efficacy of AB0799 in a human-derived pancreatic cancer cell line xenograft mouse model, specifically showing the results for the Capan-2 cell line.

[0082] Figure 30 shows the evaluation of the efficacy of AB0799 in a human-derived pancreatic cancer cell line xenograft mouse model, specifically showing the results for the CFPAC-1 cell line.

[0083] Figure 31 shows the results of the anti-tumor activity of AB0799 in 36 PDX mouse models, representing the rate of change relative to baseline upon administration of AB0799 for each model and each dosage. Figure 32 shows the IHC results confirming the expression of EGFR and MUC1 in PDX models. The expression of EGFR and MUC1 was confirmed in all colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, head and neck cancer, and bladder cancer samples.

[0084] Figure 33 shows the IHC results confirming the expression of EGFR and MUC1 in PDX models. Specifically, it shows the IHC results for EGFR and MUC1, respectively, in the LU5371, CR5082, HN9296, PA3139, PA3127, HN0696, CR1197, CR6225, CR3595, PA6258, PA3142, LU5351, ES0110, LU11855, LU1206, BL5439, HN3533, and PA6238 models.

[0085] Figure 34 relates to the results confirming the binding ability of AB0799 to cells expressing EGFR.

[0086] Figure 35 shows the results of the in vitro cytotoxicity analysis of AB0799, AB0709, MRG-003, and naked MRG-003 against breast cancer cell line (MDA-MB-468).

[0087] Figure 36 shows the results of the in vitro cytotoxicity assay of AB0799, AB0709, MRG-003, and naked MRG-003 on human epidermal cells (HEKn).

[0088] Figure 37 shows the results of the in vitro cytotoxicity analysis of AB0799 and BSA01 in HEKa cells.

[0089] Figure 38 shows the results of the analysis of EGFR expression in each cancer using a human cancer tissue microarray.

[0090] Figure 39 shows the results of the analysis of MUC1 expression in each cancer using a human cancer tissue microarray.

[0091] Figure 40 shows the results of analyzing the antitumor activity of AB0799 alone and AB0799 in combination with sotor as ib in the NCI-H1373 CDX model. Figure 41 shows the results of analyzing the antitumor activity of AB0799 and the Benchmark ADC in the NCI-H1975 CDX model.

[0092] Figure 42 shows the results of analyzing the antitumor activity of AB0799 and Dato-Dxd in the CTG-4001 model.

[0093] Figure 43 shows the results of the cytotoxicity analysis of AB0799 against the tumor cell line NCI-H1373.

[0094] Figure 44 shows the results of confirming the binding affinity of AB0799 to EGFR according to the storage period in monkey plasma.

[0095] Figure 45 shows the results of confirming the binding affinity of AB0799 to MUC1 according to the storage period in monkey plasma.

[0096] Figure 46 shows the results of confirming the cytotoxicity of AB0799 against the tumor cell line HCC827 according to the storage period in monkey plasma.

[0097] Figure 47 shows the results of confirming the in vitro stability of AB0799 according to the storage period in human and monkey plasma. Specifically, in vitro stability was analyzed based on the ratio of the amount of released exatecan according to the plasma storage period.

[0098]

Form for carrying out the invention

[0099] Hereinafter, the content of the invention disclosed in the present disclosure will be explained in more detail through embodiments and examples. The invention disclosed by the present disclosure may be embodied in various ways and is not limited to the specific embodiments or examples described herein.

[0100] A person skilled in the art to which the invention disclosed in this disclosure belongs would be able to conceive of various modifications and other embodiments of the content of the invention disclosed in this disclosure. Accordingly, it will be clear to a person skilled in the art that the content of the invention disclosed in this disclosure is not limited to the specific embodiments or examples described herein, and that substitutions and modifications may be made within the scope of this disclosure and the scope of the inventive spirit disclosed in this disclosure.

[0101] As used in this disclosure, the term "comprising" and its derivatives refer to the specified feature(s),

[0102] It is an open-ended term specifying the existence of elements(s), components(s), groups(s), integers(s), and / or steps(s), but not excluding the existence of other unspecified features(s), elements(s), components(s), groups(s), integers(s), and / or steps(s). This also applies to words with similar meanings, such as "including," "having," and their derivatives.

[0103] The term “consisting of” and its derivatives used in the present disclosure are closed-ended terms specifying the presence of specified feature(s), element(s), component(s), group(s), integer(s), and / or step(s), and are used to exclude the presence of other unspecified feature(s), element(s), component(s), group(s), integer(s), and / or step(s).

[0104] The term “consisting essentially of” as used in this disclosure is used to specify elements that do not substantially affect the fundamental and novel characteristics of the specified feature(s), element(s), component(s), group(s), integer(s), and / or step(s), as well as the existence of such feature(s), element(s), component(s), group(s), integer(s), and / or step(s).

[0105] It should be understood that the contents of the disclosure described exemplarily in this disclosure can be suitably practiced in the absence of any element(s) or limitation(s) not specifically presented in this disclosure.

[0106] All publications, patents, and other references mentioned in this disclosure are incorporated by reference in their entirety.

[0107] 1. Definition of Terms

[0108] Unless otherwise stated, all technical and scientific terms used in this disclosure have the meaning generally understood by those skilled in the art related to this disclosure.

[0109] The term “about” as used in the present disclosure means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0110] As used in this disclosure, the term "disease" refers to any condition or disorder that impairs or / or interferes with the normal function of a cell, tissue, organ, or organism. A disease may be, for example, a tumor or cancer. As used in this disclosure, the term "treatment" means to alleviate, reduce, or / or inhibit the progression of a disease and / or symptoms, etc., and encompasses all such meanings. For example, treatment may mean a partial or complete recovery of the disease and / or symptomatic condition to a normal state. For example, said treatment may mean a change in condition in which symptoms are improved or beneficially altered by the antibody-drug conjugate or composition, etc. of this disclosure, or any act that causes symptoms to be improved or altered.

[0111] As used in this disclosure, the term "therapeutic effective amount" refers to an amount sufficient to influence a beneficial or desired clinical outcome during treatment. The therapeutic effective amount may be administered to a subject on a single or more occasions. The therapeutic effective amount may be an amount sufficient to alleviate, improve, stabilize, reverse, or slow the progression of the disease, or to reduce the pathological outcomes of the disease. The compositions of this disclosure (e.g., pharmaceutical compositions) contain a therapeutic effective amount of an active substance (e.g., antibody-drug).

[0112] It may include conjugates.

[0113] The terms “protein,” “peptide,” and “polypeptide” as used in this disclosure are used interchangeably and encompass naturally occurring proteins, etc. and unnaturally occurring proteins, etc. Unnaturally occurring proteins, etc. may be, for example, fusion proteins, recombinant proteins, or synthetic proteins, etc. For example, a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. For example, a protein, peptide, or polypeptide may be naturally occurring. For example, a protein, peptide, or polypeptide may be recombinant or synthetic, or a combination of recombinant or synthetic and naturally occurring. Representative examples of proteins in this disclosure include antibodies and antigens, etc.

[0114] The term "amino acid" as used in this specification may be used to refer to both amino acids not combined with other amino acids and amino acid residues combined with other amino acids contained in a protein or peptide, and may be appropriately interpreted according to the content or context of the paragraph in which the term amino acid is used. For example, alanine may be used to refer to alanine and / or alanine residues. For example, arginine may be used to refer to arginine and / or arginine residues.

[0115] As used herein, the term "amino acid residue" refers to a structure derived from an amino acid that is contained in a compound, peptide, and / or protein (e.g., antibody, etc.) and is covalently connected to another part of said compound, peptide, and / or protein (e.g., an adjacent amino acid residue). Unless otherwise stated, amino acid sequences described in this disclosure are written using amino acid single-letter or triple-letter notation, from the N-terminal to the C-terminal. For amino acids that cannot be represented by the single-letter notation, other letters are used, and further supplementary explanations are provided. The amino acids and the triple-letter or single-letter abbreviations used in the art for each amino acid are as follows:

[0116] Alanine: Ala, A; Arginine: Arg, R;

[0117] Asparagine: Asn, N; Aspartic acid: Asp, D; Cysteine: Cys, C; Glutamic acid: Glu, E; Glutamine: Gin, Q; Glycine: Gly, G; Histidine: His, H; Isoleucine: I le, I; Leucine: Leu, L;

[0118] Lysine: Lys, K; Methionine: Met, M;

[0119] Phenylalanine: Phe, F; Proline: Pro, P; Serine: Ser, S; Threonine: Thr, T; Tryptophan: Trp, W; Tyrosine: Tyr, Y; Valine: Vai, V.

[0120] As used in this disclosure, the term “nucleic acid” encompasses naturally occurring nucleic acids and unnaturally occurring nucleic acids. Unnaturally occurring nucleic acids may be, for example, recombinant nucleic acids or synthetic nucleic acids. For example, nucleic acids may be naturally occurring, recombinant or synthetic, or any combination thereof. For example, nucleic acids may be naturally occurring. For example, nucleic acids may be recombinant or synthetic, or a combination of recombinant or synthetic and naturally occurring. In this disclosure, the term nucleic acid may be used to refer to the molecule itself or a part of a molecule. For example, nucleic acid may refer to a DNA molecule or a part or region thereof. For example, nucleic acid may refer to an RNA molecule or a part or region thereof. For example, nucleic acid may refer to a DNA-RNA hybrid molecule or a part or region thereof.

[0121] Unless otherwise stated, nucleic acids or nucleic acid sequences disclosed in this disclosure (e.g., DNA sequences, RNA sequences, DNA / RNA hybrid sequences) should be understood as being described in the 5' to 3' direction.

[0122] The term "sequence identity" as used in this specification is a term used in relation to the degree of similarity between two or more sequences. For example, the term "sequence identity" is used in conjunction with a term referring to a reference sequence and a term indicating a ratio (e.g., percentage). For example, the term "sequence identity" may be used to describe a sequence that is similar or substantially identical to a reference amino acid sequence. The method of calculating and / or determining the percentage of sequence identity is not otherwise limited and may be calculated and / or determined through reasonable methods or algorithms available to a person skilled in the art.

[0123] The term “linked” as used in this disclosure means that two or more conceptualizable elements are connected directly or indirectly (e.g., through other elements such as a linker), and is not intended to imply that no other additional elements can exist between said two or more elements. For example, a description such as “element B connected to element A” is intended to encompass both cases where one or more other elements exist between element A and element B (i.e., element A is connected to element B through one or more other elements such as a linker) and cases where one or more other elements do not exist between element A and element B (i.e., element A and element B are directly connected), and is not to be interpreted as being limited to such cases.

[0124] As used in this disclosure, the term “antigen-binding fragment” is used interchangeably with the term “antigen-binding domain” and refers to a molecule, compound, protein itself or a part thereof, domain, region, or fragment or a complex thereof having the ability to bind to some (or specific) antigen. The antigen-binding fragment may have the ability to specifically and / or selectively bind to at least one type of antigen (e.g., some antigen) or to specifically and / or selectively recognize at least one type of antigen. Examples of known antigen-binding fragments include, but are not limited to, antibodies, Fv (variable fragment), Fab (fragment antigen-binding), Fab', single chain variable fragment (scFv), Fab2 (e.g., monospecific Fab2 or bispecific Fab2), F(ab')2, minibodies, nanobodies, and diabodies. For example, antibodies may include antigen-binding fragments. For example, a bispecific antibody may include one or more (e.g., one or two) first antigen-binding fragments capable of binding to a first target antigen and one or more (e.g., one or two) second antigen-binding fragments capable of binding to a second target antigen.

[0125] As used in this disclosure, the term "single-chain variable fragment (scFv)" refers to a fusion protein in which the variable region of heavy chain (VH) and the variable region of light chain (VL) of an antibody (e.g., immunoglobulin) are covalently linked, and scFv is well known in the art. In scFv, the variable region of the heavy chain and the variable region of the light chain are directly linked to each other or are linked through a short peptide linker (e.g., a peptide of about 5 to 30 amino acids in length). In this case, the N-terminus of the variable region of the heavy chain and the C-terminus of the variable region of the light chain are linked (e.g., the directionality of VL-VH or VL-linker-VH), or the C-terminus of the variable region of the heavy chain and the N-terminus of the variable region of the light chain are linked (e.g., V『 The directionality of VL or VH-1 linker-VL is achieved. For example, the linker used in scFv (i.e., the scFv linker) can be designed to be glycine-rich for flexibility and serine or threonine-rich for solubility. Despite the removal of the constant region and the introduction of the linker, this protein is known to possess the specificity of the original immunoglobulin (see literature [U.S. Patent No. US10538588B]). For example, like this immunoglobulin, it may possess specificity for at least one specific antigen. scFv is used for various applications, such as flow cytometry, immunohistochemistry, bispecific antibodies, and the antigen-binding domain of artificial receptors (e.g., chimeric antigen receptors). As a non-limiting example, the linker may have any one of the following amino acid sequences:

[0126] GGGGSGGGGSGGGGSGGGGS ( (GGGGS)4 or (G4S)4, column number 46 ) ; and GGGGSGGGGSGGGGS ( (GGGGS)3 or (G4S)3, column number 47 ) .

[0127] In the present disclosure, “target antigen” is used to refer to an antigen to which any antigen-binding fragment can bind (e.g., specifically and / or selectively bind), and / or is specifically and / or selectively recognized by any antigen-binding fragment. The relationship between antigen-binding fragments such as antibodies or Fabs and scFvs and their target antigens is well known in the art.

[0128] As used in this disclosure, the term “tumor antigen” is used interchangeably with “cancer antigen,” and a tumor antigen refers to an antigenic substance (i.e., an antigen) produced or expressed in tumor (or cancer) cells (or tissues). Tumor antigens are useful as tumor markers for identifying cancer or tumor cells in diagnostic tests and are potential candidate targets (or candidate substances) for the treatment of cancer or tumors. In this disclosure, the term tumor antigen is used to encompass tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). Tumor-associated antigens refer to antigens that are expressed at high levels in tumor cells or tumor tissues but at low levels in healthy cells or normal tissues, while tumor-specific antigens refer to antigens that are found only in tumor cells and not in healthy cells.Tumor antigens known in the art include, for example, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL12A), CD7, CD20, B7-H3 (CD276), CD5, CD138, CD70, CD4, TACI, CD33, BAFF, Claudin, T4, EGFR, GD2, GD3, BCMA, TnAg, PSMA, R0R1, FLT3, NKG2D, TAG72, CD38, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, Mesothelin, IL-IIRa, PSCA, PRSS21, VEGFR2, Lewi These include sY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor or alpha, ERBB2 (Her2 / neu), MUC1, NCAM, Prostase, PAP, ELF2M, Ephr in B2, FAP, IGF-I receptor, CAIX, LMP2, gplOO, and bcr-abl, etc. For example, EGFR and MUC1 are tumor antigens.

[0129] Tumor (or cancer) cells (or tissues) express tumor antigens. Depending on the expressed tumor antigens, tumors (or cancers) can be described as tumors associated with certain tumor antigens, tumors expressing certain tumor antigens, tumors associated with the expression of certain tumor antigens, etc. The types of tumor antigens expressed vary depending on the type of tumor and / or the condition of the patient, and which tumor antigens are expressed in a patient's tumor can be identified through diagnostic methods known in the art.

[0130] In this disclosure, the expression “have” used in relation to sequences and chemical structures (e.g., chemical formulas) is an open-ended term that does not exclude the possibility of having additional elements other than the described sequence and chemical structure. On the other hand, the expression “have” encompasses the meaning of “is expressed” which cannot have additional elements other than the described sequence and chemical structure. Accordingly, in certain embodiments, the expressions “have a certain sequence” and “have a certain chemical structure” described in this disclosure may be reproduced, to the extent that the context and scientifically permissible, as expressions such as “is represented or made of a certain sequence” and “is represented or made of a certain chemical formula,” respectively. Where a sequence is disclosed in this disclosure, it may be understood that a sequence into which insignificant variations (e.g., substitution, deletion, and / or addition of amino acid residues) are introduced that do not have a significant effect on the function and / or activity of the nucleic acid or protein containing the sequence is also disclosed.

[0131] Without being bound by theory, a tumor is a mass of tissue or associated cells that may be benign (not cancerous) or malignant (cancer), whereas cancer refers to a disease resulting from the uncontrolled growth of abnormal cells. In some embodiments, the tumor in the present disclosure may be a benign tumor and / or a malignant tumor. In some embodiments, the tumor in the present disclosure may be cancer. For example, the term tumor used in the present disclosure may be replaced with the term cancer. For example, tumor cells may be cancer cells. For example, tumor tissue may be cancer tissue. For example, treatment of a tumor may be treatment of cancer. For example, anti-tumor activity or tumor-suppressing ability may be used interchangeably with anticancer activity or cancer-suppressing ability.

[0132] Strain-promoted azide-alkyne cycloaddition (SPAAC) is a cyclochemical reaction (or bioorthogonal reaction) well known in the art, in which an azide group and a cyclooctine group participate. A representative example of SPAAC is the reaction of azide with} BCN (Bicyclononyne), and a schematic diagram of this reaction is shown below.

[0133] [Reaction Schematic 01]

[0134]

[0135] Here, A and B are each arbitrary groups, symbols used to describe the reaction between azide and BCN.

[0136] As used in this disclosure, the term "drug" refers to a compound or a portion of a compound having the ability to kill cells. Meanwhile, a non-conjugated drug may be referred to as a free drug, and a conjugated drug may be referred to as a drug moiety, but is not otherwise limited. The term "drug" is used to encompass both free drugs and drug moietys and is to be appropriately interpreted and understood according to the context. Examples of drugs include auristatin, eribulin, tubulysin, geldanamycin,

[0137] Maytansinoid, cali cheami cin,

[0138] Mertansine, daunomycin, doxorubicin, methotrexate, vindesine, SG2285,

[0139] Dolastatin (dolastat in), dolastatin analog auristatin (dolastat in analogs aur i stat in), cryptophycin, camptothecin, camptothecin analog (e.g., SN38, FL118, or exatecan), rhizoxin derivative (rhizoxin derivative), CC 1065 analog or derivative,

[0140] Duocarmycin, enediyne antibiotic, esperamicin, epotilone, and

[0141] There are pyrrolobenzodiazepines (PBD) or derivatives.

[0142] The term "antibody" in this disclosure is used as a general term for proteins that specifically bind to a particular antigen. Antibodies may be proteins produced within the immune system in response to stimulation by an antigen, or proteins produced by chemical synthesis or recombinant synthesis thereof, and their types are not otherwise limited. Any antibody

[0143] It may mean an intact immunoglobulin of an isotype, or an antigen-binding fragment capable of competing with an intact antibody for binding to a target antigen. An antibody is itself a type of antigen-binding protein. An intact antibody may generally comprise at least two full-length heavy chains and two full-length light chains. In this disclosure, the antibody or antigen-binding fragment may be unnaturally generated, for example, recombinant or synthetically generated. For example, the antibody or its antigen-binding fragment may be produced by hybridomas, recombinant DNA technology, or enzymatic or chemical cleavage of an intact antibody, etc. In this disclosure, unless otherwise noted, the antibody may include an antibody comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and / or mutants thereof. The antibody may be an animal antibody (e.g., mouse antibody, etc.), a chimeric antibody (e.g., mouse-human antibody), a humanized antibody, or a human antibody. The term antibody encompasses both monospecific antibodies and bispecific antibodies. In some embodiments, the antibody may be a monoclonal antibody or a polyclonal (or multiple-clonal) antibody, but is not otherwise limited.

[0144] In the antibodies provided in this disclosure, the defined antigen-binding fragment, heavy chain CDR and light chain CDR, or the remaining portion excluding the heavy chain variable region and the light chain variable region may be derived from any isotype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgGl, IgG2, IgG3, or IgG4), IgM, etc.), and may be derived, for example, from the framework region and / or light chain constant region and / or heavy chain constant region of all said isotypes of immunoglobulin. For example, the antibodies provided in this disclosure may be human IgG type antibodies, e.g., IgGl, IgG2, IgG3, or IgG4 type antibodies, but are not limited thereto.

[0145] As used in this disclosure, the term “common light chain” refers to a light chain capable of interacting with two or more different heavy chains to form different antigen-binding sites, each of which may specifically bind to different antigens or epitopes. Similarly, the term “common light chain variable region” refers to a light chain variable region capable of interacting with two or more different heavy chain variable regions to form different antigen-binding sites, each of which may specifically bind to different antigens or epitopes. In some embodiments, the antibody or antigen-binding fragment may comprise a common light chain. In some embodiments, the antibody or antigen-binding fragment may comprise a common light chain variable region. In some embodiments, the anti-EGFR / MUC1 bispecific antibody or its antigen-binding fragment may comprise a common light chain or a common light chain variable It may include a territory.

[0146] As used in this disclosure, the term “bispecific antibody” is used to refer to an antibody designed to bind to two different types of antigens (i.e., designed to target two types of antigens). Here, the two types of antigens may be referred to as a first target antigen and a second target antigen. That is, the bispecific antibody comprises an antigen-binding fragment capable of binding to a first target antigen (e.g., a first antigen-binding fragment) and an antigen-binding fragment capable of binding to a second target antigen (e.g., a second antigen-binding fragment). Formats of bispecific antibodies include, but are not limited to, a bispecific antibody format having two first antigen-binding fragments and two second antigen-binding fragments (also referred to as a 2+2 bispecific antibody, which is divalent to the first antigen and may have divalent characteristics to the second antigen), a bispecific antibody format having one first antigen-binding fragment and one second antigen-binding fragment (also referred to as a 1+1 bispecific antibody, which may have monovalent characteristics to the first antigen and may have monovalent characteristics to the second antigen), and a bispecific antibody format having two first antigen-binding fragments and one second antigen-binding fragment (also referred to as a 2+1 bispecific antibody, which may have divalent characteristics to the first antigen and monovalent characteristics to the second antigen).

[0147] A bispecific antibody having one first antigen-binding fragment and one second antigen-binding fragment will be understood as a monovalent antibody for each of the two types of antigens.

[0148] The term "Knobs-into-holes (KIH)" as used in this disclosure refers to a method or technique for manipulating the CH3 domain of an antibody to create a "knob" or a "hole" in each heavy chain to promote heterodimerization or heavy chain dimer formation, and is a well-known technique primarily used in the production of asymmetric bispecific antibodies. For example, one heavy chain may contain one or more of Y349C (e.g., substitution of Y to C at the 349th position), T366W (or T366Y), and S354C substitutions (using the EU numbering system) for the knob, and the other heavy chain may contain one or more of Y349C, E356C, T366S, L368A, and Y407V (or Y407T) substitutions (using the EU numbering system) for the hole. An example of an amino acid sequence of the human IgGl-derived heavy chain constant region is described in SEQ No. 43 (in the amino acid sequence of SEQ No. 43, CH3 is the region consisting of the 224th to 330th amino acid residues), which can be referenced to identify the amino acids subject to substitution. For example, the heavy chain of the knob structure contains S354C and T366W substitutions, and the heavy chain of the hole structure contains Y349C, T366S, L368A, and Y407V substitutions. As another example, one heavy chain may contain one or more of Y349C and T366W substitutions, and the other heavy chain may contain one or more of E356C, T366S, L368A, and Y407V substitutions. An example of a 1+1 format bispecific antibody produced via KIH technology is shown in FIG. 01. As a non-limiting example, an example of a Knob-Hol pair of KIH technology that promotes or enables the formation of dimers is shown in Table 01 below.In Table 01 below, the Eu numbering system is used to indicate the position numbers of amino acids. 0 ] is used. For example, if one heavy chain contains knob mutations of S354C and T366W in the CH3 domain and the other heavy chain contains hole mutations of Y349C, T366S, L368A, and Y407V in the CH3 domain, these two heavy chains can form an intended heterodimer during the manufacturing process of an antibody (or bispecific antibody).

[0149] [Table 01] Pairs of known knob mutations and hole mutations

[0150] Name Knob mutation Hole mutation References

[0151] Pair 1 S354C, T366W Y349C, T366S, Carter, P. (2001). Bispecific L368A, Y407V human IgG by design. Journal of immunology

[0152] methods, 248(\-2) , 7-15.

[0153] Pair 2 T366W T366S, L368A, Atwel 1 , S. , Ridgway, J . B. , Y407V Wel ls, J . A. , & Carter , P .

[0154] (1997) . Stable heterodimers

[0155]

[0156] from remodel ing the domaininterface of a homodimer using a phage display

[0157] 1 ibrary. Journal of molecular biology, 270(1), 26-35.

[0158] Pair 3 T366Y Y407T Ridgway, J . B., Presta, L. G.,

[0159] & Carter, P. (1996). 'Knobs— into— holes' engineering of antibody CH3 domains for heavy chain heterodimerization.

[0160] Protein Engineering, Design and Selection, 9(7), 617-621.

[0161]

[0162] In this specification, when expressing the structure (substructure) of a compound, when indicating the part where a group of said substructure is connected to another group, it is approximately perpendicular to the bond.

[0163] Wavy lines drawn in the direction (wavy line) (e.g. 0 Use ■, X, and ). For example, X

[0164] When expressed as X, it indicates that group X within a substance, molecule, or compound is connected to other parts through bonds. For example, group X / VWV tV

[0165]

[0166] When expressed as such, it indicates that group X within a substance, molecule, or compound is connected to other parts through bonds. For example, in a compound having the structure "A-X", if only the structure of group X is depicted, structure X

[0167] It can be expressed as. For example, in a compound having the structure "A-X-B",

[0168] When only the x structure is depicted, it can be represented as s. If necessary, wavy lines drawn nearly perpendicular to the bonds may be indicated by additional notations. For example, in a compound having the structure A-X-B (where A, X, and B are each arbitrary groups, symbols for explaining the wavy lines drawn approximately perpendicular to the bonds), when only the structure of the X group is depicted, the structure of the X group is,

[0169] If necessary, they may be illustrated together, and information about which part each wavy line indicates a connection to can be provided by mentioning, for example, "* is the part connected to A (or the attachment part with A), and ** is the part connected to B (or the attachment part with B)."

[0170] The compounds of the present disclosure may have specific geometric or stereoisomer forms. Where a compound is disclosed in the present disclosure unless otherwise specified, the cyst and trans isomers, (-)- and (+)- enantiomers of said compound.

[0171] Isomers such as isomers (enan-VWWt *iomers), (R)- and (S)- enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemics are included in the scope of the present disclosure. That is, where there are no indications related to isomers (e.g., ', 's, and 'a', etc.) in the formula or structure disclosed in the present disclosure, the disclosed formula or structure may encompass all possible isomers.

[0172] Where compounds (e.g., small compounds, proteins (e.g., antibodies), and conjugates, etc.) are disclosed in this specification, it will be understood that the form of their salts is also disclosed. The ions forming the salts of the compounds are, for example, ammonium, calcium, sodium, potassium, acetate (CH3COO-), carbonate (CO3 2 Examples include chloride (CP), citrate, cyanide, fluoride (F“), nitrate (NO2'), nitrite (PO3'), and sulfate (SO₄'), and are not otherwise limited. Ions that form salts commonly used in the art may, if necessary, be used to form salts of the compound. The salt may be, for example, a pharmaceutically acceptable salt, wherein a pharmaceutically acceptable salt refers to a salt that possesses the efficacy of the parent agent and is not biologically undesirable (e.g., having little or no toxicity). Suitable salts include, for example, salts that can be formed by mixing a solution of the parent agent with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, phosphoric acid, sulfuric acid, or acetic acid. For example, When a compound involves an acidic residue, pharmaceutically acceptable salts may include salts formed with suitable organic ligands such as alkali metal ions (sodium or potassium), alkaline earth metal ions (calcium or magnesium), and ammonium ions.

[0173] 2. Background - Understanding Antibody Structure

[0174] In the following, to aid in understanding antibody structures, the structure of antibodies is described in detail based on what is generally known in the art, and the scope of the present disclosure is not limited by the description below.

[0175] The structure of antibodies is divided into heavy chains and light chains depending on the type of chain. Antibodies are generally known to contain two heavy chains and two light chains.

[0176] The structure of an antibody is divided into a variable region and a constant region based on the variability of its amino acid sequence. For example, the heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH). For example, the light chain includes a light chain variable region (VL) and a light chain constant region (CL).

[0177] The variable regions (VH and VL) are regions that include the antigen-binding portion, and within these variable regions, there is a hypervariable region with the greatest variability, which is called the complementarity-determining region (CDR). The heavy chain variable region and the light chain variable region each contain three CDRs. The three CDRs of the heavy chain variable region are designated as HCDR1 (heavy chain complementarity-determining region 1), HCDR2 (heavy chain complementarity-determining region 2), and HCDR3 (heavy chain complementarity-determining region 3), respectively, and the three CDRs of the light chain variable region are designated as LCDR1 (heavy chain complementarity-determining region 1), LCDR2 (heavy chain complementarity-determining region 2), and LCDR3 (heavy chain complementarity-determining region 3), respectively. These CDRs can provide major contact residues for the binding of antibodies (or antigen-binding fragments) to antigens or epitopes. The complementarity determining site (CDR) described in the present disclosure may be determined based on the definition of the CDR according to any one of the kabat numbering system, the Chothi numbering system, the IMGT numbering system, and the Martin numbering system.For example, any one of the numbering systems, such as the Kabat numbering system, the Chothi numbering system, and the Martin numbering system, may be used to refer to the position of a CDR sequence.

[0178] The portion of the variable region that is not [CDR°] is referred to as the framework region (FR). The constant region of the antibody is a region separated from the antigen-binding site, and it is known that said constant region can interact with cells or molecules of the immune system. For example, said constant region can interact (bind or connect) with the cell membrane of immune cells (e.g., lymphocytes, neutrophils, dendritic cells, and / or macrophages, etc.). Specifically, the hinge region and / or CH2 portion of said constant region can bind to a receptor (FCERIII, etc.) on the cell membrane of said immune cells. In another specific example, said constant region can bind to FcRn.

[0179] Antibodies are functionally divided into a fragment antigen-binding region (Fab region) that binds to antigens and a fragment crystallizable region (Fc region). For example, an antibody includes Fab and Fc regions. For example, Fc may include heavy chain constant region 2 and heavy chain constant region 3.

[0180] It is known that antibody types can be broadly classified into five types (or classes, or isotypes). These five types are determined by the type of heavy chain constant domain. The five types of antibodies mentioned above include immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). For example, each heavy chain of IgG is known to contain four immunoglobulin domains (VH, CHI, CH2, and CH3). Furthermore, IgG is known to be classified into more subdivided subtypes, for example, when describing the case where the antibody is a human antibody, if the type of heavy chain constant region of the antibody is gamma 1 (x 1), the type of antibody is UG1; if it is gamma 2 (x 2), the type of antibody is IgG2; if it is gamma 3 (x 3), the type of antibody is IgG3; and if it is gamma 4 (x 4), the type of antibody is IgG4.

[0181] It is known that the Fc region of each heavy chain of IgG-type antibodies contains a single N-glycosylation site. This N-glycosylation site is specifically located within the CH2 domain and is designated as site 297 according to the EU numbering system; it is commonly referred to as Asn297 (N297 or asparagine 297) or the conserved N-glycosylation site. Antibodies can contain N-glycans (or Asn297-1 linked glycans) through this N-glycosylation site. Meanwhile, many studies are being conducted to improve antibody function or confer drug-binding capabilities to antibodies by engineering this Asn297 site and / or N-glycan, and such engineering is referred to as glyco-engineering.

[0182] Meanwhile, a bispecific antibody, and in particular a 1+1 bispecific antibody format related to the bispecific antibody of the present disclosure, is designed to include one first antigen-binding fragment capable of binding to one target antigen (i.e., a first target antigen) and one second antigen-binding fragment capable of binding to another target antigen (i.e., a second target antigen) so as to be able to target additional target antigens compared to a monospecific antibody. That is, the 1+1 bispecific antibody format has one first antigen-binding fragment and one second antigen-binding fragment. The 1+1 bispecific antibody may be monovalent for each antigen (i.e., for each of the two types of antigens). For example, a variable region of one of the two heavy chains and a variable region of one of the common light chains form a first antigen-binding fragment, and a variable region of the other of the two heavy chains and a variable region of the other of the common light chains form a second antigen-binding fragment. The heavy chain or heavy chain variable region associated with the first antigen-binding fragment may be referred to as the first heavy chain or first heavy chain variable region, the light chain or light chain variable region associated with the first antigen-binding fragment may be referred to as the first light chain or first light chain variable region, the heavy chain or heavy chain variable region associated with the second antigen-binding fragment may be referred to as the second heavy chain or second heavy chain variable region, and the light chain or light chain variable region associated with the second antigen-binding fragment may be referred to as the second light chain or second heavy chain variable region. For example, the KIH technology described above may be used in the preparation of the bispecific antibody of the present disclosure. For example, the first chain may be a chain containing a knob, and the second chain may be a chain containing a hole. As another example, the first chain may be a chain containing a hole, and the second chain may be a chain containing a knob.

[0183] 3. Background - Difficulties in ADC Development

[0184] 3.1. Overview

[0185] The contents described in this section are provided to aid in understanding the difficulties in design as well as the difficulties in predicting effects due to the process and complexity of antibody-drug conjugate (ADC) development, and do not limit the scope of the disclosure.

[0186] An antibody-drug conjugate (ADC) refers to a conjugate of an antibody and a drug formed by linking the antibody and the drug through a linker so that the antibody selectively binds only to cells of interest (i.e., target cells) expressing a target antigen, and the drug that induces cell death can be selectively released in the intracellular environment.

[0187] ADCs treat cancer by selectively killing target cells (i.e., cancer cells) through the process of (i) binding of the antibody to the surface protein (target antigen) of the target cell, (ii) internalization of the ADC induced by the binding of the target antigen to the antibody, (iii) release of the drug from the conjugate by the action of a specific enzyme or environment within the cell (e.g., release of the drug through the release of conjugation between the drug and the antibody), and (iv) the action of the drug's intrinsic function (apoptosis).

[0188] 3.2. ADC Design Process

[0189] ADCs involve multiple elements such as antibodies, target antigens, drugs (payloads), linkers, drug-antibody conjugation ratios (DARs), and conjugation sites, and these elements do not act independently but interact in a non-linear manner. Since these interactions cause each element to simultaneously affect multiple pharmacological variables (e.g., binding affinity, internalization ability, stability, toxicity, anticancer activity, etc.), the process of designing a drug expected to achieve the goal of in vivo treatment inherently involves a high degree of complexity.

[0190] The following describes the representative factors and considerations generally taken into account when designing a mechanism to achieve objectives. However, the following does not limit the processes or considerations in ADC design. In practice, to design a mechanism to achieve objectives, ADC developers consider far more features and their relationships than those described below, explore vast amounts of information, and design the ADC through complex thinking and stepwise experimental verification processes. 3.2.1. Selection of Target Antigens

[0191]

[0192] A target antigen is selected. The target antigen must be a cell surface-expressed protein that is expressed on the cell surface and, simultaneously, allows the antibody to be internalized into the cell when it binds to that protein. Furthermore, the target antigen needs to be selected as one that is expressed more in tumor tissue (or cells) than in normal tissue, or / and is expressed in tumor tissue in a manner different from that in normal tissue. In addition, during this process, both the expression characteristics and biological properties of the target antigen are considered. For example, since the level of expression of the target antigen in normal tissue is strongly correlated with the on-target toxicity of the ADC (toxicity caused by the ADC acting on normal cells expressing the target antigen), the ADC is designed with this in mind. Additionally, if the target antigen exhibits shedding characteristics, the ADC is designed taking this into account.

[0193] 3.2.2. Design of Antibodies

[0194]

[0195] In antibody design, the design of the antibody's antigen-binding fragment is important because the binding affinity and binding site to the target antigen vary depending on the antigen-binding fragment (or antigen-binding domain) (particularly, CDRs) that confers binding affinity to the antibody. The characteristics of the selected target antigen are considered in the design of the antigen-binding fragment. For example, if the target antigen is an antigen with characteristics expressed in normal cells, a high affinity of the antibody for that antigen does not always guarantee a positive result; instead, the affinity for the antigen is determined through CDRs, or regarding the corresponding antigen

[0196] It is necessary to lower the affinity for the antigen by regulating the binding valency. However, while lowering the affinity for the antigen can reduce on-target toxicity to normal cells, there is a problem in that the anticancer activity against tumor cells is simultaneously reduced. In addition, the characteristics of the target antigen and the antigen binding fragment affect the internalization ability of dairy cows, and internalization ability, like binding affinity, is involved in both the anticancer activity of the antibody and toxicity in terms of side effects.

[0197] 3.2.3. Drug Design

[0198]

[0199] In drug design, the drug's inherent mechanism of action (MoA) and potency are considered. The characteristics of the target antigen and the target cells (tumor cells) expressing it are taken into account when selecting the drug's MoA. Some known drugs are known to be unsuitable for certain types of target cells. Furthermore, depending on the type of tumor cells expressing the target antigen or the tumor associated with the target antigen, a potent drug may be required. As such, a drug's MoA and potency are important considerations regarding anticancer activity. However, a drug's MoA and potency are also strongly linked to the toxicity of ADCs. Normal cells expressing certain target antigens are vulnerable to some known drugs. Additionally, drugs may act on unintended tissues, in which case a drug with high potency increases the toxicity of the ADC. Meanwhile, since drug activity is influenced not only by the inherent characteristics of the drug but also by the ADC's tumor target specificity, binding affinity to target antigens, internalization ability, and linker release capability, other characteristics of the ADC must also be considered. Furthermore, as the drug structure can affect the stability of the ADC, consideration of the drug structure is also required. 3.2.4. Linker Design

[0200]

[0201] Linkers affect the binding affinity, internalization ability, stability, toxicity, and anticancer activity of ADCs to target antigens. Therefore, it is necessary to design or select an appropriate linker that takes these factors into account, and a linker capable of releasing the drug under appropriate conditions must also be designed or selected.

[0202] 3.2.5. Others

[0203]

[0204] In ADCs, DAR (Drug-to-Antibody Ratio) is associated with the activity of the ADC. DAR is determined by considering the aforementioned selected target antigen, binding affinity to the antigen, internalization ability, and drug characteristics in combination. An increase in DAR allows for the expectation of increased anticancer activity. However, an increase in DAR also induces side effects such as increased toxicity. Furthermore, DAR affects the stability of the ADC. In addition, the conjugation method and conjugation location, which can affect the activity of the ADC, must also be considered in combination with other factors in ADC design. Meanwhile, DAR, as well as the conjugation method and location, can also be associated with the linker.

[0205] 3.3. Unpredictability of ADC Effects and Design Difficulties

[0206] In the development of antibody-drug conjugates, whether the antibody-drug conjugate can exhibit excellent anticancer activity in vivo while maintaining a low level of toxicity is an important consideration. However, since the anticancer activity and toxicity of antibodies are not derived from a single variable or simple structural characteristics, but are complex results resulting from the intertwining of multiple factors such as the target antigen, antibody, linker, drug, and the drug-antibody conjugation ratio (DAR), it is difficult to reasonably predict in advance whether a specific ADC will achieve the desired level of anticancer activity and low toxicity.

[0207] First, anticancer activity is influenced by various variables, such as antigen characteristics, antigen binding affinity and specificity of the ADC, ADC internalization ability, ADC cytorepressive ability, the cytorepressive ability of the antibody itself, ADC stability, drug release characteristics, mechanism of action, drug potency, and antibody-drug interaction (DAR). These variables do not act independently of each other but interact in a complex manner. Furthermore, these variables are influenced by the structural elements constituting the antibody; however, each structural element does not act individually on a specific variable, but rather influences each variable through non-linear interactions among multiple structural elements. For this reason, there are inherent limitations in predicting the magnitude of the anticancer activity of an ADC based solely on structural elements.

[0208] Toxicity is also influenced by a complex interplay of multiple variables. Representative examples include non-specific ADC accumulation in non-target tissues, the stability of the milk, the characteristics of the target antigen, its action on generative cells expressing the target antigen, the biological characteristics of the generative cells, the mechanism of action of the drug, the potency of the drug, and drug release characteristics, all of which have a direct impact on toxicity. Meanwhile, variables affecting anticancer activity, such as the antigen-binding affinity and specificity of the milk, the internalization capacity of the milk, the cytorepressive capacity of the milk, and the cytorepressive capacity of the antibody itself, also influence toxicity when the ADC acts on generative cells expressing the target antigen. Variables affecting toxicity are also influenced by the structural elements constituting the milk; however, rather than each structural element acting individually on a specific variable, multiple structural elements interact non-linearly to influence each variable. Therefore, there are inherent limitations to predicting the magnitude of toxicity of ADCs based solely on structural factors, just as there are limitations to anticancer activity.

[0209] Furthermore, anticancer activity and toxicity are not completely distinct and independent characteristics. While an increase in anticancer activity and a decrease in toxicity represent changes in both in a positive direction, anticancer activity and toxicity tend to respond in different ways to the same structural change (for example, a single structural change may have a positive effect on one of the two but a negative effect on the other), meaning there exists an impossible trade-off between the two. For instance, designing an antibody to increase its binding affinity to a target antigen can enhance anticancer activity, but it may also increase the potential for toxicity to normal cells expressing the same antigen. Additionally, designs that increase DAR or use potent drugs to enhance the anticancer activity of an ADC also increase toxicity. Conversely, designs that reduce binding affinity to the target antigen, reduce DAR, or use mild drugs to lower toxicity hinder the ADC from maintaining anticancer activity.

[0210] As such, the effect of yow is closer to an emergent property—a complex attribute that is difficult to explain solely by a simple structure-activity relationship. Therefore, it is difficult to predict at the structural design stage whether a specific ADC will exhibit sufficient anticancer activity and toxicity to achieve the goal of treatment through administration. In fact, determining whether a developed ADC is suitable for clinical trials requires extensive experimental performance evaluations, including numerous anticancer activity and toxicity tests. This supports the fact that it is difficult to predict whether an ADC can be used for treatment based solely on structural design, and that this can only be confirmed through experimental verification.

[0211] In addition, as previously explained, the anticancer activity and toxicity of dairy products are determined by the non-linear interaction of multiple variables and structural factors; therefore, careful consideration during the design phase is essential to increase the likelihood that the ADC will exhibit its intended effect. However, the design process itself is not simple. This is because the elements of an ADC, such as the target antigen, the antigen-binding site of the antibody, the linker, the drug (payload), the binding site, and the drug-antibody conjugation ratio (DAR), do not act independently of each other but are complexly linked with other components to influence multiple variables.

[0212] Furthermore, in the case of UPOS based on bispecific antibodies, additional factors must be considered compared to UPOS based on single-specific antibodies due to the structural characteristic of the antibody recognizing two types of antigens. Since these additional factors also interact complexly with other factors to affect multiple variables, the design of UPOS based on bispecific antibodies has a higher level of complexity compared to ADCs based on single-specific antibodies.

[0213] Consequently, the design of an ADC expected to achieve its intended purpose requires extensive technical knowledge, relevant information, the accumulated experience of highly skilled developers, and careful and complex considerations, and the process of designing the ADC is understood to entail substantially high design difficulty.

[0214] 4. Background - Target Antigens EGFR and MUC1

[0215] 4.1. EGFR One of the target antigens of the antibody (e.g., bispecific antibody) or antibody-drug conjugate of the present disclosure is EGFR (Epidermal growth factor receptor). EGFR, also referred to as ErbB or HER1, is a Type 1 transmembrane glycoprotein of about 170 kDa that is encoded by the c-erbBl proto-oncogene. The epidermal growth factor receptor is a member of the ErbB receptor family, which includes four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), HER3 (ErbB-3), and Her4 (ErbB-4). A reference sequence of the full human EGFR is provided in the present disclosure as sequence number 44 (see UniProt IN NO. P00533).

[0216] In various cancer types, EGFR expression or mutations can lead to cancer. EGFR signaling is initiated via ligand binding, leading to structural changes, homodimerization, or heterodimerization with other ErbB family members of the receptor, followed by trans-autophosphorylation of the receptor. This process ultimately forms a signaling cascade that affects various cellular functions, including cell proliferation and survival.

[0217] It initiates. Since increased EGFR expression levels or kinase activity are associated with various human cancers, EGFR is recognized as a promising target for therapeutic intervention. For example, in non-small cell lung cancer, increases in both EGFR gene copy number and protein expression are associated with a good response to the EGFR tyrosine kinase inhibitor IRESSA™ (gefit inib). Representative examples of antibodies for cancer treatment targeting EGFR include,

[0218] Examples include cetuximab, panitumumab, and necitumumab. For a detailed review of EGFR, refer to the literature [Sabbah, DA, Haj jo, R., & Swe i dan, K. (2020). Review on epidermal growth factor receptor (EGFR) structure, signaling pathways, input signaling pathways, and recent updates of EGFR input signaling pathways. Current topics in medicinal chemistry.] °].

[0219] EGFR plays an important role in cancer biology, including tumor growth and progression. Expression (or overexpression) of EGFR is observed in various types of cancer. As a non-limited example, EGFR expression is known to be observed in head and neck cancer, esophageal cancer, liver cancer, kidney cancer, bladder cancer, skin cancer, lung cancer, uterine cancer, thyroid cancer, prostate cancer, colorectal cancer, gastric cancer, breast cancer, ovarian cancer, etc.

[0220] Meanwhile, although conventional tyrosine kinase inhibitors and anti-EGFR antibodies are effective treatments for cancers expressing EGFR, there are problems such as the potential for on-target toxicity caused by targeting and acting on normal cells expressing EGFR, and the possibility that they may not show effective therapeutic effects against mutant tumors. Mutations of tumors related to EGFR include mutant tumors resulting from mutations within EGFR (i.e., mutations occurring in EGFR itself) (e.g., T790M mutation) and mutant tumors resulting from mutations in EGFR downstream signaling (e.g., KRAS mutation or BRAF mutation).

[0221] Known EGFR variants observed in tumors of some patients with EGFR-positive tumors include exon 19 deletions, indels in exon 19, exon 20 insertions (e.g., EGFR exon 20 insertions of 1–7 amino acids), L718Q / V, G719C / S / A (G719C, G719S, or G719A), S768I, L769X (substitution of L at position 769 with various amino acids; e.g., L769Q, etc.), T790M, L792X (substitution of L at position 792 with various amino acids; e.g., L792F, L792H, L792Y, etc.), G796S, and C797X (various substitutions of L at position 797). Amino acid substitutions (e.g., C797S, C797G, etc.), G834L, L858R, and the truncation mutation EGFRvIII are examples. These EGFR mutations are known to limit the therapeutic effects of some conventional TKIs and anti-EGFR antibodies, or to induce drug resistance to these agents. For the role of EGFR in tumors and mutations within EGFR, the literature [Uribe, M. L., Marrocco, I., & Yarden, Y. (2021). EGFR in cancer: Signaling mechanisms, drugs, and acquired resistance. Cancers, 7 <?(11) , 2748. ]에서 상세히 설명된다.

[0222] KRAS (Kirsten rat sarcoma viral oncogene homologue) is a well-known oncogene with a high mutation rate in cancer and is associated with lethal cancers, including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer, and colorectal cancer. Well-known KRAS mutations are predominantly single nucleotide missense mutations, the majority of which are found at codon 12 (e.g., G12), codon 13 (e.g., G13), or codon 61 (e.g., Q61). Non-limiting examples of KRAS mutations include the G12C mutation (a mutation in which glycine at codon 12 is replaced by cysteine), G12D mutation, G12V mutation, G13D mutation, and Q61H mutation. The literature [Huang, L. , Guo, Z. , Wang, F. , & Fu , L. (2021). KRAS mutation: from undruggable to druggable in cancer. Signal transduction and targeted therapy, O (1), 386.] describes KRAS mutations in cancer in detail, and the entire contents of this literature are incorporated by reference into the present disclosure. The reference amino acid sequence for the human KRAS protein is provided via sequence number 58 (refer to Unit ID No. P01116-1). BRAF belongs to the rapidly accelerated fibrosarcoma (RAAF) kinase family of mammalian cytosolic serine / threonine kinases (ARAF, BRAF, CRAF) and transmits signals downstream of RAS via the mitogen-acting induced protein kinase (MAPK) pathway.BRAF is one of the kinases that frequently mutates in various human cancers, including melanoma, thyroid cancer, colorectal cancer (CRC), and NSCLC. BRAF mutations can be classified into V600 mutations and non-V600 mutations. Non-limiting examples of BRAF mutations include V600D / E / K / R / G (i.e., V600D mutation, V600E mutation, V600K mutation, V600R mutation, or V600G mutation), G464E / V / R mutation, G469A / V / S mutation, and G469E mutation. [Dankner, M., Rose, AA, Rajkumar, S., Siegel, P. M., & Watson, I. R. (2018). Classifying BRAF alterations in cancer: new rational therapeut ic strategies for act ionab le mutat ions. Oncogene, <?7(24) , 3183— 3199. ]은 암에서의 BRAF 변이에 대하여 상세히 설명하며 , 이의 전체 내용은 본 개시에 참조로 포함된다. 인간 BRAF 단백질의 레퍼 런스 아미노산 서 열은 서 열 번호 59 (Uniprot ID No . P15056 참고) 을 통해 제공된다 .

[0223] 4.2. MUC1

[0224] One of the target antigens of the antibody (e.g., bispecific antibody) or antibody-drug conjugate of the present disclosure is MUC1. The membrane-penetrating glycoprotein mucin 1 (MUC1), also known as Episialin, PEM, H23Ag, EMA, CA15-3, and MCA, is a component of the mucin family, which performs different functions in normal cells and cancer cells. Due to its structural and biochemical properties, MUC1 can serve as a lubricant, moisturizer, and physical barrier in normal cells. However, in tumor cells, MUC1 often undergoes abnormal glycosylation and overexpression. MUC1 is involved in tumor invasion, metastasis, angiogenesis, and apoptosis by participating in intracellular signaling processes and the regulation of related biomolecules.

[0225] MUC1 is a single-pass type 1 transmembrane protein with a highly glycosylated extracellular domain extending approximately 200 to 500 nm from the cell surface. MUC1 is the most easily recognized transmembrane protein in the mucin family and possesses a highly glycosylated extracellular domain. Under normal conditions, MUC1 covers the surface of epithelial cells, forming a dense mesh and creating a protective barrier to shield the cells from harsh environmental conditions. However, in cancer cells, MUC1 performs intracellular signaling functions and plays a crucial role in cancer development. Expression (or overexpression) of MUC1 is commonly observed in various epithelial adenocarcinomas, including lung, liver, colon, breast, pancreatic, and ovarian cancers. It is a well-known oncogene that participates in various signaling pathways to regulate various aspects of cancer (cell growth, proliferation, metastasis, apoptosis, development, etc.).

[0226] MUC1 has a varying number of highly glycosylated 20-amino acid tandem repeats, a sperm protein-enterokinase-agar-in (SEA) domain (extrace l lular), a transmembrane domain, and a 72-amino acid tail domain. MUC1 is also known to be encoded as a single polypeptide chain and, immediately after translation, self-protectively cleaved at the GSVVV (SEVENTEEN 63) motif (located within the SEA domain) to be converted into the following two peptide segments: a longer N-terminal subunit (MUC1-N) and a shorter C-terminal subunit (MUC1-C). These two subunits are connected by stable hydrogen bonds, and the three-dimensional structure of the SEA domain where the cleavage site is located is maintained within the complex. That is, MUC1-N and MUC1-C form a heterodimer (or a complex of MUC1-N and MUC1-C) in which the complex is maintained by non-covalent bonding. A reference sequence for the full length of human MUC1 is provided in this disclosure as sequence number 45 (see UniProt ID NO. P15941). The SEA domain is known to be located at 1039–1148 in the amino acid sequence of sequence number 45, which is 1255aa in length.

[0227] It has been found that MUC1 in tumors (e.g., cancer-associated MUC1) undergoes structural changes, such as overexpression due to loss of polarity in epithelial cells, incomplete carbohydrate side chains, and exposure of new carbohydrate side chains (Thomsen-Fr Iedenreich (TF or T), Tn, and silyl-Tn (STn)) or core peptides. Structurally altered MUC1 in tumors can be referred to as Tumor-associated MUC1 (TA-MUC1).

[0228] MUC1-N contains a PTS (Proline, Threonine, Serine-rich) domain, which consists of amino acid tandem repeats. These repeats are referred to as Variable Number Tandem Repeat (VNTR) regions at the DNA level and are encoded by highly polymorphic exons that encode 20–21 amino acid repeat sequences (e.g., HGVTSAPDTRPAPGSTAPPA (SEQ No. 64)). The N-terminus is extensively modified by O-linked glycans. In Northern Europeans, VNTRs consist of 20–120 repeats, with 40–80 repeats being the most common. The amino acid sequences of the VNTR region vary across different cancer cell lines, reflecting the high polymorphism of this motif. Both ends of the VNTR region are surrounded by short degenerate sequences that exhibit subtle sequence similarity to the VNTR region. MUC1-C is a short subunit comprising a 58-amino acid extracellular domain (ECD), a 28-amino acid transmembrane domain (TMD), and a 72-amino acid cytoplasmic tail (CT). Depending on the N-glycosylation status of the ECD, the molecular weight of MUC1-C can vary from 17 to 25 kDa. Furthermore, in some cancer cells, cleavage and release of MUC1-N from MUC1-C are observed in some MUC1s. A detailed review of MUC1 can be found in the literature [Chen, W. , Zhang, Z. , Zhang, S. , Zhu, P. , Ko , J. KS , & Yung, KKL (2021). MUC1: structure, funct ion, and cl inic applicat ion in epithelial cancers. International journal of molecular sciences, %'(12), 6567.] is referenced.

[0229] 4.3. Co-expression of EGFR and MUC1

[0230] The target antigens of the antibody of the present disclosure (e.g., a bispecific antibody) or antibody-drug conjugate are EGFR and MUC1. The antibody or antibody-drug conjugate of the present disclosure comprises an antigen-binding fragment capable of binding to EGFR and an antigen-binding fragment capable of binding to MUC1, and thus can bind to EGFR and MUC1.

[0231] Co-expression of EGFR and MUC1 is observed in various tumors. For example, co-expression of EGFR and MUC1 is reported in lung cancer such as ESCC (Esophageal cancer), sq NSCLC (squamous NSCLC) and nsq NSCLC (non-squamous NSCLC), HNSCC (Head and Neck Squamous Cell Carcinoma), UC (Urothelial Carcinoma), Gastric Cancer, TNBC (Triple-negative breast cancer), Pancreatic Cancer, CRC (Colorectal cancer), Ampullary Cancer, Esophageal Cancer, and Head and Neck Cancer.

[0232] Strategies targeting dual tumor-associated antigens (TAAs) can improve tumor cell selectivity and reduce drug toxicity by binding more strongly to tumor cells expressing dual TAAs. For example, the literature [Cui, H., Yu, Q., Xu, Q., Lin, C., Zhang, L., Ye, W., . . . & Sun, R. (2024). EGFR and MUC1 as dual -TAAdrug targets for lung cancer and correctal cancer. Frontiers in Oncology, 14, 1433033. ] suggests that strategies targeting EGFR and MIJC1 together may be useful. This study confirmed that these two targets do not coexist in normal tissues, and therefore suggests the possibility that toxicity can be minimized when targeting both antigens together. In addition, this literature reports that co-expression of EGFR and MUC1 was observed in many patient-derived LUAD (Lung Adenocarcinoma) and CRC samples, confirming that co-expression of EGFR and MUC1 for LUAD and CRC can have a negative impact on the prognosis of patients, and suggesting that a treatment targeting these two antigens together could be an effective treatment option for patients with LUAD or CRC.

[0233] 5. Antibody-drug conjugate

[0234] 5.1. Antibody-Drug Conjugate - Overview

[0235] The present disclosure provides an antibody-drug conjugate or a salt thereof. An antibody-drug conjugate is a conjugate of an antibody and a drug, wherein the antibody and the drug are connected via a linker. Herein, the drug (or linker) may be specifically or selectively connected to a specific site on the antibody. An antibody-drug conjugate may be described as comprising, but is not otherwise limited to, an antibody portion (e.g., antibody), a linker portion (e.g., linker), and a drug portion (e.g., drug).

[0236] The antibody-drug conjugate of the present disclosure is a bispecific antibody-drug conjugate. That is, it is a conjugate of a bispecific antibody and a drug. The antibody-drug conjugate of the present disclosure is prepared by the reaction of a bispecific antibody that has been glyco-engineered (or glyco-engineered) to have an azide and a linker-payload compound comprising BCN capable of reacting bioorthogonally with the azide. The antibody-drug conjugate of the present disclosure may be expressed as an antibody-drug conjugate comprising a bispecific antibody, and is not otherwise limited.

[0237] The bispecific antibody of the antibody-drug conjugate of the present disclosure is a bispecific antibody capable of binding to EGFR and MUC1. That is, the target antigens of the bispecific antibody of the antibody-drug conjugate of the present disclosure are EGFR and MUC1. In some embodiments, the bispecific antibody of the antibody-drug conjugate of the present disclosure may be monovalent to EGFR and monovalent to MUC1. The bispecific antibody confers the antibody-drug conjugate the ability to bind to antigen(s), and accordingly, the target antigens of the antibody-drug conjugate may be expressed as EGFR and MUC1, but are not limited thereto. For example, the antibody-drug conjugate of the present disclosure is an antibody-drug conjugate capable of binding to EGFR and MUC1. For example, the antibody-drug conjugates of the present disclosure may be referred to as antibody-drug conjugates against EGFR and MUC1, anti-EGFR and anti-MUC1 antibody-drug conjugates, anti-EGFR / MUC1 antibody-drug conjugates, and are not otherwise limited.

[0238] In the following, each element of the antibody-drug conjugate of the present disclosure and the technology used in the manufacture of the conjugate will be described in detail.

[0239] 5.2. Elements of the Antibody-Drug Conjugate

[0240] 5.2.1. Antibodies: Anti-EGFR and anti-MUC1 bispecific antibodies (Anti-EGFR / MUC1 bispecific antibodies) (1) Bispecific antibodies - Overview

[0241] The antibody-drug conjugate of the present disclosure is an antibody-drug conjugate of a bispecific antibody. The antibody involved in or used in the manufacture of the antibody-drug conjugate of the present disclosure is a bispecific antibody. In some embodiments, the antibody-drug conjugate of the present disclosure may comprise a bispecific antibody.

[0242] Some embodiments of the present disclosure provide a bispecific antibody or a salt thereof. The bispecific antibody may be used in the manufacture of antibody-drug conjugates after glycan manipulation. A bispecific antibody of the present disclosure according to some embodiments is described in detail in PCT patent application no. PCT / CN2024 / 071122 (publication no. W02024 / 149195A1), which is incorporated herein by reference in its entirety.

[0243] The target antigens of the bispecific antibodies of the present disclosure are the EGFR protein (i.e., EGFR) and the MUC1 protein (i.e., MUC1). That is, the bispecific antibodies may be bispecific antibodies that target EGFR and MUC1. The bispecific antibodies of the present disclosure that target EGFR and MUC1 may be referred to as anti-EGFR and anti-MUC1 bispecific antibodies, anti-EGFR / anti-MUC1 bispecific antibodies, anti-EGFR / MUC1 bispecific antibodies, etc., and are not otherwise limited. In some embodiments, the bispecific antibodies of the present disclosure may be referred to as bispecific antibodies that target EGFR and MUC1, bispecific antibodies that bind to EGFR and MUC1, etc., and are not otherwise limited.

[0244] In some embodiments, the bispecific antibody of the present disclosure or the antibody-drug conjugate of the present disclosure may have the ability to bind to EGFR. In some embodiments, EGFR may be human EGFR or a fragment thereof. Human EGFR is known to have an amino acid sequence of sequence number 44 with a length of 1210aa (see UniProt IN NO. P00533), but is not limited thereto. In some embodiments, EGFR may be a variant of human EGFR or a fragment thereof. For example, a variant of human EGFR or a fragment thereof may have an amino acid sequence having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the amino acid sequence of sequence number 44. In some embodiments, the variant of human EGFR may include one or more amino acid variants, wherein each amino acid variant may be independently selected from insertions, deletions, and substitutions. Some

[0245] In an embodiment, the human EGFR variant may include one or more variants of human EGFR (e.g., wild type), an exon 19 deletion, an indel in exon 19, an exon 20 insertion (e.g., an EGFR exon 20 insertion of 1-7 amino acids), L718Q / V, G719C / S / A (G719C, G719S, or G719A), S768I, L769X, T790M, L792X, G796S, C797X, G834L, and L858R. In some embodiments, the human EGFR variant may be EGFRvI II. In some embodiments, the human EGFR variant may comprise one or more substitutions of T790M, C797S, and L858R from human EGFR, but is not limited thereto. In some embodiments, the human EGFR variant may comprise amino acid substitutions of T790M and C797S, T790M and L898R, or C797S and L858R, but is not limited thereto. In some embodiments, the bispecific antibody or antibody-drug conjugate may have the ability to bind to human EGFR or a fragment thereof, a variant of human EGFR or a fragment thereof.

[0246] In some embodiments, EGFR may comprise human EGFR doma in III or a portion thereof. In some embodiments, human EGFR or a fragment thereof, a variant of human EGFR or a fragment thereof may comprise human EGFR doma in III or a portion thereof. Human EGFR doma in III is known to be located at position 334-504 based on the amino acid sequence of human EGFR 1210aa (SEQ No. 44). In some embodiments, a bispecific antibody or antibody-drug conjugate may have the ability to bind to human EGFR doma in III or a portion thereof. In some embodiments, the bispecific antibody or antibody-drug conjugate of the present disclosure may bind to one or more amino acids within 1-405 of human EGFR.

[0247] In some embodiments, the bispecific antibody or antibody-drug conjugate may bind to the reference human EGFR of sequence number 44. In some embodiments, the bispecific antibody or antibody-drug conjugate may bind to a variant of human EGFR. In some embodiments, the bispecific antibody or antibody-drug conjugate may bind to a fragment of human EGFR or a variant thereof. Variants of human EGFR may have one or more of the following:, for example, exon 19 deletions from human EGFR, indels in exon 19, exon 20 insertions (e.g., EGFR exon 20 insertions of 1-7 amino acids), L718Q, G719C / S / A (G719C, G719S, or G719A), S768I, L769X, T790M, L792X, G796S, C797X, G834L, and L858R. In some embodiments, the amino acid position number of any one or more of the aforementioned EGFR variants may be based on sequence number 44, which is an EGFR reference sequence. Since all of these EGFR variants are located in the intracellular domain of EGFR and the bispecific antibody of the present disclosure binds to a portion of EGFR that is far from the intracellular domain where these variants occur, it can also bind to EGFR variants containing these variants.

[0248] In some embodiments, the binding affinity of the bispecific antibody to EGFR (e.g., human EGFR) may be about 1 On M to 200 NM based on the dissociation constant (KD) [e.g., equilibrium dissociation constant]. In some embodiments, the binding affinity of the bispecific antibody to EGFR may be approximately 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 90 nM, or 100 nM based on the dissociation constant, or within the range of two values ​​selected from the above values. In some embodiments, the binding affinity of the bispecific antibody to EGFR may be 20 nM to 100 nM or 25 nM to 80 nM based on the dissociation constant, but is not limited thereto.

[0249] In some embodiments, the bispecific antibody of the present disclosure or the antibody-drug conjugate of the present disclosure may have the ability to bind to MUC1. In some embodiments, MUC1 may be human MUC1 or a fragment thereof. Human MUC1 is known to have an amino acid sequence of sequence number 45 with a length of 1255aa (see UniProt ID NO. P15941), but is not limited thereto. In some embodiments, MUC1 may be a variant of human MUC1 or a fragment thereof. For example, a variant of human MUC1 may have an amino acid sequence having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity of the amino acid sequence of sequence number 45. In some embodiments, MUC1 may be a complex of MUC1-N and MUC1-C (i.e., a non-covalent heterodimer of MUC1-N and MUC1-C). In some embodiments, MUC1 may be a form of MUC1 expressed in MUCK tumor cells. In some embodiments, MUC1 may be TA-MUC1.

[0250] In some embodiments, MUC1 may include the SEA domain of human MUC1 or a portion thereof. In some embodiments, human MUC1 or a fragment thereof, or a variant of human MUC1 or a fragment thereof, may include the SEA domain or a portion thereof. The SEA domain is known to be located at positions 1039–1148 based on the amino acid sequence of human MUC1 of 1255aa (sequence number 45). In some embodiments, a bispecific antibody or antibody-drug conjugate may have the ability to bind to the SEA domain of human MUC1 or a portion thereof.

[0251] In some embodiments, the bispecific antibody or antibody-drug conjugate may bind to the reference human MUC1 of sequence number 45. In some embodiments, the bispecific antibody or antibody-drug conjugate may bind to a variant of human MUC1. In some embodiments, the bispecific antibody or antibody-drug conjugate may bind to a fragment of human MUC1 or a variant thereof.

[0252] In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to a complex of MUC1-N and MUC1-C. In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to MUC1 in a form expressed in a tumor. In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to TA-MUC1.

[0253] In some embodiments, the binding affinity of the bispecific antibody to MUC1 (e.g., human MUC1) may be about 0.0M to 50NM based on the dissociation constant (KD) [e.g., equilibrium dissociation constant]. In some embodiments, the binding affinity of the bispecific antibody to MUC1 is approximately InM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 1OnM, 10nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, or 20nM based on the dissociation constant, or may be within the range of two values ​​selected from the above values. In some embodiments, the binding affinity of the bispecific antibody to MUC1 may be 2 nM to 20 nM, 4 nM to 18 nM, or 5 nM to 15 nM based on the dissociation constant, but is not limited thereto. For example, the binding affinity of the bispecific antibody to MUC1 may be for full-length MUC1, but is not limited thereto.

[0254] In some embodiments, the bispecific antibody can bind to cells expressing EGFR and / or MUC1. For example, the bispecific antibody can bind to cells expressing EGFR on the cell surface and / or cells expressing MUC1 on the cell surface. In some embodiments, the bispecific antibody may have binding affinity for at least some of the regions of the EGFR protein that protrude to the surface of the cell membrane (i.e., outside the cell) and / or for at least some of the regions of the MUC1 protein that protrude to the surface of the cell membrane (i.e., outside the cell). Accordingly, the bispecific antibody can confer specificity to the antibody-drug conjugate to cells and / or tissues (e.g., tumor cells and / or tissues) expressing EGFR and / or MUC1.

[0255] In some embodiments, the antibody against EGFR of the present disclosure (e.g., an antibody targeting EGFR or an anti-EGFR antibody) may bind to domain III of human EGFR (or part thereof), and the antibody against MUC1 of the present disclosure (e.g., an antibody targeting MUC1 or an anti-MUC1 antibody) may bind to the SEA domain of human MUC1 (or part thereof). In some embodiments, the antibody against EGFR of the present disclosure may target domain 111 of human EGFR (or part thereof), and the antibody against MUC1 of the present disclosure may target the SEA domain of human MUC1 (or part thereof).

[0256] In some embodiments, the bispecific antibody comprises one first antigen-binding fragment capable of binding to EGFR, a first target antigen, and one second antigen-binding fragment capable of binding to MUC1, a second target antigen. In some embodiments, the first antigen-binding fragment has an antigen-binding portion capable of binding to EGFR, and the second antigen-binding fragment has an antigen-binding portion capable of binding to MUC1. In some embodiments, the bispecific antibody of the present disclosure comprises one antigen-binding portion capable of binding to EGFR and one antigen-binding portion capable of binding to MUC1.

[0257] In some embodiments, the bispecific antibody may be of an IgG class (e.g., human IgG type) and is not otherwise limited. In some embodiments, the subclass of the bispecific antibody may be IgGl (e.g., human IgGl). In some embodiments, the heavy chain constant region of the bispecific antibody may be the heavy chain constant region of IgG (e.g., human IgG). In some embodiments, the heavy chain constant region of the bispecific antibody may be the heavy chain constant region of IgGl (e.g., human IgGl). In some embodiments, the light chain constant region of the bispecific antibody may be the light chain constant region of IgG (e.g., human IgG). In some embodiments, the light chain constant region of the bispecific antibody may be the light chain constant region of IgGl (e.g., human IgGl). In some embodiments, the bispecific antibody may comprise the Fc region of IgG (e.g., human IgG). In some embodiments, the bispecific antibody may be referred to as an antibody of the IgGl type (e.g., human IgGl type) and is not otherwise limited. In some embodiments, the bispecific antibody may comprise the Fc region of IgGl (e.g., human IgGl).

[0258] In some embodiments, the bispecific antibody may be a mouse, chimeric, humanized, or human antibody. In some embodiments, the bispecific antibody may be a humanized or human antibody. In some embodiments, the antigen-binding fragment may be a humanized or human antigen-binding fragment. In some embodiments, the first antigen-binding fragment may be a humanized or human antigen-binding fragment. In some embodiments, the second antigen-binding fragment may be a humanized or human antigen-binding fragment.

[0259] In some embodiments, the bispecific antibody may be a humanized or human IgGl bispecific antibody.

[0260] In some embodiments, the Fc region of the bispecific antibody may contain KIH mutations. For example, the Fc region of either the first heavy chain or the second heavy chain of the bispecific antibody may contain knob mutations (e.g., one or more substitutions of S354C and T366W), and the other Fc region may contain hole mutations (e.g., one or more substitutions of Y349C, T366S, L368A and Y407V). In some embodiments, the heavy chain constant region 3 (CH3) of either the first heavy chain or the second heavy chain of the bispecific antibody may contain knob mutations, and the other heavy chain constant region (CH) may contain hole mutations. In some embodiments, either the first heavy chain or the second heavy chain of the bispecific antibody may have a heavy chain constant region (CH) containing a knob mutation, and the other heavy chain constant region 3 (CH3) may have a hole mutation. In some embodiments, either the first heavy chain or the second heavy chain of the bispecific antibody may have an IgG Fc region containing a knob mutation (or a heavy chain constant region or a heavy chain constant region 3), and the other of the first heavy chain or the second heavy chain may have an IgG Fc region containing a hole mutation (or a heavy chain constant region or a heavy chain constant region 3). In some embodiments, either the first heavy chain and the second heavy chain of the bispecific antibody may comprise an Fc region of IgGl containing a knob variant (or a heavy chain constant region or a heavy chain constant region 3), and the other of the first heavy chain and the second heavy chain may comprise an Fc region of IgGl containing a hole variant (or a heavy chain constant region or a heavy chain constant region 3).

[0261] Methods for producing antibodies or bispecific antibodies are widely known in the art. Furthermore, the method for producing bispecific antibodies of the present disclosure is described in detail in PCT patent application publication number W02024 / 149195A1. For example, CHO-S (Chinese hamster ovary-S) cells can be co-transformed with a vector encoding the heavy chain of an anti-EGFR antibody, a vector encoding the heavy chain of an anti-MUC1 antibody, and a vector encoding the common light chain, and a bispecific antibody can be obtained from the co-transformed CHO-S cells.

[0262] (2) First antigen-binding fragment (antigen-binding fragment for EGFR)

[0263] In some embodiments, the bispecific antibody of the present disclosure comprises a first antigen-binding fragment. In some embodiments, the bispecific antibody of the present disclosure may comprise one first antigen-binding fragment. The first antigen-binding fragment is an antigen-binding fragment designed to be able to bind to EGFR or to target EGFR, having an antigen-binding portion capable of binding to EGFR. The antigen-binding portion capable of binding to EGFR may be referred to as the first antigen-binding portion. That is, the target antigen of the first antigen-binding fragment is EGFR. In some embodiments, the first antigen-binding fragment may be referred to as an anti-EGFR antigen-binding fragment, an antigen-binding fragment for EGFR, or an antigen-binding fragment targeting EGFR. In some embodiments, the first antigen-binding fragment may be referred to as the first antigen-binding portion. In some embodiments, the bispecific antibody of the present disclosure may include a first antigen-binding portion.

[0264] The first antigen-binding fragment (or first antigen-binding portion) comprises HCDR1 having the amino acid sequence of SEQ ID NO. 1, HCDR2 having the amino acid sequence of SEQ ID NO. 2, HCDR3 having the amino acid sequence of SEQ ID NO. 3, LCDR1 having the amino acid sequence of SEQ ID NO. 4, LCDR2 having the amino acid sequence of SEQ ID NO. 5, and LCDR3 having the amino acid sequence of SEQ ID NO. 6. In some embodiments, the bispecific antibody of the present disclosure may comprise HCDR1 having the amino acid sequence of SEQ ID NO. 1, HCDR2 having the amino acid sequence of SEQ ID NO. 2, HCDR3 having the amino acid sequence of SEQ ID NO. 3, LCDR1 having the amino acid sequence of SEQ ID NO. 4, LCDR2 having the amino acid sequence of SEQ ID NO. 5, and LCDR3 having the amino acid sequence of SEQ ID NO. 6.

[0265] The CDRs of the first antigen-binding fragment may be referred to as the first HCDR1, the first HCDR2, the first HCDR3, the first LCDR1, the first LCDR2, and the first LCDR3, etc., to distinguish them from the CDRs of the second antigen-binding fragment described below, and are not otherwise limited. The amino acid sequences of the aforementioned CDRs of the first antigen-binding fragment are described in Table 02 below.

[0266] [Table 0 shows the amino acid sequences of the CDRs of the first antigen-binding fragment]

[0267] Name: Amino acid sequence

[0268] HCDR1 SGDYYWS (Sequence No. 1)

[0269] HCDR2 YIYYSGSTYYNPSLKS (Sequence No. 2)

[0270] HCDR3 ERVYSSSLDY (Sequence No. 3)

[0271] LCDR1 RASQSVSSYLA (Sequence No. 4)

[0272] LCDR2 DASNRAT (Sequence No. 5)

[0273]

[0274] LCDR3 QQRSNWPPT (Sequence No. 6)

[0275] In some embodiments, the first antigen-binding fragment (or the first antigen-binding portion) may include a heavy chain variable region and a light chain variable region. To distinguish them from the heavy chain variable region and light chain variable region of the second antigen-binding fragment described below, the heavy chain variable region and light chain variable region of the first antigen-binding fragment may be referred to as the first heavy chain variable region and the first light chain variable region, respectively, and are not otherwise limited. In some embodiments, the first heavy chain variable region may be a mouse, chimeric, humanized, or human heavy chain variable region. In some embodiments, the first heavy chain variable region may be a humanized or human heavy chain variable region. For example, the framework sequences within the first heavy chain variable region may each be independently humanized or of human origin. In some embodiments, the first light chain variable region may be a mouse, chimeric, humanized, or human light chain variable region. In some embodiments, the first light chain variable region may be a humanized or human light chain variable region. For example, the framework sequences within the first light chain variable region may each be independently humanized or human-derived.

[0276] In some embodiments, the heavy chain variable region of the first antigen-binding fragment (i.e., the first heavy chain variable region) comprises HCDR1 having the amino acid sequence of sequence number 1, HCDR2 having the amino acid sequence of sequence number 2, and HCDR3 having the amino acid sequence of sequence number 3. In some embodiments, in the first heavy chain variable region, HCDR1, HCDR2, and HCDR3 may be positioned from the N-terminus to the C-terminus in the order listed above.

[0277] In some embodiments, the heavy chain variable region of the first antigen-binding fragment (i.e., the first heavy chain variable region) may further include, in addition to the HCDRs, HFR1 having the amino acid sequence of sequence number 11, HFR2 having the amino acid sequence of sequence number 12, HFR3 having the amino acid sequence of sequence number 13, and HFR4 having the amino acid sequence of sequence number 14. In some embodiments, the first heavy chain variable region is, in addition to the HCDRs, an HFR1 having the amino acid sequence of sequence no. 11 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity with the same, the amino acid sequence of sequence no. 12 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, HFR2 having an amino acid sequence having 94, 95, 96, 97, 98, or 99% or more sequence identity, the amino acid sequence of sequence number 13 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity, and HFR3 having an amino acid sequence having 99% or more sequence identity, and the amino acid sequence of sequence number 14 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 It may further include HFR4 having an amino acid sequence having at least 99% sequence identity, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity.In some embodiments, in the first heavy chain variable region, HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4 may be positioned in the order listed, from the N-terminus to the C-terminus.

[0278] In some embodiments, the heavy chain variable region of the first antigen-binding fragment may have the amino acid sequence of sequence number 10. In some embodiments, the heavy chain variable region of the first antigen-binding fragment may have the amino acid sequence of sequence number 10 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity therewith, and may include HCDR1 having the amino acid sequence of sequence number 1, HCDR2 having the amino acid sequence of sequence number 2, and HCDR3 having the amino acid sequence of sequence number 3.

[0279] The sequences of the framework region of the heavy chain variable region of the aforementioned first antigen-binding fragment and the heavy chain variable region amino acid sequences are described in Table 03 below.

[0280] [Table 03] Amino acid sequence names of the variable region of the heavy chain of the first antigen-binding fragment

[0281] Heavy chain variable region QVQLQQWGPGLVKPSQTLSLTCTVSGGS INSGDYYWSWIRQPPGKGLES I GY I YYSGSTYYNP (VH) SLKSRVT I SADTSKNQFSLKLTSVTAADTAVYYCARERVYSSSLDYWGQGTLVTVSS (Sequence No. 10)

[0282] HFR1 QVQLQQWGPGLVKPSQTLSLTCTVSGGS IN (Sequence No. 11)

[0283]

[0284] HFR2 WIRQPPGKGLESIG (Sequence No. 12)HFR3 RVTI SADTSKNQFSLKLTSVTAADTAVYYCAR (Sequence No. 13)

[0285]

[0286] HFR4 WGQGTLVTVSS (Sequence No. 14)

[0287] In some embodiments, the light chain variable region of the first antigen-binding fragment (i.e., the first light chain variable region) comprises LCDR1 having the amino acid sequence of sequence no. 4, LCDR2 having the amino acid sequence of sequence no. 5, and LCDR3 having the amino acid sequence of sequence no. 6. In some embodiments, in the first light chain variable region, LCDR1, LCDR2, and LCDR3 may be positioned in the order listed above, from the N-terminus to the C-terminus.

[0288] In some embodiments, the light chain variable region of the first antigen-binding fragment (i.e., the first light chain variable region) may further include, in addition to the LCDRs, LFR1 having the amino acid sequence of sequence number 16, LFR2 having the amino acid sequence of sequence number 17, LFR3 having the amino acid sequence of sequence number 18, and LFR4 having the amino acid sequence of sequence number 19. In some embodiments, the first light chain modified region is, in addition to the LCDRs, an LFR1 having an amino acid sequence of sequence no. 16 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity with the same, an amino acid sequence of sequence no. 17 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, LFR2 having an amino acid sequence having 94, 95, 96, 97, 98, or 99% or more sequence identity, the amino acid sequence of sequence number 18 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity, LFR3 having an amino acid sequence having 99% or more sequence identity, and the amino acid sequence of sequence number 19 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 It may further include LFR4 having an amino acid sequence having at least 99% sequence identity, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity.In some embodiments, in the first light chain variable region, LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4 may be positioned in the order listed, from the N end to the C end.

[0289] In some embodiments, the light chain variable region of the first antigen-binding fragment may have the amino acid sequence of sequence number 15. In some embodiments, the light chain variable region of the first antigen-binding fragment may have the amino acid sequence of sequence number 15 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity therewith, and may include LCDR1 having the amino acid sequence of sequence number 4, LCDR2 having the amino acid sequence of sequence number 5, and LCDR3 having the amino acid sequence of sequence number 6. In some embodiments, the light chain variable region of the first antigen-binding fragment may be a common light chain variable region.

[0290] The sequences of the framework region of the light chain variable region of the aforementioned first antigen-binding fragment and the amino acid sequences of the light chain variable region are described in Table 04 below.

[0291] [Table 04] Amino acid sequence names of the variable region of the light chain of the first antigen-binding fragment

[0292] Light chain variable region EI VLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL I YDASNRATGIPARFS (VL) GSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK (Sequence No. 15) LFR1 EI VLTQSPATLSLSPGERATLSC (Sequence No. 16)

[0293] LFR2 WYQQKPGQAPRLLIY (Sequence No. 17)

[0294] LFR3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (Sequence No. 18)

[0295]

[0296] LFR4 FGQGTKVEIK (Sequence No. 19)

[0297] In some embodiments, the first antigen-binding fragment may be Fv. In this case, Fv includes a first heavy chain variable region and a first light chain variable region.

[0298] In some embodiments, the first antigen-binding fragment may be Fab. In some embodiments, Fab comprises a first heavy chain variable region and a heavy chain constant region 1 (heavy chain constant region 1, CH1) having the amino acid sequence of sequence number 28, and a first light chain variable region and a light chain constant region having the amino acid sequence of sequence number 38. In some embodiments, Fab comprises a heavy chain variable region-heavy chain constant region 1 (VH-CH1) having the amino acid sequence of sequence number 20, and a light chain variable region-light chain constant region (VL-CL, or light chain) having the amino acid sequence of sequence number 21. In some embodiments, the light chain of the first antigen-binding fragment may be a common light chain. In some embodiments, in Fab, the heavy chain constant region and the light chain constant region may be covalently connected through disulfide bonds by cysteine.

[0299] The amino acid sequences of VH-CH1 and VL-CL are described in Table 05 below.

[0300] [Table 05] Amino acid sequences of VH-CH1 and VL-CL

[0301] Name: Amino acid sequence

[0302] Heavy chain variable region- QVQLQQWGPGLVKPSQTLSLTCTVSGGS INSGDYYWSWIRQPPGKGLES I GY I YYSGSTYYNP Heavy chain invariant region 1 SLKSRVT I SADTSKNQFSLKLTSVTAADTAVYYCARERVYSSSLDYWGQGTLVTVSSASTKGP (VH-CH1) SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTY I CNVNHKPSNTKVDKKV (Sequence No. 20) Light chain variable region- EI VLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL I YDASNRATGIPARFS Light chain invariant region GSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK (VL-CL) or light chain SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

[0303]

[0304] KVYACEVTHQGLSSPVTKSFNRGEC (Sequence No. 21)

[0305] In some embodiments, the first antigen-binding fragment may comprise an amino acid sequence corresponding to the CDR, VH, and / or VL of an antibody targeting EGFR, such as cetuximab, panitumumab, necitumumab, or nimotuzumab, but is not otherwise limited.

[0306] (3) Second antigen-binding fragment (antigen-binding fragment for MUC1)

[0307] In some embodiments, the bispecific antibody of the present disclosure comprises a second antigen-binding fragment. In some embodiments, the bispecific antibody of the present disclosure may comprise one second antigen-binding fragment. The second antigen-binding fragment is an antigen-binding fragment designed to bind to MUC1 or to target MUC1, having an antigen-binding site capable of binding to MUC1. The antigen-binding site capable of binding to MUC1 may be referred to as the second antigen-binding site. That is, the target antigen of the second antigen-binding fragment is MUC1. In some embodiments, the second antigen-binding fragment may be referred to as an anti-MUC1 antigen-binding fragment, an antigen-binding fragment for MUC1, or an antigen-binding fragment targeting MUC1. In some embodiments, the second antigen-binding fragment may be referred to as the second antigen-binding site. In some embodiments, the bispecific antibody of the present disclosure may include a second antigen-binding portion.

[0308] In some embodiments, the second antigen-binding fragment (or second antigen-binding portion) may include HCDR1 having the amino acid sequence of sequence number 7, HCDR2 having the amino acid sequence of sequence number 8, HCDR3 having the amino acid sequence of sequence number 9, LCDR1 having the amino acid sequence of sequence number 4, LCDR2 having the amino acid sequence of sequence number 5, and LCDR3 having the amino acid sequence of sequence number 6. In some embodiments, the bispecific antibodies of the present disclosure may include HCDR1 having an amino acid sequence of sequence number 7, HCDR2 having an amino acid sequence of sequence number 8, HCDR3 having an amino acid sequence of sequence number 9, LCDR1 having an amino acid sequence of sequence number 4, LCDR2 having an amino acid sequence of sequence number 5, and LCDR3 having an amino acid sequence of sequence number 6.

[0309] The CDRs of the second antigen-binding fragment may be referred to as 2nd HCDR1, 2nd HCDR2, 2nd HCDR3, 2nd LCDR1, 2nd LCDR2, and 2nd LCDR3, etc., to distinguish them from the CDRs of the first antigen-binding fragment described above, and are not otherwise limited. The amino acid sequences of the aforementioned CDRs of the second antigen-binding fragment are described in Table 06 below.

[0310] [Table 06] Amino acid sequences of CDRs of the second antigen-binding fragment

[0311] Name Amino ac id sequence

[0312] HCDR1 SYGMH (Sequence No. 7)

[0313] HCDR2 VISYDGSNKYYADSVKG (Sequence No. 8)

[0314] HCDR3 DRRGFYESGNYYNVPFDY (Sequence No. 9)

[0315] LCDR1 RASQSVSSYLA (Sequence No. 4)

[0316] LCDR2 DASNRAT (Sequence No. 5)

[0317]

[0318] LCDR3 QQRSNWPPT (Sequence No. 6)

[0319] In some embodiments, the second antigen-binding fragment (or the second antigen-binding portion) may include a heavy chain variable region and a light chain variable region. To distinguish them from the heavy chain variable region and the light chain variable region of the first antigen-binding fragment described above, the heavy chain variable region and the light chain variable region of the second antigen-binding fragment may be referred to as the second heavy chain variable region and the second light chain variable region, respectively, and are not otherwise limited.

[0320] In some embodiments, the second heavy chain variable region may be a mouse, chimeric, humanized, or human heavy chain variable region. In some embodiments, the second heavy chain variable region may be a humanized or human heavy chain variable region. For example, the framework sequences within the second heavy chain variable region may each independently be humanized or human-derived. In some embodiments, the second light chain variable region may be a mouse, chimeric, humanized, or human light chain variable region. In some embodiments, the second light chain variable region may be a humanized or human light chain variable region. For example, the framework sequences within the second light chain variable region may each independently be humanized or human-derived.

[0321] In some embodiments, the heavy chain variable region of the second antigen-binding fragment (i.e., the second heavy chain variable region) comprises HCDR1 having the amino acid sequence of sequence number 7, HCDR2 having the amino acid sequence of sequence number 8, and HCDR3 having the amino acid sequence of sequence number 9. In some embodiments, in the second heavy chain variable region, HCDR1, HCDR2, and HCDR3 may be positioned in the order described, from the N-terminus to the C-terminus.

[0322] In some embodiments, the heavy chain variable region of the second antigen-binding fragment (i.e., the second heavy chain variable region) may further include, in addition to the HCDRs, HFR1 having the amino acid sequence of sequence no. 23, HFR2 having the amino acid sequence of sequence no. 24, HFR3 having the amino acid sequence of sequence no. 25, and HFR4 having the amino acid sequence of sequence no. 26. In some embodiments, the second heavy chain variable region is, in addition to the HCDRs, an HFR1 having the amino acid sequence of sequence no. 23 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or more of sequence identity with respect to it, and the amino acid sequence of sequence no. 24 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or more of sequence identity with respect to it. It may further include HFR2 having a sequence, HFR3 having an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity, HFR4 having an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, in the second heavy chain variable region, HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4 may be positioned in the order described, from the N-terminus to the C-terminus.

[0323] In some embodiments, the heavy chain variable region of the second antigen-binding fragment may have the amino acid sequence of sequence no. 22. In some embodiments, the heavy chain variable region of the second antigen-binding fragment may have the amino acid sequence of sequence no. 22 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity therewith, and may include HCDR1 having the amino acid sequence of sequence no. 7, HCDR2 having the amino acid sequence of sequence no. 8, and HCDR3 having the amino acid sequence of sequence no. 9.

[0324] The sequences of the framework region of the heavy chain variable region of the aforementioned second antigen-binding fragment and the heavy chain variable region amino acid sequences are described in Table 07 below.

[0325] [Table 07] Amino acid sequence names of the variable region of the heavy chain of the second antigen-binding fragment

[0326] Heavy chain variable region QVTLKESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV I SYDGSNKYYADS (VH) VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANDRRGFYESGNYYNVPFDYWGQGTLVTVS S (Sequence No. 22)

[0327] HFR1 QVTLKESGGGWQPGRSLRLSCAASGFTFS (Sequence No. 23)

[0328] HFR2 WVRQAPGKGLEWVA (Sequence No. 24)

[0329] HFR3 RFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAN (Sequence No. 25)

[0330]

[0331] HFR4 WGQGTLVTVSS (Sequence No. 26)

[0332] In some embodiments, the light chain variable region of the second antigen-binding fragment (i.e., the second light chain variable region) comprises LCDR1 having the amino acid sequence of sequence no. 4, LCDR2 having the amino acid sequence of sequence no. 5, and LCDR3 having the amino acid sequence of sequence no. 6. In some embodiments, in the second light chain variable region, LCDR1, LCDR2, and LCDR3 may be positioned in the order listed above, from the N-terminus to the C-terminus.

[0333] In some embodiments, the light chain variable region of the second antigen-binding fragment (i.e., the second light chain variable region) may further include, in addition to the LCDRs, LFR1 having the amino acid sequence of sequence number 16, LFR2 having the amino acid sequence of sequence number 17, LFR3 having the amino acid sequence of sequence number 18, and LFR4 having the amino acid sequence of sequence number 19. In some embodiments, the second light chain variable region comprises, in addition to the LCDRs, an LFR1 having the amino acid sequence of sequence no. 16 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or more of sequence identity with respect to it, and the amino acid sequence of sequence no. 17 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or more of sequence identity with respect to it. It may further include LFR2 having the amino acid sequence of sequence number 18 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity therewith, LFR3 having the amino acid sequence of sequence number 19 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity therewith. In some embodiments, in the second light chain variable region, LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4 may be positioned in the order listed, from the N-terminus to the C-terminus.

[0334] In some embodiments, the light chain variable region of the second antigen-binding fragment may have the amino acid sequence of sequence no. 15. In some embodiments, the light chain variable region of the second antigen-binding fragment may have the amino acid sequence of sequence no. 15 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with respect to it, and may include LCDR1 having the amino acid sequence of sequence no. 4, LCDR2 having the amino acid sequence of sequence no. 5, and LCDR3 having the amino acid sequence of sequence no. 6. In some embodiments, the light chain variable region of the second antigen-binding fragment may be a common light chain variable region. In some embodiments, the amino acid sequence of the light chain variable region of the second antigen-binding fragment may be identical to the amino acid sequence of the light chain variable region of the first antigen-binding fragment. The sequences of the framework region of the light chain variable region of the second antigen-binding fragment and the amino acid sequence of the light chain variable region are described in Table 08 below.

[0335] [Table 08] Amino acid sequence names of the variable region of the light chain of the second antigen-binding fragment

[0336] Light chain variable region EI VLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL I YDASNRATGIPARFS (VL) GSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK (Sequence No. 15) LFR1 EI VLTQSPATLSLSPGERATLSC (Sequence No. 16)

[0337] LFR2 WYQQKPGQAPRLLIY (Sequence No. 17)

[0338] LFR3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (Sequence No. 18)

[0339]

[0340] LFR4 FGQGTKVEIK (Sequence No. 19)

[0341] In some embodiments, the second antigen-binding fragment may be Fv. In this case, Fv includes a second heavy chain variable region and a second light chain variable region.

[0342] In some embodiments, the second antigen-binding fragment may be Fab. In some embodiments, Fab comprises a second heavy chain variable region and a heavy chain constant region 1 having the amino acid sequence of SEQ ID NO. 33, and a second light chain variable region and a light chain constant region having the amino acid sequence of SEQ ID NO. 38. In some embodiments, Fab comprises a heavy chain variable region-heavy chain constant region 1 (VH-CH1) having the amino acid sequence of SEQ ID NO. 27, and a light chain variable region-light chain constant region (VL-CL, or light chain) having the amino acid sequence of SEQ ID NO. 21. In some embodiments, the light chain of the second antigen-binding fragment may be a common light chain. In some embodiments, in Fab, the heavy chain constant region and the light chain constant region may be covalently connected through disulfide bonds by cysteine.

[0343] The amino acid sequences of VH-CH1 and VL-CL are described in Table 09 below.

[0344] [Table 09] Amino acid sequences of VH-CH1 and VL-CL

[0345]

[0346] Name Amino acid sequence Heavy chain variable region- QVTLKESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV I SYDGSNKYYADS Heavy chain constant region 1 VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANDRRGFYESGNYYNVPFDYWGQGTLVTVS (VH-CH1) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKV (Sequence No. 27)

[0347] Light chain variable region - EI VLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL I YDASNRATGIPARFS Light chain invariant region GSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK (VL-CL) or light chain SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

[0348]

[0349] KVYACEVTHQGLSSPVTKSFNRGEC (Sequence No. 21)

[0350] In some embodiments, the second antigen-binding fragment may comprise an amino acid sequence corresponding to the CDR, VH, and / or VL of gatipotuzumab or clibatuzumab, which are antibodies targeting MUC1, but is not otherwise limited.

[0351] (4) Structure of a bispecific antibody

[0352] In some embodiments, the bispecific antibody of the present disclosure may comprise two heavy chains and two light chains. In some embodiments, either of the two heavy chains and either of the two light chains of the bispecific antibody of the present disclosure may be referred to as an anti-EGFR arm, and the other of the two heavy chains and the other of the two light chains may be referred to as an anti-MUC1 arm. For example, an anti-EGFR / MUC1 bispecific antibody of the 1+1 form has two heavy chains and two light chains, wherein one heavy chain and one light chain are designed to target EGFR, and the other heavy chain and the other light chain are designed to target MUC1. Here, for example, a heavy chain and a light chain designed to target EGFR (or for EGFR) are connected to each other by one or more disulfide bonds, a heavy chain and a light chain designed to target MUC1 (or for MUC1) are connected to each other by one or more disulfide bonds, and two (other) heavy chains are connected to each other by one or more disulfide bonds. In this case, the heavy chain and light chain designed to target EGFR connected by one or more disulfide bonds may be referred to as an anti-EGFR arm, and the heavy chain and light chain designed to target MUC1 connected by one or more disulfide bonds may be referred to as an anti-MUC1 arm. The anti-EGFR arm may include a heavy chain and a light chain designed to target EGFR, and the anti-MUC1 arm may include a heavy chain and a light chain designed to target MUC1.

[0353] In some embodiments, the bispecific antibody of the present disclosure may be a 1+1 bispecific antibody. In some embodiments, the bispecific antibody of the present disclosure may be a bispecific antibody that is monovalent to EGFR and monovalent to MUC1.

[0354] In some embodiments, for the bispecific antibodies of the present disclosure, both light chains may be common light chains. The heavy chain or heavy chain and light chain for EGFR may be referred to as the first heavy chain or the first heavy chain and the first light chain, and the heavy chain or heavy chain and light chain for MUC1 may be referred to as the second heavy chain or the second heavy chain and the second light chain.

[0355] In some embodiments, the anti-EGFR cancer may comprise a first heavy chain and a first light chain, and the anti-MUC1 cancer may comprise a second heavy chain and a second light chain.

[0356] In some embodiments, the bispecific antibody may comprise one first heavy chain, one first light chain, one second heavy chain, and one second light chain. For example, the first heavy chain and the second heavy chain may be connected to each other through one or more (e.g., one, two, or three or more) disulfide bonds. For example, the first heavy chain and the first light chain may be connected through one or more disulfide bonds. For example, the second heavy chain and the second light chain may be connected through one or more disulfide bonds, but are not limited thereto. In some embodiments, the first heavy chain of the bispecific antibody may comprise a heavy chain variable region of the first antigen-binding fragment and a heavy chain constant region of the first heavy chain. In the first heavy chain of the bispecific antibody, the heavy chain variable region of the first antigen-binding fragment and the heavy chain constant region of the first heavy chain may be located from the N-terminus to the C-terminus in the order described above.

[0357] In some embodiments, the second heavy chain of the bispecific antibody may include a heavy chain variable region of the second antigen-binding fragment and a heavy chain constant region of the second heavy chain. In the second heavy chain of the bispecific antibody, the heavy chain variable region of the second antigen-binding fragment and the heavy chain constant region of the second heavy chain may be located from the N-terminus to the C-terminus in the order described above.

[0358] In some embodiments, the heavy chain constant region of the bispecific antibody (e.g., the heavy chain constant region of the first heavy chain or the heavy chain constant region of the second heavy chain) may be the heavy chain constant region of human IgG. For example, the heavy chain constant region of the bispecific antibody may be the heavy chain constant region of human IgG1. The heavy chain constant region of human IgG1 may include, for example, any one amino acid sequence selected from the amino acid sequences of sequence number 43 and sequence numbers 60 to 62, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with respect to this.

[0359] Sequence numbers 43, 60, 61, and 62 are heavy chain constant region sequences of antibodies commonly used in the art. Sequence number 43 corresponds to the heavy chain constant region sequence of the known IgGl antibody Herceptin. Sequence number 60 is the heavy chain constant region sequence of wild-type human IgGl (see UniProt Accession P01857). Sequence number 61 corresponds to the heavy chain constant region sequence of the known IgGl antibody atezolizumab. Sequence number 62 corresponds to the heavy chain constant region sequence of the known IgGl antibody durvalumab.

[0360] In some embodiments, one heavy chain of the bispecific antibody may be designed to be dimeric with another heavy chain. For example, the heavy chain constant region of the first heavy chain of the bispecific antibody (e.g., heavy chain constant region 3) and the heavy chain constant region of the second heavy chain (e.g., heavy chain constant region 3) may be designed to be dimeric with each other, and such design allows the first heavy chain and the second heavy chain to dimeric. In some embodiments, a KIH variant may be introduced into the heavy chain constant region (or heavy chain constant region 3) of the bispecific antibody. For example, heavy chain constant region 3 of one of the two heavy chains of the bispecific antibody may contain a knob mutation, and heavy chain constant region 3 of the other heavy chain may contain a hole mutation. In some embodiments, the bispecific antibody may contain a heavy chain constant region (or heavy chain constant region 3) of IgGl into which a KIH mutation has been introduced (or which has a KIH mutation). In some embodiments, one of the two heavy chains of the bispecific antibody may contain a heavy chain constant region (or heavy chain constant region 3) of IgGl into which a knob mutation has been introduced (or which has a knob mutation), and the other of the two heavy chains may contain a heavy chain constant region (or heavy chain constant region 3) of IgGl into which a hole mutation has been introduced (or which has a hole mutation).

[0361] In some embodiments, the knob variation may include one or more substitutions selected from S354C and T366W (or T366Y). In some embodiments, the hole variation may include one or more substitutions selected from Y349C, T366S, L368A and Y407V (or Y407T).

[0362] In some embodiments, the heavy chain invariant region of one of the two heavy chains of the bispecific antibody comprises any one of sequence number 43 and sequence numbers 60 to 62, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity, wherein the heavy chain invariant region 3 of the heavy chain invariant region may have a knob variation. In some embodiments, the heavy chain invariant region of the other heavy chain of the two heavy chains of the bispecific antibody comprises any one of sequence number 43 and sequence numbers 60 to 62, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity therewith, wherein the heavy chain invariant region 3 of the heavy chain invariant region may have a Hole variant.

[0363] In some embodiments, the heavy chain invariant region of one of the two heavy chains of the bispecific antibody comprises any one of sequence number 43 and sequence numbers 60 to 62, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity, wherein heavy chain invariant region 3 of the heavy chain invariant region may comprise one or more of S354C and T366W (or T366Y) substitutions (knob mutation); The heavy chain constant region of the other heavy chain of the two heavy chains of the bispecific antibody comprises any one of the amino acid sequences of sequence No. 43 and sequence Nos. 60 to 62, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity, wherein heavy chain constant region 3 of the heavy chain constant region comprises any one or more of Y349C, T366S, L368A, and Y407V (or Y407T) substitutions (hole mutation). In some embodiments, the heavy chain invariant region 3 of the first heavy chain may include a knob variation, and the heavy chain invariant region 3 of the second heavy chain may include a hole variation. In some embodiments, the heavy chain invariant region 3 of the first heavy chain may include a hole variation, and the heavy chain invariant region 3 of the second heavy chain may include a knob variation.

[0364] In some embodiments, the heavy chain constant region (or heavy chain constant region 3) of the first heavy chain may include a knob variant (e.g., one or more substitutions of S354C and T366W; or one or more substitutions of an amino acid residue at position 354 to C and an amino acid residue at position 366 to W (wherein the positions of the amino acid residues are specified according to the EU numbering system)). In some embodiments, the Fc region of the first heavy chain may include a knob variant. In some embodiments, the heavy chain constant region of the first heavy chain may include S354C and T366W substitutions. For example, if the heavy chain constant region of the first heavy chain includes a knob variant, the second heavy chain, which is the other heavy chain of the bispecific antibody, may include a hole variant. In some embodiments, the heavy chain invariant region (or heavy chain invariant region 3) of the first heavy chain may include a hole variation (e.g., one or more substitutions of Y349C, T366S, L368A, and Y407V; or one or more substitutions of an amino acid residue at position 349 to C, an amino acid residue at position 366 to A, and an amino acid residue at position 407 to V (wherein the positions of the amino acid residues are specified according to the Eu numbering system). In some embodiments, the Fc region of the first heavy chain may include a hole variation. In some embodiments, the heavy chain constant region of the first heavy chain may include Y349C, T366S, L368A, and Y407V substitutions. For example, if the heavy chain constant region of the first heavy chain includes a hole variant, the second heavy chain, which is the other heavy chain of the bispecific antibody, may include a knob variant.

[0365] In some embodiments, the second heavy chain of the bispecific antibody may include a heavy chain variable region of the second antigen-binding fragment and a heavy chain constant region of the second heavy chain. In the second heavy chain of the bispecific antibody, the heavy chain variable region of the second antigen-binding fragment and the heavy chain constant region of the second heavy chain may be located in the direction from the N-terminus to the C-terminus as described above. In some embodiments, the heavy chain constant region of the second heavy chain (or heavy chain constant region 3) may include a hole variant (e.g., one or more substitutions of Y349C, T366S, L368A, and Y407V). In some embodiments, the Fc region of the second heavy chain may include a hole variant. In some embodiments, the heavy chain constant region of the second heavy chain may include Y349C, T366S, L368A, and Y407V substitutions. For example, if the heavy chain constant region of the second heavy chain includes a hole variant, the first heavy chain, which is the other heavy chain of the bispecific antibody, may include a knob variant. In some embodiments, the heavy chain constant region of the second heavy chain (or heavy chain constant region 3) may include a knob variant (e.g., a substitution of one or more of S354C and T366W). In some embodiments, the Fc region of the second heavy chain may include a knob variant. In some embodiments, the heavy chain constant region of the second heavy chain may include S354C and T366W substitutions. For example, if the heavy chain invariant region of the second heavy chain contains a knob variant, the first heavy chain, which is the other heavy chain of the bispecific antibody, may contain a hole variant.

[0366] In some embodiments, the light chain of the bispecific antibody (e.g., the first light chain) may include a light chain constant region (the first light chain constant region) in addition to the light chain variable region (the first light chain variable region) of the first antigen-binding fragment. Here, the light chain variable region and the light chain constant region of the first antigen-binding fragment may be located in the direction from the N-terminus to the C-terminus in the order described above. In some embodiments, the light chain constant region may include the amino acid sequence of sequence number 38. In some embodiments, the light chain invariant region may include the amino acid sequence of sequence number 38 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with respect to this.

[0367] In some embodiments, the heavy chain variable region of the first antigen-binding fragment and the light chain variable region of the first antigen-binding fragment form the first antigen-binding fragment, and accordingly, the bispecific antibody of the present disclosure comprises the first antigen-binding fragment.

[0368] In some embodiments, the light chain of the bispecific antibody (e.g., the second light chain) may include a light chain constant region (the second light chain constant region) in addition to the light chain variable region (the second light chain variable region) of the second antigen-binding fragment. Here, the light chain variable region and the light chain constant region of the second antigen-binding fragment may be located in the direction from the N-terminus to the C-terminus in the order described above. In some embodiments, the light chain constant region may include the amino acid sequence of sequence number 38. In some embodiments, the light chain invariant region may include the amino acid sequence of sequence number 38 or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity with respect to this.

[0369] In some embodiments, the heavy chain variable region of the second antigen-binding fragment and the light chain variable region of the second antigen-binding fragment form the second antigen-binding fragment, and accordingly, the bispecific antibody of the present disclosure comprises the second antigen-binding fragment.

[0370] If the light chain of the bispecific antibody is a common light chain having a common light chain variable region, the sequences of the first light chain and the second light chain may be identical. In some embodiments, the first heavy chain, the second heavy chain, the first light chain, and the second light chain of the bispecific antibody may each have the following structures:

[0371] First chain : [First chain variable region] - [First chain invariant region (including knob mutat ion or ho le mutat i on)] ,

[0372] Second chain : [Second chain variable region] - [Second chain invariant region (including ho le mutat ion or knob mutat i on)] ,

[0373] 1st light chain : [1st light chain variable region (common light chain variable region) ] - [1st light chain invariant region (common light chain invariant region)] ,

[0374] Second light chain : [Second light chain variable region (common light chain variable region) ] - [Second light chain invariant region (common light chain invariant region)] ,

[0375] Here, "-" can be independently a direct linkage (e.g., a linkage through a covalent bond) or an indirect linkage (e.g., a linkage through a structure such as an amino acid sequence or a linker).

[0376] In a specific embodiment, the first heavy chain, second heavy chain, first light chain, and second light chain of the specific antibody may each have the following structure:

[0377] First heavy chain : [First heavy chain variable region] - [First heavy chain invariant region (including knob mutation)] ,

[0378] Second chain : [Second chain variable region] - [Second chain invariant region (including ho le mutat ion)] ,

[0379] 1st light chain : [1st light chain variable region (common light chain variable region) ] - [1st light chain invariant region (common light chain invariant region)] ,

[0380] Second light chain : [Second light chain variable region (common light chain variable region) ] - [Second light chain invariant region (common light chain invariant region)] ,

[0381] Here, "-" can be independently a direct linkage (e.g., a linkage through a covalent bond) or an indirect linkage (e.g., a linkage through a structure such as an amino acid sequence or a linker).

[0382] In some embodiments, the first heavy chain may include a first heavy chain variable region and a first heavy chain invariant region.

[0383] In some embodiments, the first heavy chain invariant region may include a first CH1, a first hinge, a first CH2, and a first CH3.

[0384] In some embodiments, the first heavy chain, second heavy chain, first light chain, and second light chain of the bispecific antibody may each have the following structure:

[0385] 1st heavy chain : [1st heavy chain variable region ]-[1st heavy chain invariant region 1]- [1st hinge ]- [1st heavy chain invariant region e- [1st heavy chain invariant region 3 ( including knob mutation or hole mutation)] ,

[0386] Second chain : [Second chain variable region]-[Second chain invariant region 1]-[Second hinge]-[Second chain invariant region - [Second chain invariant region 3 (including hole mutation or knob mutation)] ,

[0387] First light chain : [First light chain variable region (common light chain variable region)]-[First light chain invariant region (common light chain invariant region)] ,

[0388] Second light chain : [Second light chain variable region (common light chain variable region)]-[Second light chain invariant region (common light chain invariant region)] ,

[0389] Here, "-" can be independently a direct linkage (e.g., a linkage through a covalent bond) or an indirect linkage (e.g., a linkage through a structure such as an amino acid sequence or a linker).

[0390] In some embodiments, the first heavy chain, second heavy chain, first light chain, and second light chain of the bispecific antibody may each have the following structure:

[0391] 1st heavy chain: [1st heavy chain variable region]-[1st heavy chain invariant region 1]-[1st hinge]-[1st heavy chain invariant region 2]-[1st heavy chain invariant region 3 (including knob mutation)] , 2nd heavy chain: [2nd heavy chain variable region]-[2nd heavy chain invariant region 1]-[2nd hinge]-[2nd heavy chain invariant region - [2nd heavy chain invariant region 3 (including hole mutation)] , 1st light chain: [1st light chain variable region (common light chain variable region)]-[1st light chain invariant region (common light chain invariant region)] ,

[0392] Second light chain : [Second light chain variable region (common light chain variable region)]-[Second light chain invariant region (common light chain invariant region)] ,

[0393] Here, "-" can be independently a direct linkage (e.g., a linkage through a covalent bond) or an indirect linkage (e.g., a linkage through a structure such as an amino acid sequence or a linker).

[0394] In some embodiments, the first heavy chain invariant region may include either sequence number 32 or 37, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the first heavy chain invariant region may include the amino acid sequence of sequence number 32 or 37. In some embodiments, the first heavy chain invariant region may include the amino acid sequence of sequence number 32.

[0395] In some embodiments, the first CH1 (e.g., the first CH1 of the first heavy chain constant region) may comprise the amino acid sequence of sequence number 28 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the first CH1 (e.g., the first CH1 of the first heavy chain constant region) may comprise the amino acid sequence of sequence number 28.

[0396] In some embodiments, the first hinge (e.g., the hinge of the first heavy chain invariant region) may comprise the amino acid sequence of sequence number 29 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the first hinge (e.g., the hinge of the first heavy chain invariant region) may comprise the amino acid sequence of sequence number 29.

[0397] In some embodiments, the first CH2 (e.g., the first CH2 of the first heavy chain constant region) may comprise the amino acid sequence of sequence number 30 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the first CH2 (e.g., the first CH2 of the first heavy chain constant region) may comprise the amino acid sequence of sequence number 30.

[0398] In some embodiments, the first CH3 (e.g., the CH3 of the first heavy chain invariant region) may comprise either one of the amino acid sequences of sequence numbers 31 and 36, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity.

[0399] In an embodiment, the first CH3 (e.g., the CH3 of the first heavy chain constant region) may comprise the amino acid sequence of sequence number 31 or 36. In some embodiments, the first CH3 may comprise the amino acid sequence of sequence number 31.

[0400] In some embodiments, the first Fc region (e.g., the Fc region of the first heavy chain or the first heavy chain invariant region) may comprise an amino acid sequence of either sequence number 41 or 42 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the first Fc region (e.g., the Fc region of the first heavy chain or the first heavy chain invariant region) may include the amino acid sequence of sequence number 41 or 42. In some embodiments, the first Fc region may include the amino acid sequence of sequence number 41.

[0401] In some embodiments, the second heavy chain may include a second heavy chain variable region and a second heavy chain invariant region.

[0402] In some embodiments, the second heavy chain invariant region may include a second CH1, a second hinge, a second CH2, and a second CH3.

[0403] In some embodiments, the second heavy chain invariant region may include an amino acid sequence of either sequence number 37 or 32, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the second heavy chain invariant region may include an amino acid sequence of sequence number 37 or 32. In some embodiments, the second heavy chain invariant region may include an amino acid sequence of sequence number 37.

[0404] In some embodiments, the second CH1 (e.g., the second CH1 of the second heavy chain invariant region) may comprise the amino acid sequence of sequence number 33 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the second CH1 (e.g., the second CH1 of the second heavy chain invariant region) may comprise the amino acid sequence of sequence number 33.

[0405] In some embodiments, the second hinge (e.g., the hinge of the second heavy chain invariant region) may comprise the amino acid sequence of sequence number 34 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the second hinge (e.g., the hinge of the second heavy chain invariant region) may comprise the amino acid sequence of sequence number 34.

[0406] In some embodiments, the second CH2 (e.g., the second CH2 of the second heavy chain constant region) may comprise the amino acid sequence of sequence number 35 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity. In some embodiments, the second CH2 (e.g., the second CH2 of the second heavy chain constant region) may comprise the amino acid sequence of sequence number 35.

[0407] In some embodiments, the second CH3 (e.g., the CH3 of the second heavy chain invariant region) may comprise an amino acid sequence of either sequence number 36 or 31, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or an amino acid sequence having 99% or more sequence identity.

[0408] In an embodiment, the second CH3 (e.g., the second CH3 of the second heavy chain invariant region) may include the amino acid sequence of sequence number 36 or 31. In some embodiments, the second CH3 may include the amino acid sequence of sequence number 36. In some embodiments, the second Fc region (e.g., the Fc region of the second heavy chain or the second heavy chain invariant region) may include an amino acid sequence of either sequence number 42 or 41, or an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity. In some embodiments, the second Fc region (e.g., the Fc region of the second heavy chain or the second heavy chain invariant region) may include the amino acid sequence of SEQ ID NO. 42 or 41. In some embodiments, the second Fc region may include the amino acid sequence of SEQ ID NO. 42.

[0409] Exemplary amino acid sequences of the heavy and light chains of the bispecific antibodies of the present disclosure are described in Table 10 below. Furthermore, amino acid residues in which knobs-into-holes mutations occurred in each heavy chain are indicated by underlining and bolding.

[0410] [Table 1 shows the heavy and light chains of the bispecific antibody, and exemplary amino acid sequences of each element.]

[0411] Name Amino ac id sequence

[0412] Bispecific antibody's 1st QVQLQQWGPGLVKPSQTLSLTCTVSGGS INSGDYYWSWI RQPPGKGLES I GY I YYSGST heavy chain YYNPSLKSRVT I SADTSKNQFSLKLTSVTAADTAVYYCARERVYSSSLDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQN 39) 1st QVQLQQWGPGLVKPSQTLSLTCTVSGGS INSGDYYWSWI RQPPGKGLES I GY I YYSGST heavy chain variable region YYNPSLKSRVT I SADTSKNQFSLKLTSVTAADTAVYYCARERVYSSSLDYWGQGTLVTV SS (SEQN 10)

[0413] The heavy chain constant region of the first heavy chain of the bispecific antibody SGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL (knob) (CHl-hinge- GGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE CH2-CH3(knob) ) QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP CREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence No. 32)

[0414]

[0415] Heavy chain invariant of the first heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS Region 1 (CH1) SGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKV (SEQ No. 28) Hinge of the first heavy chain EPKSCDKTHTCPPCP (SEQ No. 29)

[0416] The first heavy chain's heavy chain invariant APELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT Region 2 (CH2) KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKTI SKAK

[0417] (Column number 30)

[0418] Heavy chain invariant of heavy chain 1 GQPREPQVYTLPPCREEMTKN2VSLWCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLD region 3 (knob) SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number (CH3(knob) ) 31)

[0419] 1st light chain (common light chain) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21) 2nd QVTLKESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKY of bispecific antibody Heavy chain YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANDRRGFYESGNYYNVPFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence No. 40) The second of a bispecific antibody QVTLKESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKY heavy chain variable region of heavy chain YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANDRRGFYESGNYYNVPFDYWG QGTLVTVSS (Sequence No. 22)

[0420] The heavy chain constant region of the second heavy chain of the bispecific antibody SGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL (hole) (CHl-hinge- GGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE CH2-CH3(hole) ) QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPP SREEMTKNQVSLSCAVKGFYPSD ​​I AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQN 37) — Second heavy chain invariant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS Region 1 (CH1) SGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKV (SEQN 33) Second heavy chain hinge EPKSCDKTHTCPPCP (SEQN 34)

[0421] The second heavy chain's heavy chain invariant APELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT Region 2 (CH2) KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKTI SKAK

[0422] (Column No. 35)

[0423] Second heavy chain invariant GQPREPQVCTLPPSREEMTKN2VSLSCAVKGFYPSDI AVEWESNGQPENNYKTTPPVLD region 3 (hole) SDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence No. (CH3(hole) ) 36)

[0424] Second light chain (common light chain) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0425]

[0426] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence No. 21)

[0427] An example of the structure of a bispecific antibody of the present disclosure is shown in FIG. 02, and FIG. 02 is referenced to understand the structure of the bispecific antibody.

[0428] In some embodiments, the bispecific antibody may further comprise a heavy chain constant region in addition to the antigen-binding fragments (a first antigen-binding fragment and a second antigen-binding fragment). For example, the bispecific antibody may further comprise heavy chain constant regions (a first heavy chain constant region and a second heavy chain constant region) in addition to the antigen-binding fragments. In some embodiments, the bispecific antibody may further comprise an Fc region in addition to the antigen-binding fragments. In some embodiments, the bispecific antibody may further comprise Fc regions (e.g., a first Fc region and a second Fc region) in addition to the antigen-binding fragments.

[0429] 5.2.2. Modified bispecific antibody

[0430]

[0431] The bispecific antibody of the present disclosure comprises an N-glycosylation site (e.g., Asn297). Here, number 297 of Asn297 is known as a position number according to EU numbering (see [Rabat, EA (1991). Sequences of proteins of immunological interest (No. 91). US Department of Health and Human Services, Public Health Service, National Institutes of Health.]). Asn297 is known to be located in the heavy chain constant region 2 (CH2). The bispecific antibody of the present disclosure comprises two heavy chains (e.g., a first heavy chain and a second heavy chain) and, accordingly, comprises two Asn297s. Asn297 can be described as being included in the heavy chain constant region, Fc region, or heavy chain constant region 2 (CH2) of a bispecific antibody, but is not otherwise limited.

[0432] Meanwhile, this N-glycosylation site and / or the glycan linked thereto (N-1 inked glycan, N-glycan, or, for example, Asn297-1 inked glycan) may be engineered to contain an azide. For example, Asn297-1 inked glycan may be enzymatically engineered to contain an azide. By engineering such a glycan, a bispecific antibody may be engineered to contain an azide group. In some embodiments, the engineering that causes a bispecific antibody to contain an azide (i.e., the engineering of the glycan) may be referred to as glyco-engineering (or glycan remodeling), but is not otherwise limited. Glyco-engineering that causes a bispecific antibody to contain an azide is [Van Geel, R., Wij deven, MA, Heesbeen, R., Verkade, J. M., Wasiel, A.A., van Berkel, S. S. , & van Delft , F . L. (2015). Chemoenzymat ic conjugat ion of toxic pay loads to the globally ly conserved N-glycan of nat i ve mAbs provides homogeneous and highly effective antibody-drug conjugates. Biocon juga te chemistry, 26(11), 2233-2242.] , [Wi j deven, M.A., van Geel, R., Hoogenboom, J. H. , Verkade , J . M., Janssen, B. M., Hurkmans, I. , . . . & van Delft, F. L. (2022, December).Enzymatic glycan remodeling-metal-free click (GlycoConnect®) provides homogenous antibodies with improved stability and therapeutic index with sequence engineering. This is described in detail in MAbs (Vol. 14, No. 1, p. 2078466). Taylor & Francis. ] and [PCT Patent Application Publication No. W02022 / 058395A1]. This glyco-engineering technology may be GlycoConnect® (Synaff ix BV) and is not otherwise limited. An example of an antibody engineered through glyco-engineering is shown in FIG. 03. In engineered antibodies or bispecific antibodies, azide can be linked to an Asm residue (e.g., Asn297) located in the heavy chain constant region 2 (CH2) (where the 297 position of Asn297 is designated according to the EU numbering system). In some embodiments, the Asn residue at position 297 according to the EU numbering system (i.e., Asn297) may correspond to the Asn at position 300 (i.e., Asn300) when based on the amino acid sequence of the heavy chain full length sequence of the bispecific antibody according to some embodiments of the present disclosure (e.g., sequence number 39), or may correspond to the Asn at position 307 (i.e., Asn307) when based on the amino acid sequence of the heavy chain full length sequence of the bispecific antibody according to some embodiments of the present disclosure (e.g., sequence number 40).In some embodiments, the Asn residue at position 297 according to the EU numbering system (i.e., Asn297) may correspond to the Asn at position 180 (i.e., Asn180) based on the amino acid sequence of the heavy chain constant region of the bispecific antibody according to some embodiments of the present disclosure (e.g., SEQ ID NO: 32 or SEQ ID NO: 37). In some embodiments, the Asn residue at position 297 according to the EU numbering system (i.e., Asn297) may correspond to the Asn at position 67 (i.e., Asn67) based on the amino acid sequence of the heavy chain constant region 2 of the bispecific antibody according to some embodiments of the present disclosure (e.g., SEQ ID NO: 30 or SEQ ID NO: 35).

[0433] In some embodiments, a bispecific antibody engineered to include an azide (i.e., a bispecific antibody comprising an azide) may be referred to as an engineered antibody, an engineered bispecific antibody, a glyco-engineered antibody (or bispecific antibody), an antibody (or bispecific antibody) comprising an azide, or an azidated antibody (or bispecific antibody), without limitation thereto.

[0434] Some embodiments of the present disclosure provide an engineered antibody or a salt thereof. In some embodiments, the engineered antibody may have the structure of formula 01 of AB-(Li-Q1) X and may have<000********><000********> it.<000********><********> [Formula 01]<********><********> AB - I - - -<********><********><********> <********> <********> L Jx <********> <********> where AB is an antibody and L 1is a linker, and is a first click functional group comprising an azide group, and x can be an integer from 1 to 4 (e.g., x is an integer of 1, 2, 3, or 4). In a specific embodiment, x can be an integer of 1 or 2. In a specific embodiment, arc can be 2. In some embodiments, is a structure

[0441] N3

[0442]

[0443] It may include. In some embodiments, a can be an azide group.

[0444] HL

[0445] There is. In some embodiments, Q 1 One can possess talent.

[0446] AB, i.e., the antibody, is a bispecific antibody according to some embodiments of the present disclosure. I? is a linker formed through glycoengineering of the antibody, which may be referred to as a glucosamine linker or a glucose linker, but the name is not limited. I? is - GlcNAc(Fuc) a It can be expressed as - Su-. Here, GlcNAc is linked to AB, and O is linked to A. That is, the engineered antibody is AB-(GlcNAc(Fuc)a- Su- Qi) x It can have the structure. Gl cNAc is an N-acetylglucosamine moiety, Fuc is a fucose moiety, a is independently 0 or 1, and Su is a monosaccharide

[0447] am .

[0448] In some embodiments, I may be connected to Asn of an antibody (i.e., a bispecific antibody) (e.g., Asm within heavy chain constant region 2). In some embodiments, I may be connected via a side chain of Asm within heavy chain constant region 2 of the antibody. In some embodiments, L 1 I can be linked to the N-glycosylation site of the antibody (e.g., Asn297). In some embodiments, I can be linked to Asn297 of the antibody. In some embodiments, when x is 1 or 2, I can be linked to Asn297 of the antibody, respectively. In some embodiments, when x is 2, each I can be linked to Asn297 of each of the two heavy chains of the antibody. In some embodiments, I can be linked through the side chain of Asn297. In some embodiments, L 1 ...is connected to the amine group of the side chain of Asn297 (the amine group of carboxamide (-C(O)NH-)). In some embodiments, ...is attached to the amine group of the side chain of Asn297 (the amine group of carboxamide).

[0449] GlcNAc is a core N-acetylglucosamine moiety that is commonly present in the glycan structure of antibodies. Here, the core N-acetylglucosamine moiety may refer to an N-acetylglucosamine moiety directly attached to a peptide chain. This core N-acetylglucosamine moiety can be selectively fucosylated (a is 0 or 1), which is a general characteristic of antibodies.

[0450] Structure of engineered antibody AB-(L 1 - Q x ) x In the case where x is 1, -L 1 - Q 1It will be understood to be linked to one of the heavy chains of the antibody (e.g., either the first heavy chain and the second heavy chain), (e.g., "-1'- is linked to Asn297 of one heavy chain), and when x is 2, two -L 1 -Q 1 Each will be understood to be linked to each of the two heavy chains of the antibody (e.g., each of the first heavy chain and the second heavy chain), (e.g., "-1'- is linked to Asn297 of each of the two heavy chains).

[0451] L 1 Specifically showing the structure of the engineered antibody shown is described below.

[0452] In some embodiments, the engineered antibody of Formula 01 may have the structure of Formula 02.

[0453] In some embodiments, the engineered antibody may have the structure of the following formula: [Formula 0

[0454] r (F r )a 1

[0455] AB + GIcNAc—Su—Q I

[0456]

[0457] I Jx

[0458] At this time,

[0459] AB is an antibody; GIcNAc is an N-acetylglucosamine moiety; Fuc is a fucose moiety; a is independently 0 or 1 each; Su is a monosaccharide; azide is azide; and x is an integer from 1 to 4 (preferably, an integer of 1 or 2). Here, Su-Q 1It can be referred to as az i dosugar.

[0460] In some embodiments, AB is an antibody, wherein the antibody is a bispecific antibody according to some embodiments of the present disclosure. In some embodiments, in Formula 02 - Gl cNAc(Fuc) a - Su- Q 1 It can be linked to Asn of a bispecific antibody (e.g., Asn within heavy chain constant region 2). In some embodiments, -GIcNAc(Fuc)—Su-Q 1 ... can be connected via the side chain of Asn. In some embodiments, in Formula 02 - GI cNAc (Fuc) — Su-Q 1 It can be linked to the N-glycosyl at i on site (e.g., Asn297) of the bispecific antibody. In some embodiments, GI cNAc in Formula 02 is linked to Asn297 of the bispecific antibody. In some embodiments, Gl cNAc in Formula 02 is linked to the bispecific antibody through Asn297 of the bispecific antibody. In some embodiments, Gl cNAc in Formula 02 is linked to the bispecific antibody through the side chain of Asn297 of the bispecific antibody. In some embodiments, Gl cNAc in Formula 02 can be attached to the amine group (amine group of carboxamide) of the side chain of Asn297 of the bispecific antibody.

[0461] Su is a monosaccharide. Su may be any one selected from galactose (Gal), mannose (Man), glucose (Gl c), N-acetylglucosamine (Gl cNAc), N-acetylgalactosamine (N-acetylgalactosamine; GalNAc), N-acetylneuraminidic acid (N-acetylneuraminidic acid) or sialic acid (sial ic acid; sial ) and fucose (Fuc). Preferably, it may be N-acetylgalactosamine (GalNAc).

[0462] In a specific embodiment, in Formula 02, the number may be 1 or 2. When the number is 1, the antibody has one azide group, wherein the one azide group is connected to Asn297 of one of the two heavy chains of the antibody. When x is 2, the antibody has two azide groups, wherein each of the two azide groups is connected to Asn297 of each of the two heavy chains of the antibody. Preferably, x may be 2.

[0463] In some embodiments, the engineered antibody of any one of Formulas 01 to 02 may have the structure of Formula 03. In some embodiments, the engineered antibody may have the structure of Formula 03 below:

[0464] [Essence 03]

[0465]

[0466] At this time,

[0467] AB is an antibody, and

[0468] a is independently 0 or 1, and

[0469] x is an integer from 1 to 4 (preferably, x is an integer of 1 or 2). Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is represented as -NHC(0)CH3.

[0470] In the previous description, it was explained in detail that GlcNAc is attached to the Asn (e.g., Asn297) site of an antibody (i.e., a bispecific antibody). In some embodiments, x is 1 or 2, and the structure attached to each AB in Formula 03 may be attached to the Asn297 of the antibody. In some embodiments, x is 2, and the structure attached to each AB in Formula 03 may be attached to the Asn297 of each of the two heavy chains of the antibody. For example, in Formula 03, the structure attached to AB may be attached to the amine group of the side chain of Asn297. In Formula 03, A

[0471]

[0472] This is B. Meanwhile, when a is 0 (i.e., when Gl cNAc is not fucosylated), the structure connected to the yue may have the following structure and is not otherwise restricted:

[0473]

[0474] In some embodiments, Formula 03 may be Formula 04.

[0475] In some embodiments, the engineered antibody may have the structure of the following chemical formula 04:

[0476] [Essence 04]

[0477]

[0478] In this case, AB is an antibody, and

[0479] a is independently 0 or 1, and

[0480] x is an integer from 1 to 4 (preferably an integer of 1 or 2).

[0481] In the previous description, it was explained in detail that GlcNAc is attached to the Asn (e.g., Asn297) site of an antibody (i.e., a bispecific antibody). In some embodiments, x is 1 or 2, and the structure attached to AB in Formula 04 can be attached to the Asn297 of the antibody. In some embodiments, x is 2, and the structure attached to each AB in Formula 04 can be attached to the Asn297 of each of the two heavy chains of the antibody. For example, in Formula 04, the structure attached to the AB can be attached to the amine group of the side chain of Asn297. The structure attached to AB

[0482]

[0483] Meanwhile, in the case of , x7} 1 or 2, if the side chain of Asn297 of the antibody is plotted together, the engineered antibody of Equation 04 can be represented by the structure of the following Equation 05:

[0484] [Food 05]

[0485]

[0486] At this time,

[0487] a is independently 0 or 1, and

[0488] x is 1 or 2 and,

[0489] -CH2C(0)NH- connected adjacent to AB represents the side chain of 811297 and is part of AB (antibody).

[0490] Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is represented as -NHC(0)CH3.

[0491] A method for preparing a engineered antibody according to some embodiments of the present disclosure may be a glyco-engineering (or glycan remodeling) method of an antibody. For example, the engineered antibody (or engineered bispecific antibody) is prepared by preparing an antibody (glycoform or mixture of glycoforms) and endoglycosidase-mediated the antibody.

[0492] It can be prepared by enzymatically remodeling various glycoforms or glycans (e.g., GO, Gl, G2, etc. (specifically, GoF, GiF, G2F, SI, 2GI, 2F, Ms, M3, etc.)) through endoglycosidase-mediated trimming, and then delivering an azido-sugar (e.g., 6-azidoGalNAc, UDP6-azidoGalNAc, etc.) to the core GlcNAc exposed by endoglycosidase via glycosyltransferase, but is not otherwise limited.

[0493] For example, a engineered antibody can be prepared by preparing an antibody, then cleaving various N-glycans (GoF, GiF, G2F, high-mannose, etc.) bound to Asn297 by an endoglycosidase (e.g., Endo-S, Endo-S2, etc.) to expose a core GlcNAc (or GlcNAc-Fuc), and then enzymatically adding an azido donor (e.g., UDP-6-azido-GalNAc, etc.) to the core GlcNAc using a glycosyltransferase.

[0494] In some embodiments, a method for producing a engineered antibody (or bispecific antibody) comprises endoglycosidase,

[0495] Glycosyltransferases, and may include a mixture or storage with an azido donor (e.g., azido-sugar). In some embodiments, the method of preparing an engineered antibody from the mixture may further include obtaining the engineered antibody. Mixing or storing the antibody with endoglycosidase, glycosyltransferase, and azido donor can be performed in various ways. For example, mixing can be performed by adding a composition containing the antibody to a container and adding a composition containing endoglycosidase, glycosyltransferase, and azido donor or each composition containing each of them to the container. For example, an antibody, endoglycosidase, glycosyltransferase, and azido donor can be added to a container, mixed or stored for an appropriate time, and then an engineered antibody can be obtained from the mixture, unless otherwise limited.

[0496] 5.2.3. Linker-payload compound

[0497]

[0498] The antibody-drug conjugate can be obtained by reacting an engineered antibody engineered to contain an azide with a linker-payload compound containing BCN. The reaction of azide and BCN is well known in the art as a click chemistry reaction and / or a bioorthogonal reaction, and the reaction scheme has been described above.

[0499] Some embodiments of the present disclosure provide a linker-payload compound. The linker payload compound may have a structure of formula 06 of Q 2 - L 2 -(I?-D)2.

[0500] [Formula 06]

[0501] Q 2 — L 2 L 3 — D

[0502]

[0503] Here, Q 2 is a second click chemical functional group (or second reactive group) containing a BCN group, and L 2 and I? are linkers, respectively, and D represents the drug (exatecan moiety). Q 2 - L 2 - (L 3 - D) As can be seen from the structure of 2, the linker-payload compound contains two drugs (or drug moiety). L 2 Since this linker-payload compound contains two drugs, it may be referred to as a branched linker, but the name of this linker is not otherwise limited. I? comprises a cleavable portion and, accordingly, may be referred to as a cleavable linker, but the name of this linker is not otherwise limited. In some embodiments, I? may comprise a cleavable portion that can be cleaved by a protease (e.g., cathepsin B). In some embodiments, L 3 It may contain a VA-PABC linker (valine-alanine-p-aminobenzyl carbamate linker).

[0504] For example, the linker-payload compound of Formula 06 can be represented by the structure of Formula 07. In some embodiments, the linker-payload compound may have the structure of Formula 07:

[0505] [Essence 07]

[0506] Q 2 — I—

[0507]

[0508] 』 、 L 3 —— ° D

[0509] For the linker-payload compound of the present disclosure, reference may be made to document [PCT Patent Application Publication No. W02022 / 058395A1]. The linker-payload compound of the present disclosure is commercialized under the trademark SYNtecan E™.

[0510] Hereinafter, the linker-payload compounds of the present disclosure are described in detail. In some embodiments, a linker-payload compound having any one of the structures of Formulas 06 to 07 may have the structure of Formula 08.

[0511] In some embodiments, the linker-payload compound of the present disclosure may have the structure of Formula 08 below:

[0512] [Method 08]

[0513] o、 ,o N=N〜° HH

[0514]

[0515] The structure of Formula 08 is the structure of the aforementioned linker-payload compound "Q 2 - It can appropriately correspond to each element of !?-(!?- D)2", and the structure of Equation 08 and "Q 2 - L 2 The correspondence examples of the -(I?- D) 2" structure are as follows:

[0516] This and here, 1* is L 2 Indicates the attachment point with.

[0517] (AA / WWV

[0518]

[0519] L 2 is here, 1* is Q 2 It indicates the attachment point with, and each 2* is two L 3 Indicates the attachment point with each.

[0520] L 3 Is

[0521]

[0522] L 2 Indicates the attachment point with, and 2* indicates the attachment point with D.

[0523]

[0524] and, here 1* represents the attachment point with I?.

[0525] In some embodiments, a linker-payload compound having any one of the structures of Formulas 06 to 08 may have the structure of Formula 09. In some embodiments, a linker-payload compound of the present disclosure may have the structure of Formula 09 below:

[0526] [Essence 09]

[0527]

[0528] 5.3. Structure of Antibody-Drug Conjugates

[0529] Some embodiments of the present disclosure provide an antibody-drug conjugate or a salt thereof (e.g., a pharmaceutically acceptable salt). In some embodiments, the antibody-drug conjugate may comprise a bispecific antibody. In some embodiments, the antibody-drug conjugate may comprise a linker. In some embodiments, the antibody-drug conjugate may comprise a drug (or payload). In some embodiments, the antibody-drug conjugate may comprise a bispecific antibody, a linker, and a drug.

[0530] Antibody-drug conjugates can be obtained through the reaction of the aforementioned engineered antibody (i.e., a glyco-engineered antibody to contain an azide) and a linker-payload compound. The engineered antibody contains an azide, and the linker-payload compound contains BCN, and the engineered antibody and the linker-payload compound are conjugated through a click chemistry reaction (or bioorthogonal reaction) between the azide and BCN.

[0531] The structure of the engineered antibody is AB- (I?- Qi) x It can have, where a is a first click functional group containing an azide group, as explained in the previous paragraphs. Linker-payload compounds have the structure Q 2 -L 2 -(L 3 -D)2 can have, and Q 2 It was explained in previous paragraphs that is a second click chemical functional group containing BCN. Antibody-drug conjugates that can be prepared by the conjugation of an engineered antibody and a linker-payload compound are AB-(L 1 - Q"- L 2 - (L 3 - D)2)y can have the structure of Equation 10.

[0532] Some embodiments of the present disclosure provide an antibody-drug conjugate having the structure of Formula 10.

[0533] [Equation 1 is

[0534] AB

[0535]

[0536] Here, y can be an integer from 1 to 4 (e.g., an integer of 1, 2, 3, or 4) (preferably, y can be an integer of 1 or 2). Here, Q" is a first click chemical functional group (Q) comprising an azide. 1) and a second click chemical functional group (Q containing BCN 2 ), which represents a structure formed by the reaction of and includes any of the following structures:

[0537]

[0538] , * •

[0539] In some embodiments, Q" can have any of the following structures:

[0540]

[0541] where 1* represents the attachment point with L 1 and 2* represents the attachment point with I?.

[0542] In the AB-(Li-Q"-L 2 -(L 3 -D)2)y structure of the antibody-drug conjugate, the remaining elements other than Q" (AB, I』, L 2 , L 3 , and D) are as described in the previous paragraphs including "5.2.1. Antibody: Anti-EGFR and anti-MUC1 bispecific antibody (anti-EGFR / MUC1 bispecific antibody)", "5.2.2. Engineered bispecific antibody", and "5.2.3. Linker-payload compound" of this disclosure.

[0543] For example, AB is an antibody, where the antibody is a bispecific antibody according to some embodiments of the present disclosure.

[0544] For example, 日 is a glucosamine linker. For example, 日 can have the structure of - GlcNAc(Fuc) a - Su-.

[0545] For example, I? is a dendritic linker. For example, I? is

[0546] 유 WW蟲

[0547]

[0548] 2 It may have the structure of *, where 1* represents the attachment point with Q", and each 2* represents the attachment points with two Ls 3 respectively.

[0549] For example, L 3 is a cutting ring connector. For example, L 3 is

[0550]

[0551] It may have the structure of 흐, where 1* represents the attachment point with L 2 and 2* represents the attachment point with D.

[0552] For example, 日 is a drug. For example, 日 is an exacatecan moiety. For

[0553] example, 日 is

[0554]

[0555] structure of

[0556]

[0557] It can have, and here it represents the attachment point with I?

[0558] For example, y may be an integer from 1 to 4. In a specific embodiment, y may be an integer of 1 or 2. In a specific embodiment, y may be 2. For example, if y is 4, the antibody-to-drug ratio (drug-to-antibody rat io; DAR) is 8. For example, if y is 3, the DAR is 6. For example, if y is 2, the DAR is 4. For example, if y is 1, the DAR is 2. In a specific embodiment, the DAR of the antibody-drug conjugate may be 2 or 4. An antibody-drug conjugate with a DAR of 4 (i.e., AB-(L 1 - Q"- L 2 - (L 3 - D)2)y, y=2) is a engineered antibody having two azide groups (i.e., AB-(I』-Qi) x It can be formed by conjugating two linker-payload compounds to , x=2). In antibody-drug conjugates, when y is 2, the structure - (L 1 - Q"- L 2 - (L 3 - D)2) is connected to each of the two heavy chains of the antibody (e.g., the first heavy chain and the second heavy chain, respectively) (e.g., connected to Asn297 on each of the two heavy chains). An example of a DAR4 antibody-drug conjugate is shown in FIG. 04.

[0559] When y is 1, the antibody-to-antibody ratio (drug-to-antibody rat io; DAR) is 2. The antibody-drug conjugate of DAR 2 (i.e., AB-(L 1 -Q"-L 2 - (L 3 -D)2)y, y=l) is a engineered antibody having two azide groups (i.e., AB-(I?-Qi) xIt is formed by conjugating one linker-payload compound to , x=2), or a engineered antibody having one azide group (i.e., AB-(L 1 - Q 1 ) ). It can be formed by conjugating one linker-payload compound to x=l), and is not otherwise limited. When y is 1, the structure - (L 1 - Q"- L 2 - (L 3 - 0)2) is connected to either of the two heavy chains of the antibody. Examples of DAR2 antibody-drug conjugates are disclosed in FIGS. 05 and 06. FIG. 05 is an example A of a DAR2 antibody-drug conjugate, showing an example of a DAR2 antibody-drug conjugate made by conjugating one linker-payload compound to an engineered antibody having two azide groups. FIG. 06 is an example B of a DAR2 antibody-drug conjugate, showing an engineered antibody having one azide group (one heavy chain of the engineered antibody having one azide group has an azide group, and the other heavy chain may have a natural glycan (or N-glycan) in the absence of glyco-engineering). An example of a DAR2 antibody-drug conjugate formed by conjugating one linker-payload compound to ) is shown.

[0560] In some embodiments, I may be connected to Asn of an antibody (i.e., a bispecific antibody) (e.g., Asn within heavy chain constant region 2). In some embodiments, I may be connected via a side chain of Asn within heavy chain constant region 2 of the antibody. In some embodiments, L 1It can be linked to the N-glycosylate site of the antibody (e.g., Asn297). In some embodiments, I’ can be linked to Asn297 of the antibody. In some embodiments, y is 1 or 2, and il can each be linked to Asn297 of the antibody. In some embodiments, y is 2, and each I’ can be linked to Asn297 of each of the two heavy chains of the antibody. In some embodiments, il can be linked through the side chain of Asn297. In some embodiments, il is linked to the amine group of the side chain of Asn297 (the amine group of carboxamide). In some embodiments, L 1 It is attached to the amine group of the side chain of Asn297 (the amine group of carboxamide). In some embodiments, - (L 1 - Q"- L 2 - (L 3 - D)2) can be linked to Asn of an antibody (i.e., a bispecific antibody). In some embodiments, - (L 1 - Q"- L 2 - (L 3 - D)2) can be linked to the N-glycosylation site of an antibody (e.g., Asn297). In some embodiments, - (I』- Q"- L 2 - (L 3 - D)2) can be linked to Asn297 of the antibody. In some embodiments, - (I?- Q"- L 2 - (I?- D)2) is connected via the side chain of Asn297. In some embodiments, - (L 1 - Q"- L 2 - (L 3 - D)2) is connected to the amine group of the side chain of Asn297 (the amine group of carboxamide). In some embodiments, - (I”-Q”-L 2 - (L3 - D)2) is attached to the amine group of the side chain of Asn297 (the amine group of the carboxamide (—C(O)NH—)). In some embodiments, the Asn in the antibody that can be linked to D (e.g., Asn297) is shown as follows:

[0561]

[0562] Here, 1* and 2* each represent the attachment points of the amino acid residues adjacent to Asn (e.g., Asn297) in the antibody, and 3* represents the attachment point to L 1 and the attachment point to D. In some embodiments, the antibody-drug conjugate having the structure of Formula 10 may have the structure of Formula 11. In some embodiments, the antibody-drug conjugate may have the structure of the following Formula 11:

[0563] [Formula 11]

[0564] L 3 — D

[0565] AB-- L 1 —

[0566] “즈 으

[0567]

[0568] The antibody-drug conjugate may include an antibody (bispecific antibody), a linker (e.g., the linker of the antibody-drug conjugate), and a drug. In some embodiments, the antibody-drug conjugate may include an antibody (bispecific antibody), a linker (e.g., the linker of the antibody-drug conjugate), and two drugs. In some embodiments, the drug may be linked to the antibody through the linker. In some embodiments, two drugs may be linked to the antibody through the linker. For example, in Formula 11, AB is bispecific

[0569] I. 1 O .. L 2 to L 3 —I|-L—Q—L,,3

[0570]

[0571] an antibody, L and the following is a linker of the antibody-drug conjugate, and in Formula 11, each 日 is a different drug.

[0572] In some embodiments, an antibody-drug conjugate having the structure of any one of Formulas 10 to 11 may have the structure of Formula 12. In some embodiments, the antibody-drug conjugate may have the structure of the following Formula 12:

[0573]

[0574] At this time,

[0575] AB is an antibody, and at this time, the antibody is a bispecific antibody according to some embodiments of the present disclosure,

[0576] GlcNAc is an N-acetylglucosamine moiety,

[0577] Su is a monosaccharide,

[0578] Fuc is a fucose moiety,

[0579] a is independently 0 or 1 respectively,

[0580] y may be an integer from 1 to 4. In certain embodiments, y may be an integer of 1 or 2. In certain embodiments, y may be 2.

[0581] In some embodiments, in Formula 12, the structure connected to [AM] (i.e., the structure other than AB in Formula 12) may be connected to Asn of the antibody (e.g., Asm within heavy chain constant region 2). In some embodiments, in Formula 12, the structure connected to [Yu] may be connected to N-glycosylatione site of the antibody. In some embodiments, in Formula 12, the structure connected to AB may be connected to Asn297 of the antibody. In some embodiments, in Formula 12, y is 1 or 2, and the structure connected to each AB may be connected to Asn297 of the antibody. In some embodiments, in Formula 12, y is 2, and the structure connected to each AB may be connected to Asn297 of each of the two heavy chains of the antibody. In some embodiments, in Formula 12, the structure connected to AB may be attached to the amine group of the side chain of Asn297. In Formula 12, the structure connected to AB is as follows:

[0582]

[0583] In some embodiments, an antibody-drug conjugate having any one of the structures of Formulas 10 to 12 may have the structure of Formula 13. In some embodiments, the antibody-drug conjugate may have the structure of Formula 13 below:

[0584] [Equation 13]

[0585]

[0586] At this time,

[0587] AB is an antibody (e.g., a bispecific antibody), and

[0588] a is independently 0 or 1, and

[0589] ymay be an integer from 1 to 4. In a specific embodiment, y may be 1 or 2. In a specific embodiment, y may be 2.

[0590] Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is expressed as -NHC(0)CH3.

[0591] In some embodiments, in Formula 13, the structure connected to [AM] (i.e., the structure other than AB in Formula 13) may be connected to Asn of the antibody (e.g., Asn within heavy chain constant region 2). In some embodiments, in Formula 13, the structure connected to [Yu] may be connected to the N-glycosylatione site of the antibody. In some embodiments, in Formula 13, the structure connected to AB may be connected to Asn297 of the antibody. In some embodiments, in Formula 13, y is 1 or 2, and the structure connected to each AB may be connected to Asn297 of the antibody. In some embodiments, in Formula 13, y is 2, and the structure connected to each AB may be connected to Asn297 of each of the two heavy chains of the antibody. In some embodiments, in Formula 13, the structure connected to AB can be attached to the amine group of the side chain of Asn297. In Formula 13, the structure connected to AB is as follows:

[0592]

[0593] In some embodiments, an antibody-drug conjugate having any one of the structures of Formulas 10 to 13 may have the structure of Formula 14. In some embodiments, the antibody-drug conjugate may have the structure of Formula 14 below:

[0594] [Equation 14]

[0595]

[0596] At this time,

[0597] AB is an antibody (e.g., a bispecific antibody), and

[0598] a is independently 0 or 1, and

[0599] y y can be an integer from 1 to 4. In certain embodiments, y can be 1 or 2. In certain embodiments, y can be 2. Ac refers to an acetyl group, and per is acetamide. In some embodiments, -NHCAc is expressed as -NHC(0)CH3.

[0600] In some embodiments, in Formula 14, the structure connected to [AM] (i.e., the structure other than AB in Formula 14) may be connected to Asn of the antibody (e.g., Asn within heavy chain constant region 2). In some embodiments, in Formula 14, the structure connected to [Yu] may be connected to the N-glycosylatione site of the antibody. In some embodiments, in Formula 14, the structure connected to AB may be connected to Asn297 of the antibody. In some embodiments, in Formula 14, y is 1 or 2, and the structure connected to each AB may be connected to Asn297 of the antibody. In some embodiments, in Formula 14, y is 2, and the structure connected to each AB may be connected to Asn297 of each of the two heavy chains of the antibody. In some embodiments, in Formula 14, the structure connected to AB can be attached to the amine group of the side chain of Asn297. In Formula 14, the structure connected to AB is as follows:

[0601]

[0602] An example of the antibody-drug conjugate of Formula 14 (y=2, DAR4 ADC) is shown in Fig. 07.

[0603] To aid the understanding of those skilled in the art, the structure of an antibody-drug conjugate is disclosed below, with the side chain of Asn297 of the antibody also depicted. In some embodiments, y may be 1 or 2, and the antibody-drug conjugate may have the structure of Formula 15 below:

[0604] [Equation 15]

[0605]

[0606] At this time,

[0607] AB is an antibody (e.g., a bispecific antibody), and

[0608] a is independently 0 or 1, and

[0609] y is 1 or 2 and,

[0610] - CH2C(0)NH- connected adjacent to AB represents the side chain of 811297 and is part of AB (antibody).

[0611] Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is represented as -NHC(0)CH3.

[0612] For the benefit of those skilled in the art, Table 11 below shows examples of amino acid sequences of antibodies (i.e., bispecific antibodies) according to some embodiments of the present disclosure, and indicates the Asn297 position in the amino acid sequence. In the following sequences, Asn297 is indicated by underlining and bolding. As previously mentioned, Asn297 is located within the Fc region or within the CH2 domain.

[0613] [Table 11] Example of amino acid sequence of antibody according to some embodiment of the present disclosure and indication of Asn297 position

[0614] chain Amino acid sequence

[0615] 제 1 중쇄 QVQLQQWGPGLVKPSQTLSLTCTVSGGS INSGDYYWSWIRQPPGKGLES I GY IYYSGSTYYNPSLKS (knob) RVTISADTSKNQFSLKLTSVTAADTAVYYCARERVYSSSLDYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLWKPKDTLM I SRTPEVTCVWD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 39) 제 1 경쇄 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGS GTDFTLTI SSLEPEDFAVYYCQQRSNWPPTFGQGTKVE I KRTVAAPSVF I FPPSDEQLKSGTASWC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC (서열번호 21)

[0616] 2nd heavy chain QVTLKESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV I SYDGSNKYYADSVKGR (hole) FTISRDNSKNTLYLQMNSLRAEDTAVYYCANDRRGFYESGNYYNVPFDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVfflFPAVLQSSGLYSLSSWTVPSS SLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM I SRTPE VTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAP I EKTI SKAKGQPREPQVCTLPPSREEMTKN2VSLSCAVKGFYPSD ​​IAVEWESNGQPENNY KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0617] 2nd light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGS GTDFTLTI SSLEPEDFAVYYCQQRSNWPPTFGQGTKVE I KRTVAAPSVF I FPPSDEQLKSGTASWC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

[0618]

[0619] SSPVTKSFNRGEC (Sequence No. 21)

[0620] 5.4. Preparation of Antibody-Drug Conjugates

[0621] The antibody-drug conjugate of the present disclosure can be prepared by the reaction of the aforementioned engineered antibody (e.g., a bispecific antibody engineered to include an azide) and a linker-payload.

[0622] An example of a schematic diagram of the reaction between an engineered antibody and a linker-payload compound to produce an antibody-drug conjugate according to some embodiments of the present disclosure is provided through the reaction schematic diagram 02 below.

[0623] [Reaction Schematic 0

[0624] Formula 06

[0625]

[0626] Here, each expression and each variable (AB, L 1 , Q 1 , x , Q 2 , L 2 , L 3 , D, Q", y, etc.) are as described in the previous paragraph. For example, when two linker-payload compounds of Formula 06 react with one engineered antibody of Formula 01 (where x is 2), y can be 2.

[0627] A schematic diagram of the reaction for preparing an antibody-drug conjugate through a specific example of a engineered antibody (Chemical Formula 04) and a specific example of a linker-payload compound (Chemical Formula 09) is shown in reaction schematic diagram 03 below.

[0628] [Reaction Schematic Diagram 03]

[0629]

[0630] Formula 14

[0631] Here, each equation and each variable (AB, a, x, y, etc.) are as described in the previous paragraph. For example, if two linker-payload compounds of Equation 09 react with one engineered antibody of Equation 04 (where arc is 2), y can be 2.

[0632] The antibody-drug conjugate of the present disclosure may be prepared through the following two processes: preparation of a engineered antibody through antibody engineering (glyco-engineering); and reaction of the engineered antibody with a linker-payload compound.

[0633] In some embodiments, a method for preparing an antibody-drug conjugate of the present disclosure may include the following:

[0634] Contact or react the engineered antibody with the linker-payload compound.

[0635] It has been explained in detail in the preceding paragraphs of this disclosure that engineered antibodies containing azide can be obtained through glyco-engineering of antibodies. Meanwhile, the engineered antibody contains azide, and the linker-payload compound contains a BCN group capable of reacting with the azide (e.g., bioorthogonal reaction or click chemistry reaction) as described above.

[0636] In contact or reaction between a engineered antibody and a linker-payload compound, an antibody-drug conjugate is produced in which a drug is linked to the antibody through a linker by a click chemistry reaction between the azide of the engineered antibody and the BCN of the linker-payload compound.

[0637] Contact or reaction between the engineered antibody and the linker-payload compound may be performed in various ways and is not otherwise limited. For example, contact or reaction between the engineered antibody and the linker-payload compound may be performed by mixing or reacting a composition containing one or more engineered antibodies with a composition containing multiple linker-payload compounds.

[0638] In some embodiments, the method for preparing an antibody-drug conjugate of the present disclosure may further comprise: purifying the product of the reaction of the engineered antibody and the linker-payload compound. Such purification may be performed to obtain an antibody-drug conjugate of the desired DAR (e.g., DAR4, or a mixture of DAR2 and DAR4) in high purity, and the method of purification may be any method widely known in the art, which is not otherwise limited. For example, purification may be performed through one or more of Size Exclusion Chromatography (SEC), Hydrophobic Interaction Chromatography (HIC), Ion Exchange Chromatography (IEX), High-Performance Liquid Chromatography (HPLC), and Action Chromatography, but is not otherwise limited. For example, a crude product of an antibody-drug conjugate can be obtained by mixing a composition containing a engineered antibody with a composition containing a linker-payload compound, and by purifying this crude product through a method known in the art to obtain a high-purity DAR2 ADC composition, a high-purity DAR4 ADC composition, or a mixed composition of DAR2 and DAR4 ADCs.

[0639] In some embodiments, the method for preparing an antibody-drug conjugate may be performed in vitro or ex vivo.

[0640] Furthermore, some embodiments of the present disclosure provide a composition or kit for preparing an antibody-drug conjugate. In some embodiments, the kit (or composition) for preparing an antibody-drug conjugate may comprise an antibody and / or engineered antibody according to some embodiments of the present disclosure, and a linker-payload compound according to some embodiments of the present disclosure. In some embodiments, the kit (or composition) for preparing an antibody-drug conjugate may comprise an antibody, an endoglycosidase, a glycosyltransferase, an azido donor (e.g., azido-sugar), and a linker-payload compound.

[0641] 5.5. Targets of Antibody-Drug Conjugates 5.5.1. Targets of Antibody-Drug Conjugates - EGFR and MUC1

[0642]

[0643] The antibody-drug conjugate of the present disclosure comprises a bispecific antibody (i.e., an anti-EGFR / MUC1 bispecific antibody) according to some embodiments of the present disclosure, and accordingly, can target EGFR and MUC1. The antibody-drug conjugate of the present disclosure can bind to either EGFR or MUC1. The antibody-drug conjugate of the present disclosure can bind to EGFR. The antibody-drug conjugate of the present disclosure can bind to MUC1. The target antigens of the antibody-drug conjugate of the present disclosure are EGFR and MUC1. Below, the characteristics of these target antigens and the antibody-drug conjugate are described in detail.

[0644] 5.5.2. Target Antigen 1 of Antibody-Drug Conjugate - EGFR

[0645]

[0646] In some embodiments, the antibody-drug conjugate may have the ability to bind to EGFR, and EGFR as a target antigen is described in detail in the present disclosure, including Section “4. Background - Target Antigens EGFR and MUC1” and “5.2.1. Antibody: Anti-EGFR and Anti-MUC1 Bispecific Antibody (Anti-EGFR / MUC1 Bispecific Antibody)”, specifically “(1) Bispecific Antibody - Overview”.

[0647] In some embodiments, the binding affinity of the antibody-drug conjugate to EGFR (e.g., human EGFR) may be about 0.0M to 500NM based on the dissociation constant (KD) [e.g., equilibrium dissociation constant]. In some embodiments, the binding affinity of the antibody-drug conjugate to EGFR based on the dissociation constant may be about 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180 nM, or 200 nM, or may be within the range of two values ​​selected from the aforementioned values. In some embodiments, the binding affinity of the antibody-drug conjugate to EGFR based on the dissociation constant may be 40 nM to 100 nM, or 50 nM to 80 nM. In some embodiments, the binding affinity of the antibody-drug conjugate to EGFR based on the dissociation constant may be about 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, or 70 nM. In some embodiments, the antibody-drug conjugate may bind to cells expressing EGFR (e.g., human tumor cells). For example, the antibody-drug conjugate may bind to cells expressing EGFR on the cell surface. For example, the antibody-drug conjugate may have specificity for cells and / or tissues expressing EGFR (e.g., tumor cells and / or tissues).

[0648] 5.5.3. Target Antigen 2 of Antibody-Drug Conjugate - MUC1

[0649]

[0650] In some embodiments, the antibody-drug conjugate may have the ability to bind to MUC1, and MUC1 as a target antigen is described in detail in the present disclosure, including in Section “4. Background - Target Antigens EGFR and MUC1” and “5.2.1. Antibody: Anti-EGFR and Anti-MUC1 Bispecific Antibody (Anti-EGFR / MUC1 Bispecific Antibody)” (1) Bispecific Antibody - Overview).

[0651] In some embodiments, the binding affinity of the antibody-drug conjugate to MUC1 (e.g., human MUC1) may be about 0.0M to 50NM based on the dissociation constant (KD) [e.g., equilibrium dissociation constant]. In some embodiments, the binding affinity of the antibody-drug conjugate to MUC1 based on the dissociation constant may be approximately InM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 1OnM, 10nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, or 20nM, or may be within a range of two values ​​selected from the above values. In some embodiments, the binding affinity of the antibody-drug conjugate to MUC1 may be 2 nM to 20 nM, 4 nM to 18 nM, or 5 nM to 15 nM based on the dissociation constant, but is not limited thereto. In some embodiments, the binding affinity of the antibody-drug conjugate to MUC1 may be about 5.0 nM, 5.2 nM, 5.4 nM, 5.8 nM, 6.0 nM, 6.2 nM, 6.4 nM, or 6.6 nM based on the dissociation constant.

[0652] In some embodiments, the antibody-drug conjugate can bind to cells expressing MUC1 (e.g., human tumor cells). For example, the antibody-drug conjugate can bind to cells expressing MUC1 on the cell surface. For example, the antibody-drug conjugate may have specificity for cells and / or tissues expressing MUC1 (e.g., tumor cells and / or tissues).

[0653] 5.5.4. Dual specificity for EGFR and MUC1

[0654]

[0655] In some embodiments, the antibody-drug conjugate can bind to both EGFR and MUC1.

[0656] In some embodiments, the antibody-drug conjugate is capable of binding to cells (e.g., human tumor cells) that express one or more of EGFR and MUC1 (e.g., cell-surface expression). In some embodiments, the antibody-drug conjugate has the ability to bind to cells that express one or more of EGFR and MUC1.

[0657] In some embodiments, the antibody-drug conjugate can bind to cells expressing both EGFR and MUC1 (e.g., human tumor cells). For example, the antibody-drug conjugate can bind to cells expressing EGFR on the cell surface and MUC1 on the cell surface. For example, the antibody-drug conjugate may have specificity for cells and / or tissues expressing both EGFR and MUC1 (e.g., tumor cells and / or tissues).

[0658] 5.6. Target Tumors of Antibody-Drug Conjugates

[0659] An antibody-drug conjugate or a composition containing the same (e.g., a pharmaceutical composition) according to some embodiments of the present disclosure may be used for the treatment of cancer or tumors. In some embodiments, the antibody-drug conjugate or a composition containing the same (e.g., a pharmaceutical composition) may have a therapeutic effect on cancer or tumors. In some embodiments, the antibody-drug conjugate or a composition containing the same (e.g., a pharmaceutical composition) may have anticancer or antitumor activity against cancer or tumors. In some embodiments, the antibody-drug conjugate or a composition containing the same (e.g., a pharmaceutical composition) may have the ability to suppress cancer or tumors. In some embodiments, the cancer or tumor may be a solid tumor or a blood cancer. In the present disclosure, a tumor that can be targeted or treated using an antibody-drug conjugate or a composition containing the same (e.g., a pharmaceutical composition) according to some embodiments of the present disclosure may be referred to as a target tumor and is not otherwise limited.

[0660] In some embodiments, the tumor (or target tumor) is colon cancer, rectal cancer, renal cancer, renal cell carcinoma, urothelial cancer, breast cancer (e.g., breast cancer includes triple-negative breast cancer or epithelial breast cancer), liver cancer, lung cancer (e.g., lung cancer includes small cell lung cancer (SCLC), non-smal lung cancer (NSCLC), squamous non-smal lung cancer (sq-NSCLC), or non-squamous non-smal lung cancer (NSQ-NSCLC), gastric cancer, colorectal cancer (e.g., colorectal cancer includes colorectal adenocarcinoma), prostate cancer, cancer of the small intestine, cancer of the esophagus, hepatocellular carcinoma, melanoma, orthotopic cancer, bone cancer, ampullary cancer, pancreatic cancer (e.g., pancreatic cancer includes pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma,or including pancreatic ductal adenocarcinoma), skin cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma or head and neck adenocarcinoma), neuroendocrine tumors (carcinoid), cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, epithelial ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, Endometrial cancer, Cervical cancer, Vaginal cancer (carcinoma of the vagina), Vulvar cancer (carcinoma of the vulva), Hodgkin's Disease, Non-Hodgkin's lymphoma, Lymphoma, Cancer of the endocrine system, Thyroid cancer, Cancer of the parathyroid gland, Adrenal cancer, Adrenocortical carcinoma, Mesothelioma, CholangiocarcinomaEsophageal cancer (e.g., esophageal cancer includes esophageal squamous-cell carcinoma (ESCC) or esophageal adenocarcinoma), salivary gland cancer, sarcoma, soft tissue sarcoma, cancer of the urethra, urothelial carcinoma, cancer of the penis, pediatric solid tumors, bladder cancer (e.g., bladder cancer includes bladder squamous-cell carcinoma (bladder SCC)), cancer of the kidney or ureter, renal pelvis cancer (carcinoma of the renal pelvis), neurological cancer, neoplasm of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, G1 ioma, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, combinations of the above cancers, and one or more selected from metastatic lesions of the above cancers, but not limited thereto.

[0661] In a specific embodiment, the tumor (or target tumor) is head and neck cancer, head and neck squamous-cell carcinoma (HNSCC), head and neck adenocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), esophageal adenocarcinoma (EAC), liver cancer, renal cancer, skin cancer, G1 ioma, neuroendocrine tumor (Carcinoid), ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma (PASC), pancreatic ductal adenocarcinoma (PDAC), lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous non-smal cell lung cancer (sq-NSCLC), non-squamous non-smal cell lung cancer (nsq-NSCLC), cervical cancer, endometrial cancer, cervical cancer, thyroid cancer, prostate cancer, bladder cancer, urothelial carcinomaIt may be one or more selected from bladder squamous cell carcinoma (bladder SCC), colorectal cancer, colorectal adenocarcinoma, stomach cancer, breast cancer, ovarian cancer, testicular cancer, combinations of the above cancers, and metastatic lesions of the above cancers, but is not limited thereto.

[0662] In some embodiments, the tumor (or target tumor) may be a primary tumor. In some embodiments, the tumor may be a secondary tumor. In some embodiments, the tumor may be a recurrent tumor. In some embodiments, the tumor may be a metastatic tumor or metastatic cancer. For example, the target tumor may be a metastatic tumor generated from or induced from any one of the aforementioned tumors (or cancers).

[0663] In some embodiments, the tumor (or target tumor) may be an EGFR-expressing tumor. In some embodiments, the tumor (or target tumor) may be an EGFR-overexpressing tumor. In some embodiments, the tumor (or target tumor) may be an EGFR-positive tumor. In some embodiments, the tumor (or target tumor) may be an EGFR-associated tumor. In some embodiments, the EGFR-expressing tumor may be an EGFR-overexpressing tumor. In some embodiments, the EGFR-expressing tumor may be an EGFR-positive tumor. In some embodiments, the expression level of EGFR in a tumor expressing EGFR may be a low expression level, an intermediate expression level, and a high expression level, but is not limited thereto. In some embodiments, a tumor expressing EGFR may have a high EGFR expression level compared to normal cells or tissues.

[0664] In some embodiments, the tumor (or target tumor) may be a tumor resistant to an EGFR drug. In some embodiments, the tumor may be a tumor resistant to a tyrosine kinase inhibitor. In some embodiments, the tumor may be a tumor resistant to osimert inib and / or cetuximab, pan itumumab. Such resistance may be mutations that interfere with the binding of the inhibitor, such as T790M and C797S; mutations that induce persistent kinase activation, such as exon20 insert ion; or genetic mutations that amplify EGFR expression to counteract the effect of the inhibitor. In some embodiments, the tumor (or target tumor) may be a tumor with mutations in EGFR downstream signaling pathways, such as KRAS and BRAF. An antibody-drug conjugate according to some embodiments of the present disclosure binds to the extracellular domain of EGFR that is independent of the aforementioned mutation, and is more effective as EGFR expression increases, and since it operates independently of EGFR activity, it can be active even against tumors that have such resistance.

[0665] In some embodiments, the tumor (or target tumor) may be a tumor expressing MUC1 (MUCl-expressing tumor). In some embodiments, the tumor (or target tumor) may be a tumor overexpressing MUC1 (MUC1-overexpressing tumor). In some embodiments, the tumor (or target tumor) may be a MUC1-positive tumor (MUCl-positive tumor). In some embodiments, the tumor (or target tumor) may be a tumor associated with MUC1. In some embodiments, the tumor expressing MUC1 may be a tumor overexpressing MUC1. In some embodiments, the tumor expressing MUC1 may be a MUC1-positive tumor. In some embodiments, the expression level of MUC1 in the tumor expressing MUC1 may be a low expression level, an intermediate expression level, and a high expression level, but is not limited thereto. In some embodiments, tumors expressing MUC1 may have high levels of MUC1 expression compared to normal cells or tissues.

[0666] In some embodiments, the tumor (or target tumor) may be a tumor expressing either EGFR or MUC1. In some embodiments, the target tumor may be a tumor associated with either one or more of the expression of EGFR and the expression of MUC1. Some

[0667] In some embodiments, the target tumor may be a tumor that expresses both EGFR and MUC1. In some embodiments, the tumor may be a tumor that expresses EGFR but does not express or barely expresses MUC1. In some embodiments, the tumor may be a tumor that expresses MUC1 but does not express or barely expresses EGFR. In some embodiments, the tumor may be a tumor that expresses EGFR at a low expression level, an intermediate expression level, and a high expression level, and expresses MUC1 at a low expression level, an intermediate expression level, and a high expression level. In some embodiments, the tumor may be an EGFR-positive and / or MUC1-positive tumor.

[0668] In some embodiments, the tumor (or target tumor) may be, for example, a tumor expressing both EGFR and MUC1. In some embodiments, the target tumor may be an EGFR-positive and MUC1-positive tumor. In some embodiments, the target tumor may be a tumor associated with both EGFR and MUC1. In some embodiments, the target tumor may be a tumor associated with both EGFR expression and MUC1 expression. In some embodiments, the target tumor may be a tumor co-expressing EGFR and MUC1. In some embodiments, the tumor expressing both EGFR and MUC1 may be an EGFR-positive and MUC1-positive tumor.

[0669] In some embodiments, the tumor may be a mutational tumor. The mutational tumor may have one or more of KRAS mutations, BRAF mutations, and EGFR mutations.

[0670] For example, the mutational tumor may be a KRAS mutational tumor having a KRAS mutation. The KRAS mutation may include one or more of the G12C mutation, G12D mutation, G12V mutation, G13D mutation, and Q61H.

[0671] For example, the mutational tumor may be a BRAF mutational tumor having a BRAF mutation. The BRAF mutation may include, for example, one or more of the V600D / E / K / R / G mutation, G464E / V / R mutation, G469A / V / S mutation, and G469E mutation.

[0672] For example, a mutational tumor may be an EGFR mutational tumor having an EGFR mutation. EGFR mutations include, for example, exon 19 deletions, indels in exon 19, exon 20 insertions (e.g., EGFR exon 20 insertions of 1–7 amino acids), L718Q / V mutations, G719C / S / A (G719C, G719S, or G719A) mutations, S768I mutations, L769X mutations (substitution of L at position 769 with various amino acids; e.g., L769Q, etc.), T790M mutations, L792X mutations (substitution of L at position 792 with various amino acids; It may include one or more of the cleavage mutations such as L792F, L792H, L792Y, etc.), G796S mutation, C797X mutation (substitution of the 797 position with various amino acids; e.g., C797S, C797G, etc.), G834L mutation, L858R mutation, and EGFRvIII.

[0673] In some embodiments, the tumor may be a tumor resistant to EGFR drugs. In some embodiments, the tumor may be a tumor resistant to tyrosine kinase inhibitors. In some embodiments, the tumor may be a tumor resistant to one or more of osmiter inib, cetuximab, and panitumumab. In some embodiments, the tumor may be a tumor resistant to conventional chemotherapy agents. The chemotherapy agents may be selected from, for example, 5-FU, fol inate, oxal iplatin, Karenitecine, Gemcitabine, palbociclib, erlotinib, and combinations thereof, but are not limited thereto.

[0674] In some embodiments, the tumor (or target tumor) may be a tumor expressing EGFR and / or MUC1 (or an EGFR-positive and / or MUC1-positive tumor). For example, the tumor (or target tumor) is EGFR-positive and / or MUC1-positive colon cancer, rectal cancer, renal cancer, renal cell carcinoma, urothelial cancer, breast cancer, triple-negative breast cancer, epithelial breast cancer, liver cancer, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous non-smal cell lung cancer (sq-NSCLC), or non-squamous non-smal cell lung cancer (nsq-NSCLC), Gastric cancer, Colorectal cancer, Colorectal adenocarcinoma, Prostate cancer, Cancer of the small intestine, Esophageal cancer, Hepatic cystic carcinoma, Melanoma, Orthopedic cancer, Bone cancer, Ampullary cancer, Pancreatic cancer, Pancreatic adenocarcinomaPancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, skin cancer, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), head and neck adenocarcinoma, neuroendocrine tumor (Carcinoid), cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, epithelial ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, fallopian tube cancer (carcinoma of the fallopian tube) lopian tubes), Endometrial cancer, Cervical cancer, Vaginal cancer (carcinoma of the vagina), Vulvar cancer (carcinoma of the vulva), Hodgkin's Disease, Non-Hodgkin's lymphoma, Lymphoma, Cancer of the endocrine system, Thyroid cancer, Cancer of the parathyroid gland, Adrenal cancer, Adrenocortical carcinoma, Mesothelioma, Cholangiocarcinoma,Esophageal cancer, esophageal squamous-cell carcinoma (ESCC), esophageal adenocarcinoma, salivary gland cancer, sarcoma, soft tissue sarcoma, cancer of the urethra, urothelial carcinoma, cancer of the penis, pediatric solid tumors, bladder cancer, bladder squamous-cell carcinoma (bladder SCC), cancer of the kidney or ureter, carcinoma of the renal pelvis, nervous system cancer It may be one or more selected from neurological cancer, neoplasm of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, glioma, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, and T-cell lymphoma.

[0675] Tumors expressing EGFR, tumors expressing MIJC1, and tumors (or cancers) co-expressing EGFR and MIJC1 are well known in the art, and known literature or databases may be referenced or utilized. For example, various companies conducting research on tumors provide information on tumors and related antigens, such as the database provided by Crown Bioscience International (San Diego, California, United States), the database provided by Merck Group (Darmstadt, Germany), and the database provided by Champions Oncology, Inc. (Hackensack, New Jersey, United States). 0 ] may be referenced, but is not limited thereto.

[0676] Whether a tumor expresses EGFR or MIJC1 can be identified through methods known in the art. For example, whether a tumor expresses EGFR (or MIJC1) can be identified by examining the relevant tumor tissue or tumor cells through immunohistochemistry, FISH (Fluorescence in situ hybridization), PCR (polymerase chain reaction), NGS (Next-generation sequencing), etc. For example, whether a tumor expresses EGFR (or MUC1) can be identified through biopsy (or tissue biopsy).

[0677] In some embodiments, whether a tumor expresses EGFR (or MUC1) can be confirmed by performing immunohistochemistry according to a known protocol on a tumor (or suspected tumor tissue) obtained by collecting tumor tissue (or suspected tumor tissue) from a patient requiring examination (e.g., tissue isolated from a patient) or on pre-prepared tumor tissue (or suspected tumor tissue). IHC testing methods are well known in the art. For example, an EGFR (or MUC1) IHC test involves collecting tumor tissue (or suspected tumor tissue) from a patient; preparing a specimen from the tumor tissue; treating the specimen with an anti-EGFR (or anti-MUC1) antibody (primary antibody); This can be performed by a method comprising treating a sample treated with the primary antibody with a secondary antibody labeled with an enzyme or a type mineral capable of recognizing the primary antibody (e.g., capable of binding to the Fc region of the primary antibody). The degree of expression of EGFR (or MUC1) in the tumor may be expressed as an IHC grade or H-score. For example, without limitation, if faint, partial staining is observed in more than 10% of tumor cells, it may be evaluated as a 1+ IHC grade; if week to moderate staining is observed in more than 10% of tumor cells, it may be evaluated as a 2+ IHC grade; and if strong, complete staining is observed in more than 10% of tumor cells, it may be evaluated as a 3+ IHC grade. For example, a tumor that is evaluated as having an immunostaining score or IHC grade of 1+ or higher (e.g., 1+, 2+, or 3+) based on IHC test results may be an EGFR (or MUC1) positive tumor.For example, a tumor evaluated by IHC test results or an H-score of 100 or higher may be an EGFR (or MUC1) positive tumor, but is not limited thereto.

[0678] 5.7. Composition containing an antibody-drug conjugate

[0679] Some embodiments of the present disclosure provide a composition comprising an antibody-drug conjugate (or a salt thereof). In some embodiments, the composition comprising the antibody-drug conjugate may be a pharmaceutical composition. Some embodiments of the present disclosure provide a kit comprising an antibody-drug conjugate (or a salt thereof).

[0680] In some embodiments, a composition (or kit) containing an antibody-drug conjugate may contain one or more antibody-drug conjugates.

[0681] In some embodiments, a composition containing an antibody-drug conjugate may contain, in addition to the antibody-drug conjugate, an antibody that is not an antibody-drug conjugate. In some embodiments, a composition containing an antibody-drug conjugate may contain only antibody-drug conjugates. In some embodiments, a composition containing an antibody-drug conjugate may contain one or more of antibody-drug conjugates of DAR2 and antibody-drug conjugates of DAR4. In some embodiments, a composition containing an antibody-drug conjugate may contain only antibody-drug conjugates of DAR4.

[0682] In some embodiments, the composition containing the antibody-drug conjugate may contain purified antibody-drug conjugate. In some embodiments, the composition containing the antibody-drug conjugate may contain purified antibody-drug conjugate of DAR4.

[0683] In some embodiments, the composition may comprise an antibody-drug conjugate at a concentration of 0.0lnM to 100M, but is not limited thereto. In some embodiments, the composition may comprise an antibody-drug conjugate at a concentration of 0.1mg / mL to 200g / mL, but is not limited thereto.

[0684] In some embodiments, the average DAR of antibody-drug conjugates present in a composition containing antibody-drug conjugates may be 0.5 to 8. In some embodiments, the average DAR of antibody-drug conjugates present in a composition containing antibody-drug conjugates may be 0.5 to 4.5. Specific

[0685] In an embodiment, the average DAR of antibody-drug conjugates present in a composition containing antibody-drug conjugates may be 2.0 to 4.0, or 3.0 to 4.0, or 3.5 to 4.0. In some embodiments, the average DAR may be calculated based on antibodies and antibody-drug conjugates other than antibody-drug conjugates present in the composition. In some embodiments, the average DAR may be calculated based only on antibody-drug conjugates present in the composition. In some embodiments, the average DAR of a composition containing an antibody-drug conjugate may be about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4. In some embodiments, the average DAR of a composition containing an antibody-drug conjugate may be 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4. In a specific embodiment, the average DAR of a composition containing an antibody-drug conjugate may be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4. In a specific embodiment, the average DAR of a composition containing an antibody-drug conjugate may be 4.

[0686] In some embodiments, a composition containing an antibody-drug conjugate may contain a therapeutically effective amount of antibody-drug conjugate. In some embodiments, a composition containing an antibody-drug conjugate may contain antibody-drug conjugate as an active ingredient.

[0687] In some embodiments, a composition containing an antibody-drug conjugate may be purified to substantially contain the antibody-drug conjugate. For example, a composition containing an antibody-drug conjugate may be purified to substantially contain the antibody-drug conjugate of DAR4.

[0688] In some embodiments, a composition comprising an antibody-drug conjugate may further comprise an antibody-drug conjugate of a different type from the antibody-drug conjugate of the present disclosure, but is not limited thereto.

[0689] In some embodiments, the composition comprising the antibody-drug conjugate may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable carrier may comprise, for example, excipients, diluents and / or adjuvants. The carrier may be one or more selected from, for example, lactose, dextrose, sucrose, sorbitol, polysorbate (e.g., polysorbate 80), mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, arginine hydrochloride, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, physiological saline, PBS, buffer solutions such as tris(hydroxymethyl)aminomethane buffer, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The above carrier may include a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof. For example, a composition comprising an antibody-drug conjugate may, in addition to the antibody-drug conjugate, include tris(hydroxymethyl)aminomethane buffer, arginine

[0690] It may further include one or more of hydrochloride (arginine hydrochloride), sucrose, and polysorbate (e.g., polysorbate 80).

[0691] In some embodiments, the pH of the composition containing the antibody-drug conjugate may be 4.5 to 9. In some embodiments, the pH of the composition containing the antibody-drug conjugate may be 7 to 8. In some embodiments, the pH of the composition containing the antibody-drug conjugate may be about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, but is not limited thereto.

[0692] In some embodiments, a composition comprising an antibody-drug conjugate may comprise 10 mg / mL of antibody-drug conjugate according to some embodiments of the present disclosure, 20 mM tris(hydroxymethyl)aminomethane buffer (e.g., 2.42 mg / mL), 50 mM arginine hydrochloride (e.g., 10.53 mg / mL), 5% (w / v) sucrose (e.g., 50 mg / mL), and 0.02% (w / v) polysorbate 80 (e.g., 0.2 mg / mL), wherein the pH of the composition may be 7.5.

[0693] In some embodiments, a composition (or kit) comprising an antibody-drug conjugate may be used to treat a disease (e.g., tumor or cancer) of a subject (or patient, i.e., a human patient). In some embodiments, a composition comprising an antibody-drug conjugate may be used for the manufacture of a pharmaceutical for the treatment of the subject's disease. In some embodiments, a composition comprising an antibody-drug conjugate may be a pharmaceutical composition (e.g., a pharmaceutical composition for treating tumors or cancer).

[0694] 5.8. Functions / Effects of Antibody-Drug Conjugates

[0695] 5.8.1. Overview of Functions / Effects of Antibody-Drug Conjugates

[0696]

[0697] In some embodiments, the antibody-drug conjugate of the present disclosure may have the ability to bind to EGFR and / or MUC1. In some embodiments, the antibody-drug conjugate may bind to EGFR (e.g., human EGFR or a variant thereof). In some embodiments, the antibody-drug conjugate may bind to MUC1 (e.g., human MUC1 or a variant thereof).

[0698] In some embodiments, the antibody-drug conjugate may bind to EGFR expressed on the surface (or cell membrane) of a cell (e.g., human cell). Some

[0699] In an embodiment, the antibody-drug conjugate can bind to MUC1 expressed on the surface (or cell membrane) of the cell.

[0700] In some embodiments, the antibody-drug conjugate may bind to EGFR-positive cells (e.g., EGFR-positive human cells or EGFR-positive human cancer cells). In some embodiments, the antibody-drug conjugate may bind to MUC1-positive cells (e.g., MUC1-positive human cells or MUC1-positive human cancer cells).

[0701] In some embodiments, the antibody-drug conjugate can bind to cells expressing both EGFR and MUC1. In some embodiments, the antibody-drug conjugate is designed to target both EGFR and MUC1 together, and because EGFR has a low expression level in normal tissues and a high expression level in tumor cells, and MUC1 has a low expression level in normal tissues and a high expression level in tumor cells, it can bind more specifically to tumor cells, and accordingly, the toxicity of the antibody-drug conjugate to normal tissues may be low.

[0702] In some embodiments, targeting of MUC1 can play a role in enhancing tumor targeting and specificity in cancers expressing EGFR that co-express MUC1, and accordingly, the antibody-drug conjugate of the present disclosure comprising a dual-specific antibody targeting EGFR and MUC1 can have a potent anticancer effect in cancers expressing EGFR. In some embodiments, targeting of EGFR can play a role in enhancing tumor targeting and specificity in cancers expressing MUC1 that co-express EGFR, and accordingly, the antibody-drug conjugate of the present disclosure comprising a dual-specific antibody targeting EGFR and MUC1 can have a potent anticancer effect in cancers expressing MUC1.

[0703] In some embodiments, the antibody-drug conjugate may be internalized into the cell after binding to a target antigen expressed in the cell. In some embodiments, the internalized antibody-drug conjugate may be cleaved by a protease (e.g., cathepsin B).

[0704] In some embodiments, the antibody-drug conjugate may have an antitumor effect (or anticancer effect). In some embodiments, the antibody-drug conjugate may kill tumor cells. In some embodiments, the antibody-drug conjugate may attack tumor cells. In some embodiments, the antibody-drug conjugate may have an antitumor effect (anticancer effect) in vivo. In some embodiments, the antibody-drug conjugate may have an excellent in vivo anticancer effect. The antitumor effect (or anticancer effect) may mean an effect that inhibits the occurrence, growth, and / or metastasis of tumor cells (or cancer cells) or tumor tissue (or cancer tissue).

[0705] In some embodiments, the antibody-drug conjugate may have a bystander effect. In some embodiments, the antibody-drug conjugate may have an immunogenic cell death (ICD) effect. Cancer or tumors may be inhibited by any one or more of [apoptosis, bystander effect, immunogenic cell death, and apoptosis induction by a drug released into the cell], but are not limited thereto.

[0706] In some embodiments, the antibody-drug conjugate may have low toxicity. In some embodiments, the antibody-drug conjugate may have an excellent toxicity profile. Toxicity may refer, for example, to adverse effects or unintended effects that may occur upon treatment and / or administration of the antibody-drug conjugate. In some embodiments, the antibody-drug conjugate may have low endotoxicity. In some embodiments, the antibody-drug conjugate may have excellent safety. In some embodiments, the antibody-drug conjugate may have excellent stability.

[0707] In some embodiments, the antibody-drug conjugate may have a high maximum dose. In some embodiments, the antibody-drug conjugate may have a high HNSTD (highest non-severe ly toxic dose).

[0708] In some embodiments, the antibody-drug conjugate may have a broad (or excellent) therapeutic index (TI). In some embodiments, the antibody-drug conjugate may have a broad (or excellent) therapeutic window (TW).

[0709] In some embodiments, the antibody-drug conjugate may have a good toxicity profile while simultaneously having high tumor suppression ability in vivo. In some embodiments, the antibody-drug conjugate may be an antibody-drug conjugate having a good balance of efficacy / toxicity.

[0710] 5.8.2. Targeting

[0711]

[0712] The antibody-drug conjugate of the present disclosure may have the ability to bind to EGFR and / or MUC1. In some embodiments, the antibody-drug conjugate may bind to EGFR (e.g., human EGFR or a variant thereof). In some embodiments, the antibody-drug conjugate may bind to MUC1 (e.g., human MUC1 or a variant thereof).

[0713] In some embodiments, the antibody-drug conjugate can bind to EGFR expressed on the surface (or cell membrane) of a cell (e.g., human cell).

[0714] In some embodiments, the antibody-drug conjugate can bind to MUC1 expressed on the surface (or cell membrane) of the cell.

[0715] In some embodiments, the antibody-drug conjugate may bind to cells expressing EGFR. In some embodiments, the cells expressing EGFR may be cells that overexpress EGFR. In some embodiments, the cells expressing EGFR may be EGFR-positive cells.

[0716] In some embodiments, the antibody-drug conjugate may bind to cells expressing MUC1. In some embodiments, the cells expressing MUC1 may be cells that overexpress MUC1. In some embodiments, the cells expressing MUC1 may be MUC1-positive cells.

[0717] In some embodiments, the antibody-drug conjugate may bind to cells co-expressing EGFR and MUC1. In some embodiments, the cells co-expressing EGFR and MUC1 may be cells that overexpress EGFR and express MUC1. In some embodiments, the cells co-expressing EGFR and MUC1 may be cells that express EGFR and overexpress MUC1. In some embodiments, the cells co-expressing EGFR and MUC1 may be cells that overexpress EGFR and overexpress MUC1. In some embodiments, the cells co-expressing EGFR and MUC1 may be EGFR- and MUC1-positive cells.

[0718] In some embodiments, the cell may be a human cell. In some embodiments, the cell may be a cancer cell. In some embodiments, the cell may be a human cancer cell.

[0719] In some embodiments, the antibody-drug conjugate may bind to a tumor (e.g., cancer) or tumor tissue (e.g., cancer tissue) expressing EGFR. In some embodiments, the antibody-drug conjugate may target a tumor (e.g., cancer) or tumor tissue (e.g., cancer tissue) expressing EGFR. In some embodiments, the tumor expressing EGFR may be a tumor that overexpresses EGFR. In some embodiments, the tumor expressing EGFR may be an EGFR-positive tumor. In some embodiments, the antibody-drug conjugate may bind to a tumor (e.g., cancer) or tumor tissue (e.g., cancer tissue) expressing MUC1. In some embodiments, the antibody-drug conjugate may target a tumor (e.g., cancer) or tumor tissue (e.g., cancer tissue) expressing MUC1. In some embodiments, the tumor expressing MUC1 may be a tumor overexpressing MUC1. In some embodiments, the tumor expressing MUC1 may be a MUC1-positive tumor.

[0720] In some embodiments, the antibody-drug conjugate is designed to target EGFR and MUC1 together, and since EGFR has a low expression level in normal tissues and a high expression level in tumor cells and MUC1 has a low expression level in normal tissues and a high expression level in tumor cells, it can bind more specifically to tumor cells, and accordingly, the toxicity of the antibody-drug conjugate to normal tissues may be low.

[0721]

[0722] In some embodiments, the antibody-drug conjugate may be internalized into the cell after binding to a target antigen expressed in the cell. In some embodiments, the antibody-drug conjugate may have excellent cell internalization ability. Some

[0723] In an embodiment, the internalized antibody-drug conjugate may be cleaved by a protease (e.g., cathepsin B). As described in previous paragraphs, the antibody-drug conjugate contains a cleavable portion that can be cleaved by a protease. Through this cleavage process, the drug of the antibody-drug conjugate (e.g., exatecan moiety) may be released from the antibody-drug conjugate. Exatecan or its derivatives are known as DNAtosomersase I inhibitor, and DNAtosomersase I inhibitor is known to be capable of inducing damage to the DNA of cells and cell death.

[0724] 5.8.4. Additional Cell Death Mechanisms

[0725]

[0726] In some embodiments, the antibody-drug conjugate of the present disclosure may have various tumor cell death mechanisms or tumor suppression mechanisms. These various cell death mechanisms (e.g., tumor cells) or tumor suppression mechanisms may be additional tumor cell death mechanisms or tumor suppression mechanisms in addition to cell death caused by the action of the drug after internalization of small cells.

[0727] In some embodiments, the antibody-drug conjugate may induce exposure of calreticulin to the cell membrane of a cell (e.g., a target cell). Exposure of calreticulin to the cell membrane of a target cell, such as a tumor cell, may trigger an immune response, which may help suppress cancer or tumors. For a method to confirm the effect of the antibody-drug conjugate inducing calreticulin cell membrane exposure, reference may be made to “Example 9. Induction of calreticulin cell membrane exposure in MDA-MB-468 cells by a bispecific antibody-drug conjugate” of the present disclosure.

[0728] In some embodiments, the antibody-drug conjugate may induce HMGB1 release in cells (e.g., target cells). Induction of HMGB1 release in target cells may trigger an immune response, which may help suppress cancer or tumors. For a method to confirm the HMGB1 release-inducing effect of the antibody-drug conjugate, reference may be made to “Example 10. Induction of HMGB1 release in MDA-MB-468 cells by a bispecific antibody-drug conjugate” of the present disclosure. In some embodiments, the antibody-drug conjugate may induce cell cycle arrest in cells (e.g., target cells). In some embodiments, the antibody-drug conjugate may have the effect of arresting the cell cycle progression of cells at the S or G2 phase. Cell cycle arrest can effectively inhibit the proliferation of tumor cells, which can help suppress cancer or tumors. For a method to confirm the cell cycle arrest-inducing effect of an antibody-drug conjugate, reference may be made to “Example 11. Induction of cell cycle arrest in MDA-MB-468 cells by a bispecific antibody-drug conjugate” of the present disclosure.

[0729] In some embodiments, the antibody-drug conjugate may induce caspase-3 / 7 activity in cells (e.g., target cells). Activation of Caspase-3 / 7 in target cells, such as tumor cells, may induce apoptosis, which may help suppress cancer or tumors. For a method to confirm the caspase-3 / 7 activity-inducing effect of the antibody-drug conjugate, reference may be made to “Example 12. Induction of caspase-3 / 7 activity in MDA-MB-468 cells by a bispecific antibody-drug conjugate” of the present disclosure.

[0730] In some embodiments, the antibody-drug conjugate may induce apoptosis in cells (e.g., target cells). The effect of inducing apoptosis in target cells, such as tumor cells, may help in the suppression of cancer or tumors. For a method to confirm the apoptosis-inducing effect of the antibody-drug conjugate, reference may be made to “Example 13. Induction of apoptosis in MDA-MB-468 cells by a bispecific antibody-drug conjugate” of the present disclosure.

[0731] In some embodiments, the antibody-drug conjugate may have a bystander killing effect. Due to the bystander killing effect, not only cells expressing the target antigen (e.g., EGFR, MUC1) to which the antibody-drug conjugate binds, but also non-target cells adjacent to those cells may be killed, and this bystander killing effect may be helpful in suppressing tumors in which the target is expressed heterogeneously. For a method to confirm the bystander killing effect of the antibody-drug conjugate, reference may be made to the embodiment of the present disclosure "Example 14. Evaluation of bystander killing effect by bispecific antibody-drug conjugate."

[0732] 5.8.5. Antitumor effect

[0733] (1) Overview of Antitumor Effects

[0734] In some embodiments, the antibody-drug conjugate or a composition containing the same (e.g., a pharmaceutical composition) may have an antitumor effect. In some embodiments, the antitumor effect may be an anticancer effect. In some embodiments, the antibody-drug conjugate may kill tumor cells (e.g., cancer cells). In some embodiments, the antibody-drug conjugate may attack tumor cells (e.g., cancer cells).

[0735] An antitumor effect (or anticancer effect) may mean an effect that inhibits the development, growth, and / or metastasis of tumor cells (or cancer cells) or tumor tissue (or cancer tissue).

[0736] In some embodiments, the antibody-drug conjugate may have an antitumor effect (or anticancer effect) against a tumor (or cancer). The tumor may be, for example, a variant tumor. The variant tumor may be, for example, a variant tumor having an EGFR mutation. The variant tumor may be, for example, a KRAS variant tumor having a KRAS mutation. The variant tumor may be, for example, a BRAF variant tumor having a BRAF mutation. The variant tumor may be, for example, a tumor having one or more of an EGFR mutation, a KRAS mutation, and a BRAF mutation. The EGFR mutation may be, for example, one or more of L858R, T790M, and C797S. The KRAS variant may be, for example, one or more of the KRAS G12D variant, G12C variant, G12V variant, G13D variant, and Q61H variant. The BRAF variant may be, for example, one or more of the G469E variant and V600E variant.

[0737] (2) Antitumor effect

[0738] In some embodiments, the antibody-drug conjugate or a composition containing the same may have an antitumor effect (anticancer effect). In some embodiments, the antitumor effect may be measured in vitro or in vivo. In some embodiments, the antibody-drug conjugate or a composition containing the same may have an excellent in vivo antitumor effect. In some embodiments, the antibody-drug conjugate may have an excellent in vivo antitumor effect against various tumors. In some embodiments, the antibody-drug conjugate may show an effect of complete remission or partial remission against various tumors. In some embodiments, the antibody-drug conjugate may show a tumor regression effect against various tumors.

[0739] In some embodiments, the antibody-drug conjugate of the present disclosure may exhibit a superior antitumor effect compared to a comparative drug (e.g., an antibody other than the antibody-drug conjugate of the present disclosure or another antibody-drug conjugate). In some embodiments, the antibody-drug conjugate of the present disclosure may exhibit a superior antitumor effect compared to a comparative drug in cancer cell lines (e.g., human-derived cancer cell lines) or in a human-derived cancer cell line xenograft mouse model. In some embodiments, the comparative drug may be one or more of AB0796, AB0797, AB0798, AB0800, and AB0900 (DS-3939a) and is not otherwise limited. Information regarding the comparative drug is described in detail in the examples of the present disclosure. In some embodiments, human-derived cancer cell lines may be one or more of MDA-MB-468, CAL 27, HSC-4, 647-V, NUGC-4, CFPAC-1, Capan-2, HPAF-II, HCC827, and NCI-H1975, and are not otherwise limited. Information regarding human-derived cancer cell lines is described in detail in the examples of the present disclosure.

[0740] TGI (tumor growth inhibition) is a key metric used to measure the antitumor effect of a drug (e.g., antibody-drug conjugate) in vivo. A TGI of 50% or higher indicates an excellent antitumor effect. In some embodiments, the TGI (%) measured in in vivo experiments of the antibody-drug conjugate of the present disclosure may be 50% or higher. In some embodiments, the TGI (%) measured in in vivo experiments of the antibody-drug conjugate of the present disclosure may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% or greater, or within any two of the above-mentioned percentage ranges.

[0741] TGI can be calculated using the following formula:

[0742] TGI = ( (mean (Ct) -mean (C0 ) ) - (mean (Tt) -mean (TO ) ) ) / (mean(Ct)- mean(CO) ) x 100%,

[0743] At this time,

[0744] mean(Ct) is the tumor size (or average tumor size) of the control group (e.g., vehicle administration group) at the time of measurement, and mean(CO) is the tumor size of the control group at the time of administration (e.g., the time of the first administration of the antibody-drug conjugate), and

[0745] mean(Tt) is the tumor size of the experimental group (e.g., antibody-drug conjugate administration group) at the time of measurement, and

[0746] mean(TO) is the tumor size of the experimental group at the time of administration (e.g., the time of the first administration of the antibody-drug conjugate).

[0747] In some embodiments, the antibody-drug conjugate may exhibit excellent antitumor effects in a mouse xenograft model (e.g., CDX or PDX). In some embodiments, the TGI of the antibody-drug conjugate may be measured using a mouse xenograft model. In some embodiments, the mouse xenograft model may be a cell line-derived xenograft (CDX) model or a patient-derived xenograft (PDX) model. In some embodiments, the antibody-drug conjugate may have antitumor effects against various carcinomas. In some embodiments, the TGI of the antibody-drug conjugate may be measured in various carcinomas expressing EGFR and / or MUC1.

[0748] In some embodiments, the TGI of the antibody-drug conjugate may be the TGI measured in any one of the following cancer types: breast cancer, pancreatic cancer, bladder cancer, colorectal cancer, esophageal cancer, head and neck cancer, and lung cancer. In some embodiments, the subtype of bladder cancer may be urothelial carcinoma or squamous cell carcinoma. In some embodiments, the subtype of colorectal cancer may be adenocarcinoma. In some embodiments, the subtype of esophageal cancer may be esophageal squamous cell carcinoma or esophageal adenocarcinoma. In some embodiments, the subtype of head and neck cancer may be head and neck squamous cell carcinoma. In some embodiments, the subtype of lung cancer may be squamous non-smal 1-cell 1-lung cancer (sq-NSCLC). In some embodiments, the subtype of pancreatic cancer may be adenocarcinoma or adenosquamous carcinoma, ductal adenocarcinoma.

[0749] In some embodiments, the tumor may be a mutational tumor. The mutational tumor may have one or more of KRAS mutations, BRAF mutations, and EGFR mutations. In some embodiments, the tumor may be a KRAS mutational tumor (or cancer). In some embodiments, the KRAS mutation may be one or more of G12D mutation, G12C mutation, G12V mutation, G13D mutation, and Q61H. In some embodiments, the tumor may be a BRAF mutational tumor (or cancer). In some embodiments, the BRAF mutation may be one or more of G469E mutation and V600E mutation. In some embodiments, the tumor may be an EGFR mutational tumor (or cancer). The EGFR variant may be one or more of L858R, T790M, and C797S. In some embodiments, the tumor may have multiple variants. For example, the tumor may have BRAF G469E and KRAS G12D mutations. For example, the tumor may have L858R and T790M mutations of EGFR or T790M and C797S mutations of EGFR.

[0750] Methods for evaluating in vivo antitumor effects in mouse xenograft models are well known in the art. For methods to evaluate in vivo antitumor effects in mouse xenograft models, reference may be made to "Example 16. Comparative evaluation of the efficacy of AB0799 in a mouse model of human-derived breast cancer cell line xenograft," "Example 17. Evaluation of in vivo anticancer efficacy of monovalent, bivalent antibody ADCs and dual antibody ADCs in a mouse model of human-derived pancreatic cancer cell line CFPAC-1 xenograft," "Example 18. Evaluation of efficacy of dual antibody ADCs using a patient-derived tumor model," and "Example 24. Evaluation of the efficacy of AB0799 in a mouse model of human-derived lung cancer cell xenograft" of the present disclosure. 5.8.6. Excellent toxicity profile - Low toxicity

[0751]

[0752] The antibody-drug conjugate of the present disclosure may have low toxicity. In some embodiments, the antibody-drug conjugate of the present disclosure may exhibit low toxicity upon administration in vivo. For example, the antibody-drug conjugate of the present disclosure may have low toxicity at a level suitable for administration to humans.

[0753] There is a possibility that normal cells or tissues expressing EGFR at normal levels exist in the body, and antibodies or antibody-drug conjugates targeting EGFR may target these normal cells or tissues while primarily targeting cancer cells expressing EGFR, and targeting of normal cells or tissues can cause toxicity in the body. Additionally, there is a possibility that normal cells or tissues expressing MUC1 at normal levels exist in the body, and antibodies or antibody-drug conjugates targeting MUC1 may target these normal cells or tissues while primarily targeting cancer cells expressing MUC1, and targeting of normal cells or tissues can cause toxicity in the body. Such toxicity resulting from antibody-drug conjugates targeting normal cells or tissues can be referred to as on-target toxicity.

[0754] In some embodiments, the antibody-drug conjugate may have low on-target toxicity.

[0755] In some embodiments, the bispecific antibody of the antibody-drug conjugate of the present disclosure may be monovalent to each antigen (EGFR and MUC1, respectively). This characteristic of monovalent to each antigen may positively control the toxicity that occurs when acting on normal cells or tissues expressing EGFR or MUC1. For example, the antibody-drug conjugate of the present disclosure may have lower toxicity (e.g., on-target toxicity) than an antibody-drug conjugate comprising an antibody designed to be divalent to each antigen.

[0756] In some embodiments, the bispecific antibody of the antibody-drug conjugate of the present disclosure may have a reduced EGF signaling inhibitory effect. For example, the bispecific antibody of the antibody-drug conjugate may not have an effect of inhibiting EGF signaling. In some embodiments, the bispecific antibody of the antibody-drug conjugate of the present disclosure may inhibit EGF signaling less than a control antibody targeting another EGFR (e.g., cetuximab). The antibody-drug conjugate of the present disclosure may have no or few side effects (e.g., inflammatory side effects) resulting from the inhibition of EGF signaling. For a method of evaluating the EGF signaling inhibitory effect, refer to "Example 15. Evaluation of EGF signaling inhibitory effect by ADC" of the present disclosure.

[0757] In some embodiments, the antibody-drug conjugate of the present disclosure may have low dermal toxicity. In some embodiments, the antibody-drug conjugate may have low dermal toxicity compared to other comparator drugs. Comparator drugs may be, for example, one or more of cetuximab and MRG-003, and are not otherwise limited. Cetuximab, an antibody that binds to EGFR, has shown grade 3-4 dermal toxicity in about 60% of patients in colorectal cancer clinical trials. In some embodiments, the antibody-drug conjugate may have low toxicity to human epidermal keratinocytes. Human epidermal keratinocytes may be, for example, adult human epidermal keratinocytes or neonatal human epidermal keratinocytes. Human epidermal keratinocytes expressing EGFR are cells frequently used to verify the dermal toxicity of EGFR inhibitors. In some embodiments, the antibody-drug conjugate may have lower toxicity to human epidermal keratinocytes than the control drug. The control drug may be, for example, one or more of cetuxidab, MRG003, and BSA01. In some embodiments, the viability of human epidermal keratinocytes according to the treatment concentration of the antibody-drug conjugate may be 90% (or 95%) or more when the treatment concentration of the antibody-drug conjugate is 10 nM or less, and 80% or more when the treatment concentration is 10 nM or less. In some embodiments, the antibody-drug conjugate may have a level of low toxicity (i.e., significantly low toxicity to human epidermal keratinocytes) such that the IC50 for toxicity to human epidermal keratinocytes cannot be measured within the test concentration range. For example, the test concentration range is IO' 3 IO in nM 3It may be in the nM range. Methods for evaluating toxicity to human epidermal keratinocytes are well known in the art. Methods for evaluating toxicity to human epidermal keratinocytes include "Example 8 of the present disclosure regarding the toxicity of AB0799 to human epidermal keratinocytes in

[0758] vitro evaluation (using Human, Neonatal Foreskin cell model) and Example 22.

[0759] The safety of AB0799 for EGFR expressing primary cell 1 may be referenced.

[0760] 5.8.7. Low toxicity and high dosage

[0761]

[0762] In some embodiments, the antibody-drug conjugate of the present disclosure may have an excellent toxicity profile (i.e., low toxicity). In some embodiments, the antibody-drug conjugate of the present disclosure may have a level of toxicity suitable for administration to humans (e.g., human patients).

[0763] In some embodiments, the highest non-severely toxic dose (HNSTD) of an antibody-drug conjugate in monkeys (e.g., cynomolgus monkeys) is 60 mpk (mg / kg (body weight)), 50 mpk, 40 mpk, or 30 mpk

[0764] It may be greater than or equal to, or within the range of two values ​​selected from the aforementioned values. In some embodiments, the HNSTD in cynomolgus monkeys upon two administrations of the antibody-drug conjugate at 3-week intervals may be 60 mpk, 50 mpk, 40 mpk, or 30 mpk or greater. In a specific embodiment, the HNSTD may be 40 mpk. Meanwhile, a dose of 40 mpk in monkeys corresponds to a dose of 12.97 mg / kg in humans. This dose of approximately 13 mg / kg is significantly higher than the standard clinical dose of 1.8-2.5 mpk for MMAE-based antibody-drug conjugates. For a method to identify HNSTD in monkeys from antibody-drug conjugates and a method to calculate human HNSTD based on HNSTD identified in monkey toxicity tests, "Example 19. Toxicity test in monkeys" of the present disclosure may be referenced.

[0765] Meanwhile, the advantage that antibody-drug conjugates can be administered at high doses can have a positive effect on the tumor penetration ability of antibody-drug conjugates. Administration at high doses can increase the absolute number of antibody-drug conjugates reaching the tumor and penetrating the tumor (e.g., solid tumor), and excellent anti-tumor effects can be expected through this excellent tumor penetration ability.

[0766] 5.8.8. Excellent stability

[0767]

[0768] In some embodiments, the antibody-drug conjugate of the present disclosure may have excellent stability. For example, the antibody-drug conjugate may have excellent plasma stability.

[0769] In some embodiments, when the antibody-drug conjugate is stored in the plasma of a monkey (e.g., cynomolgus monkey) for 3 days, the target cell killing ability based on IC50 may be maintained at 90%, 95%, or 98% or higher (e.g., 100%). For example, IC50(day0) / IC50(day3) may be 90% (i.e., 0.9), 95%, 98% or higher, or 100% (wherein IC50(day0) is the IC50 value of the antibody-drug conjugate measured on day 0 of plasma storage, and IC50(day3) is the IC50 value of the antibody-drug conjugate measured on day 3 of plasma storage). In some embodiments, the antibody-drug conjugate may maintain a target cell killing ability of 85% or 90% or more based on IC50 when stored in the plasma of a cynomolgus monkey for 14 days. For a method to determine the change in the target cell killing ability of the antibody-drug conjugate depending on the storage period in plasma, refer to “Example 26. In vitro plasma stability test (1)” of the present disclosure.

[0770] In some embodiments, the antibody-drug conjugate may maintain an ability to bind to EGFR based on EC50 of 50%, 60%, or 65% or more when stored in the plasma of a monkey (e.g., a cynomolgus monkey) for 3 days. The antibody-drug conjugate may maintain an ability to bind to EGFR based on EC50 of 45%, 50%, or 55% or more when stored in the plasma of a monkey (e.g., a cynomolgus monkey) for 14 days. For a method to determine the change in the EGFR binding ability of the antibody-drug conjugate depending on the storage period in plasma, refer to “Example 26. In vitro plasma stability test (1)” of the present disclosure.

[0771] In some embodiments, the antibody-drug conjugate may maintain an ability to bind to MUC1 based on EC50 of 60%, 65%, or 70% or more compared to day 0 when stored in the plasma of a monkey (e.g., cynomolgus monkey) for 3 days. In some embodiments, the antibody-drug conjugate may maintain an ability to bind to MUC1 based on EC50 of 45%, 50%, or 55% or more when stored in the plasma of a monkey (e.g., cynomolgus monkey) for 14 days. For a method to determine the change in the MUC1 binding ability of the antibody-drug conjugate according to the storage period in plasma, reference may be made to “Example 26. In vitro plasma stability test (1)” of the present disclosure. In some embodiments, the payload release from the plasma of the antibody-drug conjugate may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0% or less. In some embodiments, the payload release of the antibody-drug conjugate in plasma may be measured in vitro in a 1% BSA / PBS solution, human plasma, or monkey plasma. In some embodiments, the payload release rate of the antibody-drug conjugate in plasma may be measured on day 0, day 1, day 6, or day 14 of storage in plasma (or BSA / PBS).In some embodiments, when measuring the payload release rate of the antibody-drug conjugate in plasma, the initial concentration of the antibody-drug conjugate may be 20 ii g / mL or 200 ug / mL, but is not limited thereto. For a method to determine the payload release of the antibody-drug conjugate according to the storage period in plasma, refer to “Example 27. In vitro plasma stability test (2)” of the present disclosure.

[0772] In some embodiments, the plasma half-life of the antibody-drug conjugate in monkey pk (pharmacokinet ics) experiments may be 80 to 160 hours. In some embodiments, the plasma half-life of the antibody-drug conjugate in monkey pk experiments may be about 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150 hours or more, or may be within a range set to any two values ​​selected from the aforementioned values. In some embodiments, the plasma half-life of the antibody-drug conjugate in monkey pk experiments may be the plasma half-life when administered to monkeys (e.g., cynomolgus monkeys) at a dose of 10 mg / kg.

[0773] 6. Use of Antibody-Drug Conjugates

[0774] 6.1. Overview of Antibody-Drug Conjugate Uses

[0775] Antibody-drug conjugates according to some embodiments of the present disclosure have uses for treating tumors or cancer. In some embodiments, antibody-drug conjugates may be used to treat a tumor in a subject (e.g., a human patient) having a tumor (or cancer). In some embodiments, antibody-drug conjugates may be used to treat a tumor in a subject (e.g., a human patient) who has been diagnosed or confirmed to have a tumor (or cancer). In some embodiments, antibody-drug conjugates have uses for manufacturing tumor or cancer therapeutic agents. For example, antibody-drug conjugates may be used for manufacturing therapeutic agents to treat a tumor in a subject (e.g., a human patient) who has a tumor, or who has been diagnosed or confirmed to have a tumor.

[0776] Some embodiments of the present disclosure provide a use of an antibody-drug conjugate or a composition (or pharmaceutical composition) containing the same for treating a target cancer or tumor.

[0777] Some embodiments of the present disclosure provide a pharmaceutical composition for treating a tumor of a target comprising an antibody-drug conjugate.

[0778] Some embodiments of the present disclosure provide a method for treating cancer or a tumor of a subject, comprising administering an antibody-drug conjugate or a composition (or pharmaceutical composition) containing the same to the subject.

[0779] Some embodiments of the present disclosure provide a use for manufacturing a medicine (or therapeutic agent) for treating a target cancer or tumor of an antibody-drug conjugate or a composition (or pharmaceutical composition) containing the same.

[0780] 6.2. Tumors Subject to Treatment

[0781] Examples of tumors that can be treated using the antibody-drug conjugate of the present disclosure (or compositions or pharmaceutical compositions containing the same) have been described in detail in the preceding paragraphs, including sections of the present disclosure “4. Background - Target Antigens EGFR and MUC1”, “5. Antibody-Drug Conjugate”, and “5.6. Target Tumors of Antibody-Drug Conjugate”, and these descriptions apply herein.

[0782] In some embodiments, the tumor is colon cancer, rectal cancer, renal cancer, renal cell carcinoma, urothelial cancer, breast cancer, triplenegitive breast cancer, epithelial breast cancer, liver cancer, lung cancer, small cell lung cancer (SCLC), non-smal cell lung cancer (NSCLC), squamous non-smal cell lung cancer (sq-NSCLC), non-squamous non-smal cell lung cancer (nsq-NSCLC), gastric cancer (Gastric cancer), colorectal cancer, colorectal adenocarcinoma, prostate cancer, small intestine cancer, esophageal cancer, hepatocellular carcinoma, melanoma, orthotopic cancer, bone cancer, ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, skin cancer, head and neck cancer,Head and neck squamous cell carcinoma, Head and neck adenocarcinoma, Neuroendocrine tumor (Carcinoid), Cutaneous or intraocular malignant melanoma, Uterine cancer, Ovarian cancer, Epithelial ovarian cancer, Rectal cancer, Cancer of the anal region, Stomach cancer, Testicular cancer, Carcinoma of the fallopian tubes, Endometrial cancer, Cervical cancer, Carcinoma of the vagina, Vulvar cancer (Carcinoma of the vulva), Hodgkin's Disease, non-Hodgkin's lymphoma, lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma, cholangiocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), esophageal adenocarcinoma,Salivary gland cancer, Sarcoma, Soft tissue sarcoma, Cancer of the urethra, Urothelial carcinoma, Cancer of the penis, Pediatric solid tumors, Bladder cancer, Bladder squamous cell carcinoma (bladder SCC), Cancer of the kidney or ureter, Carcinoma of the renal pelvis, Neurological cancer, Neoplasm of the central nervous system (CNS), Primary CNS lymphoma, Spinal axis tumor, Glioma (G1 ioma), brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma), combinations of the above cancers, and one or more selected from metastatic lesions of the above cancers, but not limited thereto.

[0783] In some embodiments, the tumor (or target tumor) may be a primary tumor. In some embodiments, the tumor may be a secondary tumor. In some embodiments, the tumor may be a recurrent tumor. In some embodiments, the tumor may be a metastatic tumor or metastatic cancer. For example, the tumor may be a metastatic tumor generated from or induced from any one of the aforementioned tumors (or cancers).

[0784] In some embodiments, the tumor may be a solid tumor or a blood cancer.

[0785] In some embodiments, the tumor may be a tumor expressing EGFR. In some embodiments, the tumor may be a tumor overexpressing EGFR. In some embodiments, the tumor may be an EGFR-positive tumor. In some embodiments, the tumor may be an EGFR-associated tumor.

[0786] In some embodiments, the tumor may be a tumor expressing MUC1. In some embodiments, the tumor may be a tumor overexpressing MUC1. In some embodiments, the tumor may be a MUC1-positive tumor. In some embodiments, the tumor may be a tumor associated with MUC1.

[0787] In some embodiments, the tumor may be a tumor expressing one or more of EGFR and MUC1. In some embodiments, the tumor may be an EGFR-positive and / or MUC1-positive tumor. In some embodiments, the tumor may be a tumor associated with one or more of EGFR and MUC1. In some embodiments, the tumor may be a tumor overexpressing one or more of EGFR and MUC1.

[0788] In some embodiments, the tumor may be a tumor expressing both EGFR and MUC1. In some embodiments, the tumor may be an EGFR-positive and MUC1-positive tumor. In some embodiments, the tumor may be a tumor associated with both EGFR and MUC1. In some embodiments, the tumor may be a mutational tumor. For example, the mutational tumor may have one or more of KRAS mutations, BRAF mutations, and EGFR mutations. For example, the KRAS mutation may include one or more of G12D mutations and G13D mutations. For example, the BRAF mutation may include one or more of G469E mutations and V600E mutations. For example, the EGFR mutation may include one or more of T790M mutations, C797S mutations, and L858R mutations.

[0789] In some embodiments, the tumor may be a tumor resistant to EGFR drugs. In some embodiments, the tumor may be a tumor resistant to a tyrosine kinase inhibitor. In some embodiments, the tumor may be a tumor resistant to one or more of Osimert inib, cetuximab, and panitumumab.

[0790] In some embodiments, the tumor may be a target tumor of the antibody-drug conjugate.

[0791] 6.3. Subject for treatment or administration. The subject for treatment or administration of the antibody-drug conjugate of the present disclosure or a composition (or pharmaceutical composition) containing it may be a human. In some embodiments, the subject may be a patient. In some embodiments, the subject may be a human requiring administration of the antibody-drug conjugate or a composition (or pharmaceutical composition) containing it, or treatment of a tumor (or cancer). In some embodiments, the subject may be a human (patient) having a tumor, or diagnosed or confirmed to have a tumor.

[0792] In some embodiments, the subject may be a human identified as having a tumor expressing EGFR, a tumor overexpressing EGFR, or an EGFR-positive tumor. In some embodiments, the subject may be a human diagnosed or identified as having a tumor expressing MUC1, a tumor overexpressing MUC1, or a MUC1-positive tumor.

[0793] In some embodiments, the subject may be a human identified as having a tumor that co-expresses EGFR and MUC1, a tumor that overexpresses both EGFR and MUC1, or an EGFR-positive and MUC1-positive tumor. Whether the patient's tumor expresses EGFR and / or MUC1 can be determined by methods well known in the art and is not otherwise limited.

[0794] In some embodiments, the subject may be a subject resistant to an EGFR drug. In some embodiments, the subject may be a subject resistant to Tyrosine Kinase Inhibitor. In some embodiments, the subject may be a subject resistant to one or more of Osimertinib, cetuximab, and panitumumab.

[0795] 6.4. Pharmaceutical compositions containing antibody-drug conjugates

[0796] In some embodiments, the antibody-drug conjugate may be used to treat a tumor in the form of a composition or a pharmaceutical composition. Some embodiments of the present disclosure provide a pharmaceutical composition for treating a tumor of a target. The pharmaceutical composition of the present disclosure comprises an antibody-drug conjugate or a salt thereof (e.g., a pharmaceutically acceptable salt). In some embodiments, the pharmaceutical composition may comprise a therapeutically effective amount of the antibody-drug conjugate or a salt thereof. In some embodiments, the pharmaceutical composition may comprise the antibody-drug conjugate or a salt thereof as an active ingredient. In some embodiments, the pharmaceutical composition may comprise a therapeutically effective amount of the antibody-drug conjugate or a salt thereof as an active ingredient.

[0797] Examples of tumor types and types that can be treated using the antibody-drug conjugate of the present disclosure (or pharmaceutical compositions containing the same) have been described in detail in the preceding paragraphs, including sections of the present disclosure “4. Background - Target Antigens EGFR and MUC1”, “5. Antibody-Drug Conjugate”, and subsections therein “5.6. Target Tumors of Antibody-Drug Conjugates” and “6.2. Tumors Subject to Treatment”, and these descriptions apply herein. For example, the tumor may be a tumor expressing one or more of EGFR and MUC1. In some embodiments, the tumor may be an EGFR-positive tumor. In some embodiments, the tumor may be a MUC1-positive tumor. In some embodiments, the tumor may be an EGFR-positive and MUC1-positive tumor.

[0798] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable carrier may comprise, for example, excipients, diluents, and / or adjuvants. The carrier may be, for example, lactose, dextrose, sucrose, sorbitol, polysorbate (e.g., polysorbate 80), mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, arginine

[0799] The carrier may be one or more selected from hydrochloride, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, physiological saline, buffer solution such as tris(hydroxymethyl)aminomethane buffer, PBS, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The carrier may include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.

[0800] In some embodiments, the pharmaceutical composition may comprise 10 mg / mL of antibody-drug conjugate according to some embodiments of the present disclosure, 20 mM tris(hydroxymethyl)aminomethane buffer (e.g., 2.42 mg / mL), 50 mM arginine hydrochloride (e.g., 10.53 mg / mL), 5% (w / v) sucrose (e.g., 50 mg / mL), and 0.02% (w / v) polysorbate 80 (e.g., 0.2 mg / mL), wherein the pH of the composition may be 7.5.

[0801] Furthermore, the pharmaceutical composition may additionally contain one or more other elements suitable for treating or preventing cancer. 6.5. Treatment method using antibody-drug conjugates

[0802] Some embodiments of the present disclosure provide a method for treating a tumor using an antibody-drug conjugate according to some embodiments of the present disclosure. The treatment method may include administering a therapeutically effective amount of an antibody-drug conjugate appropriately formulated (e.g., a pharmaceutical composition) to a subject at an appropriate method of administration, an appropriate dosage, and an appropriate dosage.

[0803] Some embodiments of the present disclosure provide a method for treating a tumor of a subject comprising: administering an antibody-drug conjugate (or a salt thereof) or a pharmaceutical composition comprising the same to the subject. The subject for administration is described in detail in the preceding paragraphs, including Section 6.3. Subject for treatment or administration of the present disclosure. For example, the subject for administration may be a human having a tumor or diagnosed or confirmed to have a tumor.

[0804] Examples of tumor types and types that can be treated using the antibody-drug conjugate of the present disclosure (or pharmaceutical compositions containing the same) have been described in detail in the preceding paragraphs, including sections of the present disclosure “4. Background - Target Antigens EGFR and MUC1”, “5. Antibody-Drug Conjugate”, and subsections therein “5.6. Target Tumors of Antibody-Drug Conjugates” and “6.2. Tumors Subject to Treatment”, and these descriptions apply herein. For example, the tumor may be a tumor expressing one or more of EGFR and MUC1. In some embodiments, the tumor may be an EGFR-positive tumor. In some embodiments, the tumor may be a MUC1-positive tumor. In some embodiments, the tumor may be an EGFR-positive and MUC1-positive tumor.

[0805] In some embodiments, the recipient of the antibody-drug conjugate or the pharmaceutical composition containing the same may be a human. In some embodiments, the recipient of the tumor treatment may be a human. In some embodiments, the recipient may be a human having a tumor, or diagnosed or confirmed to have a tumor.

[0806] In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same (or an antibody-drug conjugate appropriately formulated) may be administered to a subject by various routes / methods. In some embodiments, administration of the antibody-drug conjugate or a pharmaceutical composition containing the same may be performed in an appropriate manner including injection, blood transfusion, and transplantation. In certain embodiments, administration of the antibody-drug conjugate or a composition containing the same may be performed via injection. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same may be administered to a subject by intravenous injection, via an artery, subcutaneously, intradermally, into a tumor, into a lesion, into a lymph node, into the bone marrow, into the muscle, or into the abdominal cavity. In a specific embodiment, the antibody-drug conjugate or a pharmaceutical composition containing the same may be administered to a target by intradermal or subcutaneous injection. A specific

[0807] In an embodiment, the antibody-drug conjugate or a pharmaceutical composition containing it may be administered to a subject by intravenous injection. In a specific embodiment, the antibody-drug conjugate or a pharmaceutical composition containing it may be injected directly into or implanted into a tumor site, lymph node, or site of infection. Furthermore, the antibody-drug conjugate (or a pharmaceutical composition containing it) may be administered using a controlled-release or delayed-release system, or by being contained in a formulation for controlled-release or delayed-release. In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing it may be administered to a subject one or more times. For example, the antibody-drug conjugate or a pharmaceutical composition containing it may be administered to a subject a single dose or multiple times. When an antibody-drug conjugate or a pharmaceutical composition containing it is administered to a subject multiple times, the time between administrations of the antibody-drug conjugate or the pharmaceutical composition containing it may be independently and appropriately selected, and is not otherwise limited.

[0808] In some embodiments, the antibody-drug conjugate or the pharmaceutical composition containing it may be administered to a subject at an appropriate dose. For example, the dosage of the antibody-drug conjugate or the pharmaceutical composition containing it may be determined by considering, but is not limited to, one or more selected from the type of disease (e.g., type of cancer), the site of administration, the subject's body weight, age, and the stage of disease progression. Furthermore, the dosage may be appropriately determined within the scope of medical judgment, corresponding to a reasonable benefit / risk ratio, within a range where other problems such as excessive toxicity, irritation, and allergic reactions are minimal.

[0809] In some embodiments, the antibody-drug conjugate or a pharmaceutical composition containing the same may be co-administered with other substances (e.g., therapeutic or prophylactic agents). For example, the antibody-drug conjugate or a pharmaceutical composition containing the same may be co-administered with, but is not limited to, other therapeutic agents for treating tumors other than the antibody-drug conjugate of the present disclosure (e.g., small compounds, antibodies, conjugates such as the antibody-drug conjugate). Co-administration is not limited to administering the therapies exactly simultaneously. For example, the therapies to be co-administered may be administered at appropriate intervals or simultaneously. 6.6. Examples of Co-administration

[0810] In some embodiments, an antibody-drug conjugate or a pharmaceutical composition containing the same may be used with a KRAS inhibitor (e.g., sotorasib). For example, an antibody-drug conjugate or a pharmaceutical composition containing the same may be used in combination therapy with a KRAS inhibitor.

[0811] For example, a KRAS inhibitor may be administered at the same time as the antibody-drug conjugate. For example, if a KRAS inhibitor is administered at the same time as the antibody-drug conjugate, the KRAS inhibitor may be included in a composition containing the antibody-drug conjugate, or administered to the subject in the form of a composition separate from the composition containing the antibody-drug conjugate, and is not otherwise limited.

[0812] As another example, the antibody-drug conjugate may be administered to the subject at a different time than the time of administration, and is not otherwise limited. When a KRAS inhibitor is administered at a different time than the time of administration of the antibody-drug conjugate, it may be administered earlier or later than the time of administration of the antibody-drug conjugate, and is not otherwise limited. For example, when the antibody-drug conjugate is administered multiple times, the KRAS inhibitor may be administered at a different time than each time of administration of the antibody-drug conjugate, at the same time as one or more of the times of administration of the antibody-drug conjugate, or at the same time as all of the times of administration of the antibody-drug conjugate.

[0813] In some embodiments, the KRAS inhibitor may be administered once or multiple times. The interval between the administration of the antibody-drug conjugate and the administration of the KRAS inhibitor, or the interval between administration of the KRAS inhibitor when administered multiple times, may be appropriately adjusted.

[0814] KRAS inhibitors may be administered in the form of a composition or in a form appropriately formulated thereof, and are not otherwise limited. KRAS inhibitors may be administered in the form of a pharmaceutically acceptable salt. For example, a composition or pharmaceutical composition containing a KRAS inhibitor or a pharmaceutically acceptable salt thereof may be used for administration. For example, a KRAS inhibitor or a pharmaceutically acceptable salt thereof may be formulated in the form of a tablet and used for administration (e.g., oral administration). KRAS inhibitors may be formulated with a pharmaceutically acceptable carrier (e.g., excipients, etc., and are not otherwise limited).

[0815] Administration of KRAS inhibitors can be performed in an appropriate manner, including oral administration, injection, blood transfusion, and transplantation. For example, KRAS inhibitors may be administered orally, intravenously, via artery, subcutaneously, intradermally, into a tumor, into a lesion, into a lymph node, into the bone marrow, into the muscle, or into the abdominal cavity.

[0816] In some embodiments, the KRAS inhibitor may be administered to the subject at an appropriate dose. For example, the dosage of the KRAS inhibitor may be determined by considering, but is not limited to, one or more of the type of disease (e.g., type of cancer), the site of administration, the subject's body weight, age, and the stage of disease progression. Furthermore, the dosage may be appropriately determined within the scope of medical judgment, corresponding to a reasonable benefit / risk ratio, within a range where other problems such as excessive toxicity, irritation, and allergic reactions are minimal.

[0817] KRAS inhibitors can be, for example, sot or as ib. Sot or as ib is known to target the mutated tumor KRAS G12C and is known as the first targeted therapy approved for patients with non-small cell lung cancer with KRAS mutations.

[0818] Sotorasib can be expressed by the structure of Equation 16 below.

[0819] [Equation 16]

[0820]

[0821] 7. Exemplary embodiments Hereafter, exemplary embodiments of the invention provided according to some embodiments of the present disclosure are provided. The invention provided by the present disclosure is not limited to the examples below.

[0822] A. Antibody-drug conjugate

[0823] A01. Antibody-drug conjugate having the structure of Formula 13 below or salt thereof:

[0824] [Equation 13]

[0825]

[0826] At this time,

[0827] AB is an antibody, wherein the antibody is an anti-EGFR / MUC1 bispecific antibody, and the anti-EGFR / MUC1 bispecific antibody comprises an antigen-binding fragment for EGFR and an antigen-binding fragment for MUC1, wherein the antigen-binding fragment for EGFR is referred to as the first antigen-binding fragment and the antigen-binding fragment for MUC1 is referred to as the second antigen-binding fragment, and

[0828] At this time, the first antigen-binding fragment comprises HCDR1 having the amino acid sequence of SEQ ID NO. 1, HCDR2 having the amino acid sequence of SEQ ID NO. 2, and HCDR3 having the amino acid sequence of SEQ ID NO. 3, and

[0829] At this time, the second antigen-binding fragment comprises HCDR1 having the amino acid sequence of SEQ ID NO. 7, HCDR2 having the amino acid sequence of SEQ ID NO. 8, and HCDR3 having the amino acid sequence of SEQ ID NO. 9, and

[0830] a is independently 0 or 1, and

[0831] y is 1 or 2.

[0832] A02. In A01, the anti-EGFR / MUC1 bispecific antibody is an antibody-drug conjugate having one first antigen-binding fragment and one second antigen-binding fragment.

[0833] A03. An antibody-drug conjugate or a salt thereof, wherein in any one of A01 to A02, the first antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence of sequence number 10, and the second antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence of sequence number 22.

[0834] A04. In any one of A01 to A03,

[0835] The first antigen-binding fragment further comprises LCDR1 having the amino acid sequence of sequence number 4, LCDR2 having the amino acid sequence of sequence number 5, and LCDR3 having the amino acid sequence of sequence number 6, and

[0836] The above second antigen-binding fragment further comprises LCDR1 having the amino acid sequence of sequence number 4, LCDR2 having the amino acid sequence of sequence number 5, and LCDR3 having the amino acid sequence of sequence number 6,

[0837] Antibody-drug conjugate or its salt.

[0838] A05. An antibody-drug conjugate or a salt thereof, wherein in any one of A01 to A04, the first antigen-binding fragment comprises a heavy chain variable region and a common light chain variable region of SEQ ID NO. 10, and the second antigen-binding fragment comprises a heavy chain variable region and a common light chain variable region of SEQ ID NO. 22.

[0839] A06. In A05, the common light chain variable region is an antibody-drug conjugate or a salt thereof having the amino acid sequence of SEQ ID NO. 15.

[0840] A07. In any one of A01 to A06, the anti-EGFR / MUC1 bispecific antibody is an antibody-drug conjugate or a disodium that is a human IgG type antibody.

[0841] A08. In any one of A01 to A07, the anti-EGFR / MUC1 dual-specific antibody is an antibody-drug conjugate or a salt thereof, which is a human IgGl-type antibody.

[0842] A09. In any one of A07 to A08, the Fc region of the anti-EGFR / MUC1 bispecific antibody is an antibody-drug conjugate or a salt thereof having a KIH mutation (knobs in to holes mutation).

[0843] A10. An antibody-drug conjugate or a salt thereof, wherein the anti-EGFR / MUC1 bispecific antibody comprises a heavy chain constant region having a knob mutation (e.g., a human IgG or human IgGl heavy chain constant region) and a heavy chain constant region having a Hole mutation (e.g., a human IgG or human IgGl heavy chain constant region).

[0844] All . In any one of A01 to A10,

[0845] The above anti-EGFR / MUC1 bispecific antibody comprises a first heavy chain and a second heavy chain, wherein the first heavy chain comprises a heavy chain variable region of the first antigen-binding fragment and a heavy chain constant region of the first heavy chain, and

[0846] At this time, the second heavy chain includes a heavy chain variable region of the second antigen-binding fragment and a heavy chain invariant region of the second heavy chain, and

[0847] At this time, the heavy chain invariant region of the first heavy chain has a knob variation or a hole variation, and

[0848] At this time, the heavy chain constant region of the second heavy chain has a Hole mutation or a knob mutation, provided that if the heavy chain constant region of the first heavy chain has a knob mutation, the heavy chain constant region of the second heavy chain has a Hole mutation, and if the heavy chain constant region of the first heavy chain has a Hole mutation, the heavy chain constant region of the second heavy chain has a knob mutation, an antibody-drug conjugate or a salt thereof.

[0849] A12. In any one of A01 to All,

[0850] The above anti-EGFR / MUC1 bispecific antibody comprises a first heavy chain and a second heavy chain, and two common light chains, and

[0851] At this time, the first heavy chain includes a heavy chain variable region of the first antigen-binding fragment and a heavy chain invariant region of the first heavy chain, and

[0852] At this time, the second heavy chain includes a heavy chain variable region of the second antigen-binding fragment and a heavy chain invariant region of the second heavy chain, and

[0853] The heavy chain variable region of the first antigen-binding fragment and the light chain variable region of either of the two common light chains form the first antigen-binding fragment, and the heavy chain variable region of the second antigen-binding fragment and the light chain variable region of the other of the two common light chains form the second antigen-binding fragment, wherein the heavy chain invariant region of the first heavy chain has a knob variation or a hole variation,

[0854] At this time, the chain invariant region of the second chain mentioned above has a hole variation or a knob variation, and

[0855] However, an antibody-drug conjugate or its salt, wherein if the heavy chain constant region of the first heavy chain has a knob mutation, the heavy chain constant region of the second heavy chain has a Hole mutation, and if the heavy chain constant region of the first heavy chain has a Hole mutation, the heavy chain constant region of the second heavy chain has a knob mutation.

[0856] A13. In any one of All to A12,

[0857] The heavy chain invariant region of the first heavy chain mentioned above is the heavy chain invariant region of human IgG having a knob variant or a Hole variant, and

[0858] The heavy chain invariant region of the second heavy chain mentioned above is the heavy chain invariant region of human IgG having a hole mutation or a knob mutation, and

[0859] An antibody-drug conjugate or its salt, wherein if the heavy chain constant region of the first heavy chain has a knob mutation, the heavy chain constant region of the second heavy chain has a Hole mutation, and if the heavy chain constant region of the first heavy chain has a Hole mutation, the heavy chain constant region of the second heavy chain has a knob mutation.

[0860] A14. In any one of All to A13,

[0861] The heavy chain invariant region of the first heavy chain mentioned above is the heavy chain invariant region of human IgGl having a knob variant or a Hole variant, and

[0862] The heavy chain invariant region of the second heavy chain mentioned above is the heavy chain invariant region of human IgGl having a hole mutation or a knob mutation, and

[0863] An antibody-drug conjugate or its salt, wherein if the heavy chain constant region of the first heavy chain has a knob mutation, the heavy chain constant region of the second heavy chain has a Hole mutation, and if the heavy chain constant region of the first heavy chain has a Hole mutation, the heavy chain constant region of the second heavy chain has a knob mutation.

[0864] A15. In any one of A10 to A14,

[0865] The above knob variation includes one or more substitutions of S354C and T366W, and the above hole variation includes one or more substitutions of Y349C, T366S, L368A and Y407V, and

[0866] In this case, the number of the above-mentioned substituted amino acid residue is determined according to the Eu number i ng system,

[0867] Antibody - drug conjugate or its inflammation.

[0868] A16. In any one of A01 to A15,

[0869] The above anti-EGFR / MUC1 bispecific antibody is an antibody-drug conjugate or a salt thereof comprising a heavy chain having the amino acid sequence of sequence number 39 (first heavy chain), a heavy chain having the amino acid sequence of sequence number 40 (second heavy chain), and a light chain having the amino acid sequence of sequence number 21 (common light chain).

[0870] A17. In A16, the anti-EGFR / MUC1 bispecific antibody is an antibody-drug conjugate or a salt thereof having one heavy chain having the amino acid sequence of sequence number 39, one heavy chain having the amino acid sequence of sequence number 40, and two common light chains having the amino acid sequence of sequence number 21.

[0871] A18. In any one of A01 to A17, in Formula 13, the structure connected to AB is an antibody-drug conjugate or a salt thereof connected to Asn297 of the anti-EGFR / MUC1 bispecific antibody.

[0872] A19. In any one of A01 to A18,

[0873] Formula 13 above is Formula 14 below, an antibody-drug conjugate or its salt:

[0874] [Equation 14]

[0875]

[0876] At this time,

[0877] AB is an anti-EGFR / MUC1 bispecific antibody, and

[0878] a is independently 0 or 1, and

[0879] y is 1 or 2.

[0880] A20. An antibody-drug conjugate or a salt thereof, wherein in any one of A01 to A19, y is 2, and each of the two AB-linked structures is linked to each of the two heavy chains of the anti-EGFR / MUC1 bispecific antibody (e.g., to each of the first heavy chain and the second heavy chain).

[0881] A21. An antibody-drug conjugate or a salt thereof, wherein in any one of A01 to A20, y is 1, and the structure connected to AB is connected to only one of the two heavy chains of the anti-EGFR / MUC1 bispecific antibody (e.g., only one of the first heavy chain and the second heavy chain).

[0882] B. Pharmaceutical composition containing an antibody-drug conjugate

[0883] B01. A pharmaceutical composition for treating a target tumor comprising an antibody-drug conjugate or a salt thereof of any one of A01 to A21.

[0884] B02. A pharmaceutical composition in which, in B01, the tumor is a tumor expressing one or more of EGFR and MUC1.

[0885] B03. A pharmaceutical composition in which, in any one of B01 to B02, the tumor is a tumor expressing both EGFR and MUC1.

[0886] B04. A pharmaceutical composition in any one of B02 to B03, wherein the EGFR is human EGFR.

[0887] B05. A pharmaceutical composition in any one of B02 to B04, wherein the MIJC1 is human MIJC1.

[0888] B06. In any one of B01 to B05, the tumor is head and neck cancer, head and neck squamous-cell carcinoma (HNSCC), head and neck adenocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), esophageal adenocarcinoma (EAC), liver cancer, renal cancer, skin cancer, G1 ioma, neuroendocrine tumor (Carcinoid), ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, lung cancer, small 1-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous non-small 1-cell lung cancer (sq-NSCLC), non-squamous non-smal 1-cell lung cancer (nsq-NSCLC), cervical cancer, endometrial cancer, cervical cancer, thyroid cancer, prostate cancer, bladder cancer, bladder squamous cel 1-cell carcinoma (bladder SCC),A pharmaceutical composition comprising one or more selected from urothelial carcinoma, colorectal cancer, colorectal adenocarcinoma, stomach cancer, breast cancer, ovarian cancer, and testicular cancer.

[0889] B07. A pharmaceutical composition in any one of B01 to B06, wherein the tumor is a primary tumor or a metastatic tumor.

[0890] c. Methods for treating subjects with tumors

[0891] C01. A method for treating a subject having a tumor or diagnosed or confirmed to have a tumor, comprising: administering the pharmaceutical composition of B01 to the subject.

[0892] C02. In C01, the tumor is a tumor expressing one or more of EGFR and MUC1, method.

[0893] C03. A method in which, in any one of C01 to C02, the tumor is a tumor expressing both EGFR and MUC1.

[0894] C04. Method in any one of C02 to C03, wherein the EGFR is human EGFR.

[0895] C05. A method in any one of C02 to C04, wherein the MUC1 is a human MUC1.

[0896] C06. In any one of C01 to C05, the tumor is head and neck cancer, head and neck squamous-cell carcinoma (HNSCC), head and neck adenocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), esophageal adenocarcinoma (EAC), liver cancer, renal cancer, skin cancer, G1 ioma, neuroendocrine tumor (Carcinoid), ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreat adenosquamous carcinoma, pancreatic ductal adenocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer (sq-NSCLC), non-squamous non-smal cell lung cancer (nsq-NSCLC), cervical cancer, endometrial cancer, cervical cancer, thyroid cancer, prostate cancer, bladder cancer, bladder squamous cell carcinoma (bladder SCC),One or more selected from urothelial cancer, colorectal cancer, colorectal adenocarcinoma, stomach cancer, breast cancer, ovarian cancer, and testicular cancer, method.

[0897] D. Uses of Antibody-Drug Conjugates

[0898] DOI. Use of an antibody-drug conjugate or a salt thereof for treating a tumor of the target according to any one of A01 to A21.

[0899] D02. Use for treating a tumor of a target antibody-drug conjugate or salt thereof according to any one of A01 to A21.

[0900] D03. In any one of D01 to D02, the tumor is a tumor expressing one or more of EGFR and MUC1.

[0901] D04. In any one of D01 to D03, the tumor is a tumor that expresses both EGFR and MUC1.

[0902] D05. In any one of D02 to D04, the use is that the EGFR is human EGFR.

[0903] D06. In any one of D02 to D05, the MIJC1 is used as a human MIJC1.

[0904] D07. In any one of D01 to D06, the tumor is head and neck cancer, head and neck squamous cell carcinoma, head and neck adenocarcinoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, liver cancer, renal cancer, skin cancer, G1 ioma, neuroendocrine tumor (Carcinoid), ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, lung cancer, small 1-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous non-smal 1-cell lung cancer (sq-NSCLC), non-s...

Claims

【Scope of Claim】 【Claim 11 Antibody-drug conjugate having the structure of Formula 13 below or a pharmaceutically acceptable salt thereof: [Equation 13] At this time, AB is an antibody, wherein the antibody is an anti-EGFR / MUC1 bispecific antibody, and wherein the anti-EGFR / MUC1 bispecific antibody comprises a first antigen-binding fragment targeting EGFR and a second antigen-binding fragment targeting MUC1, and a is independently 0 or 1, and y is 1 or 2.

2. In Paragraph 1, The first antigen-binding fragment comprises HCDR1 having the amino acid sequence of sequence no. 1, HCDR2 having the amino acid sequence of sequence no. 2, HCDR3 having the amino acid sequence of sequence no. 3, LCDR1 having the amino acid sequence of sequence no. 4, LCDR2 having the amino acid sequence of sequence no. 5, and LCDR3 having the amino acid sequence of sequence no. 6, and the second antigen-binding fragment comprises HCDR1 having the amino acid sequence of sequence no. 7, HCDR2 having the amino acid sequence of sequence no. 8, and HCDR3 having the amino acid sequence of sequence no. 9, LCDR1 having the amino acid sequence of sequence no. 4, LCDR2 having the amino acid sequence of sequence no. 5, and LCDR3 having the amino acid sequence of sequence no. 6, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof. 【claim Paragraph 3] In any one of paragraphs 1 to 2, The first antigen-binding fragment comprises a first heavy chain variable region of SEQ ID NO. 10 and a light chain variable region of SEQ ID NO. 15, and The second antigen-binding fragment comprises the second heavy chain variable region of SEQ ID NO. 22 and the light chain variable region of SEQ ID NO. 15, Antibody-drug conjugate or a pharmaceutically acceptable salt thereof. 【claim Paragraph 4] In any one of paragraphs 1 to 3, The structure connected to AB in Formula 13 above is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof connected to an Asn residue located within the heavy chain constant region 2 (CH2) of the anti-EGFR / MUC1 bispecific antibody. [Claim] Paragraph 5] In any one of paragraphs 1 through 4, The structure connected to AB in Formula 13 above is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof connected to Asn297 of the anti-EGFR / MUC1 bispecific antibody. 【claim Paragraph 6] In any one of paragraphs 1 through 5, The above antibody-drug conjugate is an antibody-drug conjugate having the structure of Formula 14 below, or a pharmaceutically acceptable salt thereof: [Equation 14] In this case, AB is the above-mentioned anti-EGFR / MUC1 bispecific antibody, and a is independently 0 or 1, and y is 1 or 2.

7. In any one of Claims 1 through 6, y is a 2-substance, antibody-drug conjugate or a pharmaceutically acceptable salt thereof. 【claim Paragraph 8] A pharmaceutical composition for treating a tumor of a target, comprising an antibody-drug conjugate according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition according to claim 8, wherein the tumor is a tumor expressing one or more of EGFR and MUC1.

10. A pharmaceutical composition according to any one of claims 8 to 9, wherein the tumor is a tumor expressing both EGFR and MUC1.

11. In any one of claims 8 to 10, the tumor is colon cancer, rectal cancer, renal cancer, renal cell carcinoma, urothelial cancer, breast cancer, triple-negative breast cancer, epithelial breast cancer, liver cancer, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-smal cell lung cancer (sq-NSCLC), non-squamous non-smal cell lung cancer (nsq-NSCLC), gastric cancer. cancer), colorectal cancer, colorectal adenocarcinoma, prostate cancer, small intestine cancer, esophageal cancer, hepatocelular carcinoma, melanoma, orthotopic cancer, bone cancer, ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, skin cancer,Head and neck cancer, Head and neck squamous cell carcinoma, Head and neck adenocarcinoma, Neuroendocrine tumor (Carcinoid), Cutaneous or intraocular malignant melanoma, Uterine cancer, Ovarian cancer, Epithelial ovarian cancer, Rectal cancer, Cancer of the anal region, Stomach cancer, Testicular cancer, Carcinoma of the fallopian tubes, Endometrial cancer, Cervical cancer, Vaginal cancer (Carcinoma of the vagina), carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma, cholangiocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC),Esophageal adenocarcinoma, Salivary gland cancer, Sarcoma, Sarcoma of soft tissue, Cancer of the urethra, Urothelial carcinoma, Cancer of the penis, Solid tumors of childhood, Bladder cancer, Bladder squamous cell carcinoma (bladder SCC), Cancer of the kidney or ureter, Carcinoma of the renal pelvis, Neurological cancer, Neoplasm of the central nervous system (CNS), Primary CNS lymphoma, A pharmaceutical composition comprising one or more selected from spinal axis tumor, G1 ioma, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, and T-cell lymphoma.

12. A method for treating a tumor of a subject, comprising administering the pharmaceutical composition of claim 8 to the subject.

13. In Paragraph 12, The above tumor is a tumor that expresses one or more of EGFR and MUC1, method.

14. A method according to any one of claims 12 to 13, wherein the tumor is a tumor expressing both EGFR and MUC1.

15. In any one of claims 12 to 14, the tumor is colon cancer, rectal cancer, renal cancer, renal cell carcinoma, urothelial cancer, breast cancer, triple-negative breast cancer, epithelial breast cancer, liver cancer, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-smal cell lung cancer (sq-NSCLC), non-squamous non-smal cell lung cancer (nsq-NSCLC), gastric cancer (Gastric cancer), colorectal cancer, colorectal adenocarcinoma, prostate cancer, small intestine cancer, esophageal cancer, hepatocelular carcinoma, melanoma, orthotopic cancer, bone cancer, ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, skin cancer,Head and neck cancer, Head and neck squamous cell carcinoma, Head and neck adenocarcinoma, Neuroendocrine tumor (Carcinoid), Cutaneous or intraocular malignant melanoma, Uterine cancer, Ovarian cancer, Epithelial ovarian cancer, Rectal cancer, Cancer of the anal region, Stomach cancer, Testicular cancer, Carcinoma of the fallopian tubes, Endometrial cancer, Cervical cancer, Vaginal cancer (Carcinoma of the vagina), carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma, cholangiocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC),Esophageal adenocarcinoma, Salivary gland cancer, Sarcoma, Sarcoma of soft tissue, Cancer of the urethra, Urothelial carcinoma, Cancer of the penis, Solid tumors of the pediatric pediatric urinary tract, Bladder cancer, Bladder squamous cell carcinoma (bladder SCC), Cancer of the kidney or ureter, Carcinoma of the renal pelvis, Neurological cancer, Neoplasm of the central nervous system (CNS), Primary CNS lymphoma , spinal axis tumor, glioma (G1 ioma), brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, and T-cell lymphoma, one or more selected from the above.

16. Use in the manufacture of a therapeutic agent for treating a tumor of a target antibody-drug conjugate or salt thereof according to any one of claims 1 to 7.

17. In Clause 16, the above tumor is a tumor expressing one or more of EGFR and MUC1, for use.

18. In any one of claims 16 to 17, the use is that the tumor is a tumor expressing both EGFR and MUC1.

19. In any one of claims 16 to 18, the tumor is colon cancer, rectal cancer, renal cancer, renal cell carcinoma, urothelial cancer, breast cancer, triple-negative breast cancer, epithelial breast cancer, liver cancer, lung cancer, small cell lung cancer (SCLC), non-smal cell lung cancer, squamous non-smal cell lung cancer (sq-NSCLC), non-squamous non-smal cell lung cancer (nsq-NSCLC), gastric cancer (Gastr ic cancer), colorectal cancer, colorectal adenocarcinoma, prostate cancer, small intestine cancer, esophageal cancer, hepatocelular carcinoma, melanoma, orthotopic cancer, bone cancer, ampullary cancer, pancreatic cancer, pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic ductal adenocarcinoma, skin cancer,Head and neck cancer, Head and neck squamous cell carcinoma, Head and neck adenocarcinoma, Neuroendocrine tumor (Carcinoid), Cutaneous or intraocular malignant melanoma, Uterine cancer, Ovarian cancer, Epithelial ovarian cancer, Rectal cancer, Cancer of the anal region, Stomach cancer, Testicular cancer, Carcinoma of the fallopian tubes, Endometrial cancer, Cervical cancer, Vaginal cancer (Carcinoma of the vagina), carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma, cholangiocarcinoma, esophageal cancer, esophageal squamous-cell carcinoma (ESCC),Esophageal adenocarcinoma, Salivary gland cancer, Sarcoma, Soft tissue sarcoma, Cancer of the urethra, Urothelial carcinoma, Cancer of the penis, Solid tumors of childhood, Bladder cancer, Bladder squamous cell carcinoma (bladder SCC), Cancer of the kidney or ureter, Carcinoma of the renal pelvis, Neurological cancer, Neoplasm of the central nervous system (CNS), Primary CNS lymphoma, One or more selected from spinal axis tumor, G1 ioma, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, and T-cell lymphoma, use.

20. A method for manufacturing an antibody-drug conjugate comprising the following: Engineered bispecific antibody having the structure of Formula 03 below - [Essence 03] At this time, AB is an antibody, wherein the antibody is an anti-EGFR / MUC1 bispecific antibody, and wherein the anti-EGFR / MUC1 bispecific antibody comprises a first antigen-binding fragment targeting EGFR and a second antigen-binding fragment targeting MUC1, and a is independently 0 or 1, and x is 1 or 2 - ; and Linker-payload compound having the structure of Formula 08 below - [Essence 08] Contacting.

21. In Article 20, At this time, the first antigen-binding fragment comprises HCDR1 having the amino acid sequence of SEQ ID NO. 1, HCDR2 having the amino acid sequence of SEQ ID NO. 2, HCDR3 having the amino acid sequence of SEQ ID NO. 3, LCDR1 having the amino acid sequence of SEQ ID NO. 4, LCDR2 having the amino acid sequence of SEQ ID NO. 5, and LCDR3 having the amino acid sequence of SEQ ID NO. 6, and At this time, the second antigen-binding fragment comprises HCDR1 having the amino acid sequence of sequence number 7, HCDR2 having the amino acid sequence of sequence number 8, and HCDR3 having the amino acid sequence of sequence number 9, LCDR1 having the amino acid sequence of sequence number 4, LCDR2 having the amino acid sequence of sequence number 5, and LCDR3 having the amino acid sequence of sequence number 6, method .

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