Antibody-drug conjugate of Anti-ROR1 / b7-h3 bispecific antibody

Abstract

The present disclosure relates to: an antibody-drug conjugate of an anti-ROR1 / B7-H3 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-R0R1 / B7-H3 bispecific antibody

[0004]

Cross-reference to related applications

[0005] The present application claims priority to Korean patent application No. 10-2025-0004253 filed on January 10, 2025, Korean patent application No. 10-2025-0142901 filed on September 30, 2025, and Korean patent application No. 10-2025-0153529 filed on 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 worldwide received market approval as of 2022. Although research on various antibodies and drugs for the development of ADCs has been conducted for approximately 20 years, the development of effective ADCs remains a challenge.

[0010] Meanwhile, RORl (NTRKRl) is expressed during embryonic and fetal development and regulates cell polarity, cell migration, and neurite growth. Its expression gradually decreases with the progression of development and is almost non-existent in adults; it is expressed transiently during B cell development, and only slight expression has been reported in adipose cells. However, as overexpression of R0R1 has been observed in various cancer cells, it has been classified as an oncofetal gene (Oncof et al. gene). In particular, R0R1 began to receive attention as a target for anticancer antibodies after it was discovered that it is overexpressed in chronic lymphocytic leukemia (CLL). In addition to Chronic Lymphocytic Leukemia (CLL), cerebrocellular leukemia, lymphoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantocellular lymphoma (MCL),

[0011] It has been reported that R0R1 is overexpressed in various solid cancers, including breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer. In these cancers, the expression of R0R1 is associated with a poor prognosis in cancer patients and is known to affect cancer metastasis. It was found that injecting cancer cells with suppressed R0R1 expression into mice increased survival time and reduced metastasis. B7-H3 (B7 Homolog3, CD276) is known to play a predominantly inhibitory role in adaptive immunity in non-malignant tissues, thereby inhibiting the activation and proliferation of T cells. In malignant tissues, B7-H3 acts as an immune checkpoint molecule to suppress immune responses specific to tumor antigens, and B7-H3 is also known to possess non-immunological tumor-promoting functions. Meanwhile, while B7-H3 mRNA is found in most normal tissues, B7-H3 protein (i.e., B7-H3) is known to exhibit very limited expression in normal tissues due to post-transcriptional regulation by microRNA. On the other hand, B7-H3 protein is expressed at a high frequency in various cancer types and is reported to appear in about 60% of all cancers.Based on selective expression in these cancers, B7-H3 has been selected as a target antigen for antibodies and ADC therapies such as enob li tuzumab (MGA271), ombur tamab, MGD009, MGC018, DS-7300a.

[0012] Anti-R0R1 and anti-B7-H3 bispecific antibodies designed to target both R0R1 and B7-H3 are disclosed in PCT patent application publication number W02024 / 014930A1, and the anti-R0R1 and anti-B7-H3 bispecific antibodies disclosed herein have been experimentally confirmed to have excellent stability, delivery opioidity, binding ability to antigens, and internalization ability.

[0013]

Description of the Invention

[0014]

Technical Challenges

[0015] The problem to be solved by the present disclosure is to provide a bispecific antibody-drug conjugate (ADC) targeting R0R14 B7-H3 that is usable for tumor treatment, and a method for treating a tumor using said antibody-drug conjugate.

[0016]

Technical Solution

[0017] The present disclosure provides a novel antibody-drug conjugate based on a bispecific antibody targeting R0R1 and B7-H3 (e.g., an anti-R0R1 and anti-B7-H3 bispecific antibody). The antibody-drug conjugate of the present disclosure may be used for tumor treatment. For example, the antibody-drug conjugate of the present disclosure may be used to treat a human with a tumor (e.g., a tumor expressing at least one of R0R1 and B7-H3).

[0018] Some embodiments of the present disclosure provide an antibody-drug conjugate or a pharmaceutically acceptable salt 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-R0R1 / B7-H3 bispecific antibody, and the anti-R0R1 / B7-H3 bispecific antibody comprises a first antigen-binding fragment targeting R0R1 and a second antigen-binding fragment targeting B7-H3, 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 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. 30, HCDR2 having the amino acid sequence of sequence no. 31, HCDR3 having the amino acid sequence of sequence no. 32, LCDR1 having the amino acid sequence of sequence no. 33, LCDR2 having the amino acid sequence of sequence no. 34, and LCDR3 having the amino acid sequence of sequence no. 35.

[0027] In some embodiments, the first antigen-binding fragment may include a heavy chain variable region of sequence number 7 and a light chain variable region of sequence number 12, and the second antigen-binding fragment may include a heavy chain variable region of sequence number 36 and a light chain variable region of sequence number 41.

[0028] In some embodiments, the second antigen-binding fragment may be scFv. In some embodiments, the second antigen-binding fragment may have the amino acid sequence of sequence number 46.

[0029] In some embodiments, the anti-R0R1 / B7-H3 bispecific antibody may comprise two first antigen-binding fragments and two second antigen-binding fragments, or two first antigen-binding fragments and one second antigen-binding fragment.

[0030] In some embodiments, the anti-R0R1 / B7-H3 bispecific antibody comprises two first antigen-binding fragments and two second antigen-binding fragments, and the anti-R0R1 / B7-H3 bispecific antibody may comprise two heavy chains of sequence number 49 and two light chains of sequence number 18.

[0031] In some embodiments, the anti-R0R1 / B7-H3 bispecific antibody comprises two first antigen-binding fragments and one second antigen-binding fragment, and the anti-R0R1 / B7-H3 bispecific antibody may comprise one heavy chain of SEQ ID NO. 59, one heavy chain of SEQ ID NO. 60, and two light chains of SEQ ID NO. 18.

[0032] 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-R0R1 / B7-H3 bispecific antibody.

[0033] In some embodiments, the structure connected to AB in Formula 13 may be connected to Asn297 of the anti-R0R1 / B7-H3 bispecific antibody.

[0034] In some embodiments, the antibody-drug conjugate may have the structure of Formula 14 below:

[0035] [Equation 14]

[0036] AB

[0037] < u V

[0038]

[0039] y where AB is the above anti-R0R1 / B7-H3 bispecific antibody, a is independently 0 or 1, and y is 1 or 2.

[0040] In some embodiments, y can be 2.

[0041] 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.

[0042] 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. Some embodiments of the present disclosure provide a use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to some embodiments of the present disclosure for manufacturing a therapeutic agent for treating a tumor of the subject.

[0043] In some embodiments, the tumor may be a tumor expressing at least one of R0R1 and B7-H3.

[0044] In some embodiments, the tumor may be an R0R1-positive and / or B7-H3-positive tumor.

[0045] In some embodiments, the tumor may be a tumor expressing both R0R1 and B7-H3.

[0046] In some embodiments, the tumor may be an R0R1-positive and B7-H3-positive tumor.

[0047] In some embodiments, the tumor is colon cancer, colorectal cancer, rectal cancer, renal cancer, renal cell carcinoma, breast cancer, triple-negative breast cancer, liver cancer, hepatocellular carcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung neuroendocrine carcinoma, gastric cancer, neuroendocrine cancer, large cell Neuroendocytic cancer (LCNEC), Prostate cancer, Cancer of the small intestine, Cancer of the esophagus, Melanoma, Orthopedic cancer, Bone cancer, Pancreatic cancer, Skin cancer, Head or neck cancer, Cutaneous or intraocular malignant melanoma, Uterine cancer, Ovarian cancer, Epithelial ovarian cancer, Rectal cancerCancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, endometrial cancer, cervical cancer, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, lymphoma, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma , Cholangiocarcinoma , Esophageal cancer , Salivary gland cancer , Sarcoma , Soft tissue sarcoma , Cancer of the urethra , Urothelial cancer , Cancer of the penis , Pediatric solid tumors , Bladder cancer , Cancer of the kidney or ureter , Carcinoma of the renal pelvis , Neurological cancer ,It may be one or more selected from neoplasm of the central nervous system (CNS), neuroendocrine tumor (carcinoid), 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.

[0048] Some embodiments of the present disclosure provide a method for manufacturing an antibody-drug conjugate comprising the following:

[0049] Engineered bispecific antibody having the structure of Formula 03 below

[0050] - [Essence 03]

[0051]

[0052] At this time, AB is an antibody, and at this time, the antibody is an anti-R0R1 / B7-H3 bispecific antibody, and the anti-R0R1 / B7-H3 bispecific antibody comprises a first antigen-binding fragment targeting R0R1 and a second antigen-binding fragment targeting B7-H3, and

[0053] a is independently 0 or 1, and

[0054] x is 1 or 2 - ; and

[0055] Linker-payload compound having the structure of Formula 08 below

[0056] - [Essence 08]

[0057]

[0058] Contacting.

[0059] In some embodiments, the first antigen-binding fragment may comprise HCDR1 having the amino acid sequence of sequence number 1, HCDR2 having the amino acid sequence of sequence number 2, HCDR3 having the amino acid sequence of sequence number 3, 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 the second antigen-binding fragment may comprise HCDR1 having the amino acid sequence of sequence number 30, HCDR2 having the amino acid sequence of sequence number 31, HCDR3 having the amino acid sequence of sequence number 32, LCDR1 having the amino acid sequence of sequence number 33, LCDR2 having the amino acid sequence of sequence number 34, and LCDR3 having the amino acid sequence of sequence number 35.

[0060]

Effects of the Invention

[0061] 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.

[0062]

Brief Description of the Drawing

[0063] Figure 01 relates to an example of a 2+2 format bispecific antibody.

[0064] Fig. 02 relates to an example of a 2+1 format bispecific antibody.

[0065] FIG. 03 relates to an example of an anti-R0R1 / B7-H3 bispecific antibody in a 2+2 format according to some embodiments of the present disclosure.

[0066] FIG. 04 relates to an example of an anti-R0R1 / B7-H3 bispecific antibody in a 2+1 format of the present disclosure.

[0067] FIG. 05 relates to an example of a 2+2 format bispecific antibody of the present disclosure expressed through a single specific antibody for R0R1 and a second antigen-binding fragment.

[0068] FIG. 06 relates to an example of a bispecific antibody of the present disclosure in a 2+1 format expressed through a single specific antibody for R0R1 and a second antigen-binding fragment.

[0069] Figure 07 relates to an example of a bispecific antibody engineered through glyco-engineering.

[0070] FIG. 08 relates to an example of an antibody-drug conjugate (DAR4) of a 2+2 bispecific antibody of the present disclosure.

[0071] FIG. 09 relates to an example of an antibody-drug conjugate (DAR4) of a 2+1 bispecific antibody of the present disclosure.

[0072] FIG. 10 relates to an example of an antibody-drug conjugate (DAR2) of a 2+2 bispecific antibody of the present disclosure.

[0073] FIG. 11 relates to an example of an antibody-drug conjugate (DAR2) of a 2+1 bispecific antibody of the present disclosure.

[0074] FIG. 12 relates to an example of an antibody-drug conjugate (DAR2) of a 2+1 bispecific antibody of the present disclosure.

[0075] Figure 13 is intended to illustrate the structure of the antibody-drug conjugate of DAR2 in antibody-drug conjugates containing 2+2 bispecific antibodies.

[0076] Figure 14 is intended to illustrate the structure of a DAR2 antibody-drug conjugate in an antibody-drug conjugate containing a 2+2 bispecific antibody.

[0077] FIG. 15 relates to an example of an antibody-drug conjugate of the present disclosure. FIG. 16 relates to an example of an antibody-drug conjugate of the present disclosure. FIG. 17 relates to the RP-UPLC chromatogram results of BA6(M12)xB5 SYNtecan E™ ADC.

[0078] Figure 18 relates to the results of the binding activity analysis of the bispecific antibody BA6xB5 on various cell lines. Specifically, Figure 18 shows the results of the binding activity analysis of BA6xB5 on CH0-huR0Rl-huB7H3, PA-1, and A549 cell lines.

[0079] Figure 19 relates to the results of the binding activity analysis of the bispecific antibody BA6xB5 on various cell lines. Specifically, Figure 19 shows the results of the binding activity analysis of BA6xB5 on MDA-MB-231, Calu-3, and NCI-H446 cell lines.

[0080] Figure 20 shows the results of the analysis of the binding activity of ABL206 on various cell lines (CHO- huRORl- huB7H3, PA-1, Calu- 3).

[0081] Figure 21 shows the results of the binding activity analysis of ABL206 for various cell lines (HCC1187, NCI-H446, NCI-H82).

[0082] Figure 22 shows the results of the binding activity analysis of ABL206 against various cell lines (MDA-MB-231, Calu-6, A549).

[0083] Figure 23 shows the results of the binding activity analysis of ABL206 on various cell lines (DU-145, KATOI II).

[0084] Figure 24 relates to the results of the internalization capability evaluation of ABL206.

[0085] Figure 25 shows the results of the in vitro cytotoxicity evaluation of ABL206 in various cell lines (Calu—3, HCC1187, PA—1, A549).

[0086] Figure 26 shows the results of the in vitro cytotoxicity evaluation of ABL206 in various cell lines (CHO- huRORl- huB7H3, CHO-huRORl, KATOI I-huB7H3, KATOI II).

[0087] Figure 27 relates to the results confirming that ABL2067 induces cell cycle arrest. Figure 28 relates to the results confirming that ABL206 induces apoptosis. Figure 29 relates to the results confirming that ABL206 induces apoptosis through caspase-3 / 7 activation.

[0088] Figure 30 shows the results of confirming the bystander effect of ABL206. Figure 31 shows the results of evaluating the antitumor activity of ABL206 in the PA-1 CDX model.

[0089] Figure 32 shows the results of the evaluation of the antitumor activity of ABL206 in the PA-1 CDX model. The antitumor activity of ABL206 was compared with benchmark drugs (VLS-101 and DS-7300). Figure 33 shows the results of the evaluation of the antitumor activity of ABL206 in the Calu-3 CDX model. The antitumor activity of ABL206 was compared with benchmark drugs (VLS-101 and DS-7300).

[0090] Figure 34 shows the results of the evaluation of the antitumor activity of ABL206 in a TNBC PDX model.

[0091] Figure 35 shows the results of the evaluation of the antitumor activity of ABL206 in an NSCLC PDX model.

[0092] Figure 36 shows the results of the evaluation of the antitumor activity of ABL206 in an NSCLC PDX model.

[0093] Figure 37 shows the results of the evaluation of the antitumor activity of ABL206 in a head and neck cancer PDX model.

[0094] Figure 38 shows the results of the evaluation of the antitumor activity of ABL206 in an ovarian cancer PDX model.

[0095] Figure 39 shows the results of the evaluation of the antitumor activity of ABL206 in an ovarian cancer PDX model.

[0096] Figure 40 shows the results of the evaluation of the antitumor activity of ABL206 in a pancreatic cancer PDX model.

[0097] Figure 41 shows the results of the evaluation of the antitumor activity of ABL206 in a sarcoma PDX model.

[0098] Figure 42 shows the results of confirming the expression of R0R1 in various human cancer types (breast cancer, TNBC, prostate cancer, lung cancer (including NSCLC and SCLC), ovarian cancer, head and neck cancer, sarcoma, neuroendocrine tumor, gastric cancer) via IHC. Figure 43 shows the results of confirming the expression of B7-H3 in various human cancer types (breast cancer, TNBC, prostate cancer, lung cancer (including NSCLC and SCLC), ovarian cancer, head and neck cancer, sarcoma, neuroendocrine tumor, gastric cancer) via IHC.

[0099] Figure 44 shows the results of the evaluation of the antitumor activity of ABL206 in the SCLC PDX model.

[0100] Figure 45 shows the results of the evaluation of the antitumor activity of ABL206 in the LCNEC PDX model.

[0101] Figure 46 shows the results of the evaluation of the antitumor activity of ABL206 in a prostate cancer PDX model.

[0102] Figure 47 shows the results of the binding affinity analysis of the anti-R0R1 antibodies BA6CM11 and BA6(M12) for human R0R1.

[0103] Figure 48 shows the results of the binding affinity analysis of the dual-specific antibodies BA6(Mll)xB5 and BA6(M12)xB5 for human R0R1 and human B7-H3.

[0104] Figure 49 shows the results of an in vitro cytotoxicity analysis of BA6(Mll)xB5 SYNtecan E™ ADC and BA6(M12)xB5 SYNtecan E™ ADC in PA-1 cancer cell lines.

[0105] Figure 50 shows the results of an in vitro cytotoxicity analysis of BA6(Mll)xB5 SYNtecan E™ ADC and BA6(M12)xB5 SYNtecan E™ ADC in PA-1 cancer cell lines.

[0106] Figure 51 shows the results of the in vitro cytotoxicity analysis of ABL206 in DU-145 and CH0-R0R1-B7H3 cell lines. The cytotoxicity of ABL206 was compared with ABL206-Dxd, which was prepared by conjugating Deruxtecan (GGFG-Dxd) to ABL206 naked Ab.

[0107] Figure 52 shows the results of an in vitro cytotoxicity analysis of BA6(Mll)xB5 SYNtecan E™ ADC, a 2+2 ADC, and BA6(Mll)xyB5 SYNtecan E™ ADC, a 2+1 ADC, in various cell lines (CHO-huRORl-B7H3, KATOI I-huB7H3, HCC1187, Calu-3). Figure 53 shows the results of an in vitro cytotoxicity analysis of BA6(Mll)xB5 SYNtecan E™ ADC, a 2+2 ADC, and BA6(Mll)xyB5 SYNtecan E™ ADC, a 2+1 ADC, in various cell lines (CHO-huRORl, KATOIII, PA-1, A549).

[0108]

Form for carrying out the invention

[0109] 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.

[0110] 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.

[0111] As used in this disclosure, the term "comprising" and its derivatives refer to the specified feature(s),

[0112] It is an open-ended term specifying the existence of element(s), component(s), group(s), integer(s), and / or step(s), but not excluding the existence of other unspecified feature(s), element(s), component(s), group(s), integer(s), and / or step(s). This also applies to words with similar meanings, such as "including," "having," and their derivatives.

[0113] As used in this disclosure, the term “consisting of” and its derivatives are closed-ended terms specifying the existence of specified feature(s), element(s), component(s), group(s), integer(s), and / or step(s), and other unspecified feature(s), element(s), component(s), group(s), integer(s), and / or step(s The existence of s) ) is used to exclude.

[0114] As used in this disclosure, the term “consisting essentially of” refers to the existence of the specified feature(s), element(s), component(s), group(s), integer(s), and / or step(s), as well as the fundamental and novel properties of such feature(s), element(s), component(s), group(s), integer(s), and / or step(s). It is used to identify factors that do not have a substantial impact.

[0115] 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.

[0116] Furthermore, the terms and expressions used in this disclosure should be understood as being used for illustrative purposes rather than for limitation. All publications, patents, and other references mentioned in this disclosure are incorporated by reference in their entirety.

[0117] 1. Definition of Terms

[0118] 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.

[0119] 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.

[0120] As used in this disclosure, the term "disease" refers to any condition or disorder that impairs and / 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 alleviating, reducing, and / or inhibiting 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 of this disclosure, or any act that causes symptoms to improve or change.

[0121] As used in this disclosure, the term "therapeutic effective dose" refers to an amount sufficient to influence a beneficial or desired clinical outcome during treatment. The therapeutic effective dose may be administered to a subject on a single or more occasions. The therapeutic effective dose 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 dose of an active substance (e.g., antibody-drug).

[0122] It may include conjugates.

[0123] 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.

[0124] As used in this specification, the term "amino acid" 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. As used in this specification, the term "amino acid residue" refers to a structure derived from an amino acid that is covalently connected to another part of a compound, peptide, and / or protein (e.g., antibody, etc.) (e.g., with an adjacent amino acid residue) contained in the compound, peptide, and / or protein. Unless otherwise stated, amino acid sequences described in this disclosure are written using amino acid single-character or three-character notation, from the N-terminal to the C-terminal. For amino acids that cannot be represented by the single-character notation, other characters are used, and further explanations are provided. The amino acids and the three-character or single-character abbreviations used in the art for each amino acid are as follows:

[0125] Alanine: Ala, A; Arginine: Arg, R;

[0126] 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;

[0127] Lysine: Lys, K; Methionine: Met, M;

[0128] Phenylalanine: Phe, F; Proline: Pro, P; Serine: Ser, S; Threonine: Thr, T; Tryptophan: Trp, W; Tyrosine: Tyr, Y; Valine: Vai, V.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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 there cannot be any other additional elements 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 this.

[0133] 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 comprise antigen-binding fragments. For example, a bispecific antibody may comprise 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.

[0134] 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 scFvs are well known in the art. In scFvs, 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『 VL or VH-1 linker- VL directionality). For example, the linker used in scFv (i.e., 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, it may have specificity for at least one specific antigen, like immunoglobulin. scFv is used for various applications, such as flow cytometry, immunohistochemistry, bispecific antibodies, and antigen-binding domains 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:

[0135] GGGGSGGGGSGGGGSGGGGS (also denoted as (GGGGS)4 or (G4S)4, sequence number 47); and GGGGSGGGGSGGGGS (also denoted as (GGGGS)3 or (G4S)3, sequence number 48).

[0136] In this disclosure, “target antigen” is used to refer to an antigen to which an antigen-binding fragment can bind (e.g., specifically and / or selectively bind), and / or an antigen that is specifically and / or selectively recognized by an antigen-binding fragment. The relationship between an antibody or an antigen-binding fragment, such as Fab, and scFv, and its target antigen is well known in the art.

[0137] 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 are, 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, GD2, GD3, BCMA, TnAg, PSMA, R0R1, FLT3, NKG2D, TAG72, CD38, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, Mesothelin, IL-IIRa, PSCA, PRSS21, VEGFR2, LewisY, There are CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor a α, ERBB2 (Her2 / neu), MUC1, NCAM, Prostase, PAP, ELF2M, Ephr in B2, FAP, IGF-I receptor, EGFR, CAIX, LMP2, gplOO, and bcr-abl, etc. For example, R0R1 and B7-H3 are tumor antigens.

[0138] 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.

[0139] 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. Meanwhile, 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 expressed or made up of a certain sequence” and “is expressed or made up of a certain chemical formula,” respectively.

[0140] Where a sequence is disclosed in the present disclosure, it may be understood that a sequence to 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.

[0141] 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.

[0142] 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.

[0143] [Reaction Schematic 01]

[0144]

[0145] Here, A and B are each arbitrary groups and are symbols used to describe the reaction between azide and BCN.

[0146] 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 interpreted and understood appropriately according to the context. Examples of drugs include auristatin, eribulin, tubulysin, geldanamycin,

[0147] Maytansinoid, cali cheami cin,

[0148] Mertansine, daunomycin, doxorubicin, methotrexate, vindesine, SG2285,

[0149] 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,

[0150] Duocarmycin, enediyne antibiotics, esperamycin, epotilone, and

[0151] There are pyrrolobenzodiazepines (PBD) or derivatives.

[0152] 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

[0153] 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, antibodies or antigen-binding fragments may be unnaturally generated, for example, recombinant or synthetically generated. For example, antibodies or their antigen-binding fragments may be produced by hybridomas, recombinant DNA technology, or enzymatic or chemical cleavage of intact antibodies, etc. In this disclosure, unless otherwise noted, antibodies may include antibodies 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.

[0154] In the antibodies provided in the present 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 the present disclosure may be human IgG type antibodies, e.g., IgGl, IgG2, IgG3, or IgG4 type antibodies, but are not limited thereto.

[0155] 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). Herein, 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).

[0156] A bispecific antibody having two first antigen-binding fragments and two second antigen-binding fragments will be understood as an antibody that is bivalent to each of the two types of antigens.

[0157] A bispecific antibody having two first antigen-binding fragments and one second antigen-binding fragment will be understood as an antibody that is divalent to the first antigen and monovalent to the second antigen. A representative example of a bispecific antibody having two first antigen-binding fragments and two second antigen-binding fragments is a monospecific antibody having two first antigen-binding fragments in which two second antigen-binding fragments (e.g., scFv) are connected to the C-terminus of each heavy chain of said monospecific antibody.

[0158] The term "Knobs-into-holes (KIH)" as used in this disclosure is a method or technique for facilitating heterodimerization or heavy chain dimer formation by manipulating the CH3 domain of an antibody to create a "knob" or a "hole" in each heavy chain, 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. Examples of amino acid sequences of the human IgGl-derived heavy chain constant region are described in SEQ No. 21 (in the amino acid sequence of SEQ No. 21, CH3 is the region consisting of the 224th to 330th amino acid residues), which may be referenced to identify the amino acids subject to substitution. For example, the heavy chain of the knob structure contains the T366W substitution, and the heavy chain of the hole structure contains the T366S, L368A, and Y407V substitutions. As another example, one heavy chain may contain one or more of the substitutions Y349C and T366W, and another heavy chain may contain one or more of the substitutions E356C, T366S, L368A, and Y407V. As a non-limiting example, examples of knob-hole pairs of KIH technology that promote or enable dimer formation are shown in Table 01 below. In Table 01 below, the Eu numbering system is used to indicate the position numbers of amino acids.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 a bispecific antibody).

[0159] [Table 01] Pairs of known knob mutations and hole mutations

[0160] Name Knob mutation Hole mutation References

[0161] Pair 1 S354C, T366W Y349C, T366S, Carter, P. (2001). Bispecific L368A, Y407V

[0162]

[0163] human IgG by design. Journal of immunology

[0164] methods, 248(\-2) , 7-15.

[0165] Pair 2 T366W T366S, L368A, Atwel 1 , S. , Ridgway, J . B. , Y407V Wel ls, J . A. , & Carter , P .

[0166] (1997). Stable heterodimers from remodeling the domain interface of a homodimer using a phage display

[0167] 1 ibrary. Journal of molecular biology, 270(1), 26-35.

[0168] Pair 3 T366Y Y407T Ridgway, J . B., Presta, L. G.,

[0169] & Carter, P. (1996). 'Knobs— into— holes' engineering of antibody CH3 domains for heavy chain heterodimerization.

[0170] Protein Engineering, Design and Selection, 9(7), 617-621.

[0171]

[0172] 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, the Xss cs' is approximately perpendicular to the bond.

[0173] Wavy lines drawn in the direction (wavy line) (e.g. 0 Use ■, , ) for example.

[0174] When expressed as such, it indicates that Group X within a substance, molecule, or compound is connected to other parts through bonds. For example,

[0175] 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 the X group is depicted, the structure

[0176]

[0177] X

[0178] It can be expressed as follows. For example, in a compound having the structure "A-X-B", when only the X structure is depicted,

[0179]

[0180] It can be expressed as. If necessary, wavy lines drawn nearly perpendicular to the bonds may be represented through stock price indications. For example, in a compound having the structure A-X-B (where A, X, and B are each arbitrary groups, symbols for explaining wavy lines drawn approximately perpendicular to the bonds), if only the structure of the X group is drawn, the structure of the X group is,

[0181] I

[0182] X

[0183] If necessary, it can be city, and in this case "* is arc WWV connected to A".

[0184] By mentioning things like "it is a part (or attachment part with A) and ** is a part connected to B (or attachment part with B)," you can provide X information about which part each wavy line indicates a connection to.

[0185] The compounds of AAAA* in the present disclosure may have specific geometric or stereoisomer forms. Where a compound is disclosed in the present disclosure unless otherwise specified, the cis and trans isomers, (-)- and (+)- enantiomers of said compound.

[0186] Isomers such as enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemics are included within the scope of this disclosure. That is, isomers related to isomers in the chemical formulas or structures disclosed in this disclosure

[0187] In the absence of a notation (e.g., , , s and 'hae', etc.), the disclosed chemical formula or structure may encompass all possible isomers.

[0188] Where compounds (e.g., small compounds, proteins (e.g., antibodies), and conjugates, etc.) are disclosed in this specification, it shall 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 ') , chloride (CP) , citrate (citrate ) , cyanide (cyanide) , fluoride (F“) ,

[0189] Examples include nitrate (NO2'), nitrate (PO3'), and sulfate (SO₄'), and are not otherwise limited. Ions that form salts commonly used in the art may be used to form the salt of the compound as needed. 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, if the 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.

[0190] 2. Background - Understanding Antibody Structure

[0191] In the following, the structure of antibodies is described in detail based on what is generally known in the art to aid in understanding antibody structures, and the scope of the present disclosure is not limited by the description below.

[0192] The structure of an antibody is divided into a heavy chain and a light chain depending on the type of chain. Antibodies are generally known to contain two heavy chains and two light chains. 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).

[0193] 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 CDR according to any one of the kabat numbering system, the Chothi numbering system, and the Mart-in numbering system.For example, any one of the numbering systems, such as the Kabat numbering system, Chothia numbering system, IMGT numbering system, and Mart-in numbering system, may be used to refer to the location of a CDR sequence.

[0194] The part of the variable region that is not CDR is referred to as the framework region (FR).

[0195] The constant region of an 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 be connected) 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.

[0196] 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.

[0197] 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, and 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.

[0198] 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 position 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 through this N-glycosylation site. Meanwhile, many studies are being conducted to engineer this Asn297 site and / or N-glycan (or Asn297-linked glycan) to improve antibody function or confer drug conjugation to the antibody, and such engineering is referred to as glyco-engineering.

[0199] Meanwhile, bispecific antibodies, particularly the 2+2 bispecific antibody format or 2+1 bispecific antibody format associated with the bispecific antibody of the present disclosure, are designed to further include an antigen-binding fragment capable of binding to this additional target antigen so as to be able to target an additional target antigen compared to a monospecific antibody. This antigen-binding fragment may be referred to as a second antigen-binding fragment. In some embodiments, the bispecific antibody of the present disclosure may be understood to further include one or two second antigen-binding fragments in addition to the monospecific antibody. For example, each second antigen-binding fragment (e.g., scFv) is attached to the C-terminus of the heavy chain (or Fc region) of the monospecific antibody. Specifically, with respect to the bispecific antibody as a whole, the second antigen-binding fragment may be understood to be attached to the C-terminus of the heavy chain constant region. This second antigen-binding fragment may be linked to the heavy chain constant region via a short peptide linker (e.g., a peptide linker having a length of 5 to 50 aa). At least one of the two heavy chains of the bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment in the direction from the N-terminus to the C-terminus. For example, a 2+2 format bispecific antibody may have each of the two heavy chains including a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment in the direction from the N-terminus to the C-terminus. As another example, a 2+1 format bispecific antibody may have one of the two heavy chains containing a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment from the N-terminus to the C-terminus, and the other of the two heavy chains containing a heavy chain variable region and a heavy chain constant region from the N-terminus to the C-terminus.For example, the heavy chain of a bispecific antibody containing the aforementioned second antigen-binding fragment can be described by the following structure, which is illustrated from the N-terminus to the C-terminus: [VH] - [CH] - [second antigen-binding fragment], where each indicates that the indicated domains are covalently linked and encompasses modes of direct linkage and indirect linkage (e.g., linked through structures such as amino acid sequences or linkers). For example, "-" may each independently indicate direct linkage or indirect linkage. For example, "-" may indicate direct linkage (e.g., linked through covalent or peptide bonds).

[0200] 3. Background - Format of Bispecific Antibodies 3.1. Overview of Bispecific Antibody Formats

[0201] In this section "3. Background - Format of Bispecific Antibodies," the format of bispecific antibodies is described in detail based on what is generally known in the art to aid in understanding bispecific antibodies, and the scope of the present disclosure is not limited by the description in this section.

[0202] Bispecific antibodies are antibodies designed to target two different types of antigens, and various formats of bispecific antibodies are known in the art. For example, formats of bispecific antibodies include 2+2 format bispecific antibodies having two binding sites for each of the two types of antigens (also referred to as 2+2 bispecific antibodies), 2+1 format bispecific antibodies having two binding sites for one of the two types of antigens and one binding site for the other antigen (also referred to as 2+1 bispecific antibodies), and 1+1 format bispecific antibodies having one binding site for one of the two types of antigens and one binding site for the other antigen (also referred to as 1+1 bispecific antibodies). This section describes examples of 2+2 format bispecific antibodies and 2+1 format bispecific antibodies. In this section "3. Background - Format of Bispecific Antibodies," for convenience, one of the two types of antigens targeted by the bispecific antibody is referred to as Antigen A, and the other of the two types of antigens is referred to as Antigen B. For example, Antigen A may be R0R1 and Antigen B may be B7-H3.

[0203] 3.2. 2+2 Format Bispecific Antibodies

[0204] Bispecific antibodies can be designed to have two binding sites for each of two different types of antigens. That is, a bispecific antibody can be designed to have two binding sites for antigen A and two binding sites for antigen B. A bispecific antibody designed in this way has a total of four binding sites (i.e., two binding sites for one antigen and two binding sites for the other antigen). Such a bispecific antibody is referred to as a 2+2 format bispecific antibody, a 2+2 type bispecific antibody, or a 2+2 bispecific antibody.

[0205] A 2+2 format bispecific antibody may have a total of four antigen-binding fragments, two of which are antigen-binding fragments for antigen A, and the remaining two of which are antigen-binding fragments for antigen . That is, a 2+2 format bispecific antibody may contain two antigen-binding fragments for antigen A and two antigen-binding fragments for antigen .

[0206] For example, a 2+2 format bispecific antibody can be designed to include an intact IgG-shaped antibody against antigen A (i.e., a Y-shaped, full 1-length IgG-shaped antibody) and two antigen-binding fragments against antigen A. Here, the antibody against antigen A having an intact IgG-shaped antibody has two antigen-binding fragments against antigen A. Here, the antigen-binding fragments against antigen A can be attached to the C-terminus of each of the two heavy chains of the intact IgG-shaped antibody. Additionally, the antigen-binding fragments against antigen A can be scFv. That is, a 2+2 format bispecific antibody can include a single-specific intact IgG-shaped antibody against antigen A and two antigen-binding fragments against antigen A.

[0207] A 2+2 bispecific antibody comprising a monospecific antibody against antigen A in the intact IgG form and two antigen-binding fragments against antigen B may comprise two heavy chains of bispecific antibodies and two light chains of bispecific antibodies. Here, the heavy chains of the two bispecific antibodies may have a symmetric (or identical) structure. For example, each of the heavy chains of the two bispecific antibodies may comprise a heavy chain variable region, a heavy chain constant region, and an antigen-binding fragment against antigen B (e.g., scFv) of the antigen-binding fragment against antigen A. Here, the antigen-binding fragment against antigen B may be connected to the C-terminus of the heavy chain constant region via a linker (e.g., a peptide linker). The heavy chains of the bispecific antibodies may be described as comprising the heavy chain of the antibody against antigen A (i.e., the heavy chain of the antibody in the intact IgG form) and the antigen-binding fragment against antigen B, but are not otherwise limited. The light chain of a bispecific antibody may include a light chain variable region and a light chain constant region of the antigen-binding fragment for antigen A. The heavy chain variable region of the antigen-binding fragment for antigen A and the variable region of the light chain of the bispecific antibody (i.e., the light chain variable region) may form an antigen-binding fragment for antigen A.

[0208] An example of such a 2+2 bispecific antibody structure is shown in FIG. 01. FIG. 01 shows a portion corresponding to the antibody against antigen A in the aforementioned intact IgG form, a portion corresponding to the antigen-binding fragment against antigen A, a portion corresponding to the antigen-binding fragment against antigen A, a light chain of the bispecific antibody, and a heavy chain of the bispecific antibody.

[0209] 3.3. 2+1 Format Bispecific Antibodies

[0210] Bispecific antibodies can be designed to have two binding sites for one of two different types of antigens and one binding site for the other. That is, a bispecific antibody can be designed to have two binding sites for antigen A and one binding site for antigen B. A bispecific antibody designed in this way has a total of three binding sites (i.e., two binding sites for one antigen and one binding site for the other antigen). Such a bispecific antibody is referred to as a 2+1 format bispecific antibody, a 2+1 type bispecific antibody, or a 2+1 bispecific antibody.

[0211] A 2+1 format bispecific antibody may have a total of three antigen-binding fragments, two of which are antigen-binding fragments for antigen A, and the remaining one of which may be an antigen-binding fragment for antigen . That is, a 2+1 format bispecific antibody may include two antigen-binding fragments for antigen A and one antigen-binding fragment for antigen .

[0212] For example, a 2+1 format bispecific antibody can be designed to include an intact IgG-shaped antibody against antigen A (i.e., a Y-shaped, full l-length IgG shape) and an antigen-binding fragment against one antigen. Here, the antibody against antigen A having an intact IgG shape has two antigen-binding fragments against antigen A. Here, the antigen-binding fragment against antigen A may be attached to the C-terminus of either of the two heavy chains of the intact IgG-shaped antibody. Additionally, the antigen-binding fragment against antigen A may be a scFv. That is, a 2+1 format bispecific antibody may include a single-specific intact IgG-shaped antibody against antigen A and one antigen-binding fragment against antigen A.

[0213] A 2+1 bispecific antibody containing a single specific antibody for antigen A in the shape of an intact IgG and one antigen-binding fragment for antigen A may comprise two heavy chains of bispecific antibodies and two light chains of bispecific antibodies. Here, the heavy chains of the two bispecific antibodies may have structures that are not symmetric to each other. For example, one of the two heavy chains of the 2+1 bispecific antibody may have an antigen-binding fragment for antigen A on the C-terminal side, while the other of the two heavy chains may not have an antigen-binding fragment for antigen A on the C-terminal side. For example, one of the two heavy chains of the 2+1 bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and an antigen-binding fragment for antigen A (e.g., scFv), and the other of the two heavy chains may include a heavy chain variable region and a heavy chain constant region of the antigen-binding fragment for antigen A. In a heavy chain containing an antigen-binding fragment for an antigen, the antigen-binding fragment for the antigen may be linked to the C-terminus of the heavy chain constant region via a linker (e.g., a peptide linker). The heavy chain of a bispecific antibody containing an antigen-binding fragment for an antigen may be described as containing the heavy chain of the antibody against antigen A (i.e., the heavy chain of an intact IgG-shaped antibody) and the antigen-binding fragment for antigen B, but is not otherwise limited. The light chain of the bispecific antibody may contain a light chain variable region and a light chain constant region of the antigen-binding fragment for antigen A. The heavy chain variable region of the antigen-binding fragment for antigen A and the variable region of the light chain of the bispecific antibody may form the antigen-binding fragment for antigen A.

[0214] An example of such a 2+1 bispecific antibody structure is illustrated in FIG. 02. FIG. 02 shows a portion corresponding to the antibody against antigen A in the shape of the aforementioned intact IgG, a portion corresponding to the antigen-binding fragment against antigen A, a portion corresponding to the antigen-binding fragment against antigen B, a light chain of the bispecific antibody, and among the two heavy chains of the bispecific antibody, a heavy chain containing the antigen-binding fragment against antigen B and a heavy chain without the antigen-binding fragment against antigen B. Meanwhile, a design may be introduced into the heavy chain of the bispecific antibody having an asymmetric heavy chain, such as the 2+1 bispecific antibody, to induce the bispecific antibody to be formed as intended. For example, the heavy chain invariant region of one heavy chain of a bispecific antibody (e.g., heavy chain invariant region 3) and the heavy chain invariant region of another heavy chain (e.g., heavy chain invariant region 3) can be designed to be dimerizable with each other, and this design allows one heavy chain and another heavy chain of a bispecific antibody with an asymmetric structure to pair and dimerize as intended. A representative example of a technique for the dimerization of two heavy chains with an asymmetric structure is "Knobs-into-holes (KIH)". For example, one heavy chain of a bispecific antibody may contain a knob mutation, and the other heavy chain may contain a hole mutation.

[0215] 4. Background - Difficulties in ADC Development

[0216] 4.1. Overview

[0217] 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 present disclosure.

[0218] 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 drug, which induces cell death, can be selectively released into the intracellular environment by selectively binding only to cells of interest (i.e., target cells) that express a target antigen.

[0219] 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).

[0220] 4.2. ADC Design Process

[0221] 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.

[0222] The following describes the representative factors and considerations that can generally be taken into account when designing a device capable of achieving objectives. However, the following does not limit the process or factors considered in ADC design. In reality, to design a device capable of achieving objectives, ADC developers consider 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.

[0223] 4.2.1. Selection of Target Antigens

[0224]

[0225] A target antigen is selected. The target antigen must be a cell surface-expressed protein that is expressed on the cell surface and, at the same time, allows the antibody to be internalized into the cell when it binds to that protein. Additionally, the target antigen needs to be selected as an antigen 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. Furthermore, in this process, both the expression characteristics and biological characteristics of the target antigen are considered. For example, since the degree of expression of the target antigen in normal tissue is related to the on-target toxicity of the antibody (toxicity caused by the action of an ADC on normal cells expressing the target antigen), the antibody is designed with this in mind.

[0226] 4.2.2. Design of Antibodies

[0227]

[0228] 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) (especially, CDRs) that confers binding affinity to the antibody for the antigen. The characteristics of the selected target antigen are taken into account in the design of the antigen-binding fragment.

[0229] 4.2.3. Drug Design

[0230]

[0231] 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. It is known that some known drugs are unsuitable for certain types of target cells. Furthermore, depending on the type of tumor cell expressing the target antigen or the tumor associated with the target antigen, a potent drug may be required. As such, the drug's MoA and potency are important factors to consider regarding anticancer activity. However, the drug's MoA and potency are also strongly linked to the toxicity of the ADC. While drugs with high potency increase the anticancer activity of the ADC, they also increase toxicity. Meanwhile, since the activity of a drug is influenced not only by the characteristics of the drug itself but also by the tumor target specificity, binding affinity to target antigens, internalization ability, and release ability of the linker, other characteristics of the ADC must also be considered. Furthermore, since the structure of the drug can affect the stability of the ADC, consideration of the drug structure is also required.

[0232] 4.2.4. Linker Design

[0233]

[0234] 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.

[0235] 4.2.5. Others

[0236]

[0237] 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.

[0238] 4.3. Unpredictability of ADC Effects and Design Difficulties

[0239] 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 a drug 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 binding 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.

[0240] First, anticancer activity is influenced by various variables, such as antigen characteristics, antigen binding affinity and specificity of the ADC, ADC internalization ability, ADC cellular inhibitory ability, the antibody's own cellular inhibitory ability, ADC stability, drug release characteristics, mechanism of action, drug potency, and antibody-drug interaction (DAR). These variables do not act independently 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 an ADC's anticancer activity based solely on structural elements.

[0241] 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, characteristics of the target antigen, action on normal cells expressing the target antigen, the mechanism of action, the potency, and drug release characteristics, all of which directly affect toxicity. Meanwhile, variables affecting anticancer activity—such as the antigen-binding affinity and specificity of the milk, the milk's internalization capacity, the ADC's cellular inhibitory ability, and the potency—also influence toxicity when the ADC acts. 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, just as with anticancer activity, there are inherent limitations to predicting the magnitude of ADC toxicity based solely on structural factors.

[0242] Furthermore, anticancer activity and toxicity are not completely distinct and independent characteristics. While an increase in anticancer activity and a decrease in toxicity signify 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, the use of a drug with high potency can increase anticancer activity but simultaneously increase the toxicity of the ADC. Conversely, the use of a drug with low potency can reduce the toxicity of the ADC but lowers its anticancer activity. Additionally, designs aimed at increasing binding affinity to target antigens or increasing DAR to enhance anticancer activity in dairy cows also lead to increased toxicity. Conversely, designs that lower binding affinity to target antigens to reduce toxicity, designs that lower DAR, etc., hinder ADCs from maintaining anticancer activity.

[0243] As such, the effect of yo is closer to a complex property, or emergent property, 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 anticancer activity and toxicity sufficient to achieve the goal of treatment through administration. In fact, to determine whether a developed ADC is suitable for entering clinical trials, extensive experimental performance evaluations, including multiple anticancer activity and toxicity tests, are essential. 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.

[0244] In addition, as explained earlier, since the anticancer activity and toxicity of dairy products are determined by the non-linear interaction of multiple variables and structural factors, careful consideration during the design phase is essential to increase the likelihood that the ADC will exhibit its intended effect. However, this 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, 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.

[0245] 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.

[0246] 5. Antibody-drug conjugate

[0247] 5.1. Antibody-Drug Conjugate - Overview

[0248] 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).

[0249] 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.

[0250] The bispecific antibody of the antibody-drug conjugate of the present disclosure is a bispecific antibody capable of binding to R0R1 and B7-H3. That is, the target antigens of the bispecific antibody of the antibody-drug conjugate of the present disclosure are R0R1 and B7-H3. In some embodiments, the bispecific antibody of the antibody-drug conjugate of the present disclosure is ROIt may be divalent with respect to RI and divalent with respect to B7-H3. In some embodiments, the bispecific antibody of the antibody-drug conjugate of the present disclosure may be divalent with respect to R0R1 and monovalent with respect to B7-H3. 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 R0R1 and B7-H3, but are not limited thereto. For example, the antibody-drug conjugate of the present disclosure is an antibody-drug conjugate capable of binding to R0R1 and B7-H3. For example, the antibody-drug conjugate of the present disclosure may be referred to as an antibody-drug conjugate against R0R1 and B7-H3, an anti-R0R1 and anti-B7-H3 antibody-drug conjugate, an anti-R0R1 / B7-H3 antibody-drug conjugate, and is not otherwise limited.

[0251] In the following, each component of the antibody-drug conjugate of the present disclosure and the technology used in the manufacture of the antibody-drug conjugate will be described in detail.

[0252] 5.2. Elements of the Antibody-Drug Conjugate

[0253] 5.2.1. Antibodies: Anti-R0R1 and anti-B7-H3 bispecific antibodies (anti-R0R1 / B7-H3 bispecific

[0254]

[0255] Antibody

[0256] (1) Bispecific antibodies - Overview

[0257] 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.

[0258] 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 number PCT / KR2023 / 010149 (publication number W02024 / 014930A1), which is incorporated by reference in its entirety into the present disclosure.

[0259] The target antigens of the bispecific antibodies of the present disclosure are the R0R1 protein (i.e., R0R1) and the B7-H3 protein (i.e., B7-H3). That is, the bispecific antibodies may be bispecific antibodies targeting R0R1 and B7-H3. Bispecific antibodies targeting R0R1 and B7-H3 may be referred to as anti-R0R1 and anti-B7-H3 bispecific antibodies, anti-R0R1 / anti-B7-H3 bispecific antibodies, anti-R0R1 / B7-H3 bispecific antibodies, R0R1 / B7-H3 bispecific antibodies, etc., and are not otherwise limited. In some embodiments, the bispecific antibody of the present disclosure may be referred to as a bispecific antibody targeting R0R1 and B7-H3, a bispecific antibody binding to R0R1 and B7-H3, etc., and is not otherwise limited.

[0260] 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 R0R1. In some embodiments, R0R1 may be human R0R1 or a fragment thereof. Human R0R1 is known to have an amino acid sequence of sequence number 51 with a length of 937aa (see NCBI Accession No. NP_005003.2), but is not limited thereto. Human R0R1 is encoded by the human R0R1 gene (e.g., see NCBI Accession No. Gene ID: 4919). In some embodiments, R0R1 may be a variant of human R0R1 or a fragment thereof. For example, variants of human R0R1 may have amino acid sequences having sequence identity of 80% or more and less than 100% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more and less than 100%) of the amino acid sequence of sequence number 51. In some embodiments, the bispecific antibodies of the present disclosure may have the ability to bind to monkey R0R1, mouse R0R1 (see sequence number 52, GenBank Accession No. BAA75480.1), or rat R0R1.

[0261] In some embodiments, R0R1 may include an Ig-like domain. In some embodiments, a bispecific antibody or antibody-drug conjugate may have the ability to bind to the Ig-like domain of R0R1. In some embodiments, a bispecific antibody or antibody-drug conjugate may have the ability to bind to R0R1 containing an Ig-like domain. In some embodiments, the epitope of R0R1 of the bispecific antibody (i.e., the R0R1 epitope) may be a discrete epitope. Some

[0262] In an embodiment, the R0R1 epitope of the bispecific antibody may comprise one or more of Gly72, Gln73, Thr74, Glu76, His78, Argl03, Argl04, Serl06, Phel07, Argl08, ThrllO, I lelll, Tyrll2, Argl5, Argl7, Argl9, and Asnl20. In some embodiments, the R0R1 epitope of the bispecific antibody may comprise Gly72, Gln73, Thr74, Glu76, His78, Argl03, Argl04, Serl06, Phel07, Argl08, ThrllO, I lelll, Tyrll2, Argl5, Argl7, Argl9, and Asnl20. The numbers of the aforementioned amino acids may be set based on the amino acid sequence of sequence number 51, which is the reference amino acid sequence of human R0R1. For example, Gly72 may refer to Gly located at the 72nd position in the amino acid sequence of sequence number 51. In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to R0R1 comprising Gly72, Gln73, Thr74, Glu76, His78, Argl03, Argl04, Serl06, Phel07, Argl08, Thrll0, Ilelll, Tyrll2, Argll5, Argll7, Argll9, and Asnl20. In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to R0R1 comprising a region comprising Gly72, Gln73, Thr74, Glu76, His78, Argl03, Argl04, Serl06, Phel07, Argl08, ThrllO,I lelll, Tyrll2, Argl5, Argl7, Argl9, and Asnl20 (e.g., a region comprising the 72nd to 120th amino acid in the amino acid sequence of sequence number 51).

[0263] In some embodiments, the binding affinity of the bispecific antibody to R0R1 (e.g., human R0R1) may be about 10 nM 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 R0R1 may be approximately 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM 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 R0R1 may be 60 nM to 160 nM, 70 nM to 140 nM, 80 nM to 100 nM, or 85 nM to 95 nM based on the dissociation constant, but is not limited thereto.

[0264] In some embodiments, a bispecific antibody or antibody-drug conjugate may have the ability to bind to B7-H3. In some embodiments, B7-H3 may be human B7-H3 or a fragment thereof. Human B7-H3 is known to have an amino acid sequence of sequence number 53 with a length of 534aa (see NCBI Accession No. NP_001019907.1), but is not limited thereto. Human B7-H3 is encoded by the human B7-H3 gene (see, e.g., NCBI Accession No. Gene ID: 80381). In some embodiments, B7-H3 may be a variant of human B7-H3 or a fragment thereof. For example, a variant of human B7-H3 may have an amino acid sequence having sequence identity of 80% or more and less than 100% of the amino acid sequence of sequence number 53 (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more and less than 100%). In some embodiments, the bispecific antibody of the present disclosure may have the ability to bind to monkey B7-H3 (see NCBI Accession No. XP_005560056.1), white monkey B7-H3 (see NCBI Accession No. NP_598744.1), or rat B7-H3.

[0265] In some embodiments, a bispecific antibody or antibody-drug conjugate may have the ability to bind to the IgVl domain of human B7-H3 (also referred to as the Ig-1-like-VI domain or IgV-like domain). In some embodiments, B7-H3 may contain the IgVl domain. In some embodiments, a bispecific antibody or antibody-drug conjugate may have the ability to bind to the IgVl domain of B7-H3. In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to B7-H3 containing the IgVl domain. In some embodiments, the epitope of B7-H3 of the bispecific antibody (i.e., the B7-H3 epitope) may be a discontinuous epitope. In some embodiments, the B7-H3 epitope of the specific antibody may include one or more of Ala61, Gln62, Asn64, I le66, Gln68, Lys73, Leu75, Ser78, Ala80, Glu81, Gly82, Gln85, Phel23, Serl25, I lel26, Argl27, Aspl28, Phel29, Glyl30, and Serl31. In some embodiments, the B7-H3 pintops of the bispecific antibody may include Ala61, Gln62, Asn64, I le66, Gln68, Lys73, Leu75, Ser78, Ala80, Glu81, Gly82, Gln85, Phel23, Serl25, I lel26, Argl27, Aspl28, Phel29, Glyl30, and Serl31. The numbers of the aforementioned amino acids may be set based on the amino acid sequence of sequence number 53, which is the reference amino acid sequence of human B7-H3. For example, Ala61 may refer to Ala at the 61st position in the amino acid sequence of sequence number 53.In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to B7-H3 comprising Al a61, G ln62, Asn64, I l e66, G ln68, Lys73, Leu75, Ser78, Al a80, G lu81, G ly82, G ln85, Phel23, Serl25, I l el26, Argl27, Aspl28, Phel29, G lyl30, and Serl31. In some embodiments, a bispecific antibody or antibody-drug conjugate may bind to B7-H3 comprising a region comprising Al a61, Gln62, Asn64, I l e66, G ln68, Lys73, Leu75, Ser78, Al a80, G lu81, G ly82, G ln85, Phel23, Ser 125, I l el26, Argl27, Aspl28, Phel29, Glyl30, and Ser l31 (e.g., a region comprising the 61st to 131st amino acids in the amino acid sequence of sequence number 53).

[0266] In some embodiments, the binding affinity of the bispecific antibody to B7-H3 (e.g., human B7-H3) 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 B7-H3 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, 20nM, 22nM, 24nM, 26nM, 28nM, or 30nM, 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 B7-H3 may be 2 nM to 20 nM, 2 nM to 15 nM, 3 nM to 12 nM, 4 nM to 10 nM, or 4 nM to 8 nM based on the dissociation constant, but is not limited thereto.

[0267] In some embodiments, the bispecific antibody can bind to cells expressing R0R1 and / or B7-H3. For example, the bispecific antibody can bind to cells expressing R0R1 on the cell surface and / or B7-H3 on the cell surface. To this end, the bispecific antibody may have binding affinity for at least some of the regions of the R0R1 protein that protrude to the surface of the cell membrane (i.e., outside the cell) and / or at least some of the regions of the B7-H3 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 R0R1 and / or B7-H3.

[0268] In some embodiments, the bispecific antibody comprises a first antigen-binding fragment capable of binding to a first target antigen R0R1 and a second antigen-binding fragment capable of binding to a second target antigen B7-H3. In some embodiments, the first antigen-binding fragment has an antigen-binding portion capable of binding to R0R1, and the second antigen-binding fragment has an antigen-binding portion capable of binding to B7-H3. In some embodiments, the bispecific antibody may comprise an antigen-binding portion capable of binding to R0R1 and an antigen-binding portion capable of binding to B7-H3.

[0269] In some embodiments, the bispecific antibody may comprise two first antigen-binding fragments (i.e., antigen-binding fragments capable of binding to R0R1) and two second antigen-binding fragments (i.e., antigen-binding fragments capable of binding to B7-H3). For example, the bispecific antibody may have two first antigen-binding fragments and two second antigen-binding fragments. In some embodiments, the bispecific antibody of the present disclosure may have two antigen-binding portions capable of binding to R0R1 and two antigen-binding portions capable of binding to B7-H3. In some embodiments, the bispecific antibody may have a 2+2 format (or form). For example, the bispecific antibody may be a 2+2 bispecific antibody. In some embodiments, the bispecific antibody may be divalent to R0R1 and divalent to B7-H3. In some embodiments, the bispecific antibody may comprise two first antigen-binding fragments (i.e., antigen-binding fragments capable of binding to R0R1) and one second antigen-binding fragment (i.e., antigen-binding fragments capable of binding to B7-H3). For example, the bispecific antibody may have two first antigen-binding fragments and one second antigen-binding fragment. In some embodiments, the bispecific antibody of the present disclosure may have two antigen-binding portions capable of binding to R0R1 and one antigen-binding portion capable of binding to B7-H3. In some embodiments, the bispecific antibody may have a 2+1 format (or form). For example, the bispecific antibody may be a 2+1 bispecific antibody. In some embodiments, the bispecific antibody may be divalent to R0R1 and monovalent to B7-H3.

[0270] In some embodiments, the bispecific antibody may include a heavy chain constant region in addition to antigen-binding fragments. In some embodiments, the bispecific antibody may include a light chain constant region in addition to antigen-binding fragments. In some embodiments, a second antigen-binding fragment may be connected to the C-terminus of the heavy chain constant region. In some embodiments, the second antigen-binding fragment may be connected to the C-terminus of the heavy chain constant region of at least one of the two heavy chains. In some embodiments, the second antigen-binding fragment may be connected to the C-terminus of the heavy chain constant region of each of the two heavy chains. In some embodiments, the bispecific antibody may include a peptide linker connecting the heavy chain constant region and the second antigen-binding fragment.

[0271] In some embodiments, the bispecific antibody may include an Fc region. In some embodiments, the bispecific antibody may have two heavy chains and two light chains. In some embodiments, at least one of the two heavy chains of the bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment.

[0272] In some embodiments, the bispecific antibody may be a 2+2 format bispecific antibody. For example, both heavy chains of the bispecific antibody may each include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment.

[0273] In some other embodiments, the bispecific antibody may be a 2+1 format bispecific antibody. For example, one of the two heavy chains of the bispecific antibody may comprise a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment; and the other of the two heavy chains of the bispecific antibody may comprise a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment. In some embodiments, one of the two heavy chains of the bispecific antibody may comprise a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment; and the other of the two heavy chains of the bispecific antibody may comprise a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment and may not comprise a second antigen fragment.

[0274] In some embodiments, the bispecific antibody may comprise a monospecific antibody against R0R1 (or an antibody targeting R0R1 or an anti-R0R1 antibody) and one or more (e.g., one or two) second antigen-binding fragments. In some embodiments, the monospecific antibody against R0R1 may be an intact IgG (e.g., intact IgG1) shaped antibody. In some embodiments, the monospecific antibody against R0R1 may comprise two first antigen-binding fragments. In some embodiments, the bispecific antibody may further comprise a peptide linker (e.g., a peptide linker for linking the second antigen-binding fragment and the monospecific antibody against R0R1). In some embodiments, the second antigen-binding fragment may be connected to the C-terminus of the heavy chain of the monospecific antibody against R0R1 (e.g., the C-terminus of the heavy chain constant region). In some embodiments, the second antigen-binding fragment may be connected to the C-terminus of the heavy chain of the monospecific antibody against R0R1 via a peptide linker. For example, if two second antigen-binding fragments are present in the bispecific antibody, each second antigen-binding fragment may be connected to the C-terminus of each of the two heavy chains of the monospecific antibody against R0R1. For example, if one second antigen-binding fragment is present in the bispecific antibody, the second antigen-binding fragment may be connected to the C-terminus of one of the two heavy chains of the monospecific antibody against R0R1.

[0275] 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 IgGl type antibody (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).

[0276] In some embodiments, the bispecific antibody may be a mouse, chimeric, humanized, or human antibody. In some embodiments, the bispecific antibody may be a human antibody. In some embodiments, the antigen-binding fragment may be a human antigen-binding fragment. For example, the antigen-binding fragment may be of human origin. In some embodiments, the first antigen-binding fragment may be a human antigen-binding fragment. In some embodiments, the second antigen-binding fragment may be a human antigen-binding fragment.

[0277] In some embodiments, the bispecific antibody may be a human IgGl bispecific antibody.

[0278] 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 / 014930A1. For example, the bispecific antibody according to some embodiments of the present disclosure may be a monoclonal antibody, and the monoclonal antibody may be produced according to methods widely known in the art. For example, CHO-S (Chinese hamster ovary-S) cells may be co-transformed with a vector encoding the heavy chain of the bispecific antibody and a vector encoding the light chain of the bispecific antibody, and the bispecific antibody may be obtained from the co-transformed CHO-S cells.

[0279] (2) First antigen-binding fragment (antigen-binding fragment for [ROW]) 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 two first antigen-binding fragments. The first antigen-binding fragment is an antigen-binding fragment designed to be able to bind to R0R1 or to target R0R1, having an antigen-binding portion capable of binding to R0R1. The antigen-binding portion capable of binding to R0R1 may be referred to as the first antigen-binding portion. That is, the target antigen of the first antigen-binding fragment is R0R1. In some embodiments, the first antigen-binding fragment may be referred to as an antigen-binding fragment for R0R1 or an antigen-binding fragment targeting R0R1. 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 comprise the first antigen-binding portion.

[0280] 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.

[0281] The CDRs of the first antigen-binding fragment may be referred to as 1st HCDR1, 1st HCDR2, 1st HCDR3, 1st LCDR1, 1st LCDR2, and 1st LCDDR3, etc., to distinguish them from the CDRs of the second antigen-binding fragment described below, but are not otherwise limited. The amino acid sequences of the aforementioned CDRs of the first antigen-binding fragment are described in Table 02 below.

[0282] [Table 0 shows the amino acid sequences of the CDRs of the first antigen-binding fragment]

[0283] Name: Amino acid sequence

[0284] HCDR1 NYDMS (Sequence No. 1)

[0285] HCDR2 AIYHSGSSKYYADSVKG (Sequence No. 2)

[0286] HCDR3 GGSGAWDTGFDY (Sequence No. 3)

[0287] LCDR1 SGSSSNIGSNDVS (Sequence No. 4)

[0288] LCDR2 YENNRPS (Sequence No. 5)

[0289]

[0290] LCDR3 GAWDDSLSGYV (Sequence No. 6)

[0291] In some embodiments, the first antigen-binding fragment (or the first antigen-binding portion) includes a heavy chain variable region and a light chain variable region. To distinguish it from the heavy chain variable region and the light chain variable region of the second antigen-binding fragment described below, the heavy chain variable region and the 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.

[0292] 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 human heavy chain variable region. For example, the framework sequences within the first heavy chain variable region may be 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 human light chain variable region. For example, the framework sequences within the first light chain variable region may be of human origin. 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 no. 1, HCDR2 having the amino acid sequence of sequence no. 2, and HCDR3 having the amino acid sequence of sequence no. 3. In some embodiments, in the first heavy chain variable region, HCDR1, HCDR2, and HCDR3 may be positioned in the order described, from the N-terminus to the C-terminus.

[0293] 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 no. 8, HFR2 having the amino acid sequence of sequence no. 9, HFR3 having the amino acid sequence of sequence no. 10, and HFR4 having the amino acid sequence of sequence no. 11. 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. 8 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 of sequence identity therewith, or the amino acid sequence of sequence no. 9 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 of sequence identity therewith It may further include HFR2, HFR3 having an amino acid sequence of sequence number 10 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 HFR4 having an amino acid sequence of sequence number 11 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 with respect to it. In some embodiments, in the first 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.

[0294] In some embodiments, the heavy chain variable region of the first antigen-binding fragment may have the amino acid sequence of SEQ ID NO. 7. In some embodiments, the heavy chain variable region of the first antigen-binding fragment may have the amino acid sequence of SEQ ID NO. 7 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 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.

[0295] 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 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, in the first light chain variable region, LCDR1, LCDR2, and LCDR3 may be located in the order listed above, from the N-terminus to the C-terminus.

[0296] 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 SEQ ID NO. 13, LFR2 having the amino acid sequence of SEQ ID NO. 14, LFR3 having the amino acid sequence of SEQ ID NO. 15, and LFR4 having the amino acid sequence of SEQ ID NO. 16. In some embodiments, the first light chain modified region is, in addition to the LCDRs, an LFR1 having the amino acid sequence of sequence no. 13 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 of sequence identity therewith, or the amino acid sequence of sequence no. 14 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 of sequence identity therewith. It may further include LFR2, LFR3 having an amino acid sequence of sequence number 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 of sequence identity therewith, and LFR4 having an amino acid sequence of sequence number 16 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 of sequence identity therewith.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-terminus to the C-terminus.

[0297] In some embodiments, the light chain variable region of the first antigen-binding fragment may have the amino acid sequence of sequence number 12. In some embodiments, the light chain variable region of the first antigen-binding fragment may have the amino acid sequence of sequence number 12 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.

[0298] The sequences of the framework region of the variable region of the first antigen-binding fragment described above, and the amino acid sequences of the heavy chain variable region and light chain variable region are described in Table 03 below.

[0299] [Table 03] Amino acid sequences of the heavy chain variable region and light chain variable region of the first antigen-binding fragment

[0300] Name Amino ac id sequence

[0301] Heavy chain variable region EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYY (VH) ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFDYWGQGTLVTVS S (Sequence No. 7)

[0302] HFR1 EVQLLESGGGLVQPGGSLRLSCAASGFTFS (Sequence No. 8)

[0303] HFR2 WVRQAPGKGLEWVS (Sequence No. 9)

[0304] HFR3 RFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAR (Sequence No. 10)

[0305] HFR4 WGQGTLVTVSS (Sequence No. 11)

[0306] Light chain variable region QSVLTQPPSASGTPGQRVTI SCSGSSSN IGSNDVSWYQQLPGTAPKLL I YYENNRPSGVP (VL) DRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGG7KLTVL (Sequence No. 12)

[0307] LFR1 QSVLTQPPSASGTPGQRVT I SC (Sequence No. 13)

[0308] LFR2 WYQQLPGTAPKLLIY (Sequence No. 14)

[0309]

[0310] LFR3 GVPDRFSGSKSGTSASLAI SGLRSEDEADYYC (Sequence No. 15)

[0311]

[0312] LFR4 FGGGTKLTVL (Sequence No. 16)

[0313] In some embodiments, the first antigen-binding fragment may comprise an amino acid sequence corresponding to each CDR, VH, and / or VL of Cirmtuzumab, an antibody targeting R0R1, but is not otherwise limited.

[0314] 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.

[0315] 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 (CHI) having the amino acid sequence of SEQ ID NO. 22, and a first light chain variable region and a light chain constant region (CL) having the amino acid sequence of SEQ ID NO. 26. In some embodiments, the amino acid sequence of SEQ ID NO. 26 may be the amino acid sequence of SEQ ID NO. 27 or SEQ ID NO. 28. In some embodiments, Fab may comprise a first heavy chain variable region and a heavy chain constant region 1 having the amino acid sequence of SEQ ID NO. 22, and a first light chain variable region and a light chain constant region having the amino acid sequence of SEQ ID NO. 27 or SEQ ID NO. 28. 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 17, and a light chain variable region-light chain constant region (VL-CL) having the amino acid sequence of sequence number 18 (e.g., sequence number 19 or sequence number 20). In some embodiments, in Fab, the heavy chain constant region and the light chain constant region may be covalently connected through a disulfide bond formed by cysteine.

[0316] The amino acid sequences of VH-CH1 and VL-CL are described in Table 04 below. [Table 04] Amino acid sequences of VH-CH1 and VL-CL

[0317] Name: Amino acid sequence

[0318] Heavy chain variable region- EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYY Heavy chain invariant region 1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFDYWGQGTLVTVS (VH-CH1) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKV (Sequence No. 17) Light chain variable region- QSVLTQPPSASGTPGQRVTI SCSGSSSN IGSNDVSWYQQLPGTAPKLL I YYENNRPSGVP Light chain invariant region DRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVT (VL-CL) or light chain LFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPXECS (Sequence No. 18) (where , X is A or T)

[0319] Light chain variable region - QSVLTQPPSASGTPGQRVTI SCSGSSSN IGSNDVSWYQQLPGTAPKLL I YYENNRPSGVP Light chain constant region DRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVT (VL-CL) or light chain LFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS (Example of YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS of Sequence No. 18 (Sequence No. 19))

[0320] Light chain variable region- QSVLTQPPSASGTPGQRVTI SCSGSSSN IGSNDVSWYQQLPGTAPKLL I YYENNRPSGVP Light chain constant region DRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVT (VL-CL) or light chain LFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS (YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS of sequence number 18 (sequence number 20)

[0321]

[0322] example)

[0323] (3) Second antigen-binding fragment (antigen-binding fragment for B7-H3)

[0324] 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 or two second antigen-binding fragments. The second antigen-binding fragment is an antigen-binding fragment designed to be capable of binding to B7-H3 or to target B7-H3, having an antigen-binding portion capable of binding to B7-H3. The antigen-binding portion capable of binding to B7-H3 may be referred to as the second antigen-binding portion. That is, the target antigen of the second antigen-binding fragment is B7-H3. In some embodiments, the second antigen-binding fragment may be referred to as the antigen-binding fragment for B7-H3 or the antigen-binding fragment targeting B7-H3. In some embodiments, the second antigen-binding fragment may be referred to as the second antigen-binding portion. In some embodiments, the bispecific antibody of the present disclosure may comprise a second antigen-binding portion. In some embodiments, the bispecific antibody of the present disclosure may comprise one or two second antigen-binding portions.

[0325] The second antigen-binding fragment (or second antigen-binding portion) comprises HCDR1 having the amino acid sequence of SEQ NO. 30, HCDR2 having the amino acid sequence of SEQ NO. 31, HCDR3 having the amino acid sequence of SEQ NO. 32, LCDR1 having the amino acid sequence of SEQ NO. 33, LCDR2 having the amino acid sequence of SEQ NO. 34, and LCDR3 having the amino acid sequence of SEQ NO. 35. In some embodiments, the bispecific antibody of the present disclosure may comprise HCDR1 having the amino acid sequence of SEQ NO. 30, HCDR2 having the amino acid sequence of SEQ NO. 31, HCDR3 having the amino acid sequence of SEQ NO. 32, LCDR1 having the amino acid sequence of SEQ NO. 33, LCDR2 having the amino acid sequence of SEQ NO. 34, and LCDR3 having the amino acid sequence of SEQ NO. 35.

[0326] 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, but are not otherwise limited. The amino acid sequences of the aforementioned CDRs of the second antigen-binding fragment are described in Table 05 below.

[0327] [Table 05] Amino acid sequences of CDRs of the second antigen-binding fragment

[0328] Name Amino ac id sequence

[0329] HCDR1 DYAMS (Sequence No. 30)

[0330] HCDR2 SISSGSGSIYYADSVKG (Sequence No. 31)

[0331] HCDR3 NLIPLDY (Sequence No. 32)

[0332] LCDR1 SGSSSNIGSNAVS (Sequence No. 33)

[0333] LCDR2 YNSHRPS (Sequence No. 34)

[0334]

[0335] LCDR3 GSWDASLNAYV (Sequence No. 35)

[0336] In some embodiments, the second antigen-binding fragment (or second antigen-binding portion) includes a heavy chain variable region and a light chain variable region. For distinction from the heavy chain variable region and light chain variable region of the first antigen-binding fragment described above, the heavy chain variable region and 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.

[0337] 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 human heavy chain variable region. For example, the framework sequences within the second heavy chain variable region may be of human origin. 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 human light chain variable region. For example, the framework sequences within the second light chain variable region may be of human origin. 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 no. 30, HCDR2 having the amino acid sequence of sequence no. 31, and HCDR3 having the amino acid sequence of sequence no. 32. In some embodiments, in the second heavy chain variable region, HCDR1, HCDR2, and HCDR3 may be positioned in the N-terminus to C-terminus direction in the order described above.

[0338] 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. 37 (e.g., the second HFR1), HFR2 having the amino acid sequence of sequence no. 38, HFR3 having the amino acid sequence of sequence no. 39, and HFR4 having the amino acid sequence of sequence no. 40. In some embodiments, the second heavy chain variable region is, in addition to the HCDRs, an amino acid sequence of sequence no. 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 of sequence identity with the same, an HFR1, an amino acid sequence of sequence no. 38 or the same} at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, HFR2 having an amino acid sequence having 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity, the amino acid sequence of sequence number 39 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 40 or at least 80, 81, 82, 83, 84, 85, 86, 87 It may further include HFR4 having an amino acid sequence having at least 99% sequence identity, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% 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 listed, from the N-terminus to the C-terminus.

[0339] In some embodiments, the heavy chain variable region of the second antigen-binding fragment may have the amino acid sequence of sequence number 36. In some embodiments, the heavy chain variable region of the second antigen-binding fragment may have an amino acid sequence of sequence number 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, and may include HCDR1 having the amino acid sequence of sequence number 30, HCDR2 having the amino acid sequence of sequence number 31, and HCDR3 having the amino acid sequence of sequence number 32.

[0340] 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 number 33, LCDR2 having the amino acid sequence of sequence number 34, and LCDR3 having the amino acid sequence of sequence number 35. In some embodiments, in the second light chain variable region, LCDR1, LCDR2, and LCDR3 may be positioned from the N-terminus to the C-terminus in the order listed above. 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 42 (e.g., second LFR1), LFR2 having the amino acid sequence of sequence number 43, LFR3 having the amino acid sequence of sequence number 44, and LFR4 having the amino acid sequence of sequence number 45.In some embodiments, the second light chain variable region is, in addition to the LCDRs, an LFR1 having an amino acid sequence of sequence no. 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 therewith, an amino acid sequence of sequence no. 43 or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, LFR2 having an amino acid sequence having 92, 93, 94, 95, 96, 97, 98, or 99% or more sequence identity, the amino acid sequence of sequence number 44 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, and LFR3 having an amino acid sequence having the amino acid sequence of sequence number 45 and at least 80, 81, 82, 83, 84, 85, 86, 87 It may further include LFR4 having an amino acid sequence having at least 99% sequence identity, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity. 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.

[0341] In some embodiments, the light chain variable region of the second antigen-binding fragment may have the amino acid sequence of sequence number 41. In some embodiments, the light chain variable region of the second antigen-binding fragment may have an amino acid sequence of sequence number 41 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, and may include LCDR1 having the amino acid sequence of sequence number 33, LCDR2 having the amino acid sequence of sequence number 34, and LCDR3 having the amino acid sequence of sequence number 35.

[0342] The sequences of the framework region of the variable region of the aforementioned second antigen-binding fragment and the amino acid sequences of the heavy chain variable region and light chain variable region are described in Table 06 below.

[0343] [Table 06] Amino acid sequences of the heavy chain variable region and light chain variable region of the second antigen-binding fragment

[0344] Name Amino ac id sequence

[0345] Heavy chain variable region EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKCLEWVSSISSGSGSIYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLIPLDYWGQGTLVTVSS

[0346] (Sequence No. 36)

[0347] HFR1 EVQLLESGGGLVQPGGSLRLSCAASGFTFS (Sequence No. 37)

[0348] HFR2 WVRQAPGKCLEWVS (Sequence No. 38)

[0349] HFR3 RFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAK (Sequence No. 39)

[0350] HFR4 WGQGTLVTVSS (Sequence No. 40)

[0351] Light chain variable region QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVSWYQQLPGTAPKLLIYYNSHRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAYVFGCGTKLTVL (Sequence No. 41)

[0352] LFR1 QSVLTQPPSASGTPGQRVT I SC (Sequence No. 42)

[0353] LFR2 WYQQLPGTAPKLLIY (Sequence No. 43)

[0354] LFR3 GVPDRFSGSKSGTSASLAI SGLRSEDEADYYC (Sequence No. 44)

[0355]

[0356] LFR4 FGCGTKLTVL (Sequence No. 45)

[0357] In some embodiments, the second antigen-binding fragment may comprise an amino acid sequence corresponding to each CDR, VH, and / or VL of Enoblituzumab, an antibody targeting B7-H3, and is not otherwise limited.

[0358] 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.

[0359] In some embodiments, the second antigen-binding fragment may be a scFv. The scFv comprises a heavy chain variable region of the second antigen-binding fragment (i.e., the second heavy chain variable region) and a light chain variable region of the second antigen-binding fragment (i.e., the second light chain variable region). In some embodiments, the scFv may further comprise a peptide linker (e.g., referred to as a short peptide linker or scFv linker) connecting the second heavy chain variable region and the second light chain variable region. In some embodiments, the scFv linker may have a length of 5 to 50 aa. For example, the scFv linker may have the amino acid sequence of SEQ ID NO. 47. For example, the scFv linker may have the amino acid sequence of sequence number 47 or at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 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 antigen-binding fragment may be referred to as anti-B7-H3 scFv or B7-H3 scFv, but is not otherwise limited.

[0360] In some embodiments, in scFv, the C end of the second heavy chain variable region and the C end of the second light chain variable region may be connected (e.g., via a scFv linker). In some embodiments, the C end of the second light chain variable region and the N end of the second heavy chain variable region may be connected (e.g., via a scFv linker).

[0361] In some embodiments, B7-H3 scFv may have any one of the following structures, and the following structure is illustrated from the N-terminus to the C-terminus:

[0362] [VH]- [VL];

[0363] [VH]-[scFv 1 inker ] -[VL] :

[0364] [VL]-[VH] ; and

[0365] [VL]-[scFv 1 inker ] -[VH] ,

[0366] Here, "-" can each independently be 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).

[0367] In some embodiments, the scFv may comprise, from the N-terminus to the C-terminus, a light chain variable region having the amino acid sequence of sequence number 41, a scFv linker having the amino acid sequence of sequence number 47, and a heavy chain variable region having the amino acid sequence of sequence number 36. In some embodiments, the scFv may have the amino acid sequence of sequence number 46. Exemplary amino acid sequences of the aforementioned B7-H3 scFv and scFv linker are disclosed in Table 07 below.

[0368] [Table 07] Exemplary amino acid sequences of B7-H3 scFv and scFv linker

[0369] Name Amino ac id sequence

[0370] scFv (VL-scFv QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVSWYQQLPGTAPKLLIYYNSHRPSGVPD 1 inker-VH) RFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAYVFGCGTKLTVLGGGGSGGGGSGG GGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKCLEWVSSI SS GSGSI YYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKNL I PLDYWGQGTLVTV SS (SEQ ID NO: 46)

[0371] scFv within scFv GGGGSGGGGSGGGGSGGGGS (sequence number 47)

[0372]

[0373] Linker

[0374] (4) Heavy chain constant region and light chain constant region of bispecific antibody

[0375] In some embodiments, the bispecific antibody of the present disclosure may comprise a heavy chain and a light chain. For example, the bispecific antibody may comprise two heavy chains and two light chains. In this case, the two heavy chains may be connected to each other through one or more (e.g., one or more) disulfide bonds, any one of the two heavy chains and any one of the two light chains may be connected through one or more disulfide bonds, and the other of the two heavy chains and the other of the two light chains may be connected through one or more disulfide bonds. In some embodiments, any one of the two heavy chains of the bispecific antibody may be referred to as the first heavy chain of the bispecific antibody, and the other of the two heavy chains of the bispecific antibody may be referred to as the second heavy chain of the bispecific antibody. In some embodiments, one of the two light chains of the bispecific antibody may be referred to as the first light chain of the bispecific antibody, and the other of the two light chains of the bispecific antibody may be referred to as the second light chain of the bispecific antibody.

[0376] In some embodiments, the bispecific antibody may comprise a first heavy chain of one bispecific antibody, a first light chain of one bispecific antibody, a second heavy chain of one bispecific antibody, and a second light chain of one bispecific antibody. For example, the first heavy chain of the bispecific antibody and the second heavy chain of the bispecific antibody 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 of the bispecific antibody and the first light chain of the bispecific antibody may be connected to each other through one or more disulfide bonds. For example, the second heavy chain of the bispecific antibody and the second light chain of the bispecific antibody may be connected to each other through one or more disulfide bonds.

[0377] In some embodiments, the heavy chain of the bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment. In the heavy chain of the bispecific antibody, the heavy chain variable region, the heavy chain constant region, and the second antigen-binding fragment of the first antigen-binding fragment may be located from the N-terminus to the C-terminus in the order described above.

[0378] In some embodiments, the heavy chain of the bispecific antibody may include a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment. In the heavy chain of the bispecific antibody, the heavy chain variable region and the heavy chain constant region of the first antigen-binding fragment may be located in the order described above from the N-terminus to the C-terminus.

[0379] In some embodiments, at least one heavy chain of the bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment.

[0380] In some embodiments, both heavy chains of the bispecific antibody may each include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment. In this way, when the second antigen-binding fragment is present in both heavy chains of the bispecific antibody, the bispecific antibody may have a 2+2 format. For example, the first heavy chain of the bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment, and the second heavy chain of the bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment (e.g., B7-H3 scFv) of the first antigen-binding fragment.

[0381] In some embodiments, either of the two heavy chains of the bispecific antibody comprises a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment; and the other of the two heavy chains of the bispecific antibody may not comprise the second antigen-binding fragment and may comprise a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment. In this way, if the second antigen-binding fragment is present in only one of the two heavy chains, the bispecific antibody may have a 2+1 format. For example, the first heavy chain of the bispecific antibody may comprise the first antigen-binding fragment and a heavy chain constant region, and not the second antigen-binding fragment; and the second heavy chain of the bispecific antibody may comprise a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment (e.g., B7-H3 scFv) of the first antigen-binding fragment. In some embodiments, the heavy chain constant region of the bispecific antibody may include heavy chain constant region 1 (CH1), a hinge, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3). In some embodiments, the heavy chain constant region may include the amino acid sequence of sequence number 21. In some embodiments, CH1 may include the amino acid sequence of sequence number 22. In some embodiments, the hinge may include the amino acid sequence of sequence number 23. In some embodiments, CH2 may include the amino acid sequence of sequence number 24. In some embodiments, CH3 may include the amino acid sequence of sequence number 25. In some embodiments, the Fc region (e.g., the Fc region of a bispecific antibody, heavy chain, or heavy chain invariant region) may include the amino acid sequence of SEQ ID NO. 50.

[0382] In some embodiments, the heavy chain of the bispecific antibody may further comprise a peptide linker, wherein the second antigen-binding fragment (e.g., anti-B7-H3 scFv) may be connected to the C-terminus of the heavy chain constant region through the peptide linker. In some embodiments, the peptide linker may have the amino acid sequence of sequence number 48.

[0383] In some embodiments, the light chain of the bispecific antibody may include a light chain variable region of the first antigen-binding fragment. In some embodiments, the light chain of the bispecific antibody may include a light chain constant region in addition to the 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 26, 27, or 28. In some embodiments, the light chain invariant region may include any one of sequence numbers 26, 27 and 28, 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.

[0384] 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.

[0385] In some embodiments, the heavy chain constant region of the heavy chain of the bispecific antibody (e.g., the heavy chain constant region of the first heavy chain of the bispecific antibody or the heavy chain constant region of the second heavy chain of the bispecific antibody) 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 IgG. The heavy chain constant region of human IgGl may include any one amino acid sequence selected from the amino acid sequences of SEQ ID NO. 21, SEQ ID NOs 61 to 63, and SEQ ID NOs 66 to 67, 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.

[0386] Sequence No. 21, Sequence No. 61, Sequence No. 62, and Sequence No. 63 are sequences of the heavy chain constant regions of antibodies commonly used in the art. Sequence No. 21 corresponds to the sequence of the heavy chain constant region of Herceptin, a known IgGl antibody. Sequence No. 61 is the heavy chain constant region sequence of wild-type human IgGl (see UniProt Accession P01857). Sequence No. 62 corresponds to the heavy chain constant region sequence of atezolizumab, a known IgGl antibody. Sequence No. 63 is a known IgGl antibody

[0387] The durvalumab heavy chain constant region corresponds to the sequence. In some embodiments, one heavy chain of the bispecific antibody may be designed to be dimerizable 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 dimerizable with each other. This design allows the first heavy chain and the second heavy chain to dimerize when the first heavy chain and the second heavy chain have different structures (for example, when the first heavy chain and the second heavy chain have different structures, such as in a 2+1 bispecific antibody), and is useful for the production of bispecific antibodies in cases where the first heavy chain and the second heavy chain have different structures, such as in a 2+1 bispecific antibody.

[0388] In some embodiments, a KIH mutation may be introduced into the heavy chain constant region (or heavy chain constant region 3) of the bispecific antibody. For example, the heavy chain constant region 3 of one of the two heavy chains of the bispecific antibody may contain a knob mutation, and the 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 the KIH mutation has been introduced (or which has the KIH mutation). In some embodiments, either of the two heavy chains of the bispecific antibody may include 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 include 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).

[0389] 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). In some embodiments, the knob variation may include T366W. In some embodiments, the hole variation may include T366S, L368A, and Y407V. Pairs of knob variations and hole variations known to the art are shown in Table 01. In some embodiments, the heavy chain invariant region of one of the two heavy chains of the bispecific antibody comprises any one of the amino acid sequences of sequence number 21, sequence numbers 61 to 63, and sequence numbers 66 to 67, 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 the same, 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 the amino acid sequences of sequence number 21, sequence numbers 61 to 63, and sequence numbers 66 to 67, 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, wherein the heavy chain invariant region 3 of the heavy chain invariant region may have a hole variation.

[0390] In some embodiments, the heavy chain invariant region of one of the two heavy chains of the bispecific antibody comprises any one of the amino acid sequences of sequence number 21, sequence numbers 61 to 63, and sequence numbers 66 to 67, 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, wherein heavy chain invariant region 3 of the heavy chain invariant region may comprise one or more of S354C and T366W (or T366Y) substitutions (knobmutation); 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 SEQ ID NO. 21, SEQ ID NOs 61 to 63 and SEQ ID NOs 66 to 67, 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, wherein the heavy chain constant region 3 of the heavy chain constant region may comprise one or more of the Y349C, T366S, L368A, and Y407V (or Y407T) substitutions (hole mutation).

[0391] 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.

[0392] In some embodiments, the heavy chain constant region (or heavy chain constant region 3) of the first heavy chain of the bispecific antibody may include a knob mutation (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 amino acid residue positions are designated according to the EU numbering system)). In some embodiments, the Fc region of the first heavy chain may include a knob mutation. In some embodiments, the heavy chain constant region of the first heavy chain may include a T366W substitution. For example, if the heavy chain constant region of the first heavy chain includes a knob mutation, the second heavy chain, which is the other heavy chain of the bispecific antibody, may include a hole mutation.

[0393] In some embodiments, the heavy chain constant region (or heavy chain constant region 3) of the first heavy chain of the specific antibody may include a hole variation (e.g., one or more substitutions of Y349C, T366S, L368A, and Y407V; or one or more of the substitution of an amino acid residue at position 349 to C, a substitution of an amino acid residue at position 366 to A, and a substitution of an amino acid residue at position 368 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 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.

[0394] In some embodiments, the heavy chain constant region (or heavy chain constant region 3) of the second heavy chain of the bispecific antibody may contain 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 contain a hole variant. In some embodiments, the heavy chain constant region of the second heavy chain may contain T366S, L368A, and Y407V substitutions. For example, if the heavy chain constant region of the second heavy chain contains a hole variant, the first heavy chain, which is the other heavy chain of the bispecific antibody, may contain a knob variant.

[0395] In some embodiments, the heavy chain constant region (or heavy chain constant region 3) of the second heavy chain of the bispecific antibody may contain a knob mutation (e.g., a substitution of one or more of S354C and T366W). In some embodiments, the Fc region of the second heavy chain may contain a knob mutation. In some embodiments, the heavy chain constant region of the second heavy chain may contain a T366W substitution. For example, if the heavy chain constant region of the second heavy chain contains a knob mutation, the first heavy chain, which is the other heavy chain of the bispecific antibody, may contain a hole mutation.

[0396] In some embodiments, a KIH variant may not be introduced into the heavy chain of the bispecific antibody. For example, in the case of a 2+2 bispecific antibody, since the heavy chains of the two bispecific antibodies may have the same (or symmetric) structure, the manufacture of the 2+2 bispecific antibody may not require a design that allows heavy chains of different structures, such as the KIH variant, to dimerize. In some embodiments, the first heavy chain of the bispecific antibody may not have a knob variant and / or a hole variant. In some embodiments, the second heavy chain of the bispecific antibody may not have a hole variant and / or a knob variant.

[0397] In some embodiments, the heavy chain of the bispecific antibody may optionally include a KIH variant. In some embodiments, the heavy chain constant region of the first heavy chain of the bispecific antibody may optionally include a knob variant, and the heavy chain constant region of the second heavy chain of the bispecific antibody may optionally include a Hole variant. In some embodiments, the heavy chain constant region of the first heavy chain of the bispecific antibody may optionally include a Hole variant, and the heavy chain constant region of the second heavy chain of the bispecific antibody may optionally include a knob variant.

[0398] In some embodiments, the heavy chain constant region of the bispecific antibody may include one or more of the heavy chain constant region 1 (CH1), the hinge, the heavy chain constant region 2 (CH2), and the heavy chain constant region 3 (CH3). For example, the heavy chain constant region of the bispecific antibody may include the heavy chain constant region 1 (CH1), the hinge, the heavy chain constant region 2 (CH2), and the heavy chain constant region 3 (CH3). In the heavy chain constant region, the heavy chain constant region 1, the hinge, the heavy chain constant region 2, and the heavy chain constant region 3 may be positioned from the N-terminus to the C-terminus in the order described above.

[0399] In some embodiments, the heavy chain constant region of the first heavy chain of the bispecific antibody may include a heavy chain constant region 1 (CH1), a hinge, a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3). Here, the heavy chain constant region 1, the hinge, the heavy chain constant region 2 (CH2), and the heavy chain constant region 3 (CH3) of the heavy chain constant region of the first heavy chain may be referred to as the first heavy chain constant region 1 (first CH1), the first hinge, the first heavy chain constant region 2 (first CH2), and the first heavy chain constant region 3 (first CH3), respectively. In some embodiments, the heavy chain constant region of the second heavy chain of the bispecific antibody may include a heavy chain constant region 1 (CH1), a hinge, a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3). Here, the heavy chain constant region 1, hinge, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3) of the heavy chain constant region of the second heavy chain may be referred to as the second heavy chain constant region 1 (second CH1), the second hinge, the second heavy chain constant region 2 (second CH2), and the second heavy chain constant region 3 (second CH3), respectively.

[0400] In some embodiments, heavy chain invariant region 1 may include the sequence of sequence number 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 with the same. In some embodiments, the heavy chain invariant region 1 of the first heavy chain of the bispecific antibody may include the sequence of sequence number 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 with the same. In some embodiments, the heavy chain invariant region 1 of the second heavy chain of the bispecific antibody may include the sequence of sequence number 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.

[0401] In some embodiments, the hinge may comprise the sequence of sequence number 23 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 hinge of the first heavy chain of the bispecific antibody may comprise the sequence of sequence number 23 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 hinge of the second heavy chain of the bispecific antibody may comprise the sequence of sequence number 23 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.

[0402] In some embodiments, heavy chain invariant region 2 may include the sequence of sequence number 24 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. In some embodiments, the heavy chain invariant region 2 of the first heavy chain of the bispecific antibody may include the sequence of sequence number 24 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. In some embodiments, the heavy chain invariant region 2 of the second heavy chain of the bispecific antibody may include the sequence of sequence number 24 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.

[0403] In some embodiments, heavy chain invariant region 3 may include the sequence of sequence number 25 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 the same. In some embodiments, the heavy chain invariant region 3 of the first heavy chain of the bispecific antibody may include the sequence of sequence number 25 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 heavy chain invariant region 3 of the second heavy chain of the bispecific antibody may include the sequence of SEQ ID NO. 25 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.

[0404] In some embodiments, heavy chain constant region 3 may include a knob variation or a hole variation. In some embodiments, heavy chain constant region 3 of the first heavy chain of the bispecific antibody may include the amino acid sequence of sequence number 64 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. In some embodiments, the heavy chain invariant region 3 of the second heavy chain of the bispecific antibody may include the amino acid sequence of sequence number 65 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.

[0405] Exemplary sequences of each of the aforementioned regions are provided in the table below.

[0406] [Table 08] Exemplary sequences of each region of the heavy chain

[0407] Name: Amino acid sequence

[0408] Heavy chain invariant region 1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKV (sequence number

[0409]

[0410] 22) Hinge EPKSCDKTHTCPPCP (Sequence No. 23)

[0411] Heavy chain invariant region 2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I SKAK

[0412] (Column No. 24)

[0413] Heavy chain invariant region 3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Column number 25)

[0414] Heavy chain PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK containing T366W substitution (knob GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP variant) invariant region 3 (SEQN 64)

[0415] Heavy chain invariant containing T366S, L368A, and Y407V GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP substitutions (hole variants) PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Column No. 65)

[0416]

[0417] Area 3

[0418] (5) Structure of a bispecific antibody

[0419] In some embodiments, the bispecific antibody may comprise two heavy chains and two light chains of the bispecific antibody. In some embodiments, the bispecific antibody may comprise a first heavy chain of the bispecific antibody, a second heavy chain of the bispecific antibody, a first light chain of the bispecific antibody, and a second light chain of the bispecific antibody.

[0420] In some embodiments, the first heavy chain may include a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment (i.e., an antigen-binding fragment for R0R1). In some embodiments, within the first heavy chain, the heavy chain variable region and the heavy chain constant region of the first antigen-binding fragment are located in the order described from the N-terminus to the C-terminus. In some embodiments, the first heavy chain may further include a second antigen-binding fragment (i.e., an antigen-binding fragment for B7-H3; e.g., B7-H3 scFv). For example, the first heavy chain may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment. In some embodiments, within the first heavy chain, the heavy chain variable region, the heavy chain constant region, and the second antigen-binding fragment are positioned in the order described above from the N-terminus to the C-terminus. In some embodiments, the first heavy chain may further include a peptide linker. In some embodiments, the peptide linker is positioned within the first heavy chain between the heavy chain constant region and the second antigen-binding fragment to connect the heavy chain constant region and the second antigen-binding fragment.

[0421] In some embodiments, the second heavy chain may include a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment (i.e., an antigen-binding fragment for R0R1). In some embodiments, within the second heavy chain, the heavy chain variable region and the heavy chain constant region of the first antigen-binding fragment are located in the order described from the N-terminus to the C-terminus. In some embodiments, the second heavy chain may further include a second antigen-binding fragment (i.e., an antigen-binding fragment for B7-H3; e.g., B7-H3 scFv). For example, the second heavy chain may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment. In some embodiments, within the second heavy chain, the heavy chain variable region, the heavy chain constant region, and the second antigen-binding fragment are positioned in the order described, from the N-terminus to the C-terminus. In some embodiments, the first heavy chain may further include a peptide linker. In some embodiments, the peptide linker is positioned within the first heavy chain between the heavy chain constant region and the second antigen-binding fragment, so as to connect the heavy chain constant region and the second antigen-binding fragment. In some embodiments, the peptide linker may include the amino acid sequence of sequence number 48 or an amino acid sequence having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 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.

[0422] In some embodiments, at least one of the first heavy chain and the second heavy chain may include a second antigen-binding fragment. In some embodiments, both the first heavy chain and the second heavy chain may include a second antigen-binding fragment.

[0423] In some embodiments, the first light chain of the bispecific antibody may include a light chain variable region and a light chain constant region of the first antigen-binding fragment. In some embodiments, within the first light chain, the light chain variable region and the light chain constant region of the first antigen-binding fragment may be located in the order described from the N-terminus to the C-terminus.

[0424] In some embodiments, the second light chain of the bispecific antibody may include a light chain variable region and a light chain constant region of the first antigen-binding fragment. In some embodiments, within the second light chain, the light chain variable region and the light chain constant region of the first antigen-binding fragment are positioned from the N-terminus to the C-terminus in the order described. In some embodiments, the first light chain and the second light chain may have the same sequence.

[0425] In some embodiments, the bispecific antibody may be a 2+2 bispecific antibody. In some embodiments, the first heavy chain of the 2+2 bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment. In some embodiments, the second heavy chain of the 2+2 bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment. In some embodiments, the first light chain of the 2+2 bispecific antibody may include a light chain variable region and a light chain constant region of the first antigen-binding fragment. In some embodiments, the second light chain of the 2+2 bispecific antibody may include a light chain variable region and a light chain constant region of the first antigen-binding fragment. An example of a 2+2 bispecific antibody with a heavy chain and a light chain is shown in FIG. 03.

[0426] In some embodiments, the bispecific antibody may be a 2+1 bispecific antibody. In some embodiments, the first heavy chain of the 2+1 bispecific antibody may include a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment. In some embodiments, the second heavy chain of the 2+1 bispecific antibody may include a heavy chain variable region, a heavy chain constant region, and a second antigen-binding fragment of the first antigen-binding fragment. In some embodiments, the first heavy chain of the 2+1 bispecific antibody may not include a second antigen-binding fragment. In some embodiments, the heavy chain constant region 3 of the first heavy chain of the 2+1 bispecific antibody may include a knob variant or a hole variant. In some embodiments, the heavy chain constant region 3 of the second heavy chain of the 2+1 bispecific antibody may include a hole mutation or a knob mutation. In some embodiments, when the heavy chain constant region 3 of the first heavy chain of the 2+1 bispecific antibody includes a knob mutation, the heavy chain constant region 3 of the second heavy chain may include a hole mutation. In some embodiments, when the heavy chain constant region 3 of the first heavy chain of the 2+1 bispecific antibody includes a hole mutation, the heavy chain constant region 3 of the second heavy chain may include a knob mutation. In some embodiments, the first light chain of the 2+1 bispecific antibody may include a light chain variable region and a light chain constant region of the first antigen-binding fragment. Some

[0427] In an embodiment, the second light chain of the 2+1 bispecific antibody may include a light chain variable region and a light chain constant region of the first antigen-binding fragment. An example of a 2+1 bispecific antibody with a heavy chain and a light chain is shown in FIG. 04.

[0428] (6) Examples of bispecific antibodies 1 - Specific examples of 2+2 bispecific antibodies

[0429] In some embodiments, the bispecific antibody may be a 2+2 bispecific antibody.

[0430] The 2+2 bispecific antibody may include the first heavy chain of the bispecific antibody, the second heavy chain, the first light chain, and the second light chain of the bispecific antibody.

[0431] In some embodiments, the first heavy chain, second heavy chain, first light chain, and second light chain of the 2+2 bispecific antibody may each have the following structures, but are not limited thereto:

[0432] 1st Heavy Print :

[0433] [heavy chain variable region (heavy chain variable region of the first antigen-binding fragment)]-[heavy chain constant region]-[peptide link-[scFv (second antigen-binding fragment)] ;

[0434] Second heavy chain: [heavy chain variable region (heavy chain variable region of the first antigen-binding fragment)]- [heavy chain constant region]-[ scFv (second antigen-binding fragment)] ;

[0435] First light chain: [light chain variable region (light chain variable region of the first antigen-binding fragment)]- [light chain invariant region] ;

[0436] Second light chain: [light chain variable region (light chain variable region of the first antigen-binding fragment)]- [light chain invariant region] ;

[0437] 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).

[0438] In some embodiments, the heavy chain constant region of each heavy chain of the 2+2 bispecific antibody may have the structure of [heavy chain constant region 1] - [hinge] - [heavy chain constant region - [heavy chain constant region 3].

[0439] In some embodiments, the heavy chain of the bispecific antibody may have an amino acid sequence of sequence number 49. In some embodiments, the light chain of the bispecific antibody may have an amino acid sequence of sequence number 18, 19, or 20.

[0440] In some embodiments, each of the two heavy chains of the bispecific antibody may have an amino acid sequence of sequence number 49, and each of the two light chains may have an amino acid sequence of sequence number 18. In some embodiments, each of the two heavy chains of the bispecific antibody may have an amino acid sequence of sequence number 49, and each of the two light chains of the bispecific antibody may have an amino acid sequence of sequence number 19. In some embodiments, each of the two heavy chains of the bispecific antibody may have an amino acid sequence of sequence number 49, and each of the two light chains of the bispecific antibody may have an amino acid sequence of sequence number 20.

[0441] The aforementioned heavy chains and light chains, and exemplary amino acid sequences of each element, are described in Table 09 below.

[0442] [Table 09] Heavy and light chains of bispecific antibodies, and exemplary amino acid sequences of each element

[0443] Name Amino ac id sequence

[0444] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGS heavy chain full length of bispecific antibody SKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKETLM I SRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKWSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLS PGKGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTI SCSGSSSN I GSNAVSWYQ QLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDA SLNAYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSRLL SCAASGFTFSDYAMSWVRQAPGKCLEWVSSISSGSGSIYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCAKNLIPLDYWGQGTLVTVSS (SEQ ID NO: 49) of the bispecific antibody EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGS 1st antigen binding of heavy chain SKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFDYWG variable region of heavy chain of fragment QGTLVTVSS (SEQ No. 7)

[0445] (1st heavy chain variable region)

[0446] The heavy chain constant region of the heavy chain of the bispecific antibody ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPC (CHl-hinge-CH2-CH3) PAPELLGGPSVFLFPPKPKDTLM I SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I SKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0447] (Column No. 21)

[0448] Heavy chain invariant region 1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0449]

[0450] LQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKV (Sequence No. 22)Hinge EPKSCDKTHTCPPCP (Sequence No. 23)

[0451] Heavy chain invariant region 2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (Column number 24)

[0452] Heavy chain constant region 3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0453] (Column No. 25)

[0454] Fragment of the second antigen-binding SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAYVFGCGTKLTVLGG of the heavy chain of the bispecific antibody QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVSWYQQLPGTAPKLLIYYNSHRP (scFv) GGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVR QAPGKCLEWVSSI SSGSGSI YYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVY YCAKNLIPLDYWGQGTLVTVSS (SEQ ID No. 46)

[0455] The light chain full length of the bispecific antibody QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQ PKAAPSVTLFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTP SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPXECS

[0456] (Sequence No. 18) (where, X is A or T)

[0457] Full light chain length of QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP of a bispecific antibody (example of sequence number SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQ 18) PKAAPSVTLFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTP SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0458] (Sequence No. 19)

[0459] Full light chain length of the QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP of a bispecific antibody (example of sequence number SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQ 18) PKAAPSVTLFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTP SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0460] (Column number 20)

[0461] The variable region of the light chain of the first antigen-binding SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVL fragment of the QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP light chain of the bispecific antibody (SEQ No. 12)

[0462] (1st light chain variable region)

[0463] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT light chain constant region of bispecific antibody TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPXECS (Sequence No. 26) (where X is A or T)

[0464] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT light chain constant region of bispecific antibody TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS (Example of Sequence No. 26) (Sequence No. 27)

[0465] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT light chain constant region of bispecific antibody TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (Example of Sequence No. 26) (Sequence No. 28)

[0466]

[0467] Peptide linker GGGGSGGGGSGGGGS (Sequence No. 48)

[0468] (7) Examples of bispecific antibodies 2 - Specific examples of 2+1 bispecific antibodies

[0469] In some embodiments, the bispecific antibody may be a 2+1 bispecific antibody. In some embodiments, the 2+1 bispecific antibody may comprise a first heavy chain of the bispecific antibody, a second heavy chain of the bispecific antibody, a first light chain, and a second light chain. In some embodiments, the first heavy chain, the second heavy chain, the first light chain, and the second light chain of the 2+1 bispecific antibody may each have the following structures, but are not limited thereto:

[0470] First heavy chain: [heavy chain variable region (heavy chain variable region of the first antigen-binding fragment)]- [heavy chain constant region] ;

[0471] Second heavy chain: [heavy chain variable region (heavy chain variable region of the first antigen-binding fragment)]- [heavy chain constant region]-[peptide linker]-[scFv (second antigen-binding fragment)] ;

[0472] First light chain: [light chain variable region (light chain variable region of the first antigen-binding fragment)]- [light chain constant region] ;

[0473] Second light chain: [light chain variable region (light chain variable region of the first antigen-binding fragment)]- [light chain constant region],

[0474] Here, "-" can each independently be 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).

[0475] In some embodiments, the heavy chain invariant region may have a structure of [heavy chain invariant region 1]-[hinge]-[heavy chain invariant region 1-[heavy chain invariant region 3].

[0476] In some embodiments, the heavy chain invariant region 3 of the first heavy chain and the heavy chain invariant region 3 of the second heavy chain may include a design (e.g., a KIH variation) that enables the first heavy chain and the second heavy chain to be dimeric. For example, a knob variation may be introduced in the heavy chain invariant region 3 of the first heavy chain, and a hole variation may be introduced in the heavy chain invariant region 3 of the second heavy chain. As another example, a hole variation may be introduced in the heavy chain invariant region 3 of the first heavy chain, and a hole variation may be introduced in the heavy chain invariant region 3 of the second heavy chain.

[0477] In some embodiments, the first heavy chain of the bispecific antibody may have the amino acid sequence of sequence number 59. In some embodiments, the second heavy chain of the bispecific antibody may have the amino acid sequence of sequence number 60. In some embodiments, the light chain of the bispecific antibody may have the amino acid sequence of sequence number 18. In some embodiments, the first heavy chain of the bispecific antibody may have the amino acid sequence of sequence number 59, the second heavy chain may have the amino acid sequence of sequence number 60, and each of the second light chains may have the amino acid sequence of sequence number 18. In some embodiments, the first heavy chain of the bispecific antibody may have an amino acid sequence of sequence number 59, the second heavy chain may have an amino acid sequence of sequence number 60, and each of the second light chains may have an amino acid sequence of sequence number 19. In some embodiments, the first heavy chain of the bispecific antibody may have an amino acid sequence of sequence number 59, the second heavy chain may have an amino acid sequence of sequence number 60, and each of the second light chains may have an amino acid sequence of sequence number 20.

[0478] The aforementioned heavy chains and light chains, and exemplary amino acid sequences of each element, are described in Table 10 below.

[0479] [Table 1 shows the heavy and light chains of the bispecific antibody, and exemplary amino acid sequences of each element.]

[0480] Name Amino ac id sequence

[0481] The first EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGS heavy chain SKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKETLM I SRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI of bispecific antibody

[0482]

[0483] EKTISKAKGQPREPQVYTLPPSREEMTKN2VSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLS PGK (Sequence No. 59)

[0484] 이중 특이성 항체의 제 2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGS 중쇄 SKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKETLM I SRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKWSLSCAVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLVSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLS PGKGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTI SCSGSSSN I GSNAVSWYQ QLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDA SLNAYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGFTFSDYAMSWVRQAPGKCLEWVSSISSGSGSIYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCAKNLIPLDYWGQGTLVTVSS (서열번호 60) 중쇄 가변 영역 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGS SKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGFDYWG QGTLVTVSS (서열번호 7)

[0485] 1st heavy chain heavy chain invariant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV region LQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLM I SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I SKAKG QPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0486] (Column No. 66)

[0487] Second heavy chain heavy chain invariant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV region LQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLM I SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I SKAKG QPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0488] (Column No. 67)

[0489] Heavy chain invariant region 1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKKV (Sequence No. 22) Hinge EPKSCDKTHTCPPCP (Sequence No. 23)

[0490] Heavy chain constant region 2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0491] (Column No. 24)

[0492] Heavy chain invariant of the 1st heavy chain GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP Region 3 (knob variation) VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (column number 64)

[0493] Heavy chain invariant of the 2nd heavy chain GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP Region 3 (hole variation) VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Column number 65)

[0494] Second antigen of the second heavy chain QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVSWYQQLPGTAPKLLIYYNSHRP binding fragment (scFv) SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAYVFGCGTKLTVLGG

[0495]

[0496] GGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKCLEWVSSI SSGSGSI YYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVY YCAKNLIPLDYWGQGTLVTVSS (SEQ ID NO: 46)

[0497] The light chain of the bispecific antibody QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQ PKAAPSVTLFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTP SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPXECS

[0498] (Sequence No. 18) (where, X is A or T)

[0499] The light chain of the bispecific antibody QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP (example of SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQ of Sequence No. 18) PKAAPSVTLFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTP SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0500] (Sequence No. 19)

[0501] The light chain of the bispecific antibody QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP (example of SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQ of Sequence No. 18) PKAAPSVTLFPPSSEELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTP SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0502] (Column number 20)

[0503] Light chain variable region QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRP SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVL (Sequence No. 12)

[0504] Light chain constant region GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPXECS

[0505] (Sequence No. 26) (where X is A or T)

[0506] Light chain invariant region GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT (Example of Sequence No. 26) TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0507] (Column No. 27)

[0508] Light chain invariant region GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT (Example of Sequence No. 26) TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0509] (Column No. 28)

[0510]

[0511] Peptide linker GGGGSGGGGSGGGGS (Sequence No. 48)

[0512] (8) Structural description of a bispecific antibody using a monospecific antibody against R0R1. Meanwhile, bispecific antibodies (e.g., bispecific antibodies in a 2+2 format and 2+1

[0513] A bispecific antibody in the format can be described using an antibody having an intact IgG shape (e.g., a monospecific antibody against R0R1) and a second antigen-binding fragment, as described in Section 3, "Background - Format of Bispecific Antibodies" of this disclosure. For example, the bispecific antibody is a monospecific antibody against RORI (e.g., anti-R0R1

[0514] It may include an antibody (referred to as an anti-RORI IgG antibody) and a second antigen-binding fragment (e.g., an antigen-binding fragment for B7-H3). For example, the bispecific antibody may include a monospecific antibody for R0R1 containing two first antigen-binding fragments and two second antigen-binding fragments (e.g., a bispecific antibody in a 2+2 format). For example, the bispecific antibody may include a monospecific antibody for R0R1 containing two first antigen-binding fragments or one second antigen-binding fragment.

[0515] In some embodiments, the monospecific antibody for R0R1 may be of the IgG class. In some embodiments, the monospecific antibody for R0R1 may be of the IgGl, IgG2, IgG3, or IgG4 subclass. In some embodiments, the monospecific antibody for R0R1 may be of the IgGl subclass. In some embodiments, the heavy chain constant region of the monospecific antibody for R0R1 may be the heavy chain constant region of human IgG (e.g., human IgGl). In some embodiments, the monospecific antibody for R0R1 may be an antibody having an intact IgG form (e.g., an intact IgGl form). In some embodiments, the monospecific antibody for R0R1 may be a mouse, chimeric, humanized, or human antibody and is not otherwise limited. The description of the heavy chain constant region of a single-specific antibody for R0R1 or each of the elements associated therewith (e.g., heavy chain constant region 1, hinge, heavy chain constant region 2, heavy chain constant region 3, or Fc region, etc.) may be applied to the description of the aforementioned bispecific antibody. In some embodiments, in the case of a 2+1 bispecific antibody, the heavy chain constant region may contain a KIH variant.

[0516] In some embodiments, the bispecific antibody may further comprise a peptide linker in addition to a monospecific antibody for R0R1 and a second antigen-binding fragment. In a 2+2 bispecific antibody, the second antigen-binding fragment may be connected to each of the two heavy chains of the monospecific antibody for R0R1 via a peptide linker. In a 2+1 format bispecific antibody, one second antigen-binding fragment may be connected to either of the two heavy chains of the monospecific antibody for R0R1 via a peptide linker. Examples of a 2+2 bispecific antibody and a 2+1 bispecific antibody showing the monospecific antibody for R0R1 and the second antigen-binding fragment are illustrated in FIG. 5 and FIG. 6, respectively.

[0517] 5.2.2. Modified bispecific antibody

[0518]

[0519] 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 and, accordingly, comprises two Asn297s. Asn297 may be described as being contained in the heavy chain constant region, Fc region, or heavy chain constant region 2 (CH2) of the bispecific antibody, but is not otherwise limited.

[0520] 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., 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. Examples of 2+2 format and 2+1 format bispecific antibodies engineered via glyco-engineering are shown in FIG. 7. In a engineered antibody or a bispecific antibody, the azide may be linked to an Asn residue (e.g., Asn297) located in the heavy chain constant region 2 (CH2) (wherein the 297th position of Asn297 is designated according to the EU numbering system). In some embodiments, the Asn at position 297 according to the EU numbering system (i.e., Asn297) may correspond to the Asn at position 301 (i.e., Asn301) 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 49, sequence number 59, or sequence number 60). In some embodiments, Asn at position 297 according to the EU numbering system (i.e., Asn297) is the amino acid sequence of the heavy chain constant region of the bispecific antibody according to some embodiments of the present disclosure (e.g., sequence number 21, sequence number 66, or sequence number.When based on 67), it may correspond to Asn at position 180 (i.e., Asn180). In some embodiments, Asn at position 297 according to the EU numbering system (i.e., Asn297) may correspond to Asn at position 67 (i.e., Asn67) when based on the amino acid sequence of heavy chain constant region 2 of the bispecific antibody according to some embodiments of the present disclosure (e.g., sequence number 24).

[0521] In some embodiments, a bispecific antibody engineered to contain an azide (i.e., a bispecific antibody containing an azide) may be referred to as an engineered antibody, an engineered bispecific antibody, a glyco-engineered antibody (or bispecific antibody), an antibody containing an azide (or bispecific antibody), or an azidized antibody (or bispecific antibody), but is not limited thereto.

[0522] Some embodiments of the present disclosure provide a engineered antibody or a salt thereof. In some embodiments, the engineered antibody is AB-(I-Qi) x It can have the structure of Formula 01.

[0523] [Essence 01]

[0524] AB L

[0525]

[0526] 1

[0527] Here, AB is an antibody, and L 1is a linker and a first click chemistry functional group containing an azide group, and x can be an integer from 1 to 4 (for example, x can be an integer of 1, 2, 3, or 4). In certain embodiments, x can be an integer of 1 or 2. In certain embodiments, x can be 2. In some embodiments, the azide structure

[0528] In some embodiments, it can be an azide group N

[0529] exists

[0530]

[0531] is present. In some embodiments, it can have an AB stock, that is, the antibody is a bispecific antibody according to some embodiments of the present disclosure. I? is a linker formed through the glycoconjugate ring of the antibody and can be referred to as a glucosamine linker or a glucose linker, but the name of I? is not limited. I? can be represented as - GlcNAc(Fuc) a - Su-. Here, GlcNAc is connected to AB and then to the following A. That is, the engineered antibody can have the structure of AB-(GlcNAc(Fuc)a - Su - Qi) x GlcNAc is an N-acetylglucosamine moiety, Fuc is a fucose moiety, a is independently 0 or 1 respectively, and Su is a monosaccharide.

[0532] In some embodiments, I? can be connected to the Asn of the antibody (for example, Asn within the heavy chain constant region 2). In some embodiments, I? can be connected through the side chain of Asn within the heavy chain constant region 2 of the antibody. In some embodiments, L 1I 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).

[0533] 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 common characteristic of antibodies.

[0534] Structure of engineered antibody AB-(L 1 - Q x ) x In the case where x is 1, -L 1 - Q 1 It will be understood that it is connected to one heavy chain of the antibody (e.g., to Asn297 on one heavy chain), and if x is 2, each of the two -1-a will be understood to be connected to each of the two heavy chains of the antibody (e.g., to Asn297 on each of the two heavy chains).

[0535] The structure of the engineered antibody, specifically illustrated with I?, is described below.

[0536] In some embodiments, the engineered antibody of Formula 01 may have the structure of Formula 02. In some embodiments, the engineered antibody may have the structure of the following formula:

[0537] [Equation 0

[0538] 「 (F Ya 1

[0539] AB-|- GlcNAc— Su—QI

[0540]

[0541] x

[0542] Here, AB is an antibody; GlcNAc is an N-acetylglucosamine moiety; Fuc is a fucose moiety; a is independently 0 or 1; Su is a monosaccharide; a is an azide; and x is an integer from 1 to 4 (preferably an integer of 1 or 2). Here, Su-Q 1 It can be referred to as azidosugar.

[0543] 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 - GlcNAc(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, - GlcNAc (Fuc) — Su-Q 1 ... can be connected via the side chain of Asn. In some embodiments, in Formula 02 - GlcNAc (Fuc) — Su-Q 1GlcNAc can be linked to the N-glycosylation site (e.g., Asn297) of the bispecific antibody. In some embodiments, GlcNAc in Formula 02 is linked to Asn297 of the bispecific antibody. In some embodiments, GlcNAc in Formula 02 is linked to the bispecific antibody through Asn297 of the bispecific antibody. In some embodiments, GlcNAc in Formula 02 is linked to the bispecific antibody through the side chain of Asn297 of the bispecific antibody. In some embodiments, GlcNAc in Formula 02 can be attached to the amine group (amine group of carboxamide) of the side chain of Asn297 of the bispecific antibody.

[0544] Su is a monosaccharide. Su may be any one selected from galactose (Gal), mannose (Man), glucose (Glc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (N-acetylgalactosamine; GalNAc), N-acetylneuraminic acid or sialic acid (Sial), and fucose (Fuc). Preferably, it may be N-acetylgalactosamine (GalNAc).

[0545] 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.

[0546] 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:

[0547] [Essence 03]

[0548]

[0549] At this time,

[0550] AB is an antibody, and

[0551] a is independently 0 or 1, and

[0552] x is an integer from 1 to 4 (preferably, x is an integer of 1 or 2). Ac refers to an acetyl group, and per is acetamide. In some embodiments, -NHCAc is expressed as -NHC(0)CH3.

[0553] 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,

[0554] AcHN

[0555] I N3

[0556] OH

[0557]

[0558] The structure connected to AB is .

[0559] On the other hand, when a is 0 (i.e., when Gl cNAc is not fucosylated), the structure connected to eu can have the following structure and is not otherwise restricted:

[0560]

[0561] In some embodiments, Equation 03 may be Equation 04.

[0562] In some embodiments, the engineered antibody may have the structure of the following chemical formula 04:

[0563] [Essence 04]

[0564]

[0565] At this time,

[0566] AB is an antibody, and

[0567] a is independently 0 or 1, and

[0568] x is an integer from 1 to 4 (preferably an integer of 1 or 2). In the preceding 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 04 may be attached to the Asn297 of the antibody. In some embodiments, x is 2, and the structure attached to each AB in Formula 04 may be attached to the Asn297 of each of the two heavy chains of the antibody. For example, in Formula 04, the structure attached to AB may be attached to the amine group of the side chain of Asn297. Attached to AB

[0569]

[0570] The structure is this.

[0571] Meanwhile, in the case where , x7} 1 or 2, if the side chain of Asn297 of the antibody is plotted together, the engineered antibody of Formula 04 can be represented by the structure of Formula 05 below:

[0572] [Food 05]

[0573]

[0574] At this time,

[0575] a is independently 0 or 1, and

[0576] x is 1 or 2 and,

[0577] -CH2C(0)NH- connected adjacent to AB represents the side chain of 811297 and is part of AB (antibody).

[0578] Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is represented as -NHC(0)CH3.

[0579] 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.

[0580] 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.

[0581] 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.

[0582] In some embodiments, a method for producing a engineered antibody (or bispecific antibody) comprises endoglycosidase,

[0583] It may include mixing or storing with glycosyltransferase and an azido donor (e.g., azido-sugar). In some embodiments, the method for preparing a engineered antibody from a mixture may further include obtaining the engineered antibody. Mixing or storing with the antibody and the endoglycosidase, glycosyltransferase, and azido donor may be performed in various ways. For example, mixing may be performed by adding a composition containing the antibody to a container and adding a composition containing the endoglycosidase, glycosyltransferase, and azido donor, or each composition containing each of these, to said container. For example, the antibody, endoglycosidase, glycosyltransferase, and azido donor may be added to a container, and after mixing or storing for an appropriate time, the engineered antibody may be obtained from the mixture, but is not otherwise limited. 5.2.3. Linker-payload compound

[0584]

[0585] Antibody-drug conjugates can be obtained by reacting a engineered antibody engineered to contain an azide with a linker-payload compound containing BCN. The reaction between an azide and BCN is widely known in the art as a click chemical reaction and / or bioorthogonal reaction, and a reaction schematic diagram has been described above.

[0586] Some embodiments of the present disclosure provide a linker-payload compound. The linker-payload compound is Q 2 - L 2 - (L 3 - D) It can have the structure of Equation 06 of 2.

[0587] [Essence 06]

[0588] Q2_ L 2_[L 3 — D2

[0589] 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 include a VA-PABC linker (valine-alanine-p-aminobenzyl carbamate linker).

[0590] 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:

[0591] [Essence 07]

[0592] L 3 — D

[0593] Q 2 — L 2 De, L 3 — D

[0594] Reference may be made to the linker-payload compound of the present disclosure [PCT Patent Application Publication No. W02022 / 058395A1]. The linker-payload compound of the present disclosure is commercialized under the trademark SYNtecan E™. Below, the linker-payload compound of the present disclosure is described in detail.

[0595] In some embodiments, a linker-payload compound having the structure of any one of Formulas 06 to 07 may have the structure of Formula 08. In some embodiments, a linker-payload compound of the present disclosure may have the structure of Formula 08 below:

[0596] [Method 08]

[0597]

[0598] The structure of Formula 08 is the structure of the aforementioned linker-payload compound "Q 2 - L 2 - It can be appropriately corresponded to each element of (I?- D)2", and the structure of Equation 08 and "Q 2 - L 2 - (L 3 - D)2" The corresponding example of the structure is as follows:

[0599] This and here, 1* is L 2 Indicates the attachment point with.

[0600] i / WVWW

[0601]

[0602] 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.

[0603] L

[0604]

[0605] 3is this and, here, 1* is L 2 Indicates the attachment point with, and 2* indicates the attachment point with D.

[0606] Japan

[0607]

[0608] And, here 1* represents the attachment point with I?.

[0609] 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:

[0610] [Essence 09]

[0611]

[0612] 5.3. Structure of Antibody-Drug Conjugates

[0613] 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.

[0614] Antibody-drug conjugates can be obtained through the reaction of the aforementioned engineered antibody (i.e., a glyco-engineered antibody containing an azide) and a linker-payload compound. The engineered antibody contains an azide, and the linker-payload compound contains BCN; the engineered antibody and the linker-payload compound are conjugated through a click chemistry reaction (or bioorthogonal reaction) between the azide and BCN. The structure of the engineered antibody is AB-(I-Qi) x It can have, and as explained in the previous paragraphs, here is a first click chemical functional group containing an azide group. Linker-payload compounds have structure Q 2 - L 2 - (L 3 - D) Can have 2, and Q 2 It was explained in the 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-(I?-Q"-L 2 - (L 3 - D)2)y can have the structure of Equation 10.

[0615] Some embodiments of the present disclosure provide an antibody-drug conjugate having the structure of Formula 10.

[0616] [Equation 1 is

[0617]

[0618] 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 A second click chemical functional group (Q) containing ) and BCN 2) represents the structure formed by the reaction of and includes any of the following structures: In some embodiments, "" may have any of the following structures:

[0619]

[0620] where 1* represents the attachment point with L 1 and 2* represents the attachment point with I?.

[0621] 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-R0R1 and Anti-B7-H3 Bispecific Antibody (Anti-R0R1 / B7-H3 Bispecific Antibody)", "5.2.2. Engineered Bispecific Antibody", and "5.2.3. Linker-Payload Compound" of this disclosure.

[0622] For example, AB is an antibody, and in this case, the antibody is a bispecific antibody according to some embodiments of the present disclosure.

[0623] For example, 日 is a glucosamine linker. For example, 日 may have the structure of - GlcNAc(Fuc) a - Su-. For example, I? is a branched linker. For example, I?

[0624] H H

[0625] Shu V

[0626]

[0627] r may have the structure of, where 1* represents the attachment point with Q", and each 2* represents two Ls It should be noted that there may be some inaccuracies in the original text, such as "日" which is not clearly defined. This translation is based on the best understanding of the given text.3 Indicates the attachment points with each of them.

[0628] For example, L 3 is a cleavage linker. For example, L 3 is

[0629]

[0630] can have a structure after that, where 1* indicates the attachment point with L 2 and 2* indicates the attachment point with D.

[0631] For example, 日 is a drug. For example, 日 is an exatecan moiety. For

[0632] instance 日 is

[0633]

[0634] can have a structure where 1* indicates the attachment point with I?.

[0635] For example, y can be an integer from 1 to 4. In a particular embodiment, y can be an integer of 1 or 2. In a particular embodiment, y can be 2. For example, when y is 4, the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is 8. For example, when y is 3, the DAR is 6. For example, when y is 2, the DAR is 4. For example, when y is 1, the DAR is 2. In a particular embodiment, the DAR of the antibody-drug conjugate can be 2 or 4. The antibody-drug conjugate with a DAR of 4 (i.e., AB-(I?-Q"-L 2 -(I?-D)2)y, y = 2) is an engineered antibody having two azide groups (i.e., AB-(I』-Qi) xIt 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 attached to each of the two heavy chains of the antibody (e.g., attached to Asn297 on each of the two heavy chains). Examples of DAR4 antibody-drug conjugates are shown in Figs. 08 and 09. Fig. 08 shows an example of an ADC (DAR4) containing a 2+2 bispecific antibody. Fig. 09 shows an example of an ADC (DAR4) containing a 2+1 bispecific antibody.

[0636] When y is 1, the antibody-to-antibody ratio (dAR) is 2. When y is 1, structure-(L 1 - Q"- L 2 - (L 3 - D)2) is connected to either of the two heavy chains of the antibody. Examples of the structure of a DAR2 antibody-drug conjugate, where y is 1, are illustrated in FIGS. 10 to 12. FIG. 10 illustrates an example of a DAR2 antibody-drug conjugate containing a 2+2 bispecific antibody, and FIGS. 11 and 12 illustrate examples of a DAR2 antibody-drug conjugate containing a 2+1 bispecific antibody. As illustrated in FIGS. 11 and 12, for a DAR2 antibody-drug conjugate containing a 2+1 bispecific antibody, the structure - 1?- Q"- L 2 - (I?- D)2 can be connected to the heavy chain of a bispecific antibody containing the second antigen-binding fragment of the two heavy chains (Fig. 11), or to the heavy chain of a bispecific antibody that does not contain the second antigen-binding fragment of the two heavy chains (Fig. 12).

[0637] The structure of the DAR2 antibody-drug conjugate is explained here in more detail using an antibody-drug conjugate containing a 2+2 bispecific antibody as an example. DAR = antibody-drug conjugate (i.e., AB-(L 1 - Q"- L 2 - (L 3 - D)2) y , y=l) is an engineered antibody having two azide groups (i.e., AB-(I?-Qi) x , x=2) formed by conjugating one linker-payload compound, or a engineered antibody having one azide group (i.e., AB-(I?-Qi) x 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 - D) 2) is connected to either of the two heavy chains of the antibody. Examples of DAR2 antibody-drug conjugates are disclosed in FIGS. 13 and 14. FIG. 13 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. 14 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.

[0638] In some embodiments, 日 can be linked to an Asn of an antibody (i.e., a bispecific antibody), such as an Asn within the heavy chain constant region 2. In some embodiments, I』 can be linked through the side chain of an Asn within the heavy chain constant region 2 of the antibody. In some embodiments, L 1 can be linked to an N-glycosylation 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 each I』 can 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, 日 can be linked through the side chain of Asn297. In some embodiments, L 1 is linked to the amine group of the side chain of Asn297 (the amine group of carboxamide). In some embodiments, L 1 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 an 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 an N-glycosylation site of the antibody (e.g., Asn297). In some embodiments,-(L 1 - Q"- L 2 - (L 3 - D)2) can be linked to Asn297 of the antibody. In some embodiments,

[0639]

[0640] !?-(!? - D)2) is connected through the side chain of Asn297. In some embodiments, - (I?- Q"- L 2 - (I?- D)2) is connected to the amine group of the side chain of Asn297 (the amine group of carboxamide). In some embodiments, - (L 1 - Q "-!?-(!? - D)2) is attached to the amine group of the side chain of Asn297 (the amine group of carboxamide (- C(O)NH-)). In some embodiments, L 1 The Asn in the antibody (e.g., Asn297) that can be connected is shown as follows:

[0641]

[0642] Here, 1* and 2* each represent the attachment points to the amino acid residues adjacent to Asn (e.g., Asn297) in the antibody, and 3* is L 1 represents the attachment point to.

[0643] In some embodiments, the antibody-drug conjugate having the structure of formula 10 can have the structure of formula 11. In some embodiments, the antibody-drug conjugate can have the structure of the following formula 11:

[0644] [Formula 11]느!? 3 — D

[0645] AB L 1— Q"— L 2 드

[0646] 丄 L 3 — D

[0647]

[0648] y.

[0649] An antibody-drug conjugate may comprise 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 comprise 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 via a linker. In some embodiments, two drugs may be linked to the antibody via a linker. For example, in Formula 11, AB is a bispecific

[0650] i. 1 O .. L 2 to L3 — I |— L — Q - L,, 3

[0651]

[0652] antibody, L — followed by is the linker of the antibody-drug conjugate, and in Formula 11, each 日 is a drug.

[0653] 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:[[]]

[0654] [Formula 1 is

[0655]

[0656] At this time,

[0657] AB is an antibody, and at this time the antibody is a bispecific antibody according to some embodiments of the present disclosure,

[0658] GlcNAc is an N-acetylglucosamine moiety,

[0659] Su is a monosaccharide,

[0660] Fuc is a foucose moiety, and

[0661] a is independently 0 or 1, and

[0662] 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.

[0663] 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 can be attached to the amine group of the side chain of Asn297. In Formula 12, the structure connected to AB is as follows:

[0664]

[0665] In some embodiments, an antibody-drug conjugate having the structure of any one 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:

[0666] [Equation 13]

[0667]

[0668] In this case, AB is an antibody (e.g., a bispecific antibody), and

[0669] a is independently 0 or 1, and

[0670] y may 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.

[0671] Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is expressed as -NHC(0)CH3.

[0672] 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:

[0673] — No— zu

[0674] S,^ 0

[0675]

[0676] 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:

[0677] [Equation 14]

[0678]

[0679] At this time,

[0680] AB is an antibody (e.g., a bispecific antibody), and

[0681] a is independently 0 or 1, and

[0682] y may 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.

[0683] Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is represented as -NHC(0)CH3.

[0684] 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-glycosylation 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:

[0685]

[0686] Examples of antibody-drug conjugates of Equation 14 (y=2, DAR4 ADC) are shown in Figs. 15 and 16. Fig. 15 shows an example of a DAR4 ADC containing a 2+2 bispecific antibody, and Fig. 16 shows an example of a DAR4 ADC containing a 2+1 bispecific antibody.

[0687] 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 depicted. In some embodiments, y may be 1 or 2, and the antibody-drug conjugate may have the structure of Formula 15 below:

[0688] [Equation 15]

[0689]

[0690] At this time,

[0691] AB is an antibody (e.g., a bispecific antibody), and

[0692] a is independently 0 or 1, and

[0693] y is 1 or 2 and,

[0694] - CH2C(0)NH- connected adjacent to AB represents the side chain of 811297 and is part of AB (antibody).

[0695] Ac refers to an acetyl group, and NHAc is acetamide. In some embodiments, -NHAc is represented as -NHC(0)CH3.

[0696] To aid the understanding of those skilled in the art, Tables 11 and 12 below provide examples of amino acid sequences of antibodies (i.e., bispecific antibodies) according to some embodiments of the present disclosure, and indicate the Asn297 position in the amino acid sequence. In Table 11 below, the amino acid sequences of each heavy chain and each light chain of an example of a 2+2 bispecific antibody are disclosed, and in Table 12 below, the amino acid sequences of each heavy chain and each light chain of an example of a 2+1 bispecific antibody are disclosed. In Tables 11 and 12, Asn297 is indicated by underlining and bolding. Asn297 is located within the Fc region or within the CH2 domain, as previously described.

[0697] [Table 11] Amino acid sequences of each heavy chain and each light chain of the 2+2 bispecific antibody and

[0698] Asn297 digit display

[0699] chain Amino acid sequence

[0700] 제 1 중쇄 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGTOYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

[0701] DTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREFQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTI SCSGSSSNI GSNAVSW YQQLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAY VFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDY AMSWVRQAPGKCLEWVSSI SSGSGSI YYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYY CAKNLIPLDYWGQGTLVTVSS (서열번호 49)

[0702] First light chain QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSS EELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPXECS (Sequence No. 18) (where , X is A or )

[0703] 2nd heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGTOYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

[0704] DTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREFQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTI SCSGSSSNI GSNAVSW YQQLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAY VFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDY AMSWVRQAPGKCLEWVSSI SSGSGSI YYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYY CAKNLIPLDYWGQGTLVTVSS (SEQ ID NO: 49)

[0705] Second light chain QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSS EELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPXECS (Sequence No. 18) (where , X is A or

[0706]

[0707] euim ) [Table 1 shows the amino acid sequences of each heavy chain and each light chain of the 2+1 bispecific antibody and the indication of the Asn297 site

[0708] chain Amino acid sequence

[0709] 1st heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGTOYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

[0710] DTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREFQVYTLPPSREEMTKNQVSLWCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 59)

[0711] First light chain QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSS EELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPXECS (Sequence No. 18) (where , X is A or )

[0712] 제 2 중쇄 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGAWDTGTOYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

[0713] DTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREFQVYTLPPSREEMTKNQVSLSCAVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTI SCSGSSSNI GSNAVSW YQQLPGTAPKLLIYYNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAY VFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDY AMSWVRQAPGKCLEWVSSI SSGSGSI YYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYY CAKNLIPLDYWGQGTLVTVSS (서열번호 60)

[0714] Second light chain QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYENNRPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSS EELQANKATLVCL I SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPXECS (Sequence No. 18) (where , X is A or

[0715]

[0716] Euim )

[0717] 5.4. Preparation of Antibody-Drug Conjugates

[0718] 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.

[0719] 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.

[0720] [Reaction Schematic 0

[0721] X 2 Formula 01 Formula 06

[0722]

[0723] Formula 10

[0724] 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.

[0725] 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.

[0726] [Reaction Schematic Diagram 03]

[0727]

[0728] Formula 14

[0729] Here, each equation and each variable (AB, a, x, y, etc.) are as described in the previous paragraph. For example, when two linker-payload compounds of Equation 09 react with one engineered antibody of Equation 04 (where arc is 2), y can be 2.

[0730] The antibody-drug conjugate of the present disclosure may be prepared through the following two processes: preparation of a engineered antibody through the engineering (glyco-engineering) of the antibody; and reaction of the engineered antibody with a linker-payload compound.

[0731] In some embodiments, a method for preparing an antibody-drug conjugate of the present disclosure may include the following:

[0732] Contact or react the engineered antibody with the linker-payload compound.

[0733] 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.

[0734] 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.

[0735] 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.

[0736] 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 is not otherwise limited and any method widely known in the art may be used. For example, purification may be performed by Size Exclusion Chromatography (SEC), Hydrophobic

[0737] Interaction Chromatography (HIC), Ion Exchange Chromatography (IEX), High-

[0738] This may be performed via one or more methods of Performance Liquid Chromatography (HPLC) and AF first chromatography, but is not otherwise limited. For example, a crude product of an antibody-drug conjugate is obtained by mixing a composition containing an engineered antibody with a composition containing a linker-payload compound, and this crude

[0739] The product can be purified through methods 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 ADC.

[0740] In some embodiments, the method for preparing an antibody-drug conjugate may be performed in vitro or ex vivo.

[0741] 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.

[0742] 5.5. Targets of Antibody-Drug Conjugates

[0743] 5.5.1. Targets of Antibody-Drug Conjugates - R0R1 and B7-H3

[0744]

[0745] The antibody-drug conjugate of the present disclosure comprises a bispecific antibody (i.e., an anti-R0R1 / B7-H3 bispecific antibody) according to some embodiments of the present disclosure, and accordingly, can target R0R1 and B7-H3. The antibody-drug conjugate of the present disclosure can bind to either or more of R0R1 and B7-H3. The antibody-drug conjugate of the present disclosure can bind to R0R1. The antibody-drug conjugate of the present disclosure can bind to B7-H3. The target antigens of the antibody-drug conjugate of the present disclosure are R0R1 and B7-H3. Below, the characteristics of these target antigens and the antibody-drug conjugate are described in detail.

[0746] 5.5.2. Target Antigen 1 of Antibody-Drug Conjugate - R0R1

[0747]

[0748] In some embodiments, the antibody-drug conjugate may have the ability to bind to R0R1, and with respect to R0R1 as a target antigen, the above "5.2.1. Antibody: Anti-

[0749] Among the “R0R1 and anti-B7-H3 bispecific antibodies”, “(1) bispecific antibody - overview” is specifically described in the present disclosure.

[0750] In some embodiments, the binding affinity of the antibody-drug conjugate to R0R1 (e.g., human R0R1) may be about 10 nM to 200 nM based on the dissociation constant (KD) [e.g., equilibrium dissociation constant]. In some embodiments, the binding affinity of the antibody-drug conjugate to R0R1 may be about 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM based on the dissociation constant.

[0751] 85nM, 90nM, 95nM, lOOnM, HOnM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM,

[0752] It may be 180 nM, 190 nM, or 200 nM, or within the range of two values ​​selected from the above values. In some embodiments, the binding affinity of the antibody-drug conjugate to R0R1 may be 60 nM to 160 nM, 70 nM to 140 nM, 80 nM to 100 nM, or 85 nM to 95 nM based on the dissociation constant, but is not limited thereto.

[0753] In some embodiments, the antibody-drug conjugate can bind to cells expressing R0R1 (e.g., human tumor cells). For example, the antibody-drug conjugate can bind to cells expressing R0R1 on the cell surface. For example, the antibody-drug conjugate may have specificity for cells and / or tissues expressing R0R1 (e.g., tumor cells and / or tissues).

[0754] 5.5.3. Target Antigen 2 of Antibody-Drug Conjugate - B7-H3

[0755]

[0756] In some embodiments, the antibody-drug conjugate may have binding ability to B7-H3, and with respect to B7-H3 as a target antigen, the above "5.2.1. Antibody: Anti-

[0757] Among the “R0R1 and anti-B7-H3 bispecific antibodies”, “(1) bispecific antibody - overview” is specifically described in the present disclosure.

[0758] In some embodiments, the antibody-drug conjugate B7-H3 (e.g., human B7-

[0759] The binding affinity for H3) may be approximately 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 for B7-H3 is approximately 0.0M, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM based on the dissociation constant.

[0760] 8nM, 9nM, lOnM, llnM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, 20nM,

[0761] It may be 22 nM, 24 nM, 26 nM, 28 nM, or 30 nM, or within the range of two values ​​selected from the above values. In some embodiments, the binding affinity of the antibody-drug conjugate to B7-H3 may be 2 nM to 20 nM, 2 nM to 15 nM, 3 nM to 12 nM, 4 nM to 100 nM, or 4 nM to 8 nM based on the dissociation constant, but is not limited thereto.

[0762] In some embodiments, the antibody-drug conjugate can bind to cells expressing B7-H3 (e.g., human tumor cells). For example, the antibody-drug conjugate can bind to cells expressing B7-H3 on the cell surface. For example, the antibody-drug conjugate may have specificity for cells and / or tissues expressing B7-H3 (e.g., tumor cells and / or tissues).

[0763] 5.5.4. Dual specificity for R0R1 and B7-H3

[0764]

[0765] In some embodiments, the antibody-drug conjugate can bind to both R0R1 and B7-H3.

[0766] In some embodiments, the antibody-drug conjugate is capable of binding to cells (e.g., human tumor cells) that express one or more of R0R1 and B7-H3 (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 R0R1 and B7-H3.

[0767] In some embodiments, the antibody-drug conjugate can bind to cells expressing both R0R1 and B7-H3 (e.g., human tumor cells). For example, the antibody-drug conjugate can bind to cells expressing R0R1 on the cell surface and B7-H3 on the cell surface. For example, the antibody-drug conjugate may have specificity to cells and / or tissues expressing both R0R1 and B7-H3 (e.g., tumor cells and / or tissues).

[0768] 5.6. Target Tumors of Antibody-Drug Conjugates

[0769] 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.

[0770] In some embodiments, the tumor (or target tumor) is colon cancer, colorectal cancer, rectal cancer, renal cancer, renal cell carcinoma, breast cancer (e.g., examples of breast cancer include triple-negative breast cancer), liver cancer (e.g., examples of liver cancer include hepatocellular carcinoma), lung cancer (e.g., examples of lung cancer include small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), and lung neuroendocrine carcinoma (lung NEC)), stomach Cancer (Gastric cancer), Neuroendocrine cancer, Large cell neuroendocrine cancer (LCNEC), Prostate cancer, Small intestine cancer, Esophageal cancer, Melanoma, Orthopedic cancer, Bone cancer, Pancreatic cancer, Skin cancer, Head or neck cancer, Cutaneous or intraocular malignant melanoma, Uterine cancer,Ovarian cancer (e.g., examples of ovarian cancer include 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, carcinoma of the vulva, Hodgkin's Disease, lymphoma, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, Parathyroid gland cancer, Adrenal cancer, Adrenocortical carcinoma, Mesothelioma, Cholangiocarcinoma, Esophageal cancer, Salivary gland cancer, Sarcoma, Soft tissue sarcoma, Cancer of the urethra, Urothelial cancer, Penile cancer, Pediatric solid tumors, Bladder cancer, Kidney or ureter cancerIt may be, but is not limited to, one or more selected from renal pelvis carcinoma, neurological cancer, neoplasm of the central nervous system (CNS), neuroendocrine tumor (carcinoid), 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 metastatic lesions of the above cancers.

[0771] In specific embodiments, the tumor (or target tumor) may be one or more selected from the following: lymphoma, ovarian cancer, colon cancer, colorectal cancer, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer, lung neuroendocrine carcinoma (lung NEC), melanoma, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, endometrial cancer, neuroendocrine cancer, macrocephalic nerve Endocrine cancer (large cell neuroendocyte cancer: LCNEC), Prostate cancer, Adrenal cancer, Breast cancer, Triple-negative breast cancer, Mesothelioma, Cholangiocarcinoma, Esophageal cancer, Gastric cancer, Thyroid cancer, Head and neck cancer, Sarcoma, Hepatocellular carcinoma, Neurological cancer, Glioma, Adrenocortical carcinomaRenal cancer, renal cell carcinoma, cervical cancer, orthotopic cancer, and salivary gland cancer.

[0772] In a specific embodiment, the tumor (or target tumor) may be one or more selected from the following: small cell lung cancer (SCLC), ovarian cancer, non-smal cell lung cancer (NSCLC), head and neck cancer, sarcoma, breast cancer, and prostate cancer.

[0773] 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 (e.g., locally 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).

[0774] In some embodiments, the tumor (or target tumor) may be a tumor expressing R0R1 (RORl-expressing tumor). In some embodiments, the tumor (or target tumor) may be a tumor overexpressing R0R1 (R0R1-overexpressing tumor). In some embodiments, the tumor (or target tumor) may be an R0R1-positive tumor. In some embodiments, the tumor (or target tumor) may be a tumor associated with R0R1. In some embodiments, the tumor expressing R0R1 may be a tumor overexpressing R0R1. In some embodiments, the tumor expressing R0R1 may be an R0R1-positive tumor. In some embodiments, the expression level of R0R1 in the tumor expressing R0R1 may be a low expression level, an intermediate expression level, and a high expression level, but is not limited thereto. In some embodiments, the tumor expressing R0R1 may have a high R0R1 expression level compared to normal cells or tissues.

[0775] In some embodiments, the tumor (or target tumor) may be a B7-H3-expressing tumor. In some embodiments, the tumor (or target tumor) may be a B7-H3-overexpressing tumor. In some embodiments, the tumor (or target tumor) may be a B7-H3-positive tumor. In some embodiments, the tumor (or target tumor) may be a B7-H3-associated tumor. In some embodiments, the B7-H3-expressing tumor may be a B7-H3-overexpressing tumor. In some embodiments, the B7-H3-expressing tumor may be a B7-H3-positive tumor. In some embodiments, the expression level of B7-H3 in a tumor expressing B7-H3 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 B7-H3 may have a high B7-H3 expression level compared to normal cells or tissues.

[0776] In some embodiments, the tumor (or target tumor) may be a tumor expressing one or more of R0R1 and B7-H3. In some embodiments, the target tumor may be a tumor associated with one or more of the expression of R0R1 and the expression of B7-H3. In some embodiments, the target tumor may be a tumor expressing both R0R1 and B7-H3. In some embodiments, the tumor may be a tumor expressing R0R1 but not expressing or hardly expressing B7-H3. In some embodiments, the tumor may be a tumor expressing B7-H3 but not expressing or hardly expressing R0R1. In some embodiments, the tumor may be a tumor expressing both R0R1 and B7-H3. In some embodiments, the tumor may be a tumor overexpressing both R0R1 and B7-H3. In some embodiments, the tumor may be a tumor that expresses R0R1 at any one of a low expression level, an intermediate expression level, and a high expression level, and expresses B7-H3 at any one of a low expression level, an intermediate expression level, and a high expression level. Some

[0777] In an embodiment, the tumor may be an R0R1-positive and / or B7-H3-positive tumor.

[0778] In some embodiments, the tumor (or target tumor) may be a tumor expressing both R0R1 and B7-H3. In some embodiments, the target tumor may be an R0R1-positive and B7-H3-positive tumor. In some embodiments, the target tumor may be a tumor associated with both R0R1 and B7-H3. In some embodiments, the target tumor may be a tumor associated with both R0R1 expression and B7-H3 expression. In some embodiments, the target tumor may be a tumor co-expressing R0R1 and B7-H3. In some embodiments, a tumor expressing both R0R1 and B7-H3 may be an R0R1-positive and B7-H3-positive tumor.

[0779] In some embodiments, the tumor (or target tumor) may be a tumor expressing R0R1 and / or B7-H3 (or an R0R1-positive and / or B7-H3-positive tumor). For example, the tumor (or target tumor) is R0R1-positive and / or B7-H3-positive, colon cancer, colorectal cancer, rectal cancer, renal cancer, renal cell carcinoma, breast cancer, triple-negative breast cancer, liver cancer, hepatocellular carcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung neuroendocrine carcinoma, gastric cancer, neuroendocrine cancer ), large cell neuroendocrine cancer (LCNEC), prostate cancer, cancer of the small intestine, cancer of the esophagus, melanoma, orthotopic cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, 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, carcinoma of the vulva, Hodgkin's Disease, lymphoma, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, parathyroid cancer (cancer of the parathyroid gland), Adrenal cancer, Adrenocortical carcinoma, Mesothelioma, Cholangiocarcinoma, Esophageal cancer, Salivary gland cancer, Sarcoma, Soft tissue sarcoma, Cancer of the urethra, Urothelial cancer, Cancer of the penis, Pediatric solid tumors,It may be any one selected from bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neurological cancer, neoplasm of the central nervous system (CNS), neuroendocrine tumor (carcinoid), primary CNS lymphoma, spinal axis tumor, glioma, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, and T-cell lymphoma.

[0780] Whether a tumor expresses R0R1 or B7-H3 can be identified through methods known in the art. For example, whether a tumor expresses R0R1 (or B7-H3) can be determined by immunohistochemistry of the corresponding tumor tissue or tumor cells

[0781] It can be confirmed by testing through IHC (Intracellular Hydrogenation), FISH (Fluorescence in situ hybridization), PCR (polymerase chain reaction), NGS (Next-generation sequencing), etc. For example, whether a tumor expresses R0R1 (or B7-H3) can be confirmed through a biopsy (or tissue biopsy).

[0782] In some embodiments, whether a tumor expresses R0R1 (or B7-H3) can be confirmed by performing immunohistochemical testing according to a known protocol on tumor tissue (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, R0R1 (or B7-H3) IHC testing involves collecting tumor tissue (or suspected tumor tissue) from a patient; preparing a specimen from the tumor tissue; treating the specimen with an anti-R0R1 antibody (or anti-B7-H3 antibody) (primary antibody); This can be performed by a method comprising treating a specimen 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 R0R1 (or B7-H3) expression 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 weak or 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, based on IHC test results, the immunostaining score or IHC grade (IHC Tumors rated as grade 1+ or higher (e.g., 1+, 2+, or 3+) may be R0R1 (or B7-H3) benign tumors.

[0783] 5.7. Composition containing an antibody-drug conjugate

[0784] 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).

[0785] In some embodiments, a composition (or kit) containing an antibody-drug conjugate may contain one or more antibody-drug conjugates.

[0786] 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.

[0787] 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.

[0788] 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.

[0789] 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

[0790] 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 the composition containing the 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.

[0791] 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.

[0792] 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.

[0793] 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.

[0794] 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, methyl hydroxybenzoate, propyl hydroxybenzoate, 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

[0795] It may further include one or more of hydrochloride (arginine hydrochloride), sucrose, and polysorbate (e.g., polysorbate 80).

[0796] 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.

[0797] 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.

[0798] 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).

[0799] 5.8. Functions / Effects of Antibody-Drug Conjugates

[0800] 5.8.1. Overview of Functions / Effects of Antibody-Drug Conjugates

[0801]

[0802] In some embodiments, the antibody-drug conjugate of the present disclosure may have the ability to bind to R0R1 and / or B7-H3. In some embodiments, the antibody-drug conjugate may bind to R0R1 (e.g., human R0R1 or a variant thereof). In some embodiments, the antibody-drug conjugate may bind to B7-H3 (e.g., human B7-H3 or a variant thereof).

[0803] In some embodiments, the antibody-drug conjugate may bind to R0R1 expressed on the surface (or cell membrane) of a cell (e.g., human cell). In some embodiments, the antibody-drug conjugate may bind to B7-H3 expressed on the surface (or cell membrane) of a cell.

[0804] In some embodiments, the antibody-drug conjugate may bind to R0R1-positive cells (e.g., R0R1-positive human cells or R0R1-positive human cancer cells). In some embodiments, the antibody-drug conjugate may bind to B7-H3-positive cells (e.g., B7-H3-positive human cells or B7-H3-positive human cancer cells).

[0805] In some embodiments, the antibody-drug conjugate can bind to cells expressing both R0R1 and B7-H3. In some embodiments, the antibody-drug conjugate is designed to target R0R1 and B7-H3 together, and since R0R1 has a low expression level in normal tissues and a high expression level in tumor cells, and B7-H3 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.

[0806] 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).

[0807] 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).

[0808] 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 effects, immunogenic cell death, and apoptosis induction by a drug released into the cell, but are not limited thereto.

[0809] 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.

[0810] 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-severely toxic dose).

[0811] In some embodiments, the antibody-drug conjugate may have a wide (or excellent) therapeutic index (TI). In some embodiments, the antibody-drug conjugate may have a wide (or excellent) therapeutic window (TW).

[0812] In some embodiments, the antibody-drug conjugate may have a superior toxicity profile while possessing high tumor suppression ability in vivo. Some

[0813] In an embodiment, the antibody-drug conjugate may be an antibody-drug conjugate having an excellent balance of efficacy / toxicity.

[0814] 5.8.2. Targeting

[0815]

[0816] The antibody-drug conjugate of the present disclosure may have the ability to bind to RQR1 and / or B7-H3. In some embodiments, the antibody-drug conjugate may bind to R0R1 (e.g., human R0R1 or a variant thereof). In some embodiments, the antibody-drug conjugate may bind to B7-H3 (e.g., human B7-H3 or a variant thereof).

[0817] In some embodiments, the antibody-drug conjugate can bind to R0R1 expressed on the surface (or cell membrane) of a cell (e.g., human cell).

[0818] In some embodiments, the antibody-drug conjugate can bind to B7-H3 expressed on the surface (or cell membrane) of the cell.

[0819] In some embodiments, the antibody-drug conjugate may bind to cells expressing R0R1. In some embodiments, the cells expressing R0R1 may be cells overexpressing R0R1. In some embodiments, the cells expressing R0R1 are R0R1-positive (R0R1-

[0820] It may be a cell that is positive. In some embodiments, the antibody-drug conjugate may bind to a cell expressing B7-H3. In some embodiments, the cell expressing B7-H3 may be a cell that overexpresses B7-H3. In some embodiments, the cell expressing B7-H3 may be an R0R1-positive cell.

[0821] In some embodiments, the antibody-drug conjugate may bind to cells co-expressing R0R1 and B7-H3. In some embodiments, the cells co-expressing R0R1 and B7-H3 may be cells that overexpress R0R1 and express B7-H3. In some embodiments, the cells co-expressing R0R1 and B7-H3 may be cells that express R0R1 and overexpress B7-H3. In some embodiments, the cells co-expressing R0R1 and B7-H3 may be cells that overexpress R0R1 and overexpress B7-H3. In some embodiments, the cells co-expressing R0R1 and B7-H3 may be R0R1- and B7-H3-positive cells.

[0822] 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.

[0823] In some embodiments, the antibody-drug conjugate may bind to a tumor (e.g., cancer) or tumor tissue (e.g., cancer tissue) expressing R0R1. In some embodiments, the antibody-drug conjugate may target a tumor (e.g., cancer) or tumor tissue (e.g., cancer tissue) expressing R0R1. In some embodiments, the tumor expressing R0R1 may be a tumor that overexpresses R0R1. In some embodiments, the tumor expressing R0R1 may be an R0R1-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 B7-H3. In some embodiments, the antibody-drug conjugate may target a tumor (e.g., cancer) or tumor tissue (e.g., cancer tissue) expressing B7-H3. In some embodiments, the tumor expressing B7-H3 may be a tumor that overexpresses B7-H3. In some embodiments, the tumor expressing B7-H3 may be a B7-H3-positive tumor.

[0824] In some embodiments, the antibody-drug conjugate is designed to target R0R1 and B7-H3 together and can bind more specifically to tumor cells co-expressing R0R1 and B7-H3, and accordingly, the toxicity of the antibody-drug conjugate to normal tissues may be low.

[0825] In some embodiments, targeting both antigens R0R1 and B7-H3 may be effective in treating metastatic tumors. R0R1 is known to be expressed at high levels in metastatic and / or late-stage cancers. R0R1 is known to be involved in Epithelial-Mesenchymal Transmission (EMT), which has been reported to be deeply associated with tumor metastasis in cancer cells, and B7-H3 is also known to mediate EMT and the development of Cancer Stem Cells (CSCs), which have been reported to be deeply associated with tumor metastasis in cancer cells. Furthermore, it is reported that inhibiting or targeting the expression of R0R1 or B7-H3 can inhibit tumor metastasis.

[0826] In some embodiments, dual specificity for R0R1 and B7-H3 may enable one or more of the following: improved tumor-specific binding affinity due to the specificity of R0R1 and the overexpression of B7-H3, improved tumor-specific payload delivery affinity, enhanced anti-R0R1 anticancer activity, and reduced non-target toxicity.

[0827] In some embodiments, the antibody-drug conjugate targeting R0R1 and B7-H3 together may not have ADCC activity, and thus non-specific immune responses are minimized, while a therapeutic effect may be exhibited independently of immune-mediated function.

[0828] 5.8.3. Internalization and Drug Interruption

[0829]

[0830] 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

[0831] 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 comprises a cleavable portion capable of being 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 DNA

[0832] DNA topoisomerase I inhibitors are known to cause damage to the cell's solar saturation and induce cell death.

[0833] 5.8.4. Additional Cell Death Mechanisms

[0834]

[0835] 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.

[0836] 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 stopping the progression of the cell cycle at the S or G2 phase. Cell cycle arrest can effectively inhibit the proliferation of tumor cells, which may help in the suppression of cancer or tumors. For a method to confirm the cell cycle arrest-inducing effect of the antibody-drug conjugate, "Example 5" in Section 8, "Examples," of the present disclosure may be referenced.

[0837] 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 5” in Section “8. Examples” of the present disclosure.

[0838] 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 5" in Section 8, "Examples," of this disclosure.

[0839] 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 to which the antibody-drug conjugate binds (e.g., R0R1 and B7-H3) 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 occurs heterogeneously. For a method to verify the bystander killing effect of the antibody-drug conjugate, "Example 6" in Section 8. Examples of the present disclosure may be referenced.

[0840] 5.8.5. Antitumor effect

[0841]

[0842] 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).

[0843] 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).

[0844] In some embodiments, the anti-tumor 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 anti-tumor effect. In some embodiments, the antibody-drug conjugate may have an excellent in vivo anti-tumor 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.

[0845] 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 selected from BA6 SYNtecan E™ ADC, B5 SYNtecan E™ ADC, DS-7300, VLS-101, and ABL206-Dxd (a conjugate of ABL206 naked antibody and deruxtecan), and is not otherwise limited. Information regarding comparative drugs is described in detail in Section "8. Examples" of this disclosure. In some embodiments, the cancer cell line may be any one of Calu-3, HCC1187, PA-1, A549, NCI-H446, H82, MDA-MB-231, Calu-6, DU-145, KATO I II, and KATO II I (KATO I II-huB7H3) engineered to overexpress human B7-H3, but is not otherwise limited. Information regarding the cancer cell line is described in detail in Section "8. Examples" of this disclosure.

[0846] 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 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.

[0847] TGI can be calculated by the following formula: TGI = ( (mean (Ct) - mean (CO ) ) - ( mean (Tt) - mean (TO ) ) ) / (mean(Ct)-mean(CO) ) x 100% ,

[0848] At this time,

[0849] 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

[0850] mean(CO) is the tumor size (or average tumor size) of the control group at the time of administration (e.g., at the time of the first administration of the antibody-drug conjugate), and

[0851] mean(Tt) is the tumor size (or average tumor size) of the experimental group (e.g., antibody-drug conjugate administration group) at the time of measurement, and

[0852] mean(TO) is the tumor size (or average tumor size) of the experimental group at the time of administration (e.g., the time of the first administration of the antibody-drug conjugate).

[0853] In some embodiments, the antibody-drug conjugate may exhibit excellent antitumor effects in mouse xenograft models (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 line-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 R0R1 and / or B7-H3.

[0854] In some embodiments, the TGI of the antibody-drug conjugate may be the TGI measured in any one of the following carcinomas: breast cancer (e.g., triple-negative breast cancer), head and neck cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), ovarian cancer, pancreatic cancer, sarcoma, neuroendocrine cancer (e.g., pulmonary neuroendocrine cancer or large cell neuroendocrine cancer), and prostate cancer. Methods for evaluating in vivo antitumor effects in mouse xenograft models are well known in the art. For methods for evaluating in vivo antitumor effects in mouse xenograft models, reference may be made to "Example 7," "Example 8," "Example 11," and "Example 19" in Section "8. Examples" of the present disclosure.

[0855] 5.8.6. Excellent toxicity profile - Low toxicity

[0856]

[0857] 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 exhibit low toxicity upon in vivo administration. For example, the antibody-drug conjugate of the present disclosure may have low toxicity at a level suitable for administration to humans.

[0858] In some embodiments, the highest non-severely toxic dose (HNSTD) of the antibody-drug conjugate in monkeys (e.g., cynomolgus monkeys) may be 140 mpk (mg / kg(body weight)), 130 mpk, 120 mpk, 110 mpk, 100 mpk, 90 mpk, 80 mpk, 70 mpk, 60 mpk, 50 mpk, 40 mpk, or 30 mpk or higher, or may be within a range of two values ​​selected from the aforementioned mpk values. In some embodiments, when the antibody-drug conjugate is administered twice at a 3-week interval (e.g., administration on day 1 and administration on day 22), the cynomolgus monkey HNSTD may be 140 mpk, 130 mpk, 120 mpk, 110 mpk, 100 mpk, 90 mpk, 80 mpk, 70 mpk, 60 mpk, 50 mpk, 40 mpk, or 30 mpk or higher, or may be within the range of two values ​​selected from the aforementioned mpk values. In a specific embodiment, the monkey HNSTD of the antibody-drug conjugate may be about 120 mpk, 110 mpk, 100 mpk, 90 mpk, 80 mpk, 70 mpk, or 60 mpk.

[0859] In some embodiments, the antibody-drug conjugate may have excellent tolerability up to a dose of 120 mg / kg when administered intravenously to monkeys (e.g., cynomolgus monkeys) repeatedly once every three weeks (on days 1 and 22).

[0860] For a method to confirm the toxicity of an antibody-drug conjugate in monkeys, "Example 10" in Section "8. Examples" of this disclosure may be referenced.

[0861] In some embodiments, the maximum no-observed-adverse-effective dose (NOAEL) of the antibody-drug conjugate in rats (e.g., Sprague-Dawley rats) may be 80 mpk (mg / kg (body weight)), 90 mpk, 100 mpk, 110 mpk, 120 mpk, 130 mpk, 140 mpk, 150 mpk, 160 mpk, 170 mpk, or 180 mpk or higher, or may be within a range of two values ​​selected from the aforementioned mpk values. In some embodiments, when the antibody-drug conjugate is administered twice at a 2-week interval (e.g., administration on day 1 and administration on day 15), the NOAEL in Sprague-Dawley rats may be 120 mpk, 130 mpk, 140 mpk, 150 mpk, or 160 mpk or higher, or may be within the range of two values ​​selected from the aforementioned mpk values. For a method to determine the rat NOAEL of the antibody-drug conjugate, "Example 20" in Section 8, "Examples" of this disclosure may be referenced.

[0862] In some embodiments, the antibody-drug conjugate may have low on-target toxicity. On-target toxicity may refer to the toxicity that occurs when the antibody-drug conjugate acts on a normal cell (i.e., a normal cell expressing a target antigen) by targeting the target antigen.

[0863] 5.8.7. Excellent Stability and Developability

[0864]

[0865] In some embodiments, the antibody-drug conjugate of the present disclosure may have excellent (or high) stability. In some embodiments, the antibody-drug conjugate may have excellent (or high) development potential. For a method to confirm the stability and development potential of the antibody-drug conjugate, reference may be made to “Example 17” in Section “8. Examples” of the present disclosure.

[0866] In some embodiments, the antibody-drug conjugate may have excellent stress stability. Stress stability parameters (e.g., purity, charge heterogeneity, polydispersity, or pharmacological activity at the cellular level) may be evaluated under appropriate conditions. For example, stress stability parameters may be evaluated by comparing an antibody-drug conjugate stored at 40T for 2 weeks with an antibody-drug conjugate stored at 4T. In some embodiments, the difference between the purity (%) of the antibody-drug conjugate stored at 4T and the purity (%) of the antibody-drug conjugate stored at 40T may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 20% or less. Purity may be measured, for example, by Size Exclusion Chromatography — High-Performance Liquid Chromatography (SEC-HPLC), Capi 1 lary electrophoresis sodium dodecyl sulfate (CE-SDS) (Non-Reduced), or Capi 1 lary electrophoresis sodium dodecyl sulfate (CE-SDS) (Reduced), but is not otherwise limited. In some embodiments, the difference between the charge heterogeneity of antibody-drug conjugate stored at 4T and the charge heterogeneity of antibody-drug conjugate stored at 40T may be 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% or less based on the main. Charge heterogeneity can be measured, for example, through Imaged Capi 1 lary Isoelectric Focusing (icIEF), but is not otherwise limited.In some embodiments, the difference in charge heterogeneity of antibody-drug conjugates stored at 4T and the difference in polydispersity of antibody-drug conjugates stored at 40T (i.e., APDI) may be 0.1, 0.2, 0.3, or 0.4 or less. Polydispersity may be measured, for example, by dynamic light scattering (DLS), but is not otherwise limited. For a method of evaluating the stress stability of antibody-drug conjugates, reference may be made to "Example 17" in Section "8. Examples" of this disclosure.

[0867] In some embodiments, the antibody-drug conjugate may have excellent serum stability. In some embodiments, serum stability may be evaluated by adding the antibody-drug conjugate to human serum at an appropriate concentration (e.g., 100 pg / mL) and comparing the antigen (R0R1 or B7-H3) binding ability of the antibody-drug conjugate cultured in human serum for an appropriate period (e.g., one week) at 37T with that of the antibody-drug conjugate stored at 70T. Evaluation of binding ability to the antigen may be performed via ELISA, but is not limited thereto. In some embodiments, the ratio of the binding ability of the antibody-drug conjugate stored at 70T to the binding ability of the antibody-drug conjugate stored in human serum for one week at 37T to R0R1 is 70% to 130%; or it may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 106%, 108%, or 110% or more, or it may be within a range set by any two values ​​selected from the above % values. In some embodiments, in some embodiments, the ratio of the binding ability of the antibody-drug conjugate stored at -70T to B7-H3 and the binding ability of the antibody-drug conjugate stored in human serum for one week at 37T to B7-H3 may be 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, or 120% or greater, or may be within a range set by any two values ​​selected from the aforementioned % values.In some embodiments, the ratio of the binding ability of the antibody-drug conjugate stored at -70T to B7-H3 and the binding ability of the antibody-drug conjugate stored in human serum for one week at -37T to B7-H3 may be 70% to 130%; or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 106%, 108%, 110%, 115%, or 120% or more, or may be within a range set by any two values ​​selected from the above % values. - The ratio of the antigen-binding ability of an antibody-drug conjugate stored at 70°C (e.g., R0R1 or B7-H3) to the antigen-binding ability of an antibody-drug conjugate stored in human serum for one week at 37°C (e.g., EC50 at -70°C / EC50) can be calculated using the following formula, and is not otherwise limited: (EC50 at -70°C / EC50 50 at 37 °C) x 100 (%) , where , EC5o at -

[0868] 70° is the EC5O of ​​the identified antigen-binding ability of the antibody-drug conjugate stored at -70T, and EC5O at 37° is the EC5O of ​​the identified antigen-binding ability of the antibody-drug conjugate stored at 37T. For a method to evaluate the serum stability of the antibody-drug conjugate, reference may be made to “Example 17” in Section “8. Examples” of this disclosure. In some embodiments, the antibody-drug conjugate has a low drug release rate in the serum or plasma of humans, monkeys, or rats and the DAR remains constant. For example, antibody-drug conjugates have excellent stability, so even if stored in the serum or plasma of humans, monkeys, or rats for a certain period, the drug release rate of the antibody-drug conjugate may be low compared to the time of start of storage.

[0869] In some embodiments, the antibody-drug conjugate can have an excellent production yield.

[0870] 6. Use of Antibody-Drug Conjugates

[0871] 6.1. Overview of Antibody-Drug Conjugate Uses

[0872] 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.

[0873] 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.

[0874] Some embodiments of the present disclosure provide a pharmaceutical composition for treating a tumor of a target comprising an antibody-drug conjugate.

[0875] 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.

[0876] Some embodiments of the present disclosure provide a use for manufacturing a medicine (or therapeutic agent) for treating a tumor of a target with an antibody-drug conjugate or a composition (or pharmaceutical composition) containing the same.

[0877] 6.2. Tumors Subject to Treatment

[0878] 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 previous paragraphs, including Section "5. Antibody-Drug Conjugate" and subsection "5.6. Target Tumors of Antibody-Drug Conjugate" of the present disclosure, and these descriptions apply herein.

[0879] In some embodiments, the tumor is colon cancer, colorectal cancer, rectal cancer, renal cancer, renal cell carcinoma, breast cancer, triple-negative breast cancer, liver cancer, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung neuroendocrine carcinoma (lung NEC), gastric cancer, neuroendocrine cancer, large cell neuroendocrine cancer (LCNEC) , Prostate cancer , Small intestine cancer , Esophageal cancer , Hepatocyte carcinoma , Melanoma , Orthopedic cancer , Bone cancer , Pancreatic cancer , Skin cancer , Head or neck cancer , 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, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma, cholangiocarcinoma,

[0880] ( Cho 1 angi ocar ci noma ), Esophageal cancer, Salivary gland cancer, Sarcoma, Soft tissue sarcoma, Cancer of the urethra, Urothelial cancer, Cancer of the penis, Pediatric solid tumors, Bladder cancer, Cancer of the kidney or ureter, Carcinoma of the renal pelvis, Neurological cancer, Neoplasm of the central nervous system (CNS), Neuroendocrine tumor (carcinoid), Primary CNS lymphoma, Spinal axis tumor ), glioma, 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.

[0881] In a specific embodiment, the tumor (or target tumor) may be one or more selected from the following: small cell lung cancer (SCLC), ovarian cancer, non-small cell lung cancer (NSCLC), head and neck cancer, sarcoma, breast cancer, and prostate cancer.

[0882] 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).

[0883] In some embodiments, the tumor may be a solid tumor or a blood cancer.

[0884] In some embodiments, the tumor may be a tumor expressing R0R1 and / or B7-H3. In some embodiments, the tumor may be a tumor overexpressing R0R1 and / or B7-H3. In some embodiments, the tumor may be an R0R1-positive and / or B7-H3-positive tumor. In some embodiments, the tumor may be a tumor associated with R0R1 and / or B7-H3.

[0885] In some embodiments, the tumor may be a target tumor of the antibody-drug conjugate.

[0886] 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 the same 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 the same 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.

[0887] In some embodiments, the subject may be a human identified as having a tumor expressing R0R1, a tumor overexpressing R0R1, or an R0R1-positive tumor. In some embodiments, the subject may be a human diagnosed or identified as having a tumor expressing B7-H3, a tumor overexpressing B7-H3, or a B7-H3-positive tumor.

[0888] In some embodiments, the subject may be a human identified as having a tumor co-expressing R0R1 and B7-H3, a tumor overexpressing both R0R1 and B7-H3, or a tumor that is R0R1-positive and B7-H3-positive. Whether the patient's tumor expresses R0R1 and / or B7-H3 can be determined by methods well known in the art and is not otherwise limited.

[0889] 6.4. Pharmaceutical compositions containing antibody-drug conjugates. In some embodiments, antibody-drug conjugates may be used for tumor treatment in the form of a composition or a pharmaceutical composition.

[0890] 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.

[0891] Examples of tumors 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 previous paragraphs, including Section “5. Antibody-Drug Conjugate” and subsections “5.6. Target Tumors of Antibody-Drug Conjugate” and “6.2. Tumors to be Treated” of the present disclosure, and these descriptions apply herein.

[0892] For example, the tumor may be a tumor expressing one or more of R0R1 and B7-H3. In some embodiments, the tumor may be an R0R1-positive tumor. In some embodiments, the tumor may be a B7-H3-positive tumor. In some embodiments, the tumor may be an R0R1-positive and B7-H3-positive tumor.

[0893] 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 comprise, for example, lactose, dextrose, sucrose, sorbitol, polysorbate (e.g., polysorbate 80), mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, arginine

[0894] 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.

[0895] 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.

[0896] Furthermore, the pharmaceutical composition may additionally contain one or more other elements suitable for treating or preventing cancer.

[0897] 6.5. Treatment method using antibody-drug conjugates Some embodiments of the present disclosure provide a method for treating a tumor using antibody-drug conjugates according to some embodiments of the present disclosure. The treatment method may include administering a therapeutically effective amount of antibody-drug conjugates appropriately formulated (e.g., a pharmaceutical composition) to a subject at an appropriate method of administration, an appropriate dosage, and an appropriate dosage.

[0898] 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.

[0899] Examples of tumors 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 previous paragraphs, including Section “5. Antibody-Drug Conjugate” and subsections “5.6. Target Tumors of Antibody-Drug Conjugate” and “6.2. Tumors to be Treated” of the present disclosure, and these descriptions apply herein.

[0900] For example, the tumor may be a tumor expressing one or more of R0R1 and B7-H3. In some embodiments, the tumor may be an R0R1-positive tumor. In some embodiments, the tumor may be a B7-H3-positive tumor. In some embodiments, the tumor may be an R0R1-positive and B7-H3-positive tumor.

[0901] In some embodiments, the recipient of the antibody-drug conjugate or the pharmaceutical composition containing it 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.

[0902] 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

[0903] 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 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.

[0904] 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.

[0905] 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 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, etc.), but is not limited thereto. Co-administration is not limited to administering the therapies exactly at the same time. For example, the therapies to be co-administered may be administered at appropriate time intervals or at the same time.

[0906] 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.

[0907] A. Antibody-drug conjugate

[0908] A01. Antibody-drug conjugate having the structure of Formula 13 below or salt thereof:

[0909] [Equation 13]

[0910]

[0911] At this time,

[0912] AB is an anti-R0R1 / B7-H3 bispecific antibody, and

[0913] The above anti-R0R1 / B7-H3 bispecific antibody comprises an antigen-binding fragment for R0R1 and an antigen-binding fragment for B7-H3, wherein the antigen-binding fragment for R0R1 is referred to as the first antigen-binding fragment and the antigen-binding fragment for B7-H3 is referred to as the second antigen-binding fragment, and

[0914] 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

[0915] At this time, the second antigen-binding fragment comprises HCDR1 having the amino acid sequence of sequence no. 30, HCDR2 having the amino acid sequence of sequence no. 31, HCDR3 having the amino acid sequence of sequence no. 32, LCDR1 having the amino acid sequence of sequence no. 33, LCDR2 having the amino acid sequence of sequence no. 34, and LCDR3 having the amino acid sequence of sequence no. 35, and

[0916] a is independently 0 or 1, and

[0917] y is 1 or 2.

[0918] A02. In A01, ,

[0919] An antibody-drug conjugate or a salt thereof, wherein the first antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO. 7 and a light chain variable region having the amino acid sequence of SEQ ID NO. 12, and the second antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO. 36 and a light chain variable region having the amino acid sequence of SEQ ID NO. 41.

[0920] A03. In any one of A01 to A02, the second antigen-binding fragment is an antibody-drug conjugate or a salt thereof, wherein the second antigen-binding fragment is an scFv.

[0921] A04. In A03, the second antigen-binding fragment is an antibody-drug conjugate or a salt thereof having the amino acid sequence of SEQ ID NO. 46.

[0922] A05. An antibody-drug conjugate or a salt thereof, wherein in any one of A01 to A04, the second antigen-binding fragment is connected to the C-terminus of the heavy chain constant region of the anti-R0R1 / B7-H3 bispecific antibody. A06. An antibody-drug conjugate or a salt thereof, wherein in any one of A01 to A05, the anti-R0R1 / B7-H3 bispecific antibody comprises two first antigen-binding fragments and two second antigen-binding fragments.

[0923] A07. In A06, the anti-R0R1 / B7-H3 bispecific antibody is an antibody-drug conjugate or a salt thereof that is divalent to R0R1 and divalent to B7-H3.

[0924] A08. In any one of A06 to A07, the anti-R0R1 / B7-H3 bispecific antibody is an antibody-drug conjugate or a salt thereof having a 2+2 format.

[0925] A09. In any one of A06 to A08,

[0926] The above anti-R0R1 / B7-H3 bispecific antibody comprises an anti-R0R1 antibody comprising the two first antigen-binding fragments, and the two second antigen-binding fragments, and

[0927] At this time, each of the two second antigen-binding fragments is connected to the C-terminus of each of the two heavy chains of the anti-R0R1 antibody through a peptide linker (e.g., a peptide linker having an amino acid sequence of sequence number 48),

[0928] Antibody-drug conjugate or its salt.

[0929] A10. In A09, the anti-R0R1 antibody is an antibody-drug conjugate or a salt thereof that is an antibody of the human IgG class (e.g., IgG1).

[0930] All. In any one of A06 to A10, the anti-R0R1 / B7-H3 bispecific antibody comprises an antibody-drug conjugate or a salt thereof, wherein the anti-R0R1 / B7-H3 bispecific antibody comprises two heavy chains having the amino acid sequence of sequence number 49 and two light chains having the amino acid sequence of sequence number 18 (e.g....

Claims

【Scope of Claim】 【Claim 11 Antibody-drug conjugate having the structure of Formula 13 below or a pharmaceutically acceptable salt thereof: At this time, AB is an antibody, wherein the antibody is an anti-R0R1 / B7-H3 bispecific antibody, and wherein the anti-R0R1 / B7-H3 bispecific antibody comprises a first antigen-binding fragment targeting R0R1 and a second antigen-binding fragment targeting B7-H3, 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. 30, HCDR2 having the amino acid sequence of sequence no. 31, HCDR3 having the amino acid sequence of sequence no. 32, LCDR1 having the amino acid sequence of sequence no. 33, LCDR2 having the amino acid sequence of sequence no. 34, and LCDR3 having the amino acid sequence of sequence no. 35, 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 heavy chain variable region of SEQ ID NO. 7 and a light chain variable region of SEQ ID NO. 12, and The above second antigen-binding fragment comprises a heavy chain variable region of sequence number 36 and a light chain variable region of sequence number 41, Antibody-drug conjugate or a pharmaceutically acceptable salt thereof. 【claim Paragraph 4] In any one of paragraphs 1 to 3, The second antigen-binding fragment is an antibody-drug conjugate that is an scFv or a pharmaceutically acceptable salt thereof.

5. In claim 4, the second antigen-binding fragment is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having the amino acid sequence of sequence number 46. 【claim Paragraph 6] In any one of paragraphs 1 through 5, The above anti-R0R1 / B7-H3 bispecific antibody comprises two first antigen-binding fragments and two second antigen-binding fragments, or comprises two first antigen-binding fragments and one second antigen-binding fragment, an antibody-drug conjugate or a salt thereof. 【claim Paragraph 7] In any one of paragraphs 1 through 5, The above anti-R0R1 / B7-H3 bispecific antibody comprises two first antigen-binding fragments and two second antigen-binding fragments, and The above anti-R0R1 / B7-H3 bispecific antibody comprises two heavy chains of sequence number 49 and two light chains of sequence number 18, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof. 【claim Paragraph 8] In any one of paragraphs 1 through 5, The above anti-R0R1 / B7-H3 bispecific antibody comprises two first antigen-binding fragments and one second antigen-binding fragment, and The above anti-R0R1 / B7-H3 bispecific antibody comprises one heavy chain of sequence number 59, one heavy chain of sequence number 60, and two light chains of sequence number 18, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof. 【claim Paragraph 9] In any one of paragraphs 1 through 8, 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-R0R1 / B7-H3 bispecific antibody.

10. In any one of paragraphs 1 through 9, 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-R0R1 / B7-H3 bispecific antibody.

11. In any one of paragraphs 1 through 10, 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-R0R1 / B7-H3 bispecific antibody, and a is independently 0 or 1, and y is 1 or 2.

12. In any one of paragraphs 1 through 11, y is a 2-phosphorylated antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition for treating a tumor of the subject, comprising an antibody-drug conjugate according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

14. A pharmaceutical composition according to Claim 13, wherein the tumor is a tumor expressing either or more of R0R1 and B7-H3.

15. A pharmaceutical composition according to any one of claims 13 to 14, wherein the tumor is a tumor expressing both R0R1 and B7-H3.

16. In any one of claims 13 to 15, the tumor is colon cancer, colorectal cancer, rectal cancer, renal cancer, renal cell carcinoma, breast cancer, triple-negative breast cancer, liver cancer, hepatocellular carcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung neuroendocrine carcinoma, gastric cancer, neuroendocrine cancer, large cell carcinoma Endocrine cancer (large cell neuroendocytitis: LCNEC), Prostate cancer, Cancer of the small intestine, Cancer of the esophagus, Melanoma, Orthopedic cancer, Bone cancer, Pancreatic cancer, Skin cancer, Head or neck cancer, 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, carcinoma of the vulva, Hodgkin's Disease, lymphoma, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma ioma), cholangiocarcinoma, esophageal cancer, salivary gland cancer, sarcoma, soft tissue sarcoma, cancer of the urethra, urothelial cancer, penile cancer, pediatric solid tumors, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neurological cancer,A pharmaceutical composition comprising one or more selected from neoplasm of the central nervous system (CNS), neuroendocrine tumor (carcinoid), 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.

17. A method for treating a tumor of a subject, comprising administering the pharmaceutical composition of Claim 13 to the subject.

18. In Paragraph 17, The above tumor is a tumor expressing one or more of R0R1 and B7-H3, method.

19. A method according to any one of claims 17 to 18, wherein the tumor is a tumor expressing both R0R1 and B7-H3.

20. In any one of claims 17 to 19, the tumor is colon cancer, colorectal cancer, rectal cancer, renal cancer, renal cell carcinoma, breast cancer, triple-negative breast cancer, liver cancer, hepatocellular carcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung neuroendocrine carcinoma, gastric cancer, neuroendocrine cancer, macrocellular neuroendocrine Cancer (large cell neuroendocrine cancer: LCNEC), Prostate cancer, Cancer of the small intestine, Cancer of the esophagus, Melanoma, Orthopedic cancer, Bone cancer, Pancreatic cancer, Skin cancer, Head or neck cancer, 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, carcinoma of the vulva, Hodgkin's Disease, lymphoma, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma , Cholangiocarcinoma , Esophageal cancer , Salivary gland cancer , Sarcoma , Soft tissue sarcoma , Cancer of the urethra , Urothelial cancer , Penile cancer , Pediatric solid tumors , Bladder cancer , Cancer of the kidney or ureter , Carcinoma of the renal pelvis , Neurological cancer ,One selected from neoplasm of the central nervous system (CNS), neuroendocrine tumor (carcinoid), 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, method.

21. Use in the manufacture of therapeutic agents for treating tumors of the target of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12.

22. In claim 21, the above tumor is a tumor expressing one or more of R0R1 and B7-H3.

23. In any one of claims 21 to 22, the use is that the tumor is a tumor expressing both R0R1 and B7-H3.

24. In any one of claims 21 to 23, the tumor is colon cancer, colorectal cancer, rectal cancer, renal cancer, renal cell carcinoma, breast cancer, triple-negative breast cancer, liver cancer, hepatocellular carcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung neuroendocrine carcinoma, gastric cancer, neuroendocrine cancer, large cell neuroendocrine carcinoma neuroendocytic cancer (LCNEC), prostate cancer, small intestine cancer, esophageal cancer, melanoma, orthotopic cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, epithelial ovarian cancer, rectal cancerCancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, endometrial cancer, cervical cancer, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, lymphoma, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal cancer, adrenocortical carcinoma, mesothelioma , Cholangiocarcinoma , Esophageal cancer , Salivary gland cancer , Sarcoma , Soft tissue sarcoma , Cancer of the urethra , Urothelial cancer , Penile cancer , Pediatric solid tumors , Bladder cancer , Cancer of the kidney or ureter , Carcinoma of the renal pelvis , Neurological cancer ,One or more selected from neoplasm of the central nervous system (CNS), neuroendocrine tumor (carcinoid), primary CNS lymphoma, spinal axis tumor, glioma, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, and T-cell lymphoma, use.

25. A method for preparing an antibody-drug conjugate comprising the following: Engineered bispecific antibody having the structure of Formula 03 below - [Essence 03] X At this time, AB is an antibody, wherein the antibody is an anti-R0R1 / B7-H3 bispecific antibody, wherein the anti-R0R1 / B7-H3 bispecific antibody comprises a first antigen-binding fragment targeting R0R1 and a second antigen-binding fragment targeting B7-H3, 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.

26. In Article 25, 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. 30, HCDR2 having the amino acid sequence of sequence no. 31, HCDR3 having the amino acid sequence of sequence no. 32, LCDR1 having the amino acid sequence of sequence no. 33, LCDR2 having the amino acid sequence of sequence no. 34, and LCDR3 having the amino acid sequence of sequence no.

35. method .

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