Antibody-drug conjugates using a novel linker-payload system to enhance the targeting of cancer-related antigens.

Abstract

Provided are antibody-drug conjugates (ADCs) comprising an antibody conjugated to a drug via a linker, wherein the linker comprises a cleavable linker, and the drug comprises a growth inhibitor which is exatecan, deruxtecan, or a derivative thereof. The antibody-drug conjugates (ADCs) of the present invention comprise a glucuronidase-cleavable linker, for example, the linker comprises a β-glucuronide moiety, such as that shown in Formula I. The antibody-drug conjugates (ADCs) comprise a linker which is conjugated to the drug via a carbamate linkage or is modified to be conjugated via a quaternary ammonium salt linkage or is modified to be conjugated via a quaternary ammonium salt linkage. The antibody-drug conjugates (ADCs) may comprise a linker which is PEGylated with a group comprising polyethylene glycol (PEG). Novel linkers for use in ADCs are also described. [Formula 1] TIFF2026514252000080.tif56170

Description

[Technical Field]

[0001] This invention relates to the development of novel antibody-drug conjugates (ADCs) using monoclonal antibodies, functional antibody fragments thereof, or probes thereof that are specific to a group of antigens, including but not limited to, sialyl Tn (STn) antigens, which are very common in various types of cancer. Such ADCs utilize the use of exatecan-based constructs that, for example, target tumor antigens and take advantage of bystander effects observed with other exatecan-based ADCs such as trastuzumab deruxtecan, as described in International Publication No. 2022 / 048883. In addition, this invention encompasses potential applications in human and animal health, expanding the range of its therapeutic utility. [Background technology]

[0002] Sialyl Tn (STn) antigen is a cleaved O-glycan structure that plays a crucial role in various types of cancer. STn, a disaccharide, consists of sialic acid (Neu5Acα) bound to N-acetylgalactosamine (GalNAc) in a 2,6 configuration, forming an O-glycosidic bond between the GalNAc residue and either a serine or threonine amino acid residue within the polypeptide chain. Julian, Videira, & Delannoy (2012) reported that the presence of this cleaved glycan has been detected at varying frequencies in different cancer types. Notably, STn is not found in normal, healthy tissue, highlighting its importance as a target for cancer therapy.

[0003] STn has been identified as a crucial factor in metastasis, drug resistance, and high-grade tumors, and exhibits several distinctive characteristics that make it a promising target for cancer treatment.

[0004] 1. Association with early-stage and metastatic cancer cells: STn expression has been linked to early-stage cancer and metastatic cancer cells, and has been shown to play a role in tumor progression and dissemination to other parts of the body (Okasaki et al., 2012). This association highlights the potential of STn-targeted therapy to inhibit cancer progression and metastasis.

[0005] 2. Correlation with poor prognosis and reduced overall survival: Increased STn expression in patients is correlated with poor prognosis, reduced overall survival, and poor response to chemotherapy (Choi et al., 2000). This correlation suggests that STn may play a role in promoting tumor invasiveness and treatment resistance, making it an attractive target for the development of novel cancer therapies.

[0006] 3. Evasion of immune surveillance: STn has been suggested to be involved in evading immune cell surveillance, contributing to the tumor's ability to evade detection and elimination by the immune system (Carrascal et al., 2014). Novel cancer therapies targeting STn may overcome this immune evasion mechanism and thus enhance the immune system's ability to recognize and eliminate tumor cells.

[0007] 4. Antibody-drug conjugates (ADCs) in cancer therapy: ADCs are a type of therapeutic agent that combines the target specificity of an antibody with the cytotoxic efficacy of a small molecule drug. This targeted approach helps maximize the destruction of cancer cells while minimizing the impact on healthy tissue. ADCs are emerging as a promising strategy in cancer treatment, with several already approved for clinical use and many more in clinical development (Chari et al., 2014; Sievers & Senter, 2013).

[0008] 5. STn-Targeted ADCs: Several efforts are underway to develop STn-targeted ADCs for cancer therapy, including the development of ADCs using humanized anti-STn antibodies conjugated to cytotoxic drugs such as the mytansinoid DM1 (SYL-001) (Li et al., 2018). However, there is still room for improvement with regard to STn-targeted ADCs, including the development of novel linker-payload systems that will result in better stability, more efficient drug release, and improved therapeutic efficacy.

[0009] 6. Exatecan Linker-Payload System: Exatecan is a water-soluble topoisomerase I inhibitor that has shown potent antitumor activity in preclinical models and clinical trials (Kummar et al., 2006). Recently, exatecan derivatives have been explored as payloads for ADCs (e.g., DS-8201, a HER2-targeted ADC with an exatecan derivative as a payload) (Doi et al., 2017). The exatecan linker-payload system has demonstrated desirable characteristics such as high efficacy, improved stability, and efficient drug release in the tumor microenvironment, making it attractive for ADC development.

[0010] Prendergast et al. mAbs 2017, 9(4), 615-627 describes novel anti-sialyl Tn monoclonal antibodies and ADCs containing them. The ADC-linker technology used is MC-vc-PAB-MMAE. The MMAE moiety is monomethyl auristatin, a growth inhibitory agent, and the MC-vc-PAB cleavable linker contains a maleimidocaproyl moiety, a valine-citrulline dipeptide moiety, and a p-aminobenzyloxycarbonyl moiety. The document also discloses further ADCs containing an MMAF moiety which further contains a maleimidocaproyl moiety. However, none of the above linkers are cleavable with glucuronidase.

[0011] Starbuck et al. Oncotarget, 2018, 9(33), 23289-23305 describes an ADC containing an anti-STn antibody in which the growth inhibitor is monomethyl auristatin (MMAE) and the cleavable linker is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl. These linkers are also not cleavable by glucuronidase.

[0012] Burke et al., Molecular Cancer Therapeutics, 2016, 15(5), 938-945, describes glucuronide drug-linker systems whose drug release mechanism is either carbamate or quaternary ammonium. ADCs containing such linkers are also described. However, this literature does not describe linker structures containing PEG moieties.

[0013] International Publication No. 2022 / 253035 describes an ADC in which the linker contains a carbamate moiety and a PEG moiety, and part of the compound contains a glucuronide moiety. However, none of these structures terminate with a maleimide moiety. Furthermore, this document does not describe a linker moiety containing a quaternary ammonium moiety.

[0014] International Publication No. 2022 / 237884 describes linkers and linker-payload structures containing quaternary ammonium and glucuronide moieties, as well as ADCs containing this linker structure. However, this linker structure does not contain a PEG moiety.

[0015] WO 2018 / 103739 pamphlet describes compounds in which the linker contains a carbamate moiety and a glucuronide moiety, and ADCs containing this linker structure. This linker structure contains a PEG moiety and is terminated with a maleimide group. However, the maleimide-triethylene glycol moiety in the compounds described in this document is connected to the amide linker by a methylene group. Since such substances contain an ethylene group at this position, this document does not disclose compounds in which the PEGylated moiety is of formula II. Furthermore, this document does not describe linker moieties containing a quaternary ammonium moiety.

[0016] The present invention aims to address the need for improved antibody-drug conjugates (ADCs) that target cancer biomarkers by using a novel exatecan linker-payload system in combination with a novel humanized antibody or functional antibody fragment. The present invention seeks to enhance the specificity of such antibodies by fusing the cytotoxic activity and bystander effect of exatecan derivatives with two different linkers in one particular embodiment, thereby reducing the impact of cancer therapy on healthy tissues while providing enhanced therapeutic efficacy and higher tumor uptake.

[0017] ADC constructs using cancer-specific antibodies offer several advantages that contribute to effective cancer therapy and the potential as cancer diagnostics.

[0018] 1. Targeted delivery: By leveraging the specificity of the antibody, the ADC can selectively bind to antigens present on the surface of cancer cells, minimizing off-target effects and protecting healthy tissues from the cytotoxic payload.

[0019] 2. Enhanced potency: The ADC enables the delivery of very potent cytotoxic agents that are considered too toxic to administer systemically as free drugs. Targeted delivery to tumor cells allows for a higher local concentration of the cytotoxic payload, improving the therapeutic index and enhancing the overall efficacy of the treatment.

[0020] 3. Reduced systemic toxicity: Because ADCs selectively deliver cytotoxic payloads to cancer cells, systemic exposure to cytotoxic agents can be reduced, thereby lowering the risk of adverse side effects often associated with conventional chemotherapy.

[0021] 4. Synergistic effects: By combining targeted antibody binding and a potent cytotoxic payload in ADCs, synergistic effects can be achieved. The antibody not only delivers the payload but also interferes with ligand recognition by host cell receptors, which are suggested to be involved in tumor progression and immune evasion mechanisms.

[0022] 5. Customizable Properties: ADCs can be designed using various linker chemistry and payloads, allowing for optimization of properties such as stability, drug release, and cytotoxic efficacy. This flexibility enables the development of ADCs for cancer treatment and detection, including theranostics, tailored to specific cancer types, stages, or patient populations.

[0023] The present invention also envisions the use of the linker described herein in combination with other monoclonal antibodies against cancer antigens overexpressed in cancer cells. However, the present invention aims to develop more effective and targeted cancer approaches by focusing on specific cancer biomarkers and fusing the target-directing capabilities of antibodies with the cytotoxic properties of the exatecan linker-payload system. Such strategies have the potential to improve therapeutic efficacy while minimizing adverse effects on healthy tissue, thus enabling new detection methods and thus addressing the continuing demand for augmented cancer therapy.

[0024] The present invention also describes novel exatecan derivative payloads that have the potential to increase safety while maintaining an efficacy profile. [Prior art documents] [Patent Documents]

[0025]

Patent Document 1

Patent document 2

Patent document 3

Non-licensed literature

[0026] [Non-licensed document 1] Julian, Videira, & Delannoy (2012) [Non-licensed document 2] Okasaki et al., 2012 [Non-licensed document 3] Choi et al., 2000

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0027] This invention introduces an innovative antibody-drug conjugate (ADC) construct designed to address challenges in treating human cancer and to be extendable to animal health applications. These ADC constructs combine the specificity of antibodies or functional antibody fragments targeting known cancer biomarkers such as sialyl Tn (STn) and other alpha-2,6-linked sialic acid-terminated glycans with the potent cytotoxic activity of, for example, an exatecan derivative. These biomarkers are characterized by their overexpression in cancer cells but absence in healthy cells.

[0028] In a preferred embodiment, such an ADC construct uses two different types of beta-glucuronide linkers to encapsulate an antibody or functional antibody fragment in a payload such as an exatecan payload. This antibody is characterized by having a protein sequence modified to optimize its performance in therapeutic applications, taking into account factors such as immunogenicity, pharmacokinetic profile, and binding specificity to STn. This enables accurate detection of tumor cells and the ability to interfere with ligand recognition by host cell receptors that contribute to tumor progression and immune evasion mechanisms.

[0029] This invention further develops high-affinity antibodies or functional antibody fragments targeting STn by using an affinity maturation method, thereby improving the antitumor response. This method yields a series of novel and distinct antibodies or functional antibody fragments, including a clone denoted as mAb_v1, that exhibit increased affinity and binding to target STn.

[0030] ADCs with STn-targeting payloads such as exatecan enhance therapeutic efficacy by leveraging the bystander effect observed with other exatecan-based ADCs such as trastuzumab deruxtecan. The bystander effect allows the exatecan payload to diffuse into neighboring cells, effectively targeting not only cells expressing cancer biomarkers but also cells within the surrounding tumor microenvironment. This leads to improved tumor cell killing while minimizing impact on healthy tissue.

[0031] In one embodiment, the present invention discloses an alternative embodiment of the ADC construct, which includes substituting a carbamate handle / linker with a quaternary amine as the handle of the beta-glucuronide linker for a payload such as exatecan. This modification brings additional advantages and benefits to the properties and efficacy of the ADC.

[0032] By using a quaternary amine as a handle for the beta-glucuronide linker, it was found that safety is further enhanced by ensuring that the cytotoxic payload maintains its positive charge even when cleaved outside tumor cells. This positive charge effectively inhibits the internalization of the circulating payload by healthy cells, thereby reducing the risk of off-target toxicity and improving the overall safety profile of the ADC.

[0033] In addition, the quaternary amine handle / linker enhances the stability of ADCs in circulation, ensuring that the payload remains firmly bound to the antibody until it reaches the tumor microenvironment. Once inside tumor cells, the linker is cleaved by intracellular enzymes, specifically releasing the active payload into the cancer cells. This selective release mechanism ensures that the cytotoxic payload is delivered primarily to the target cells, minimizing exposure to healthy tissue and reducing the risk of adverse effects.

[0034] Furthermore, quaternary amine handles / linkers can potentially improve the pharmacokinetic properties of ADCs because their positive charge may facilitate interaction with negatively charged components within the tumor microenvironment. This could potentially lead to enhanced tumor invasion and retention of ADCs, resulting in improved therapeutic efficacy.

[0035] In summary, in one embodiment, this specification discloses the introduction of a quaternary amine handle instead of a carbamate handle / linker into the ADC construct, which offers several advantages, including improved safety, enhanced circulating stability, and potentially better pharmacokinetics. These features contribute to the overall efficacy and safety of the ADC, further supporting its potential as a promising cancer therapy for both human and animal health applications. Other embodiments are disclosed here, including further improvements to the linker design and novel exatecan derivatives.

[0036] Furthermore, the scope of the present invention extends to potential applications in animal health, where optimized antibodies or functional antibody fragments can be adapted for use in the treatment of animal cancer after necessary modifications and validation. This expands the potential impact of this novel ADC construct in addressing challenges faced in both human and animal cancer treatment.

[0037] In summary, the present invention provides a versatile ADC construct that, in preferred embodiments, uses a beta-glucuronide linker to combine an STn-targeting antibody or functional antibody fragment with an exatecan payload or derivative thereof, leveraging the bystander effect to enhance therapeutic efficacy and specificity in cancer treatment. The extensibility of the present invention to animal health applications further highlights its importance in addressing the challenges facing cancer therapy. Certain novel linkers are also provided, which have the potential to be used with a wide range of different antibodies and payloads in addition to those specifically described herein. [Brief explanation of the drawing]

[0038] [Figure 1a] This figure shows the synthetic route for the linker-payload type 1 (ExV1) synthesis of the compounds of the present invention, which contain a carbamate-bonded glucuronide drug linker. [Figure 1b] This diagram shows the synthetic route for linker-payload type 2 (ExV2) synthesis. The linker is a quaternary ammonium-based glucuronide drug linker. [Figure 2a] This figure shows the ADC characteristics, including the relevant ID, size (mg), BCA concentration (mg / ml), yield (mg), recovery rate (%), MS-DAR, HIC-DAR, SEC monomer %, free drug level %, and Endo (EU / mg). The final ADCs produced were ADC-AFI-ExV1, ADC-IgG1-ExV1, and ADC-AFI-DXd. [Figure 2b] This figure shows the final SEC profiles of ADC-AFI-ExV1, ADC-IgG1-ExV1, and ADC-AFI-DXd, with response [mAU] on the y-axis and retention time [minutes] on the x-axis. [Figure 2c-1] ~ [Figure 2c-3] This figure shows the reduction MS data for the second batch of final product ADCs, including ADC-AFI-ExV1, ADC-IgG1-ExV1, and ADC-AFI-DXd. [Figure 3-1] ~ [Figure 3-2] This figure shows an overview of endotoxin levels in ADC-AFI-ExV1, ADC-IgG1-ExV1, and ADC-AFI-DXd. [Figure 4a-b]This figure shows efficacy studies using CDX-SNU16 (gastric cancer cell model). A. Tumor growth curves of various treatment groups, including medium-treated mice, ADC-IgG1-ExV1 (isotype control), ADC-AFI-DXd, and ADC-AFI-ExV1 (novel linker payload type 1). All ADCs used were DAR4. Female BALB / c nude mice carrying established SNU-16 tumors were used. IV, PG-D0, D18; mice per group used, n=7. B. Direct comparison of ADC-AFI-ExV1 (novel linker payload type 1) versus ADC-AFI-DXd (known linker payload) using the same dosage [8 mg / kg] and the same DAR=4. Independent t-test analysis. Bars represent mean ± SEM. *P<0.01. [Figure 4c-d] This figure shows the change in D body weight (%) and D body weight increase or decrease compared to an efficacy study using mice carrying established SNU-16 tumors. IV, PG-D0, D18; mice per group used, n=7. Error bars represent the standard error of the mean (SEM). [Figure 5a-b] Figures A and B show the time-series in vivo distribution analysis and relative mAb uptake as %ID / g at 96 hours for tumor breast cancer cell line 4T1-STn and related parental WT cell line (4T1-WT) as a control. A PET image with the same heatscale. B Distribution in all groups; one-way ANOVA followed by Bonferroni post-hoc analysis. Bars represent mean ± SEM. ***P<0.0001. [Figure 6a-c] This figure summarizes various developmental feasibility analyses of first- and second-generation humanized clones compared to benchmark mAbs. A. Absorbance of DNA, LPS, lysozyme, and cell lysates detected by ELISA A450-A620 using CBS second- and first-generation antibodies, along with control palivizumab and trastuzumab. Data are normalized relative to palivizumab. B. Mean adhesion analysis compared to palivizumab. C. Antibody signal [mV] vs. retention time [min] for palivizumab, mAb_v64, and mAb_v1 using pH3 and temperature stress (45°C for 48 hours). [Figure 7a-d] This figure illustrates an antibody internalization assay using several cancer cell lines expressing different levels of STn: MDA-MB-231-STn+ (high), MDA-MB-231-WT (null; control), SNU16 (medium / high), and COLO205 (low) cell lines. Antibody internalization was reported as an internalization factor normalized to the IgG1 control mAb palivizumab. A variety of anti-STn antibodies were used, including the positive control mAb_PC (anti-STn), mAb_v57, mAb_v48, mAb_v46, mAb_v53, mAb_v25, mAb_v64, mAb_v1, and parental L2A5 mAb. [Figure 8] This figure shows EC50 assays using cancer cell lines expressing various levels of STn: COLO205 (low), SNU16 (medium / high), and OV90 (high) cell lines. Various anti-STn antibodies were used, including positive controls mAb_PC (anti-STn), mAb_v1, mAb_v46, mAb_v53, mAb_v25, and mAb_v64. [Figure 9-1] ~ [Figure 9-3] This figure shows the sequence ID numbers assigned to the variants disclosed herein. [Figure 10a] This figure shows the amino acid sequences of the VH heavy chains of the humanized variants disclosed herein. The H-CDR1, H-CDR2, and H-CDR3 sequences are highlighted in bold. [Figure 10b] This figure shows the amino acid sequences of the VL light chains of the humanized antibody variants disclosed herein. The L-CDR1, L-CDR2, and L-CDR3 sequences are highlighted in bold. [Figure 11a-1] ~ [Figure 11a-2] This figure shows the amino acid sequences of the VH heavy chains of the affinity-matured variants disclosed herein. The H-CDR1, H-CDR2, and H-CDR3 sequences are highlighted in bold. [Figure 11b-1] ~ [Figure 11b-2]This figure shows the amino acid sequences of the VL light chains of the affinity-matured variants disclosed herein. The L-CDR1, L-CDR2, and L-CDR3 sequences are highlighted in bold. [Figure 12-1] ~ [Figure 12-6] This figure shows the amino acid sequences of the variable chains (VH and VL) of the affinity-matured variants disclosed herein, along with the clonal variant names and their corresponding sequence identification numbers. [Figure 13a] This figure shows the sequence ID numbers and amino acid sequences of the VH heavy chains of the additional humanized V1 variants disclosed herein. The H-CDR1, H-CDR2, and H-CDR3 sequences are highlighted in bold. [Figure 13b] This figure shows the sequence ID numbers and amino acid sequences of the VL light chains of the additional humanized V1 variants disclosed herein. The L-CDR1, L-CDR2, and L-CDR3 sequences are highlighted in bold. [Figure 14-1] ~ [Figure 14-3] This figure shows the sequence ID number, amino acid sequence, and polynucleotide sequence of a particular mouse antibody disclosed herein. [Figure 15a-1] ~ [Figure 15a-2] This is a diagram showing the HPLC for compound 2. [Figure 15b-1] ~ [Figure 15b-3] This is a figure showing the LCMS of compound 2. [Figure 16a-1] ~ [Figure 16a-2] This is a diagram showing the HPLC for compound 3. [Figure 16b-1] ~ [Figure 16b-3] This is a figure showing the LCMS of compound 3. [Figure 17a-1] ~ [Figure 17a-2] This is a diagram showing the HPLC for compound 4. [Figure 17b-1] ~ [Figure 17b-2] This is a figure showing the LCMS for compound 4. [Figure 18a-1] ~ [Figure 18a-2] This is a diagram showing the HPLC analysis for compound 5. [Figure 18b-1] ~ [Figure 18b-2] This is a figure showing the LCMS of compound 5. [Figure 19a-1] ~ [Figure 19a-2] This is a diagram showing the HPLC for compound 6. [Figure 19b-1] ~ [Figure 19b-2] This is a figure showing the LCMS of compound 6. [Figure 20a-1] ~ [Figure 20a-2] This is a diagram showing the HPLC for compound 7. [Figure 20b-1] ~ [Figure 20b-2] This is a figure showing the LCMS of compound 7. [Figure 21a-1] ~ [Figure 21a-2] This is a diagram showing the HPLC for compound 8. [Figure 21b-1] ~ [Figure 21b-2] This is a figure showing the LCMS of compound 8. [Figure 22a-1] ~ [Figure 22a-2] This figure shows the HPLC for compounds 8 and 1a-1. [Figure 22b-1] ~ [Figure 22b-2] This figure shows the LCMS for compounds 8 and 1a-1. [Figure 23a-1] ~ [Figure 23a-2] This is a diagram showing the HPLC for compound 6a-1. [Figure 23b-1] ~ [Figure 23b-2] This is a figure showing the LCMS of compound 6a-1. [Figure 24a-1] ~ [Figure 24a-2] This is a diagram showing the HPLC for compound 9. [Figure 24b-1] ~ [Figure 24b-2] This is a figure showing the LCMS of compound 9. [Figure 25a-1] ~ [Figure 25a-2] This is a diagram showing the HPLC for compound 10. [Figure 25b-1] ~ [Figure 25b-2] This is a figure showing the LCMS of compound 10. [Figure 26a-1] ~ [Figure 26a-2] This is a diagram showing the HPLC for compound 11. [Figure 26b-1] ~ [Figure 26b-2] This is a figure showing the LCMS of compound 11. [Figure 27a-1] ~ [Figure 27a-2] This is a diagram showing the HPLC for compounds 11 and 1a-1. [Figure 27b-1] ~ [Figure 27b-2] This is a figure showing the LCMS for compounds 11 and 1a-1. [Figure 28] This figure shows a list of abbreviations related to linker-payload analysis. [Figure 29a] This figure shows an overview of the pilot conjugation including ADC-AFI-ExV1(1-01 / 02 and 03), and displays relevant TCEP / mAb ratio, drug / mAb ratio, monomer %, HIC-DAR, and reduced MS-DAR data. [Figure 29b] This figure shows HIC antibody response [mAU] on the y-axis and retention time [minutes] on the x-axis, with the relevant SEC profile of the pilot conjugation shown with response [mAU] [mAU] for ADC-AFI-ExV1 (1-01 / 02 and 03) on the y-axis and retention time [minutes] on the x-axis. [Figure 30a] This figure shows the reduced LC-MS data of the pilot conjugation of ADC-AFI-ExV1(1-01). [Figure 30b] This figure shows the reduced LC-MS data of the pilot conjugation of ADC-AFI-ExV1(1-02). [Figure 30c] This figure shows the reduced LC-MS data of the ADC-AFI-ExV1(1-03) pilot conjugation. [Figure 31] This figure shows the TCEP / mAb ratio versus reduced MS-DAR curve. [Figure 32a] This figure shows the characteristics of the ADC-AFI-ExV1 (Test 1) sample listed in the table. [Figure 32b] This figure shows the SEC profile of the ADC-AFI-ExV1 (Test 1) conjugation, with response [mAU] on the y-axis and retention time [minutes] on the x-axis. [Figure 32c] This figure shows the reduced MS data used to confirm the conjugation of ADC-AFI-ExV1 (Test 1). [Figure 33a] This figure shows the relevant ID, size (mg), TCEP / mAb ratio, drug / mAb ratio, monomer, and MS-DAR for ADC-AFI-ExV1. [Figure 33b] This figure shows the SEC profile of the final ADC-AFI-ExV1 conjugation, with response [mAU] on the y-axis and retention time [minutes] on the x-axis. [Figure 33c] This figure shows the reduced MS data to confirm the final ADC-AFI-ExV1 conjugation. [Figure 34a] This figure shows the relevant ID, size (mg), TCEP / mAb ratio, drug / mAb ratio, monomer, and MS-DAR for ADC-AFI-ExV1. [Figure 34b] This figure shows the SEC profile of the final ADC-AFI-ExV1 conjugation, with response [mAU] on the y-axis and retention time [minutes] on the x-axis. [Figure 34c] This figure shows the reduced MS data to confirm the final ADC-AFI-ExV1 conjugation. [Figure 35a]This figure shows the relevant ID, size (mg), TCEP / mAb ratio, OD at 280 nm, UV concentration (mg / ml), MS-DAR, and SEC monomer % for the ADC-AFI-ExV1 bulk conjugation product (15 mg). [Figure 35b] This figure shows the SEC profile of the final ADC-AFI-ExV1 conjugation, with response [mAU] on the y-axis and retention time [minutes] on the x-axis. [Figure 35c] This figure shows the reduced MS data for the second batch of bulk conjugation of ADC-AFI-ExV1. [Figure 36] This figure shows the RP data (370nm) of the final ADC product. [Figure 37] This figure shows the HIC for ADC-AFI-DXd. [Figure 38] This figure shows FACS staining of SNU16 using mAb_V64 versus IgG1 (isotype control) before injecting cells into nude BALB / C mice. SNU16 shows 99.5% STn staining when using mAb_V64. [Figure 39a-b] A. Figure showing the efficacy of CDX-SNU16 (gastric cancer cell model) treatment using a clinically validated vedotin platform (linker: Val-Cit-PAB, payload: MMAE, conjugation: maleimide random cys). The antibodies used were AFI (mAb_v64) and human positive control anti-STn (PC). Tumor growth curves for various treatment groups, including medium-treated mice, ADC-IgG1-MMAE (isotype control), ADC-AFI-MMAE, and ADC-PC-MMAE. All ADCs used had a DAR of 4. B. Mouse body weight for the same efficacy treatment. Female BALB / c nude mice carrying established SNU-16 tumors were used. Dosage schedule: [2 mg / Kg] PG-D0, D7, D14 (18); [3.5 mg / Kg] PG-D18, D25 (18); IP once weekly; mice per group used, n=7. [Figure 40]This figure shows the in vivo distribution analysis of various other organs in the same experimental 4T1 syngeneic mouse. The data is expressed as the relevant %ID / g at 96 hours. [Modes for carrying out the invention]

[0039] A brief explanation of the table Table 1. Relevant amounts (mg), antibodies, linker payloads, and target DARs for ADC-AFI-ExV1, ADC-IgG1-ExV1, and ADC-AFI-DXd. Table 2. SEC-HPLC method Table 3. HIC-HPLC method Table 4. LC-MS method Table 5. Creation of linker-payload standard curves. Table 6. RP-HPLC method for free drug testing. Table 7. Conjugation and formulation buffers. Table 8. Description of the experimental design. N: Number of animals per group. Dosage: Dosage is adjusted based on body weight (10 μL / g).

[0040] Outline of the invention In a broader embodiment, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody conjugated to a drug via a linker, which is bound to a glycan terminated with sialyl Tn (STn) or alpha-2,6-linked sialic acid, wherein the linker comprises a cleavable linker and the drug comprises a proliferation inhibitor. The proliferation inhibitor may be, for example, an anticancer agent such as a chemotherapeutic agent or a cytotoxic agent. The proliferation inhibitor may be, for example, exatecan or a derivative thereof, or deruxtecan or a derivative thereof.

[0041] In one embodiment, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody conjugated to a drug via a linker, which is bound to a glycan terminated with sialyl Tn (STn) or alpha-2,6-linked sialic acid, wherein the linker comprises a linker cleavable by glucuronidase, and the drug comprises a proliferation inhibitor.

[0042] In one embodiment, the linker used in the ADC includes a linker that can be cleaved with glucuronidase. The linker may be cleaved with glucuronidase derived, for example, from a human or animal source, i.e., a human enzyme or an animal enzyme.

[0043] In one embodiment, the linker includes a β-glucuronide moiety.

[0044] Antibody-drug conjugates (ADCs) are, for example, formula I': [ka] Equation (I') [In the formula, X is NH, N-CH 3、 It may also contain a linker comprising the β-glucuronide moiety shown in [or CF2].

[0045] In one embodiment of formula (I'), X is NH. In one embodiment of formula (I'), X is N-CH3. In one embodiment of formula (I'), X is CF2.

[0046] Antibody-drug conjugates (ADCs) are, for example, formula I: [ka] Equation (I) It may include a linker containing the β-glucuronide moiety shown, It will be understood that the specific formulas shown above may be modified, as long as they are cleavable by glucuronidase.

[0047] Therefore, in one embodiment, the antibody-drug conjugate (ADC) according to the present invention may include, for example, a linker shown in formula I' or I, which is bound to the drug via a carbamate bond.

[0048] In further embodiments, the carbamate bond may be modified or substituted by another chemical moiety.

[0049] In preferred embodiments, the antibody-drug conjugates (ADCs) provided herein include a linker, which is conjugated to or modified to conjugate the drug via a quaternary ammonium salt bond. Thus, for example, in Formula I above, the carbamate bond or moiety may be replaced by a quaternary ammonium salt bond or moiety. One suitable quaternary ammonium salt bond or moiety is illustrated in example in Formulas IV or VI below, and it is understood that this bond or moiety can be used in linker structures other than those specifically shown in Formulas IV or VI.

[0050] According to a further aspect of the present invention, the linker used in the antibody-drug conjugate (ADC) may be PEGylated with a group or portion containing polyethylene glycol (PEG), which is one or more polyethylene glycol (PEG) units. This group may contain one or more PEG units alone, or the one or more PEG units may be incorporated into a larger chemical group.

[0051] In one preferred embodiment, the ADC comprises a linker containing a β-glucuronide moiety PEGylated with a group containing polyethylene glycol (PEG).

[0052] In a particularly preferred embodiment, the ADC includes a linker comprising a β-glucuronide moiety PEGylated with a polyethylene glycol (PEG) group, which is also linked to the drug via a quaternary ammonium salt bond or part thereof.

[0053] In one embodiment, the linker has the following structure: [ka] It may be PEGylated with a group containing polyethylene glycol (PEG) based on the formula [wherein n is 1 to 5], If necessary, one or both of the terminal H substituents in the above structure may be substituted with another suitable chemical group, provided that the functionality of the linker is not substantially affected.

[0054] In a preferred embodiment, in the above structure, n is 2 to 4. In a more preferred embodiment, in the above structure, n is 3, meaning that PEG contains 3 individual monomer units.

[0055] In one preferred embodiment, the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody as defined herein, conjugated to a drug via a linker, wherein the linker is a base of formula IIA: [ka] Formula (IIA) The present invention provides an antibody-drug conjugate (ADC) containing [the specified ingredient].

[0056] In one preferred embodiment, the antibody-drug conjugate (ADC) is a base of formula II: [ka] Formula (II) Includes a PEGylated linker.

[0057] PEGylation may be performed, for example, on the amide group of the linker, or on the amide group of the structure shown in formula I. In a preferred embodiment, the linker containing the group of formula II is also linked to the drug via a quaternary ammonium salt bond or part thereof.

[0058] In one preferred embodiment, the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody as defined herein, conjugated to a drug via a linker, wherein the linker is of formula IIIA': [ka] Formula IIIA' The present invention provides an antibody-drug conjugate (ADC) whose group is shown in the formula [wherein X is NH, N-CH3, or CF2].

[0059] In one embodiment of formula IIIA', X is NH. In one embodiment of formula IIIA', X is N-CH3. In one embodiment of formula IIIA', X is CF2.

[0060] In one preferred embodiment, the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody as defined herein, conjugated to a drug via a linker, wherein the linker is a base of formula IIIA: [ka] Formula (IIIA) The present invention provides an antibody-drug conjugate (ADC) with the group shown.

[0061] Therefore, in one preferred example, the antibody-drug conjugate (ADC) according to the present invention is Formula III: [ka] It may include a linker which is a PEGylated linker as shown in [reference].

[0062] In one preferred embodiment, the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody as defined herein, conjugated to a drug via a linker, wherein the linker is of formula IVA': [ka] Formula IVA' The present invention provides an antibody-drug conjugate (ADC) as shown in the formula [wherein X is NH, N-CH3, or CF2].

[0063] In one embodiment of formula (IVA'), X is NH. In one embodiment of formula (IVA'), X is N-CH3. In one embodiment of formula (IVA'), X is CF2.

[0064] In one preferred embodiment, the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody as defined herein, conjugated to a drug via a linker, wherein the linker is of formula IVA: [ka] Formula (IVA) We provide antibody-drug conjugates (ADCs) as shown.

[0065] In another preferred example, the antibody-drug conjugate (ADC) according to the present invention is given by formula IV: [ka] It may include a linker which is a PEGylated linker as shown in [reference].

[0066] In one preferred embodiment, the antibody-drug conjugate (ADC) of the present invention comprises an antibody as defined herein conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is of formula VA': [ka] As shown in the formula [wherein X is NH, N-CH3, or CF2].

[0067] In one embodiment of formula VA', X is NH. In one embodiment of formula VA', X is N-CH3. In one embodiment of formula VA', X is CF2.

[0068] In one preferred embodiment, the antibody-drug conjugate (ADC) of the present invention comprises an antibody as defined herein conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is of formula VA: [ka] Formula VA As shown there.

[0069] In one preferred embodiment, the antibody-drug conjugate (ADC) of the present invention comprises an antibody as defined herein conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is given by formula VIA': [ka] Formula VIA' As shown in the formula [wherein X is NH, N-CH3, or CF2].

[0070] In one embodiment of formula VIA', X is NH. In one embodiment of formula VIA', X is N-CH3. In one embodiment of formula VIA', X is CF2.

[0071] In one preferred embodiment, the antibody-drug conjugate (ADC) of the present invention comprises an antibody as defined herein conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is given by formula VIA: [ka] formula VIA As shown there.

[0072] In one preferred embodiment, the antibody-drug conjugate (ADC) according to the present invention is of formula V or VI: [ka] It includes a drug-linker portion or payload having the structure shown.

[0073] According to the present invention, in the antibody-drug conjugate (ADC) described herein, the glycan terminated with sialyl Tn (STn) or alpha-2,6-linked sialic acid to which the ADC is bound is a human protein or an animal protein.

[0074] In a preferred embodiment, the drug or payload used in the antibody-drug conjugate (ADC) described herein is exatecan.

[0075] Drug-antibody ratio In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) that is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) that is an integer from 1 to 8. In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) that is an integer from 2 to 6. In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) that is an integer from 3 to 5. In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) of 2. In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) of 3. In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) of 4. In one embodiment, the ADC according to the present invention has a drug-antibody ratio (DAR) of 5.

[0076] In another embodiment, a composition comprising an ADC or a mixture thereof according to the present invention is provided. As will be apparent to those skilled in the art, such a composition may comprise a mixture of ADCs having different DARs, in which case the DAR of the composition is expressed as an average DAR, which may be a non-integer. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) that is an integer or decimal between 1 and 10. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) that is an integer or decimal between 1 and 8. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) that is an integer or decimal between 2 and 6. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) that is an integer or decimal between 3 and 5. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.0. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.1. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.2. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.3. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.4. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.5. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.6. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.7. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.8. In one embodiment, the composition according to the present invention has an average drug-antibody ratio (DAR) of about 2.9. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.0. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.1. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.2. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of approximately 3.3. In another embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of approximately 3.4.In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.5. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.6. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.7. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.8. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 3.9. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.0. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.1. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.2. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.3. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.4. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.5. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.6. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.7. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.8. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 4.9. In one embodiment, the ADC according to the present invention has an average drug-antibody ratio (DAR) of about 5.0.

[0077] antibody In one embodiment, the antibody used in the ADC is preferably a monoclonal antibody. The antibody may be, for example, an antibody fragment, for example, a functional antibody fragment. In one embodiment, the antibody fragment can be selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabody.

[0078] The antibodies used in the antibody-drug conjugates (ADCs) described herein may be, for example, human antibodies, animal antibodies, or fragments thereof. It is conceivable that the present invention is useful in both human and animal applications. Therefore, both medical and veterinary treatments are included within the scope of the present invention.

[0079] In one embodiment, the antibody-drug conjugate (ADC) described herein comprises an alpha-2,6-linked sialic acid-terminated glycan containing STn, 2,6-sialyl T, dicialyl T, or 2,6-sialolactosamine.

[0080] In another embodiment, in the antibody-drug conjugate (ADC) described herein, the antibody undergoes a glycan change at its glycosylation site.

[0081] In another embodiment, in the antibody-drug conjugate (ADC) described herein, the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0082] In another embodiment, in the antibody-drug conjugate (ADC) described herein, the antibody is a functional antibody fragment bound to a glycan group terminated with STn and alpha-2,6-linked sialic acid.

[0083] In a further embodiment of the present invention, one advantage of the antibody-drug conjugate (ADC) described herein is that it can maintain high efficacy while keeping the drug-antibody ratio (DAR) low. Therefore, in one embodiment, an antibody-drug conjugate (ADC) described herein is provided, wherein the DAR is about 4 or less. Preferably, the antibody component is coupled to the linker-payload by a cysteine ​​conjugate, but in principle, any suitable coupling can be used.

[0084] The inventors have found that certain antibodies are of particular interest and usefulness in the ADC of the present invention, and such antibodies specifically bind to either a glycan terminated with sialyl Tn(STn) or alpha-2,6-linked sialic acid, or to a human antigenic protein or animal antigenic protein containing a glycan terminated with sialyl Tn(STn) or alpha-2,6-linked sialic acid. Certain preferred antibodies of this type are described further below. They may be specifically coupled, for example, to the drug-linker moiety or payload described above.

[0085] Preferred antibodies include those described in the present inventors' concurrently pending patent application, International Publication No. 2023 / 249502, which describes novel antibodies with improved antibody affinity and binding affinity to the target sialyl Tn (STn). These relate to the clones described in International Publication No. 2019 / 147152. In particular, the present inventors have provided a number of different humanized antibody clones, including a humanized clone referred to herein by the abbreviation V1. Here again, the present inventors provide a series of novel and different antibodies obtained by affinity maturation using this humanized V1 clone in particular. These novel clones or variants have differences in amino acid sequences and are significantly improved compared to known antibodies due to increased affinity and binding affinity to the target sialyl Tn (STn).

[0086] Therefore, this application includes the use of such high-affinity humanized anti-STn antibodies obtained from affinity maturation methods in ADCs. In certain applications, it is highly desirable to have antibodies that have high affinity for the target antigen and enhance the antitumor response. For example, this may enable the following: - Antibody-drug conjugates or radioimmunoconjugates with increased tumor uptake and antitumor function. - To induce desired levels of T cell activation by fine-tuning bispecific T cell engagers and CAR receptors. - To improve STn blockade in vivo and restore immune cell function, thereby leading to an antitumor response.

[0087] Therefore, in a preferred embodiment, the antibody-drug conjugate (ADC) according to the present invention is (a) Heavy chain variable region (VH), where VH is (i) H-CDR1, H-CDR2, and H-CDR3 as shown in one of the sequence numbers 1-24 or 49-88, respectively. A heavy chain variable region (VH) containing a complementarity determining region (CDR) selected from the group consisting of the following: and / or, (b) Light chain variable region (VL), where the VL is (i) L-CDR1, L-CDR2, and L-CDR3 as shown in one of sequence numbers 25-48 or 89-128, respectively. A light chain variable region (VL) containing a complementarity determination region (CDR) selected from the group consisting of the following: The antibody may contain the following. Optionally, at least one CDR may contain one or two amino acid substitutions compared to the described sequence.

[0088] The fragment may be a functional antibody fragment of the disclosed antibody, that is, it may retain the ability to bind to the antigen.

[0089] In one embodiment, the antibody or fragment thereof may include a heavy chain variable region (VH) and a light chain variable region (VL). Naturally, a constant region may also be provided.

[0090] It will be understood that monoclonal antibodies (mAbs) refer to antibodies produced by a single B cell clone. MAbs can also be produced by hybridomas, which are hybrids of B cells and myeloma cells, or by cell lines that express recombinant DNA encoding immunoglobulin heavy and light chains, thus producing a single, specific antibody.

[0091] Antibodies can be expressed in the extracellular environment and then purified from there.

[0092] Antibody specificity is the ability of an antibody to react with a single antigen, or with a group of antigens that share a particular epitope. An epitope, also known as an antigenic determinant, is a part of an antigen that is recognized by an antibody.

[0093] Antibodies belong to the immunoglobulin class of proteins and are typically assembled from two identical heavy chains (approximately 50-70 kDa) and two identical light chains (approximately 25 kDa). Each heavy chain or light chain has an amino-terminus containing a sequence of 100-130 amino acids that encodes a variable region. Each heavy chain or light chain has a carboxyl-terminus containing a sequence that encodes a constant region. Typically, each antibody binds to the same antigen, i.e., is bivalent.

[0094] Antigen-binding fragments (Fabs) are antibody fragments that bind to an antigen. Each Fab consists of one constant domain and one variable domain derived from each heavy and light chain of the antibody. The crystallizable fragment (Fc) region consists of two or three domains at the carboxyl termini of the two heavy chains. The Fabs ensure binding to the antigen, and the Fc region ensures that each antibody generates an effector immune response. The Fc region binds to various cell receptors such as Fc receptors and other molecules such as complement proteins, mediating a variety of physiological effects, including opsonization that promotes phagocytosis by phagocytes, cell lysis by natural killer cells, and degranulation of mast cells, basophils, and eosinophils.

[0095] The term "variable domain" or "variable region" refers to the amino-terminal portion of an antibody's light or heavy chain that interacts with an antigen. A "variable domain" or "variable region" typically has a length of approximately 120–130 amino acids in the heavy chain and approximately 100–110 amino acids in the light chain. The sequence of each variable region is substantially variable, particularly in the complementarity-determining region (CDR) responsible for interaction with a specific antigen. CDRs are flanked by less variable framework regions (FRs). Typically, there are three CDRs in both the light and heavy chains. For example, CDRs L1, L2, and L3 are located in the light chain, while CDRs H1, H2, and H3 are located in the heavy chain.

[0096] The expression "functional antibody fragment or probe" appropriately refers to a portion of an antibody that includes the variable regions of the antibody's heavy and light chains, or includes either the variable region of the heavy chain or the variable region of the light chain. For example, a functional antibody fragment or probe retains most or all of the binding activity of the original antibody from which the fragment or probe is derived. Examples of such functional antibody fragments or probes include single-chain Fv (scFv), diabodies, triabodies, tetrabodies, and minibodies.

[0097] When the term “fragment” is used herein, it will be understood that, in particular with respect to the fragments of antibodies specifically described, they form an important aspect of the disclosure. Thus, the monoclonal or recombinant antibodies provided by the disclosure may be provided as, for example, any of the following fragments: (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of a VH domain and a CH1 domain; (iii) an Fv fragment consisting of a VL domain and a VH domain; (iv) a dAb fragment consisting of a VH domain; (v) an isolated CDR region; (vi) an F(ab')2 fragment, a bivalent fragment containing two bound Fab fragments; and (vii) a single-chain Fv molecule (scFv) in which the VH domain and VL domain are bound by a peptide linker so that the two domains can together form an antigen-binding site.

[0098] Alternatively, the antibody according to this disclosure may, of course, include the entire IgG antibody, in which case the antibody includes a variable region and a constant region.

[0099] The term "nucleotide sequence" refers to a sequence of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof.

[0100] Naturally, a nucleotide sequence can be transcribed to produce mRNA, which is then translated into polypeptides and / or fragments thereof.

[0101] Further aspects of the present invention will now be described. In one preferred embodiment, the antibody-drug conjugate (ADC) comprises an antibody or a functional antibody fragment or probe thereof, wherein the antibody or fragment or probe is a pair of heavy chain CDRs and light chain CDRs as follows: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 1 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 25; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 2, and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 26; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 3, and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 27; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 4 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 28; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 5 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 29; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 6, and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 30; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 7 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 31; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 8 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 32; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 9 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 33; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 10 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 34; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 11 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 35; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 12 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 36; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 13 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 37; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 14 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 38; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 15 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 39; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 16 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 40; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 17 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 41; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 18 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 42; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 19 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 43; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 20 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 44; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 21 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 45; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 22 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 46; Pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 23 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 47; or The pair of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 24 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 48 It includes one of the following.

[0102] Optionally, at least one CDR may contain one or two amino acid substitutions compared to the described sequence.

[0103] Sequence identity information identifying the humanization affinity mature variants provided by the present invention, including the complete VH sequence, VL sequence, and CDR region of each variant, is shown in Figures 1-6.

[0104] Therefore, the antibody-drug conjugate (ADC) of the present invention preferably contains, as the antibody moiety, a humanized affinity-mature antibody variant that exhibits excellent binding affinity and specificity to the antigen STn, but also shows reduced immunogenicity. Humanized variants are disclosed herein by SEQ ID NOs: 1-24 (variable VH region) and SEQ ID NOs: 25-48 (variable VL region), and affinity-mature antibody variants are disclosed herein by SEQ ID NOs: 49-88 (variable VH region) and SEQ ID NOs: 89-128 (variable VL region).

[0105] In one preferred embodiment, the antibody-drug conjugate (ADC) comprises an antibody or a functional antibody fragment or probe thereof, wherein the antibody or fragment or probe is a pair of heavy chain CDRs and light chain CDRs as follows: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 17 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 41; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 20 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 44; The pair of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 21 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 45 It includes one of the following.

[0106] Optionally, at least one CDR may contain one or two amino acid substitutions compared to the described sequence.

[0107] In a further embodiment, the antibody-drug conjugate (ADC) comprises an antibody or a functional antibody fragment or probe thereof, wherein the antibody or fragment or probe is a pair of heavy chain CDRs and light chain CDRs as follows: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 49 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 89; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 50 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 90; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 51 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 91; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 52 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 92; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 53 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 93; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 54 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 94; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 55 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 95; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 56 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 96; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 57 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 97; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 58 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 98; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 59 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 99; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 60 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 100; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 61 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 101; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 62 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 102; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 63 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 103; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 64 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 104; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 65 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 105; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 66 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 106; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 67 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 107; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 68 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 108; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 69 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 109; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 70 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 110; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 71 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 111; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 72 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 112; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 73 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 113; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 74 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 114; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 75 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 115; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 76 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 116; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 77 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 117; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 78 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 118; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 79 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 119; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 80 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 120; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 81 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 121; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 82 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 122; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 83 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 123; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 84 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 124; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 85 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 125; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 86 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 126; Pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 87 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 127; or The pair of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 88 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 128 It includes one of the following.

[0108] Optionally, at least one CDR may contain one or two amino acid substitutions compared to the described sequence.

[0109] In a preferred embodiment, the antibody-drug conjugate (ADC) comprises an antibody or a functional antibody fragment or probe thereof, wherein the antibody or fragment or probe is a pair of heavy chain CDRs and light chain CDRs as follows: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 49 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 89; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 70 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 110; Pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 81 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 121; or The pair of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 88 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 128 It includes one of the following.

[0110] Optionally, at least one CDR may contain one or two amino acid substitutions compared to the described sequence.

[0111] In a further aspect of the present invention, the antibody-drug conjugate (ADC) comprises an antibody or a functional antibody fragment or probe thereof, wherein the antibody or fragment or probe comprises, in addition to the CDR region described herein, (a) A heavy chain variable region (VH) in which the VH includes a humanized heavy chain framework region. and / or, (b) Light chain variable region (VL) wherein the VL includes a humanized light chain framework region. Includes.

[0112] The humanized framework region may be, for example, as further described below. The variable framework region will be understood to refer to the array surrounding the CDR region. Therefore, different combinations of the CDR region and variable framework region described herein can be fabricated as needed.

[0113] Therefore, in one aspect of the present invention, the antibody-drug conjugate (ADC) comprises an antibody or a fragment thereof, and the antibody or fragment is (a) Heavy chain variable region (VH), where VH is (i) Each of sequence numbers 1-24 or 49-88, and a sequence having at least 80% sequence identity with the sequence described. A heavy chain variable region (VH) sequence selected from the group consisting of the following, including a humanized heavy chain framework region. and / or, (b) Light chain variable region (VL), where the VL is (ii) one of sequence numbers 25-48 or 89-128, and a sequence having at least 80% sequence identity with the sequence described above. Light chain variable region (VL) sequences selected from the group consisting of the following include humanized light chain framework regions. Includes.

[0114] Therefore, in one aspect of the present invention, the antibody-drug conjugate (ADC) comprises an antibody or a fragment thereof, and the antibody or fragment is (a) Heavy chain variable region (VH), where VH is (i) Each of the following sequence numbers 1-24 or 49-88 A heavy chain variable region (VH) sequence selected from the group consisting of the following, including a humanized heavy chain framework region. and / or, (b) Light chain variable region (VL), where the VL is (ii) One of the sequence numbers 25-48 or 89-128 Light chain variable region (VL) sequences selected from the group consisting of the following include humanized light chain framework regions. Includes.

[0115] If necessary, one of the humanized heavy chain framework regions described above can be paired with any one of the humanized light chain framework regions.

[0116] A particular pair of framework regions is preferred. In one aspect of the present invention, an antibody-drug conjugate (ADC) comprising an antibody or a fragment thereof or a probe thereof, wherein the antibody or fragment comprises the following pairs of light chain framework regions and heavy chain framework regions; The pair between the heavy chain framework region of SEQ ID NO: 1 and the light chain framework region of SEQ ID NO: 25 The pair between the heavy chain framework region of SEQ ID NO: 2 and the light chain framework region of SEQ ID NO: 26; The pair between the heavy chain framework region of SEQ ID NO: 3 and the light chain framework region of SEQ ID NO: 27; The pair between the heavy chain framework region of sequence number 4 and the light chain framework region of sequence number 28; The pair between the heavy chain framework region of SEQ ID NO: 5 and the light chain framework region of SEQ ID NO: 29; The pair between the heavy chain framework region of SEQ ID NO: 6 and the light chain framework region of SEQ ID NO: 30; The pair between the heavy chain framework region of SEQ ID NO: 7 and the light chain framework region of SEQ ID NO: 31; The pair between the heavy chain framework region of sequence number 8 and the light chain framework region of sequence number 32; The pair between the heavy chain framework region of SEQ ID NO: 9 and the light chain framework region of SEQ ID NO: 33; The pair between the heavy chain framework region of sequence number 10 and the light chain framework region of sequence number 34; The pair between the heavy chain framework region of SEQ ID NO: 11 and the light chain framework region of SEQ ID NO: 35; The pair between the heavy chain framework region of sequence number 12 and the light chain framework region of sequence number 36; The pair between the heavy chain framework region of SEQ ID NO: 13 and the light chain framework region of SEQ ID NO: 37; The pair between the heavy chain framework region of SEQ ID NO: 14 and the light chain framework region of SEQ ID NO: 38; The pair between the heavy chain framework region of sequence number 15 and the light chain framework region of sequence number 39; The pair between the heavy chain framework region of sequence number 16 and the light chain framework region of sequence number 40; The pair between the heavy chain framework region of sequence number 17 and the light chain framework region of sequence number 41; The pair between the heavy chain framework region of sequence number 18 and the light chain framework region of sequence number 42; The pair between the heavy chain framework region of sequence number 19 and the light chain framework region of sequence number 43; The pair between the heavy chain framework region of sequence number 20 and the light chain framework region of sequence number 44; The pair between the heavy chain framework region of SEQ ID NO: 21 and the light chain framework region of SEQ ID NO: 45; The pair between the heavy chain framework region of sequence number 22 and the light chain framework region of sequence number 46; The pair of the heavy chain framework region of SEQ ID NO: 23 and the light chain framework region of SEQ ID NO: 47; or The pair between the heavy chain framework region of SEQ ID NO: 24 and the light chain framework region of SEQ ID NO: 48 An antibody-drug conjugate (ADC) is provided, comprising one of the above, wherein the heavy chain framework region is as shown in the heavy chain variable region (VH) sequence shown above, and the light chain framework region is as shown in the light chain variable region (VL) sequence shown above.

[0117] Optionally, the heavy chain framework region and / or light chain framework region have at least 80% sequence identity with the described sequence.

[0118] In a further aspect of the present invention, an antibody-drug conjugate (ADC) comprising an antibody or a fragment thereof or a probe thereof, wherein the antibody or fragment comprises the following pairs of light chain framework regions and heavy chain framework regions: The pair between the heavy chain framework region of SEQ ID NO: 49 and the light chain framework region of SEQ ID NO: 89; The pair of the heavy chain framework region of SEQ ID NO: 50 and the light chain framework region of SEQ ID NO: 90; The pair between the heavy chain framework region of SEQ ID NO: 51 and the light chain framework region of SEQ ID NO: 91; The pair between the heavy chain framework region of sequence number 52 and the light chain framework region of sequence number 92; The pair between the heavy chain framework region of sequence number 53 and the light chain framework region of sequence number 93; The pair between the heavy chain framework region of sequence number 54 and the light chain framework region of sequence number 94; The pair of the heavy chain framework region of SEQ ID NO: 55 and the light chain framework region of SEQ ID NO: 95; The pair between the heavy chain framework region of sequence number 56 and the light chain framework region of sequence number 96; The pair between the heavy chain framework region of SEQ ID NO: 57 and the light chain framework region of SEQ ID NO: 97; The pair between the heavy chain framework region of sequence number 58 and the light chain framework region of sequence number 98; The pair between the heavy chain framework region of SEQ ID NO: 59 and the light chain framework region of SEQ ID NO: 99; The pair of the heavy chain framework region of SEQ ID NO: 60 and the light chain framework region of SEQ ID NO: 100; The pair between the heavy chain framework region of sequence number 61 and the light chain framework region of sequence number 101; The pair between the heavy chain framework region of sequence number 62 and the light chain framework region of sequence number 102; The pair between the heavy chain framework region of sequence number 63 and the light chain framework region of sequence number 103; The pair between the heavy chain framework region of sequence number 64 and the light chain framework region of sequence number 104; Pair of the heavy chain framework region of SEQ ID NO: 65 and the light chain framework region of SEQ ID NO: 105; Pair of the heavy chain framework region of SEQ ID NO: 66 and the light chain framework region of SEQ ID NO: 106; Pair of the heavy chain framework region of SEQ ID NO: 67 and the light chain framework region of SEQ ID NO: 107; Pair of the heavy chain framework region of SEQ ID NO: 68 and the light chain framework region of SEQ ID NO: 108; Pair of the heavy chain framework region of SEQ ID NO: 69 and the light chain framework region of SEQ ID NO: 109; Pair of the heavy chain framework region of SEQ ID NO: 70 and the light chain framework region of SEQ ID NO: 110; Pair of the heavy chain framework region of SEQ ID NO: 7 and the light chain framework region of SEQ ID NO: 111; Pair of the heavy chain framework region of SEQ ID NO: 72 and the light chain framework region of SEQ ID NO: 112; Pair of the heavy chain framework region of SEQ ID NO: 73 and the light chain framework region of SEQ ID NO: 113; Pair of the heavy chain framework region of SEQ ID NO: 74 and the light chain framework region of SEQ ID NO: 114; Pair of the heavy chain framework region of SEQ ID NO: 75 and the light chain framework region of SEQ ID NO: 115; Pair of the heavy chain framework region of SEQ ID NO: 76 and the light chain framework region of SEQ ID NO: 116; Pair of the heavy chain framework region of SEQ ID NO: 77 and the light chain framework region of SEQ ID NO: 117; Pair of the heavy chain framework region of SEQ ID NO: 78 and the light chain framework region of SEQ ID NO: 118; Pair of the heavy chain framework region of SEQ ID NO: 79 and the light chain framework region of SEQ ID NO: 119; Pair of the heavy chain framework region of SEQ ID NO: 80 and the light chain framework region of SEQ ID NO: 120; Pair of the heavy chain framework region of SEQ ID NO: 81 and the light chain framework region of SEQ ID NO: 121; Pair of the heavy chain framework region of SEQ ID NO: 82 and the light chain framework region of SEQ ID NO: 122; The pair between the heavy chain framework region of sequence number 83 and the light chain framework region of sequence number 123; The pair between the heavy chain framework region of sequence number 84 and the light chain framework region of sequence number 124; The pair between the heavy chain framework region of sequence number 85 and the light chain framework region of sequence number 125; The pair between the heavy chain framework region of sequence number 86 and the light chain framework region of sequence number 126; A pair of the heavy chain framework region of sequence number 87 and the light chain framework region of sequence number 127; or The pair between the heavy chain framework region of sequence number 88 and the light chain framework region of sequence number 128. An antibody-drug conjugate (ADC) is provided, comprising one of the above, wherein the heavy chain framework region is as shown in the heavy chain variable region (VH) sequence shown above, and the light chain framework region is as shown in the light chain variable region (VL) sequence shown above.

[0119] Optionally, the heavy chain framework region and / or light chain framework region have at least 80% sequence identity with the described sequence.

[0120] In the framework region, some degree of variability in the exact sequence is acceptable, but the function, including binding affinity and specificity, can still be maintained. Accordingly, the present invention also provides an antibody-drug conjugate (ADC) comprising the described antibody or a fragment or probe thereof, wherein the heavy chain framework region and / or light chain framework region may have sequence identity of 80% or higher, preferably 85% or higher, more preferably 90% or higher, even more preferably 95% or higher, or even more preferably 99% or higher, with a particular sequence described herein. This may be achieved, for example, by substitution, addition, or deletion of amino acid residues, with substitution being preferred. The substitution may be, for example, a conservative amino acid substitution.

[0121] In a preferred embodiment of the present invention, the antibody-drug conjugate (ADC) comprises an antibody or fragment or probe described herein, wherein the heavy chain framework region and / or light chain framework region have sequence identity of 90% or higher, preferably 95% or higher, or 99% or higher, with a specific sequence described herein. This may be achieved, for example, by substitution, addition, or deletion of amino acid residues, with substitution being preferred. The substitution may be, for example, a conservative amino acid substitution.

[0122] Therefore, in a further aspect of the present invention, an antibody-drug conjugate (ADC) comprising an antibody or a fragment thereof or a probe thereof, wherein the antibody or fragment or probe is (a) Heavy chain variable region (VH), (i) Each of sequence numbers 1-24 or 49-88, and a sequence having at least 80% sequence identity with the sequence described. A heavy chain variable region (VH) selected from the group consisting of the following: and / or, (a) Light chain variable region (VL), (ii) one of sequence numbers 25-48 or 89-128, and a sequence having at least 80% sequence identity with the sequence described above. A light chain variable region (VL) selected from the group consisting of the following: An antibody-drug conjugate (ADC) containing the above is provided.

[0123] In a further aspect of the present invention, an antibody-drug conjugate (ADC) comprising an antibody, a fragment thereof, or a probe thereof, wherein the antibody, fragment, or probe is (a) Heavy chain variable region (VH), (i) Each of the following sequence numbers 1-24 or 49-88 A heavy chain variable region (VH) selected from the group consisting of the following: and / or, (a) A light chain variable region (VL), (ii) Any one selected from the group consisting of SEQ ID NOs: 25 to No. 48 or 89 to 128 respectively A light chain variable region (VL) An antibody-drug conjugate (ADC) is provided which comprises

[0124] Any one of the VH regions may pair with any one of the VL regions, but a specific pair is preferred.

[0125] Therefore, in a further aspect, the present invention is an antibody-drug conjugate (ADC) comprising an antibody or a fragment or a probe thereof as described herein, wherein the antibody or fragment or probe is a pair of the following heavy chain variable region (VH) and light chain variable region (VL): The pair of SEQ ID NO: 1 and SEQ ID NO: 25; The pair of SEQ ID NO: 2 and SEQ ID NO: 26; The pair of SEQ ID NO: 3 and SEQ ID NO: 27; The pair of SEQ ID NO: 4 and SEQ ID NO: 28; The pair of SEQ ID NO: 5 and SEQ ID NO: 29; The pair of SEQ ID NO: 6 and SEQ ID NO: 30; The pair of SEQ ID NO: 7 and SEQ ID NO: 31; The pair of SEQ ID NO: 8 and SEQ ID NO: 32; The pair of SEQ ID NO: 9 and SEQ ID NO: 33; The pair of SEQ ID NO: 10 and SEQ ID NO: 34; The pair of SEQ ID NO: 11 and SEQ ID NO: 35; The pair of SEQ ID NO: 12 and SEQ ID NO: 36; The pair of SEQ ID NO: 13 and SEQ ID NO: 37; The pair of SEQ ID NO: 14 and SEQ ID NO: 38; The pair of SEQ ID NO: 1 and SEQ ID NO: 39; The pair of SEQ ID NO: 16 and SEQ ID NO: 40; The pair of SEQ ID NO: 17 and SEQ ID NO: 41; The pair of SEQ ID NO: 18 and SEQ ID NO: 42; The pair of SEQ ID NO: 19 and SEQ ID NO: 43; The pair of SEQ ID NO: 2 and SEQ ID NO: 44; The pair of sequence number 21 and sequence number 45; The pair of sequence number 22 and sequence number 46; The pair of sequence number 23 and sequence number 47; or Pair of Sequence ID 24 and Sequence ID 48 We provide an antibody-drug conjugate (ADC) that includes one of the following.

[0126] Optionally, the heavy chain variable region (VH) and / or light chain variable region (VL) may have at least 80% sequence identity with the described sequence.

[0127] In a further aspect of the present invention, the antibody-drug conjugate (ADC) comprises an antibody or a fragment thereof or a probe thereof, wherein the antibody or fragment or probe has the following pair of heavy chain variable regions (VH) and light chain variable regions (VL): The pair of sequence number 49 and sequence number 89; The pair of sequence number 50 and sequence number 90; The pair of sequence number 51 and sequence number 91; The pair of sequence number 52 and sequence number 92; The pair of sequence number 53 and sequence number 93; The pair of sequence number 54 and sequence number 94; The pair of sequence number 55 and sequence number 95; The pair of sequence number 56 and sequence number 96; The pair of sequence number 57 and sequence number 97; The pair of sequence number 58 and sequence number 98; The pair of sequence number 59 and sequence number 99; The pair of sequence number 60 and sequence number 100; The pair of sequence number 61 and sequence number 101; The pair of sequence number 62 and sequence number 102; The pair of sequence number 63 and sequence number 103; The pair of sequence number 64 and sequence number 104; The pair of sequence number 65 and sequence number 105; The pair of sequence number 66 and sequence number 106; The pair of sequence number 67 and sequence number 107; The pair of sequence number 68 and sequence number 108; The pair of sequence number 69 and sequence number 109; The pair of sequence number 70 and sequence number 110; The pair of sequence number 71 and sequence number 111; The pair of sequence number 72 and sequence number 112; The pair of sequence number 73 and sequence number 113; The pair of sequence number 74 and sequence number 114; The pair of sequence number 75 and sequence number 115; The pair of sequence number 76 and sequence number 116; The pair of sequence number 77 and sequence number 117; The pair of sequence number 78 and sequence number 118; The pair of sequence number 79 and sequence number 119; The pair of sequence number 80 and sequence number 120; The pair of sequence number 81 and sequence number 121; The pair of sequence number 82 and sequence number 122; The pair of sequence number 83 and sequence number 123; The pair of sequence number 84 and sequence number 124; The pair of sequence number 85 and sequence number 125; Pair of Sequence ID 86 and Sequence ID 126 The pair of sequence number 87 and sequence number 127: or Pair of Sequence ID 88 and Sequence ID 128 It includes one of the following.

[0128] Optionally, the heavy chain variable region (VH) and / or light chain variable region (VL) may have at least 80% sequence identity with the described sequence.

[0129] In the same manner as described above, the present invention also provides antibody-drug conjugates (ADCs) comprising the described antibody or a fragment thereof or a probe thereof, wherein the heavy chain variable region (VH) and / or light chain variable region (VL) may have sequence identity of 80% or higher, preferably 85% or higher, more preferably 90% or higher, even more preferably 95% or higher, or even more preferably 99% or higher, with specific VH and VL sequences described herein. This may be achieved, for example, by substitution, addition, or deletion of amino acid residues, with substitution being preferred. The substitution may be, for example, a conservative amino acid substitution.

[0130] In preferred embodiments of the present invention, the antibody-drug conjugate (ADC) comprises an antibody or fragment or probe thereof as described herein, wherein the heavy chain variable region (VH) and / or light chain variable region have sequence identity of 90% or higher, preferably 95% or higher, or more preferably 99% or higher, with a specific sequence as described herein. This may be achieved, for example, by substitution, addition, or deletion of amino acid residues, with substitution being preferred. The substitution may be, for example, a conservative amino acid substitution.

[0131] In terms of functionality, the present invention provides an antibody-drug conjugate (ADC) comprising, in particular, an antibody or fragment thereof or a probe thereof conjugated to a base of STn and an alpha-2,6-linked sialic acid-terminated glycan. The alpha-2,6-linked sialic acid-terminated glycan may include, for example, STn, 2,6-sialyl T, dicialyl T, or 2,6-sialolactosamine.

[0132] The entire glycan recognized by the antibodies, fragments thereof, or probes thereof described herein is: 1. Sialyl Tn: NeuAcα--6GaINAcα / β1- 2. 2,6-Sialyl T: Gal β1-3GalNAcα1--NeuAcα2-6 3. Dithialyl T: NeuAcα2-3Galβ1-3GalNAcα1---NeuAcα2-6 4. 2,6-Sialo-N-acetyllactosamine: NeuAcα2-6Galβ1-4Glcβ1- These are some examples.

[0133] Sialyl Tn, also known as STn, sialosyl Tn, sialic acid-added Tn, Neu5Ac-α2,6GalNAcα-O-Ser / Thr, or CD175 in the "differentiation cluster" nomenclature, is the simplest sialic acid-added mucin-type O-glycan. STn is a cleaved O-glycan containing sialic acid (Neu5Ac) α-2,6-linked (via the 6th carbon) to N-acetylgalactosamine (GalNAc) alpha-O-linked to serine / threonine (Ser / Thr) (Neu5Ac-α2,6GalNAcα-O-Ser / Thr). Sial acid addition prevents the formation of various core structures otherwise found in mucin-type O-glycans.

[0134] STn is expressed in more than 80% of human cancers and is associated with poor prognosis and reduced overall survival in various cancer patients. STn antigen biosynthesis is associated with the expression of the sialyltransferase ST6GalNAc1 and mutations or loss of heterozygosity in the COSMC gene.

[0135] Antibodies that bind to STn with such specificity are particularly interesting because, in contrast to many current antibody therapies, they exhibit high tumor specificity and low or no responsiveness to normal cells.

[0136] An antibody-drug conjugate (ADC) described herein, comprising an antibody, a fragment thereof, or a probe thereof, can specifically bind to a group of STn or alpha-2,6-sialic acid-added glycan as described herein.

[0137] The antibodies or fragments thereof described herein may have glycosylated sites that have undergone glycanization.

[0138] In one aspect of the present invention, the antibodies, fragments thereof, or probes described herein may be provided in any suitable form as part of an antibody-drug conjugate (ADC). For example, the antibodies, fragments, or probes may be provided in the form of ScFv, monoclonal antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or CAR-T cells, or other forms, as will be understood by those skilled in the art.

[0139] Therefore, for example, an antibody or its fragment or probe as part of an ADC may be provided as a single-chain fragment variable antibody (scFv). This refers to a functional antibody fragment that contains only the VL and VH regions that are linked by a linker to form a monovalent antigen-binding site. Diabodies, trimos, and tetrabodies are antibodies containing dimers, trimers, or tetramers of scFv, that is, containing two, three, and four polypeptide chains, respectively, and forming two, three, and four antigen-binding sites, respectively, which may be the same or different antigen-binding sites. Such can also be used.

[0140] The antibody, its functional antibody fragment, or probe, which is a component of the ADC of the present invention, may have one or more binding sites. If there are more than one binding sites, these sites may be identical or different. If there are two different binding sites, the antibody, its functional antibody fragment, or probe is referred to as a “bispecific” antibody.

[0141] The present invention also provides a pharmaceutical composition comprising an antibody-drug conjugate (ADC) described herein, which includes an antibody or a functional antibody fragment or probe thereof, and a pharmaceutically acceptable carrier.

[0142] In another embodiment, a method is provided for detecting tumor biomarkers in a patient sample using an antibody-drug conjugate (ADC) described herein, comprising an antibody or functional antibody fragment or probe thereof, or using a pharmaceutical composition described herein. This methodology includes the step of staining a biological sample obtained from a subject with a nucleotide sequence encoding an antibody or functional antibody fragment or probe thereof, or an antibody-drug conjugate (ADC), as described herein, under conditions suitable for specific binding to the antibody. The presence or absence of antibody binding indicates tumor cells expressing cell surface STn, 2,6-sialyl T, dicialyl T, or 2,6-sialolactosamine. Examples of biological samples to be analyzed include isolated cells or tissues, or tumor-derived proteins.

[0143] The present invention also provides antibody-drug conjugates (ADCs) comprising antibodies or functional antibody fragments or probes thereof as described herein, or pharmaceutical compositions for medical use as described herein, or all of the above. In particular, antibody-drug conjugates (ADCs) comprising antibodies (as described herein) as pharmaceutical compositions for medical use are intended for use in the treatment of cancer patients. It is conceivable that various types of tumors can be treated with the variants disclosed herein.

[0144] Accordingly, in some embodiments, an antibody, its functional antibody fragment, or probe, which is part of the antibody-drug conjugate (ADC) of the present invention, may be conjugated or fused to one or more diagnostic or therapeutic agents, or any other desired molecule. The resulting conjugated antibody, its functional antibody fragment, or probe may be useful for monitoring or diagnosing the onset, development, progression, and / or severity of diseases associated with the expression of STn or alpha-2,6-sialic acid-modified glycans.

[0145] The antibody-drug conjugates (ADCs) described herein, comprising antibodies or functional fragments or probes thereof, can also be used to detect the expression of STn or alpha-2,6-sialic acid-added glycans in any biological sample using classical immunohistochemical methods (IHC or immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, and immunoblotting).

[0146] The antibody-drug conjugates (ADCs) of the present invention, comprising antibodies, functional antibody fragments, or probes described herein, may be provided at an effective concentration and exert a therapeutically useful effect with minimal side effects, provided they are provided alone, conjugated, or in combination with pharmaceutical compositions.

[0147] In further embodiments, the Disclosure includes isolated polynucleotides comprising nucleic acid sequences, wherein the nucleic acid sequences encode antibodies or functional antibody fragments or probes thereof as described herein, in particular variable heavy chain regions of antibodies, variable light chain region domains of antibodies, or functional antibody fragments or probes thereof.

[0148] In a further embodiment, the disclosure provides an expression vector comprising a polynucleotide encoding an antibody, fragment, or probe described herein. Naturally, suitable host cells comprising such an expression vector can be provided.

[0149] According to one aspect of this disclosure, a method for producing an antibody or a functional antibody fragment or probe thereof as described herein may include a step of using such suitable host cells.

[0150] In further embodiments, the present invention also provides a method for producing an antibody-drug conjugate (ADC) as described herein, comprising the steps of: preparing a drug, which is a growth inhibitor, coupled with a suitable DAR to an antibody bound to a glycan terminated with sialyl Tn (STn) or alpha-2,6-linked sialic acid; preparing a linker, which is a linker as described herein and includes a cleavable linker; and conjugating the coupled drug-antibody to the linker. The growth inhibitor may be, for example, an anticancer agent such as a chemotherapeutic agent or a cytotoxic agent. The growth inhibitor may be, for example, exatecan or a derivative thereof, or deruxtecan or a derivative thereof. The resulting ADC is cleavable and releases the drug (or payload) into the tumor or tumor cell microenvironment.

[0151] The linker may be any of the linkers described herein. Preferably, the linker is one of the linkers described in the Novel Linkers section below, and in particular, the linker comprises one of the structures shown in Formulas I to VI below.

[0152] The antibody may be, for example, any one of the antibodies described herein.

[0153] Preferably, DAR is about 4 or less.

[0154] The ADCs of this disclosure that target cancer-specific biomarkers involve innovative designs. In particular, the present invention provides novel antibody-drug conjugate (ADC) structures that combine the specificity of a cancer-specific antibody with the potent cytotoxic activity of, for example, an exatecan derivative, using a beta-glucuronide linker with improved solubility (e.g., using three PEGs).

[0155] This invention targets STn glycans, short-chain O-glycan antigens that are not present in normal healthy tissue but are cleaved and overexpressed in various types of cancer. By targeting this unique biomarker, it is expected to have a significant impact on the treatment of various types of cancer, including metastatic, drug-resistant, and highly malignant cancers.

[0156] The present invention demonstrates superior efficacy in inhibiting tumor growth in vitro and in vivo compared to controls. For example, it has been shown that using a quaternary amine as a handle for the beta-glucuronide linker increases tumor uptake and reduces payload internalization even when cleaved outside the tumor, leading to a better safety profile.

[0157] Because this invention targets cancer-specific biomarkers present in various types of animal cancers, ADCs of this disclosure and other embodiments of the invention also have potential applications in animal health. This novel approach could lead to the development of new therapies for animal cancers and provide significant benefits to the veterinary industry.

[0158] In particular, the ADCs of this disclosure combine various elements in a unique manner. The present invention combines various elements, including humanized antibodies, affinity maturation methods, beta-glucuronide linkers, PEGs, and carbamate / quaternary amine handles, and, for example, exatecan derivatives, in a novel way that results in more effective and specific cancer treatment. This unique combination of elements is expected to have a significant impact on the field of cancer therapy, particularly on the treatment of various types of cancer that have proven difficult to treat.

[0159] The inventors have found that the internal distribution of the ADCs disclosed herein is improved compared to trastuzumab-based ADCs, which provides a significant advantage in terms of efficacy. This is due to the fact that internal distribution is a crucial factor in delivering the cytotoxic payload to tumor cells. By improving internal distribution, the inventors can deliver a higher volume of the cytotoxic payload to target cells, resulting in a more potent and effective therapy. In addition, the wide range of internal distribution capabilities provided by the cancer-specific antibodies disclosed herein allows for fine-tuning of the ADC design to specific types of tumors and / or patient populations, providing further customization and improved therapeutic outcomes.

[0160] The improved uptake of the ADCs of this disclosure in the tumor microenvironment is a result of the cancer-specific antibodies disclosed herein (see in vivo distribution data). By selectively targeting cancer-specific antigens such as STn, the ADCs of this disclosure can accumulate more efficiently in the tumor microenvironment and improve therapeutic outcomes. This specificity also leads to a reduction in the possibility of off-target effects, further improving the safety of the ADCs.

[0161] Sequence liability As described in the present inventors' concurrently pending patent application, International Publication No. 2023 / 249502, the inventors also investigated the identification of sequence liabilities (i.e., post-translational modification sites) in the CDRs of the humanized V1 and affinity-mature clones described herein. The inventors identified at least one PTM site in the heavy chain and another PTM site in the light chain. To eliminate liabilities, it has been proposed to introduce a single amino acid change into each PTM, thereby altering the CDR.

[0162] The highest risk location is assessed as follows: In VL, • CDR3 position 93 / 94 "DP" aspartic acid fragmentation site In VH, • The "NS" deamidation site at position 53 / 54 of CDR2.

[0163] This risk is thought to be specific to the affinity-mature variant mAb-v53. • CDR2 position 55 / 56 "DG" aspartic acid isomerization site.

[0164] Therefore, in one aspect of the present invention, in the ADC described herein, either the L-CDR3 sequence or the VL variable light chain sequence (either humanized or affinity-matured) disclosed herein may be further mutated so that the "D" (aspartic acid / aspartate) at position 93 is replaced with one of the following amino acid residues: A, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y.

[0165] In a further embodiment, either the L-CDR3 sequence or the VL variable light chain sequence (either humanized or affinity-matured) disclosed herein may be further mutated so that the "P" (proline) at position 94 is replaced with one of the following amino acid residues: A, E, F, H, I, K, L, N, Q, R, T, V, W, or Y.

[0166] One of the changes shown for 93rd place can be paired with any one of the changes shown for 94th place.

[0167] In one preferred embodiment, either the L-CDR3 sequence or the VL variable light chain sequence (either humanized or affinity-matured) disclosed herein may be further mutated to replace the "DP" at positions 93 and 94 with one of the following pairs of amino acid residues: DA, DK, DN, EP, KP, NP, QP, RP, AA, EE, FF, GP, HH, II, KK, LL, NN, QQ, RR, SP, TT, VV, WW, or YY.

[0168] Therefore, the VL CDR3 sequence in any of the sequences disclosed herein may be modified as described above. Mutations DA, DK, DN, EP, KP, NP, QP, or RP may be preferred.

[0169] Figure 23b shows a specific preferred VL sequence incorporating the above sequence liability modification. This figure discloses variable light chains of humanized variants (v65-v88) based on V1. These are shown as SEQ ID NOs. 150-173.

[0170] Therefore, in a further aspect of the present invention, in the ADC described herein, either the H-CDR2 sequence or the VH variable heavy chain sequence (either humanized or affinity matured) disclosed herein may be further mutated so that the "D" (aspartic acid / aspartate) at position 55 is replaced with one of the following amino acid residues: A, E, F, G, H, I, K, L, P, Q, R, V, W, or Y.

[0171] In a further embodiment, either the H-CDR2 sequence or the VH variable heavy chain sequence (either humanized or affinity-matured) disclosed herein may be further mutated so that the "G" (glycine) at position 56 is replaced with one of the following amino acid residues: A, E, F, H, I, K, L, N, Q, R, T, V, W, or Y.

[0172] One of the changes shown for position 55 can be paired with any one of the changes shown for position 56.

[0173] In one preferred embodiment, either the H-CDR2 sequence or the VH variable heavy chain sequence (either humanized or affinity-matured) disclosed herein may be further mutated to replace the "DG" at positions 55 and 56 with one of the following pairs of amino acid residues: DE, DK, DA, EG, QG, RG, AA, EE, FF, GG, HH, KK, LL, DN, PQ, QQ, RR, DT, VV, WW, or YY.

[0174] Therefore, the VH CDR2 sequence in any of the sequences disclosed herein may be modified as described above. Mutations DE, DK, DA, EG, QG, or RG may be preferred.

[0175] Figure 23a shows a specific preferred VH sequence incorporating the above sequence liability modification. This figure discloses variable heavy chains of humanized variants (v65-v85) based on V1. These are shown as SEQ ID NOs. 129-149.

[0176] In a preferred embodiment, in the ADC described herein, the "DP" at positions 93 and 94 is a pair of the following amino acid residues: DA, DK, DN, EP, KP, NP, QP, RP, AA, EE, FF, GP, HH, II, KK, LL, NN, QQ, RR, SP, TT, VV, WW, or YY One of the L-CDR3 sequences or VL variable light chain sequences (either humanized or affinity matured) disclosed herein, which includes a mutation that is substituted for one of the following, The "DG" at positions 55 and 56 corresponds to the following amino acid residue pair: DE, DK, DA, EG, QG, RG, AA, EE, FF, GG, HH, KK, LL, DN, PQ, QQ, RR, DT, VV, WW, or YY It may be paired with one of the H-CDR2 sequences or VH variable heavy chain sequences (either humanized or affinity matured) disclosed herein, which include a mutation that is substituted for any one of the following.

[0177] In a further aspect of the present invention, in the affinity-mature variant mAb-v53 of the ADC described herein, it may be advantageous for the variant to be further mutated so that the "NS" (asparagine / serine) at positions 53 and 54 of the variable heavy chain CDR2 is replaced with alternative amino acids. For example, N may be replaced with any one of A, E, F, G, H, I, K, L, P, Q, R, V, W, or Y while retaining S at position 54. Alternatively, S may be replaced with A, E, F, G, H, I, K, L, P, Q, R, V, W, or Y while retaining N at position 54. Or, both N and S may be changed, i.e., combinations of the above variations may be used.

[0178] In alternative embodiments, the antibody component of the ADC described herein may be based on or incorporate the antibody described in International Publication No. 2019 / 147152. Further details can be found in this document.

[0179] Therefore, in a further aspect of the present invention, an antibody-drug conjugate (ADC) comprising an antibody having a combination of a light chain variable region (VL) and a heavy chain variable region (VH), VL includes complementarity determination regions (CDRs) L-CDR1, L-CDR2, and L-CDR3, respectively, as shown in Sequence ID Nos. 179, 181, and 183. VH includes CDR H-CDR1, H-CDR2, and H-CDR3, as shown in Sequence IDs 185, 187, and 189, respectively. Antibody-drug conjugates (ADCs) are provided.

[0180] The antibody-drug conjugate (ADC) may contain antibodies in which the VL comprises SEQ ID NOs. 178, 180, and 182, and the VH comprises SEQ ID NOs. 184, 186, and 188. In one preferred embodiment, the antibody-drug conjugate (ADC) contains the antibodies described above, in which the VL comprises SEQ ID NOs. 177 and the VH comprises SEQ ID NOs. 176.

[0181] In one preferred embodiment, the antibody-drug conjugate (ADC) comprises the antibody described above, wherein the VL amino acid sequence and VH amino acid sequence are shown for L2A5 in Figure 12.

[0182] The antibodies described herein preferably bind to a glycan group terminated with STn and alpha-2,6-linked sialic acid. Preferably, the alpha-2,6-linked sialic acid-terminated glycan includes STn, 2,6-sialyl T, dicialyl T, or 2,6-sialolactosamine. The antibodies described herein may have glycosylated sites.

[0183] The antibodies described above may be monoclonal antibodies, chimeric antibodies, or humanized antibodies.

[0184] The antibodies described above may also be in the form of functional antibody fragments that bind to STn and alpha-2,6-linked sialic acid-terminated glycan groups.

[0185] In one aspect of this disclosure, a polynucleotide encoding the antibody described above, optionally, Sequence ID 174 and / or Sequence ID 175; Sequence ID 190, GACACATCC, and Sequence ID 191; or Sequence ID 192, Sequence ID 193, and Sequence ID 194 An expression vector containing polynucleotides is also provided.

[0186] This specification also discloses host cells containing the expression vectors described above.

[0187] The method for producing the antibodies described above may include, for example, the step of using the host cells described above.

[0188] Applications of Novel ADCs The present invention also provides pharmaceutical compositions comprising antibody-drug conjugates (ADCs) and pharmaceutically acceptable carriers as described herein.

[0189] In further embodiments, antibody-drug conjugates (ADCs) described herein for use in pharmaceuticals are also provided. “Pharmaceutical” is used herein in its broadest sense, encompassing the treatment of both human and animal bodies. Therefore, in addition to the treatment of human patients, veterinary applications for the treatment of animals are particularly intended.

[0190] In a preferred embodiment, antibody-drug conjugates (ADCs) described herein are also provided for use in the treatment of cancer.

[0191] The novel ADCs described represent innovative features, including the use of antibodies targeting cancer-specific biomarkers (not present in normal tissues) conjugated with a novel linker payload that offers increased stability in circulation.

[0192] Incorporating additional PEG units, preferably three additional PEG units, into the linker structure offers several advantages, including increased solubility and stability of the ADC and enhanced pharmacokinetics. Carbamates or quaternary ammonium handles / linkers ensure stable binding between the antibody and the cytotoxic payload, allowing for precise release of the payload upon internal migration into cancer cells.

[0193] Finally, the beta-glucuronide linker combined with PEG3 increases hydrophilicity, reduces aggregation during conjugation compared to other linkers, and increases stability during circulation.

[0194] New Linker As a result of their research, the inventors have actually devised a particular linker that has not been disclosed before. In other words, such a linker is a novel linker. Such novel linkers have been found to have certain utility, including certain advantages described herein, when used in ADCs having antibodies that bind to sialyl Tn(STn) or alpha-2,6-linked sialic acid-terminated glycans, particularly including the specific antibodies described above, and especially when the drug or payload is a chemotherapeutic agent or cytotoxic agent such as exatecan or its derivatives, or deruxtecan or its derivatives. However, such linkers also have more general utility and can, in principle, be used with other types of antibodies and other drugs or payloads other than those specifically described herein, if they are expected to impart the same or similar advantages as described herein.

[0195] Accordingly, according to a further aspect of the present invention, a linker for use in an antibody-drug conjugate (ADC) suitable for the treatment of cancer is provided, comprising a linker cleavable by glucuronidase and configured to be coupled to a drug via a quaternary ammonium salt bond.

[0196] In another embodiment, a linker for use in an antibody-drug conjugate (ADC) suitable for the treatment of cancer is also provided, comprising a linker cleavable by glucuronidase and PEGylated with a group containing polyethylene glycol (PEG).

[0197] In a further embodiment, the present invention provides a linker that is cleavable by glucuronidase, configured to be coupled to a drug via a quaternary ammonium salt bond, and is PEGylated with a group containing polyethylene glycol (PEG).

[0198] In a further embodiment, the present invention provides a linker that is cleavable with glucuronidase, configured to be coupled to a drug via a carbamate bond, and which does not contain an alkyne moiety.

[0199] In a further embodiment, the present invention provides a glucuronidase-cleavable linker configured to be coupled to a drug via a carbamate bond and terminated at a maleimide moiety.

[0200] In a preferred embodiment, a linker is provided that is configured to be coupled to a drug via a quaternary ammonium salt bond and is also PEGylated with a group containing polyethylene glycol (PEG).

[0201] In one embodiment, the linker may contain a β-glucuronide moiety.

[0202] In one embodiment, the linker is given by equation I': [ka] The formula includes the β-glucuronide moiety shown in [wherein X is NH, N-CH3, or CF2].

[0203] In one embodiment of formula (I'), X is NH. In one embodiment of formula (I'), X is N-CH3. In one embodiment of formula (I'), X is CF2.

[0204] In a preferred embodiment, the linker is of formula I: [ka] Equation (I) The linker may contain a β-glucuronide moiety represented by [formula (I) is further modified to be coupled to a drug via a quaternary ammonium salt bond], or the linker may be PEGylated with a group containing polyethylene glycol (PEG). In a preferred embodiment, when PEGylation is used, the linker may have an amide moiety of formula (I') or (I) PEGylated with a group containing polyethylene glycol (PEG).

[0205] In one preferred embodiment, both formula (I') or (I) are further modified to be coupled to a drug via a quaternary ammonium salt bond, and the linker is PEGylated with a group containing polyethylene glycol (PEG). Preferably, the linker is PEGylated with a group containing polyethylene glycol (PEG) on the amide portion of formula (I') or (I).

[0206] A preferred example of a linker provided in accordance with the present invention is one in which the linker is configured to be coupled to a drug via a quaternary ammonium salt moiety or bond.

[0207] Accordingly, in a preferred embodiment, linkers are provided that are configured or modified to bind to a drug via a quaternary ammonium salt bond. For example, in formula I above, the carbamate bond or portion may be replaced by a quaternary ammonium salt bond or portion. One suitable quaternary ammonium salt bond or portion is illustrated in formula IV or VI below, and it is understood that this bond or portion can be used in linker structures other than those specifically shown in formula IV or VI.

[0208] When using PEGylation of the linker, the linker has, for example, the following structure: [ka] It may be PEGylated with a group containing polyethylene glycol (PEG) based on the formula [wherein n is 1 to 5, preferably 2 to 4], If necessary, one or both of the terminal H substituents in the above structure may be substituted with another suitable chemical group, provided that the function of the linker is not substantially affected. In a preferred embodiment, in the above structure, n is 3, i.e., PEG contains three individual monomer units.

[0209] In a preferred embodiment, the linker may be PEGylated with the base of formula II. [ka] Formula (II)

[0210] PEGylation may be performed, for example, on the amide group of the linker, or on the amide group of the structure shown in formula I. In a preferred embodiment, the linker containing the group of formula II is configured to be coupled to the drug via a quaternary ammonium salt bond or part thereof.

[0211] In one preferred embodiment, the linker is given by formula III': [ka] Formula III' As shown in the formula [wherein X is NH, N-CH3, or CF2].

[0212] In one embodiment of formula III', X is NH. In one embodiment of formula III', X is N-CH3. In one embodiment of formula III', X is CF2.

[0213] In one embodiment, a preferred linker according to the present invention is given by formula III: [ka] This is the PEGylated linker shown.

[0214] In one preferred embodiment, the linker is given by formula IV': [ka] Formula IV' As shown in the formula [wherein X is NH, N-CH3, or CF2].

[0215] In one embodiment of formula IV', X is NH. In one embodiment of formula IV', X is N-CH3. In one embodiment of formula IV', X is CF2.

[0216] In another embodiment, a preferred linker according to the present invention is given by formula IV: [ka] This is the PEGylated linker shown.

[0217] In a more preferred embodiment, the drug is coupled with formula V': [ka] Formula V' A linker is provided that forms a drug-linker payload as shown in the formula [wherein X is NH, N-CH3, or CF2].

[0218] In one embodiment of formula V', X is NH. In one embodiment of formula V', X is N-CH3. In one embodiment of formula V', X is CF2.

[0219] In a more preferred embodiment, a drug is coupled to form a drug-linker payload, and formula VI': [ka] Equation VI' A linker is provided as shown in the formula [wherein X is NH, N-CH3, or CF2].

[0220] In one embodiment of formula VI', X is NH. In one embodiment of formula VI', X is N-CH3. In one embodiment of formula VI', X is CF2.

[0221] In a more preferred embodiment, a drug is coupled to form a drug-linker payload, with formula V or VI': [ka] A linker is provided as shown.

[0222] The present invention relates to an exatecan derivative compound having a handle portion covalently bonded to the exatecan parent molecule at a position that does not significantly affect the therapeutic or pharmacokinetic properties of the parent molecule, wherein the handle portion is of the following formula: [ka] Formula (VII) We also provide exatecan derivative compounds, including quaternary ammonium salts.

[0223] In a preferred embodiment, the following general structure: [ka] Formula (VIII) Exatecan derivative compounds having the same property are also provided.

[0224] The exatecan derivative compounds provided above exhibit an improved safety profile compared to the exatecan parent molecule and retain therapeutic activity against cancer cells.

[0225] Therefore, it will be understood that the present invention also provides antibody-drug conjugates (ADCs) comprising antibodies conjugated to a drug via the linker described above. Preferably, the ADCs are for the treatment of cancer, but it will be understood that the linkers described can, in principle, be used not only for the treatment of cancer but also for other types of ADCs.

[0226] However, in a preferred embodiment, the antibody-drug conjugate (ADC) using the linker described herein comprises an antibody that binds to a glycan terminated with sialyl Tn (STn) or alpha-2,6-linked sialic acid.

[0227] In a preferred example, the antibody-drug conjugate (ADC) using the linker described herein would contain a drug that is a growth inhibitor suitable for the treatment of cancer, such as exatecan or a similar compound.

[0228] Preferably, the antibody-drug conjugate (ADC) using the linker described herein comprises an antibody that binds to a glycan terminated with sialyl Tn (STn) or alpha-2,6-linked sialic acid, and further comprises a drug that is a growth inhibitor suitable for the treatment of cancer, such as exatecan or a similar compound.

[0229] result In this invention, the inventors describe cancer-specific antibody-drug conjugates suitable for precision pharmaceuticals, with the aim of improving target specificity, bystander effect, and safety profile.

[0230] Novel linker-payload synthesis and conjugation: The linker-payload used in the present invention can be synthesized, for example, by a multi-step method that ensures the formation of a stable and cleavable bond between the cytotoxic payload and the linker, as shown in Figures 1a-1b. This synthesis method includes the following steps: 1. A step to prepare the cytotoxic payload, exatecan, in DAR4 using a well-established synthetic procedure. The selected DAR provides an optimal balance of therapeutic efficacy and safety, ensuring a sufficient number of cytotoxic molecules per antibody, thereby effectively targeting and killing tumor cells while minimizing the risk of toxicity to healthy tissue. 2. A step to synthesize a linker comprising a beta-glucuronide moiety, three polyethylene glycol (PEG) chains, and a handle (carbamate or quaternary amine) for binding to the antibody. Incorporating, for example, three additional PEG units into the linker structure provides several advantages, including increased solubility and stability of the ADC and enhanced pharmacokinetics. The carbamate handle / linker ensures stable binding between the antibody and the cytotoxic payload and also allows for accurate release of the payload upon internal migration into cancer cells. The two final linker-payloads underlined in Figures 1a-1b (final constructs) were designed to provide stability, solubility, and cleavability in the context of the ADC. 3. A step of conjugating the beta-glucuronide linker to the exatecan payload. This step includes forming a cleavable bond between the cytotoxic payload and the beta-glucuronide linker.

[0231] Stepwise synthesis of linker-payload types 1 and 2 is described in the detailed explanation sections for carbamate-linked glucuronide drug linkers (type 1) and quaternary ammonium-linked glucuronide drug linkers (type 2). HPLC and LC-MS results for each compound are shown in Figures 15–27b. A legend of relevant acronyms is provided in Figure 28. The numbering of referenced compounds is shown in the figures in the appendix.

[0232] Bioconjugation method: The bioconjugation method includes the step of conjugating a linker-payload to an antibody to produce a homogeneous and well-defined ADC having a specific drug-to-antibody ratio (DAR).

[0233] The bioconjugation method can be carried out using the following steps. 1. A step of selecting and preparing an antibody or antibody fragment that specifically recognizes the target antigen STn. This may include producing recombinant antibodies in a suitable expression system. A list of various mAb clone candidates is reported in Figures 9-14. 2. Identifying and selecting an appropriate conjugation site for the antibody, such as a cysteine ​​residue or a lysine residue, to ensure that the conjugation does not impair the antibody's binding affinity or specificity. 3. A step of conjugating the linker-payload to a selected conjugation site of the antibody using site-directed coupling chemistry. This step may include forming a stable bond, such as a thioether bond or an amide bond, between the antibody and the linker-payload. 4. Purify the obtained ADC to remove any unreacted linker-payload, unconjugated antibody, and other impurities. This step may include techniques such as size exclusion chromatography, ion exchange chromatography, or other preferred methods as described in the detailed description section. 5. Characterizing the purified ADC to confirm the drug-to-antibody ratio (DAR), antibody integrity, linker-payload stability, and retention of antigen-binding properties. This step may include various analytical techniques such as mass spectrometry, SDS-PAGE, and surface plasmon resonance (SPR).

[0234] As disclosed herein, one humanization affinity mature clone antibody (SEQ ID NO: 88(V H ) and 128(V L mAb_v64, which contains ) ), was used in conjugation with a novel linker-payload type 1 (carbamate-bound base) to obtain the named ADC-AFI-ExV1 used herein. The sequence for mAb_v64 is reported in the appendix sequence antibody list (Figure 12).

[0235] Pilot small-scale (1mg) ADC-AFI-V1 production: This method involved producing a small-scale initial pilot batch of ADC-AFI-ExV1 at a 1 mg scale. The TCEP / mAb molar equivalents were set to 1.5, 2.5, and 3.5, respectively. The results are summarized in Figure 29a. To determine the DAR, HIC-HPLC was first used, as shown in Figure 29b (left graph). When using this technique, different DAR species were not well separated by HIC-HPLC. This reflects the high hydrophilicity of linker payload type 1. This data confirms and validates the high hydrophilicity of ADC-AFI-ExV1. Next, the DAR of ADC-AFI-ExV1 was determined using reduced MS-DAR. Raw data for SEC-HPLC are shown in Figure 29b (right graph), and data for LC-MS are reported in Figures 30a-30c. A curve showing the relationship between TCEP / mAb equivalent and reduced MS-DAR is shown in Figure 31. According to the relationship between the TCEP / mAb ratio and reduced MS-DAR, the optimal TCEP / mAb ratio for ADC-AFI-ExV1 with a target DAR of 4.0 was 2.58. All ADC products showed good purity under all conditions.

[0236] Since optimal conditions were found in the pilot conjugation, a confirmation conjugation (1 mg scale) was performed to verify the conditions. The results are summarized in Figure 32a. SEC and LC-MS data are shown in Figures 32b and 32c, respectively. As shown in Figure 32b, the product showed high monomer levels, but as shown in Figures 32a and 32c, the reduction MS-DAR was lower than the expected DAR of 4.0.

[0237] To obtain an ADC product with a target DAR of 4.0, the TCEP / mAb molar equivalent was increased to 3.04 in the second confirmation conjugation. The results of the new confirmation conjugation are summarized in Figure 33a, and the SEC data and LC-MS data are shown in Figures 33b and 33c, respectively. As shown in Figure 33b, the product showed high monomer levels. As shown in Figures 33a and 33c, since the target DAR is 4 ± 0.4, an optimized TCEP / mAb ratio of 3.04 is appropriate for bulk conjugation.

[0238] Final (30mg) ADC-AFI-V1 production (bulk conjugation): To reduce deviations caused by large-scale administration, the 30 mg bulk conjugation is divided into two batches (15 mg each).

[0239] Since the confirmation conjugation was successful, the first bulk conjugation was performed using the confirmed optimal conditions, with a TCEP / mAb ratio of 3.04. The results are summarized in Figure 34a, and the SEC and LC-MS data are shown in Figures 34b and 34c, respectively. As shown in Figure 34b, the product has high monomer levels. As shown in Figures 34a and 34c, the reduction MS-DAR was slightly higher than the target of 4.0.

[0240] In the second 15 mg batch conjugation, the TCEP / mAb ratio was reduced to 2.50. The results are summarized in Figure 35a, and the SEC data and LC-MS data are shown in Figures 35b and 35c, respectively. As shown in Figure 35b, the product has high monomer levels. As shown in Figures 35a and 35c, the reduced MS-DAR meets the requirements.

[0241] Finally, the first and second batches of ADC-AFI-ExV1 were combined and dialyzed with formulation buffer. After dialyzation, the product was treated with dextran-coated charcoal to remove residual free drugs. The ADC was filtered through a 0.22 μm membrane to obtain the final product, which was submitted for characterization.

[0242] Final conjugation of three different ADC products: To evaluate the efficacy of ADC-AFI-ExV1, the same naked antibody (mAb_v64) was also conjugated with an available deruxtecan (GGFG-DXd) linker payload. In addition, an IgG1 isotype control and a control conjugated with the same novel linker-payload type 1 (V1), named ADC-IgG1-ExV1, were included. The results for all three ADCs, including the three antibodies included in the large production batch (30 mg), are shown in Figure 2a.

[0243] ADC-AFI-ExV1 (mAb v64; Exatecan carbamate-bound base "Type 1") ADC-AFI-DXd (mAb v64; GGFG-DXd "Deruxtecan") ADC-IgG1-ExV1 (IgG1 isotype control; exatecan carbamate-bound base "type 1") The SEC and reduction MS data for the three ADCs are shown in Figures 2b and 2c, respectively. Additional RP data for the same ADCs are shown in Figure 36. Finally, the data reported in Figure 3 shows that the endotoxin levels of all ADCs listed above are minimal. This indicates the safety of the test products.

[0244] Unlike ADC-AFI-ExV1 (Figure 29b; HIC graph), DAR evaluation using hydrophobic interaction chromatography (HIC) was only possible in the analysis of ADC-AFI-DXd (Figure 37). Taken together, these data highlight that the new ADC-AFI-ExV1 exhibits a higher hydrophilic profile compared to ADC-AFI-DXd (antibody linker-payload control). Additional RP data for all produced ADCs are included in Figure 37.

[0245] Accordingly, the present invention discloses a method for producing a stable and effective ADC comprising a linker-payload and a specific antibody, as well as a bioconjugation method that ensures the formation of a clearly defined ADC with a specific drug-to-antibody ratio (DAR), preferably 4 or lower. The production method and bioconjugation method described herein are easily scalable and can be applied to the production of ADCs for human and animal health applications.

[0246] SNU16-CDX In Vivo Efficacy Data The objective of this study was to evaluate the in vivo antitumor efficacy of the exatecan-V1 linker-payload described in ADC-AFI-ExV1 compared to the well-known linker-payload: deruxtecan (GGFG-DXd). The model used for efficacy evaluation was a human gastric SNU-16 subcutaneous xenograft (CDX) model in female BALB / c nude mice. SNU16 expresses high levels of the target STn, as shown in Figure 38, and is a good candidate cell line for efficacy testing.

[0247] Mice were administered two doses intravenously (iv) according to the experimental design listed in Table 8.

[0248] Analysis of tumor growth (Figure 4a) demonstrates the potential of this ADC construct to selectively target cancer cells expressing the STn antigen. This increased specificity is achieved by using cancer-specific antibodies, thereby ensuring increased tumor uptake and reduced off-target effects.

[0249] Beta-glucuronide linkers possess inherent stability and desirable release properties, further contributing to the potency and selectivity of ADCs.

[0250] According to available data, animal models treated with ADCs exhibited significant inhibition of tumor growth compared to the control group, demonstrating the advantage of targeting STn antigens with this specific ADC composition.

[0251] In addition, a direct comparison between ADC-AFI-ExV1 (exatecan, carbamate-bound base "type 1") and ADC-AFI-DXd (deruxtecan, linker-payload control) shows increased efficacy of our novel linker-payload, as reported in the enlarged view in Figure 4b. ADC-AFI-ExV1 and ADC-AFI-DXd had a DAR=4 and were administered on the same schedule and at the same dosage [8 mg / kg]. Finally, body weight analysis showed an increase over time (Figures 4c-4d), highlighting the good treatment tolerance.

[0252] Although safety data is limited, the features of this ADC construct, including cancer-specific antibodies and a well-designed linker, suggest a favorable safety profile with minimal adverse effects on normal tissue. Because this ADC offers advantages over conventional cancer therapies in terms of tumor targeting, increased efficacy, and potential safety, it warrants further investigation into its safety and efficacy in both in vitro and in vivo models.

[0253] In summary, the present invention discloses a novel ADC with a DAR of 4, having a unique combination of features comprising, in a preferred embodiment, a cancer-specific anti-STn antibody, a beta-glucuronide linker having three additional PEG units, and a carbamate handle / linker. Available tumor growth data demonstrate the potential of this ADC to improve cancer treatment. This ADC design suggests promising safety characteristics and should be further explored in future research to validate its potential for human and animal health applications.

[0254] In addition to the novel properties of the newly generated ADC-AFI-ExV1, the inventors generated data using the same antibody (mAb_v64) conjugated with a clinically validated linker-payload (vedotinin platform). In this case, the linker used was a monomethyl auristatin E (MMAE) payload using a protease-cleavable linker (Val-Cit-PAB) and maleimide conjugation (random Cys). To validate the efficacy of the inventors' antibody, a control anti-STn human mAb was used as a positive control (mAb_PC). Interestingly, the inventors found that when the inventors' novel mAb was used with a different ADC configuration named ADC-AFI-MMAE, the tumor reduction efficacy was increased compared to the control ADC-PC-MMAE, as shown in Figure 39a. Further analysis of body weight revealed that, as shown in Figure 39b, body weight increased over time, demonstrating the effectiveness of the treatment.

[0255] In summary, this data demonstrates the superiority of the inventors' novel antibody and the potential for various antibody clones (listed in the antibody list in Appendix Figures 9-13) to be used in the development of various ADC technologies.

[0256] Biomedical distribution analysis The objective of this study was to evaluate the long-term accumulation and distribution of 89Zr-Ab in mouse models of breast cancer expressing 4T1 parental cancer cell lines and 4T1 STn cancer cell lines.

[0257] The in vivo distribution of radiolabeled parental L2A5 antibodies was evaluated using a syngeneic mouse model inoculated with a 4T1-STn triple-negative breast cancer cell line modified to overexpress STn.

[0258] The data obtained from this analysis, detected by PET and shown in Figure 5a, demonstrated antibody binding specificity only in the presence of STn antigen expression in the 4T1-STn+ group compared to the control parental cell line (WT) over various exposure times. As shown in Figure 5b, high tumor uptake was detected 96 hours after antibody injection-delivery, supporting the specificity of the antibody delivery system. Finally, safety profiles were observed in several other organs (Figure 40), with relatively low uptake in well-known highly angiogenic organs, further supporting the safety of antibody therapy.

[0259] Antibody development feasibility analysis To characterize affinity-mature mAb_v64 (affinity-mature clone) and humanized mAb_v1 (humanized clone) in terms of stability, aggregation, temperature stress, and viscosity, various tests were used, data were compared, and normalized against therapeutic antibodies (palivizumab and trastuzumab).

[0260] Interestingly, mAb_v64 and mAb_v1 did not exhibit poli-reactivity or adhesion to DNA, LPS, lysozyme, and cell lysates, and showed comparable binding to therapeutic antibody controls such as palivizumab and trastuzumab, as shown in Figure 6a. Similarly, the mean adhesion analysis showed comparable safety profiles compared to the control mAbs palivizumab and trastuzumab, as shown in Figure 6b.

[0261] In addition, various stress tests, including temperature stress (45°C for 48 hours) and pH stress (pH 3 for 24 hours), showed neither aggregation nor antibody degradation, and no related increase was observed in either high-MW or low-MW fractions. This clearly indicates that these antibodies are stable and do not show any degradation (Figure 6c).

[0262] Overall, this data highlights the safety of the potential development of certain humanized antibodies.

[0263] Antibody internal distribution profiles in various cancer cell lines To evaluate the internal migration dynamics of a specific antibody, including the parent antibody, first-generation humanized clones and affinity-mature antibody clones, as shown in Figures 9-14, were tested using various cancer cell lines.

[0264] In this assay, the antibody was labeled with Zenon pHrodo fluorophores, which are activated only under low pH conditions found in early endosomes. Antibody internalization was indirectly assessed by measuring pHrodo fluorescence (MFI) using flow cytometry. All data were normalized against the control IgG1 mAb, palivizumab.

[0265] Interestingly, certain antibodies used exhibited different internal distribution profiles based on STn expression levels (Figures 7a-7d), and are therefore suitable for various antibody-based therapies.

[0266] EC50 analysis of mAb clones in various cancer cell lines To support previously generated data, the inventors produced proof-of-concept data using various mAb clones (included in Appendix Antibody Sequences, Figures 9-14) and binding affinity measurements using FACS, as disclosed in their concurrently pending patent application, International Publication No. 2023 / 249502.

[0267] This data showed that, when using various cancer cell lines, including colorectal cancer (COLO205), gastric cancer (SNU16), and ovarian cancer (OV90), the binding of various mAb clones, including mAb_v1, mAb_v64, mAb_v53, mAb_v25, and mAb_v46, was increased compared to the anti-STn human-positive control mAb_PC. Interestingly, different antibody clones exhibited different binding characteristics when using cancer cell lines that naturally expressed different levels of STn (Figure 8). In summary, these data highlight the binding specificity of the antibodies disclosed herein to their target STn, making them suitable for the treatment of various types of cancer.

[0268] Detailed explanation Antibody components, substances, and methods Generally, where applicable, the methods for producing antibodies described herein include the steps of fusing two cells to produce a hybridoma, introducing the nucleotide sequence of the present invention into host cells, culturing the host cells for a sufficient amount of time under conditions suitable for the production of the heavy and / or light chains encoding the antibody or functional fragment or probe thereof, and subsequently purifying the heavy and / or light chains of the antibody or functional fragment or probe thereof.

[0269] Recombinant expression of the antibody of the present invention, or its functional antibody fragment or probe, bound to the STn or alpha-2,6-sialic acid addition antigen base, may include constructing an expression vector containing nucleotide sequences encoding the heavy and / or light chains of the antibody of the present invention or its functional antibody fragment or probe.

[0270] The vector can be generated by recombinant DNA technology. Such a vector may also contain other coding nucleotide sequences that form the basis of the chimeric antibody sequence. For example, such a coding nucleotide sequence may include a nucleotide sequence encoding the constant region of an antibody molecule that enables the expression of a chimeric protein containing the amino acid sequence of the antibody of the present invention, its functional antibody fragment, or probe (see International Publication No. 86 / 05807 and International Publication No. 89701036), followed by the entire heavy chain or the entire light chain, or both the entire heavy chain and the entire light chain of the antibody.

[0271] The expression vector can be transfected into host cells by transfection / transduction techniques, and the resulting cells produce the antibody of the present invention or its functional antibody fragment. Therefore, the present invention includes host cells containing a nucleotide sequence encoding the antibody of the present invention or its functional antibody fragment or probe.

[0272] Host cells can be selected so that the properties of the product derived from the inserted nucleotide sequence are modified.

[0273] In one embodiment, such host cells can modify the encoded protein by adding glycosylation sites, phosphorylation sites, or other modifications. For example, the host cells can provide correct processing and cell transport / secretion of the protein.

[0274] Finally, in an attempt to increase similarity to antibodies normally produced in humans and reduce immunogenicity, the inventors have provided novel, improved, and useful antibody variants, including humanization of the parental L2A5 antibody and thorough antibody affinity maturation of a selected humanized variant, named with the acronym mAb_v1, leading to the generation of antibody variants with increased binding and affinity to target STn.

[0275] The obtained antibody clones / variants were characterized in terms of binding to various cancer cell lines, target specificity, and immunogenicity, as further explained below.

[0276] Generally speaking, those skilled in the art will fully understand the methods used by the inventors and the specific methods described, with respect to such technical details.

[0277] Humanization Variable domain analysis and CDR identification The IMGT Domain Gap Align tool was used to identify complementarity-determining regions (CDRs) and analyze the most closely related matching germline sequences: http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi.

[0278] Molecular modeling Using our own software, we constructed molecular models of the VH and VL domains based on homology with previously published antibody crystal structures. The PDB files can be viewed with any molecular visualization software. Images were created using PyMol.

[0279] Sequence Liability Analysis Based on publicly available protein motifs, antibody sequences were analyzed for specific liabilities. The analysis was performed using a custom system built in Microsoft Excel. The following motifs were used in this software. In the motifs, X represents any amino acid other than proline. [Table 1]

[0280] Gene synthesis and cloning The variable heavy and light chain domains were designed to have suitable restriction sites at the 5' and 3' ends to enable cloning into Absolute Antibody cloning and expression vectors. The variable domain sequences were codon-optimized for expression in human cells. After gene synthesis, the variable domains were cloned into suitable species and types of Absolute Antibody vectors. The correct sequences were verified by Sanger sequencing using raw data analyzed with DNASTAR Lasergene software. After verification, plasmid DNA of a suitable size was prepared to generate a sufficient amount of high-quality DNA for transfection.

[0281] Expression and Purification HEK293 (Human Fetal Kidney 293) mammalian cells were expanded to a stage optimal for transient transfection. The cells were transiently transfected with heavy and light chain expression vectors and cultured for a further 6 days. The culture was collected by centrifugation at 4000 rpm and filtered through a 0.22 M filter. The first step of purification was performed by eluting with citrate pH 3.0 buffer using protein A affinity chromatography, followed by neutralization with 0.5 M Tris, pH 9.0. The resulting eluted protein was then buffer-exchanged to PBS using a desalting column. Antibody concentrations were determined by UV spectroscopy, and antibodies were concentrated as needed.

[0282] antibody analysis Antibody purity was determined by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and HPLC (high-performance liquid chromatography). SEC-HPLC was performed using an Agilent 1100 series instrument with a suitable size exclusion column (SEC). Antibody titer was determined by Protein A HPLC.

[0283] Characterization of humanized antibodies Various assays were performed to demonstrate whether the humanized variants retained the same biophysical properties (specificity, affinity, and internal translocation) as the parent clone. These assays include the following: • Evaluation of binding to BSM mucin (STn carrier) by ELISA • Evaluation of binding to various STn+ cell lines (flow cytometry) • NMR studies to understand antibody interactions with STn-serin glycosides. · In silico immunogenicity analysis Glycan array for determining antibody specificity • Affinity measurement using SPR • TMA for evaluating mAb binding to patient-derived cancer tissue

[0284] The results of these studies (reported in International Publication No. 2023 / 249502) demonstrated that the humanized variants retained favorable biophysical properties in terms of specificity, affinity, and internal transport.

[0285] affinity maturation Library generation Bioinformatics analysis of the parent antibody was performed to generate a site-directed CDR mutation library. After homology modeling of the antibody Fv region and CDR transplantation into a template, CDR residues potentially involved in antigen binding were identified. Sixteen positions were identified in the heavy chain and fourteen in the light chain. By analyzing NGS databases, amino acids commonly used in specific germline cells were identified. Based on this, degenerate codons were designed, and mutations were introduced at the identified positions potentially involved in antigen binding. In general, amino acids with undesirable characteristics were avoided. The introduction of mutations can be described by a Gaussian distribution with an average of four mutations per antibody chain. Primers were designed based on the degenerate codons and used to introduce mutations into the antibody sequence. The mutated antibody gene was cloned into Yumab's scFv phage display vector, generating three libraries, which were then packaged into antibody phage particles. The total functional diversity was 5 × 10⁶. 8 A library larger than a cfu was generated. Antibody clones with functional open reading frames were determined by DNA sequence analysis. By packaging and purifying the antibody phage particles, at least 3 × 10⁶ particles were obtained for each library. 11 CFU / ML was obtained.

[0286] Affinity maturation by in vitro selection The generated antibody phage libraries were used for affinity maturation by in vitro selection. Each individual library used the same overall excess amount of antibody phage particles relative to its functional size. A specific amount was pooled into a single library for in vitro selection.

[0287] In the first panning round, biotinylated BSM was used. The antibody phage output from the first round of panning, generated against the biotinylated protein, was used in the second round to increase stringency by reducing the number of STn+ cells, driving output against antibodies with increased affinity. Negative selection was performed against several negative antigens in both panning rounds. Four different strategies were used for affinity maturation by in vitro selection. It was expected that the amount of antibody phage particles eluted would decrease as stringency increased from strategy 1 to 4.

[0288] Antibody screening After the second round of panning, the eluted antibody phage particles were used to infect Escherichia coli (E. coli). For antibody screening, 384 clones were randomly selected from each strategy. A total of 1536 antibody clones were used to produce monoclonal scFv antibodies in the bacterial system. The produced antibody clones were tested for binding activity to positive and negative cell lines. Prepared control antibodies (IgG) and parental scFv antibodies were used as positive controls. Since parental scFv antibodies were identified with a signal-to-noise ratio of 20, clones with a signal-to-noise ratio greater than 20 were identified as hits.

[0289] Antibody sequencing 210 clones were identified as hits and selected for DNA sequencing analysis. Sequencing revealed 40 uniquely mutated antibodies. These antibodies showed 1 to 6 mutations in the CDR. Several hotspot mutations were identified, and favorable mutations were shown at various locations.

[0290] In addition, all 40 uniquely mutated antibodies were selected and soluble scFv was produced. This product was used for ELISA screening against two positive antigens (biotinylated BSM and non-biotinylated BSM) and two negative antigens (streptavidin and BSA). For analysis, the signal-to-noise ratio between the positive and negative antigens was calculated. Most antibodies showed strong binding to both positive antigens but not to the negative antigens. Based on the generated results, antibodies were selected, converted to the final format, and produced in mammalian cell cultures.

[0291] Conversion to the final format (human IgG1): Based on the results obtained, 20 antibodies were selected for conversion to human IgG1. These antibodies were cloned into Yumab's mammalian expression vector and produced in mammalian cell culture. The antibodies were purified using protein A affinity chromatography and buffered in phosphate-buffered saline. Quality control was performed by UV / VIS spectroscopy and reduced SDS-PAGE. Successful production of 18 antibodies was achieved, demonstrating high purity and integrity. In parallel, parental antibodies were cloned using the same format and produced simultaneously.

[0292] Affinity ranking To verify antibody binding, titrations were performed on prepared positive cell lines (MDA-MB-231 STn) and negative cell lines (MDA-MB-231 WT), as well as on bovine submandibular gland mucin (BSM). Additional titration experiments were conducted using cell lines that naturally express STn (COLO205, SNU16, OV90). All antibodies showed potent and specific binding to target cells. EC50 values ​​were calculated. The best antibody showed an EC50 value of approximately 0.6 nM, while the parent antibody was calculated to have an EC50 value of 2 nM. The specificity of affinity-mature antibody clones was evaluated by glycan array. [Examples]

[0293] The preparation of a particular ADC is described below. Those skilled in the art will understand that other ADCs disclosed herein can be similarly prepared with suitable modifications depending on the identity of the antibody, linker, or drug component.

[0294] Carbamate-bound glucuronide drug linker type 1 (V1): Basic preparation procedure for compound 2 - Reduction of the NO2 group: A round-bottom flask was purged three times with Ar, and dried Pd / C (4.0 g, 10% purity) was carefully added. Then, siRNA (20 mL) was added and permeated into the dried Pd / C. A solution of compound 1 (20.0 g, 41.37 mmol) in siRNA (200 mL) was added, followed by the slow addition of TEA (627.95 mg, 6.21 mmol, 863.76 μL) under an Ar atmosphere. The resulting mixture was degassed, purged three times with Ar, and then purged three times with H2. The mixture was stirred under an H2 atmosphere (15 psi) at 25°C for 16 hours. LC-MS indicated that the reaction was complete. The mixture was filtered and concentrated under reduced pressure. Compound 2 (18 g, 38.66 mmol, yield 93.43%, purity 97.8%) was obtained as a white solid. The HPLC and LC-MS analyses of compound 2 are shown in Figures 15a and 15b, respectively.

[0295] Basic procedure for preparing compound 3 - protection of primary alcohol: To a solution of compound 2 (18 g, 39.52 mmol, 1 equivalent), imidazole (16.14 g, 237.15 mmol, 6 equivalents), DMAP (1.21 g, 9.88 mmol, 0.25 equivalents) in DMF (200 mL), and TBSCl (35.74 g, 237.15 mmol, 29.06 mL, 6 equivalents) were sequentially added. The mixture was stirred at 25°C for 1 hour. The desired compound was confirmed by TLC (petroleum ether:ethyl acetate = 2:1, R f(=0.60). The reaction mixture was then diluted with water (400 mL) and extracted twice with siRNA (400 mL). The combined organic layers were washed with brine (200 mL) and dried over Na₂SO₄. The organic portion was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 50 / 1~2 / 1) to obtain compound 3 (14.4 g, 25.28 mmol, yield 63.95%) as a white solid. The HPLC and LC-MS analyses of compound 3 are shown in Figures 16a and 16b, respectively.

[0296] Basic preparation procedure for compound 4 - coupling reaction: Compound 3 (5 g, 8.78 mmol) was dissolved in DCM (50 mL) and EEDQ (4.34 g, 17.55 mmol) and compound a (3.28 g, 10.53 mmol) were added. The mixture was stirred at 25 °C for 16 hours. LC-MS showed a main peak with the desired mass, as well as consumption of the starting material. The solvent was removed under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1~2 / 1) to obtain compound 4 (7.00 g, 8.01 mmol, yield 91.31%, purity 98.8%) as a white solid. HPLC and LC-MS analyses of compound 4 are shown in Figures 17a and 17b, respectively.

[0297] Basic preparation procedure for compound 5 - Deprotection of the TBS group: Compound 4 (5 g, 5.79 mmol) was dissolved in THF (50 mL) and HCl (1 M, 11.59 mL) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed a main peak with the desired mass, as well as consumption of the starting material. The reaction mixture was then diluted with H2O (100 mL) and extracted with ₹ (100 mL x 2). The combined organic layers were washed with brine (100 mL) and dried over Na2SO4. The organic portion was filtered and concentrated under reduced pressure. Crude compound 5 (4.00 g, 5.08 mmol, yield 87.69%, purity 95.1%) was obtained as a white solid and used directly in the next step without purification. HPLC and LC-MS analyses of compound 5 are shown in Figures 18a and 18b, respectively.

[0298] Basic preparation procedure for compound 6 - PNP activation: Compound 5 (4.00 g, 5.34 mmol, 1 equivalent) was dissolved in DMF (40 mL) and DIEA (4.14 g, 32.05 mmol, 5.58 mL, 6 equivalents) and PNP (4.88 g, 16.03 mmol, 3 equivalents) were added. The mixture was stirred at 25°C for 3 hours. LC-MS indicated that the reaction was complete. The reaction mixture was then washed with 1 M HCl (20 mL) and extracted with EtAOc (50 mL x 2). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was subjected to column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1~1 / 2, TLC: petroleum ether / ethyl acetate = 1:1, R f The compound was purified using a solution of 0.240 (0.240), and compound 6 (4.00 g, 4.29 mmol, yield 80.38%, purity 98.1%) was obtained as a white solid. The HPLC and LC-MS analyses of compound 6 are shown in Figures 19a and 19b, respectively.

[0299] Basic preparation procedure for compound 7 - Conjugation with exatecan: Compound 6 (1 g, 1.09 mmol, 1 equivalent) and exatecan (610.76 mg, 1.15 mmol, 1.05 equivalents) were dissolved in DMF (10 mL), to which HOBt (295.73 mg, 2.19 mmol, 2 equivalents) and DIEA (424.29 mg, 3.28 mmol, 571.82 μL, 3 equivalents) were added. The mixture was stirred at 25°C for 2 hours. LC-MS showed a main peak with the desired mass and consumption of compound 6. The crude product was pulverized in isopropyl ether (200 mL x 2) for 10 minutes at 25°C. Then, it was filtered and concentrated under reduced pressure to obtain crude compound 7 (1.3 g, 1.02 mmol, yield 93.26%, purity 95%) as a yellow solid. HPLC and LC-MS analyses of compound 7 are shown in Figures 20a and 20b, respectively.

[0300] Basic preparation procedure for compound 8 - Deprotection of the Fmoc group: To a solution of compound 7 (1 g, 826.33 μmol, 1 equivalent) in MeOH (10 mL), LiOH.H2O (69.35 mg, 1.65 mmol, 2 equivalents) in H2O (10 mL) was added. The reaction mixture was stirred at 0°C for 12 hours. Two reactions were carried out in parallel. Completion of the reaction was indicated by LC-MS. The two reaction mixtures were then combined and the reaction was quenched by adding CH3COOH (6 mL) at 0°C (pH=5). The residue was purified by preparative HPLC (TFA conditions) to obtain compound 8 (780 mg, 901.63 μmol, yield 54.56%, purity 98.0%) as a pale yellow solid. The HPLC and LC-MS analyses of compound 8 are shown in Figures 21a and 21b, respectively.

[0301] Basic preparation procedure for compound carbamate bond-based glucuronide drug linkers (Target 1) - Coupling of 8 and 1a-1: To a solution of compound 8 (400 mg, 471.81 μmol, 1 equivalent) in DMF (4 mL), NMM (95.44 mg, 943.63 μmol, 103.74 μL, 2 equivalents) and compound 1a-1 (206.75 mg, 518.99 μmol, 1.1 equivalents) were added. The mixture was stirred at 25°C for 3 hours. LC-MS showed a major peak with the desired mass and consumption of compound 8. The reaction mixture was then quenched with 0.1 mL of formic acid. The resulting substance was directly purified by preparative HPLC (FA conditions). Finally, a carbamate-bound glucuronide drug linker (220 mg, 194.51 μmol, yield 41.23%) was obtained as a pale yellow solid. HPLC and LC-MS analyses of compounds 8 and 1a-1 are shown in Figures 22a and 22b, respectively.

[0302] Quaternary ammonium bond-based glucuronide drug linkers: Basic preparation procedure for compound 6a-1: To a solution of compound 6a (500 mg, 940.64 μmol, 1 equivalent) in DMF (20 mL), DIC (474.83 mg, 3.76 mmol, 582.62 μL, 4 equivalents), DIEA (243.14 mg, 1.88 mmol, 327.69 μL, 2 equivalents), and HOBt (254.20 mg, 1.88 mmol, 2 equivalents) were added. The reaction mixture was stirred at 25°C for 16 hours. LC-MS showed the desired compound as the main peak. The reaction mixture was then pulverized in isopropyl ether (100 mL x 2) for 10 minutes at 25°C. After filtration, the solid was concentrated under reduced pressure to obtain compound 6a-1 (500 mg, 924.98 μmol, yield 98.34%, purity 96.3%) as a white solid. The HPLC and LC-MS analyses of compound 6a-1 are shown in Figures 23a and 23b, respectively.

[0303] Basic preparation procedure for compound 9 - Linker modification: Compound 5 (2.8 g, 3.74 mmol, 1 equivalent) was dissolved in DMF (28 mL) and PPh3 (2.94 g, 11.22 mmol, 3 equivalents) and CBr4 (1.24 g, 3.74 mmol, 1.00 equivalent) were added. The reaction mixture was stirred at 25°C for 2 hours. LC-MS detected the desired compound as the main peak. The reaction mixture was concentrated under reduced pressure to obtain the residue, which was purified by preparative HPLC (TFA conditions). Compound 9 (1.7 g, 1.83 mmol, yield 48.95%, purity 87.4%) was obtained as a white solid. The HPLC and LC-MS analyses of compound 9 are shown in Figures 24a and 24b, respectively.

[0304] Basic preparation procedure for compound 10 - Conjugation with modified exatecan: Compound 6a-1 (560.56 mg, 1.08 mmol, 1 equivalent) was dissolved in DMF (10 mL) and DIEA (139.18 mg, 1.08 mmol, 187.57 μL, 1 equivalent) and compound 9 (1 g, 1.08 mmol, purity 87.4%, 1 equivalent) were added. The mixture was stirred at 25°C for 3 hours. LC-MS detected the desired compound as the main peak. The crude product was ground in isopropyl ether (300 mL x 2) for 5 minutes at 25°C. The residue was purified by preparative HPLC (TFA conditions) to obtain compound 10 (900 mg, 657.61 μmol, yield 61.07%, purity 91.5%) as a yellow solid. HPLC and LC-MS analyses of compound 10 are shown in Figures 25a and 25b, respectively.

[0305] Basic preparation procedure for compound 11 - Deprotection of the Fmoc group LiOH.H2O (338.90 mg, 8.08 mmol, 10 equivalents) in MeOH (15 mL) was added to a solution of compound 10 (1.0 g, 807.59 μmol, 1 equivalent) in H2O (5 mL). The mixture was stirred at 0°C for 16 hours and at 25°C for 1 hour. LC-MS detected the desired compound as the main peak. The reaction mixture was then quenched with 1 mL of CH3COOH, and the crude substance was purified by preparative HPLC (TFA conditions) to obtain compound 11 (430 mg, 455.18 μmol, yield 56.36%, purity 94.2%) as a yellow solid. The HPLC and LC-MS analyses of compound 11 are shown in Figures 26a and 26b, respectively.

[0306] Basic compound preparation procedure for quaternary ammonium bond-based glucuronide drug linkers (Target 2) - Coupling of 11 and 1a-1: To a solution of compound 11 (500 mg, 561.86 μmol, 1 equivalent) in DMF (4.3 mL), NMM (113.66 mg, 1.12 mmol, 123.55 μL, 2 equivalents) and compound 1a-1 (246.21 mg, 618.05 μmol, 1.1 equivalents) were added. The mixture was stirred at 25°C for 3 hours. LC-MS detected the desired compound as the main peak. The reaction mixture was quenched by adding HCOOH (0.1 mL) at 0°C. The obtained substance was purified by preparative HPLC (FA conditions) to obtain a quaternary ammonium-bonded glucuronide drug linker (255 mg, 298.34 μmol, purity 95.1%) as a white solid. HPLC and LC-MS analyses of compound 12 are shown in Figures 27a and 27b, respectively.

[0307] Correlation analysis of the final generated ADC. ADC Sample List ADC-AFI-ExV1 (mAb_v64) and ADC-IgG1-ExV1 (IgG1 control) were generated by cysteine ​​conjugation of the linker-payload and mAb, targeting DAR4. Additional ADC-AFI-DXd (mAb_v64) were generated using the GGFG-DXd linker payload. Deruxtecan (GGFG-DXd) (catalog number HY-13631E; distributor MedChemExpress) was used for this analysis. Detailed information can be found in Table 1. [Table 2] TIFF2026514252000033.tif59170

[0308] Purification method using Zeba Spin desalting column (10 mL) The Zeba Spin desalting column was pre-treated according to the following procedure. 1) The bottom closure of the column was removed, and the storage solution was removed by centrifugation (1,000 g, 2 minutes). 2) The column was purified by adding 5 mL of 0.2 M NaOH to the top of the resin. The column was allowed to stand for 30 minutes. 3) The mixture was centrifuged (1,000g for 2 minutes), and the flow-through was discarded. 4) Add 5 mL of formulation buffer to the resin, centrifuge (1,000 g, 2 minutes), and discard the flow-through. Repeat this procedure two more times until the pH of the flow-through is the same as that of the formulation buffer. The centrifugation time for final balancing was 6 minutes. 5) Transfer the column to a new collection tube and apply the conjugation mixture to the top of the resin. 6) The mixture was centrifuged (1,000g, 4 minutes) and the flow-through containing the product was collected.

[0309] Measurement of ADC product concentration The concentration of the product was determined using a BCA protein assay from Pierce. 1) A working reagent was prepared by adding 200 μL of Pierce BCA protein assay reagent B to 10 mL of BCA protein assay reagent A and mixing thoroughly. 2) A 200 μL solution of 1.0 mg / mL mAb (mAb_V64) was diluted to 15.625 μg / mL by 2-fold serial dilution with dH2O, and a series of standards were prepared for a standard curve from 15.625 μg / mL to 1,000 μg / mL. 3) The conjugation sample was diluted with dH2O. 4) 25 μL each of the triple-duplicated standard material and sample was introduced into the designated wells of a 96-well plate, followed by the addition of 200 μL of BCA working reagent to each well. 5) After incubation at 37°C for 30 minutes using a microplate reader, the OD562nm wavelength was read. 6) A standard curve was created by plotting the average blank-corrected 562nm measurements of each antibody standard against their concentration in μg / mL units. 7) The protein concentration of the ADC sample was determined using a standard curve.

[0310] Aggregation determination by SEC-HPLC Size exclusion chromatography was performed at 25°C using an Agilent 1260 series HPLC system equipped with a TSK gel G3000SWXL size exclusion chromatography column (7.8 × 300 mm, 5 μm). The mobile phase was 78 mM KH2PO4, 122 mM K2HPO4, 250 mM KCl, 15% IPA, pH 7.0 ± 0.1. The flow rate was set to 0.75 mL / min. Sample loading was 40–50 μg per injection. The sample was detected at 280 nm using a UV detector. The retention time of the aggregation peak was recorded based on the relative molecular weight, and the aggregation level was determined by the relative area of ​​the peak. [Table 3] TIFF2026514252000035.tif69170

[0311] DAR determination by HIC-HPLC Hydrophobic interaction chromatography was performed at 25°C using an Agilent 1260 series HPLC system equipped with a TSK gel Butyl-NPR hydrophobic interaction chromatography column (4.6 mm ID × 3.5 cm, 2.5 μm). Mobile phase A consisted of 1.5 M (NH4)2SO4, 50 mM K2HPO4 3H2O, pH 7.0. Mobile phase B consisted of 21.3 mM KH2PO4, 28.6 mM K2HPO4 3H2O, 25% isopropanol, pH 7.0. The flow rate was set to 0.6 mL / min. Sample loading was 8 μL per injection. Samples were detected at 280 nm using a UV detector. Retention times of DAR species were recorded based on the associated mAb, and HIC-DAR was calculated from the peak area based on different DAR species. [Table 4] TIFF2026514252000037.tif110170

[0312] DAR determination by reduced LC-MS Sample preparation method: 6 μL of 1 M tris, pH 8.0 buffer, and 3 μL of 0.1 M DTT solution were added to 30 μg of ADC sample, and then dH2O was added to achieve a final antibody concentration of 1 mg / mL. The mixture was incubated at 37°C for 30 minutes. 2 μL of the sample was injected.

[0313] LC-MS was performed at 25°C using an Agilent 1260 series HPLC system with an Agilent PLRP-S 1000A, 8 μm, 50 × 2.1 mm microscope, and TOF mass spectrometry. 0.05% TFA-containing dH₂O was used as mobile phase A, and 0.05% TFA-containing acetonitrile was used as mobile phase B. The flow rate was set to 0.5 mL / min. Sample loading was 2–10 μg. DAR was calculated based on the peak abundance of the deconvolution mass. [Table 5] TIFF2026514252000039.tif210170

[0314] Determination of residual free drugs by RP-HPLC Determination of the free drug in the linker-payload of a type 1-carbamate-binding-based glucuronide drug linker. Residual free drug levels were determined by reverse-phase HPLC. After protein precipitation, the supernatant was loaded onto an RP-C18 HPLC column and eluted with a gradient increasing the organic mobile phase. The percentage of residual free drug was quantified by peak area by comparison with an external standard curve.

[0315] 1) Solvent preparation: a) Solvent I (for protein precipitation) 10 g of NaCl was weighed into a pre-mixed organic solvent of 30 mL of MeOH and 50 mL of ACN, mixed, and stirred for at least 1 hour. The solution was then allowed to stand for at least 1 hour before use. The supernatant was a saturated sodium chloride solution. b) Solvent II (diluent) 2 mL of DMA was added to 8 mL of formulation buffer and mixed thoroughly. Then 10 mL of solvent I was added and mixed thoroughly again.

[0316] 2) Creation of a standard curve The stock standard linker-drug solution was diluted with solvent II (diluent) to 1 mM. 10 μL of 1 mM linker-payload solution was added to 90 μL of solvent II to a final concentration of 100 μM. Then, the 100 μM standard solution was sequentially diluted to 0.2 μM according to the table below. [Table 6] TIFF2026514252000041.tif57170

[0317] 3) Sample preparation 7.5 μL of DMA was added to 42.5 μL of ADC sample and thoroughly mixed. Then, 100 μL of solvent I was added, and the mixture was vortexed in a mixer at room temperature for 10 minutes. The mixture was centrifuged at 16,000 rcf for 10 minutes at room temperature. Then, 80 μL of the supernatant was immediately pipetteed into an analytical glass HPLC vial.

[0318] 4)HPLC method [Table 7] TIFF2026514252000043.tif126170

[0319] 5) Data Analysis a) Integration of standard curve injection. The peak area (Y) was plotted as a function of concentration (X) Y = kX + b. The slope (k) and intercept (b) were calculated. b) The drug peaks associated with the sample were integrated. The total peak area (Y) was recorded and interpolated against the standard curve. Calculated concentrations were obtained.

number

[0320] Endotoxin determination Endotoxin levels were determined using Endosafe®-PTS™ (Charles River, MCS150K). 25 μL of the sample was pipetted into each of the four reservoirs of the PTS cartridge. The reader aspirated the sample in addition to the LAL reagent + positive product control in the spike channel and mixed the sample with the LAL reagent in the sample channel. The sample was combined with the chromogenic substrate and then incubated. After mixing, the optical density of the well was measured and analyzed by comparison with the standard curve stored internally.

[0321] Removal of free drug by dextran-coated charcoal Dextran-coated charcoal can be used to remove residual free drugs from some linker-payloads. The required amount of charcoal was weighed and added to an ultrafree-CL centrifuge ultrafiltration tube equipped with a microporous filter membrane. Then, 1 mL of ultrapure water was added to the charcoal, mixed well, and centrifuged at 1000 × g for 2 minutes. The flow-through in the ultrafiltration tube was discarded. The washing step was repeated twice. Then, 300 mg of charcoal each was resuspended in 1 mL of formulation buffer. The flow-through in the ultrafiltration tube was discarded. The formulation buffer was added to the charcoal and mixed well. 10% of the sample's charcoal solution was added to the ADC solution. The mixture of charcoal and ADC sample was placed in a 22°C incubator and incubated slowly with stirring for 2 hours. After 2 hours, the tube was centrifuged at 1000 × g for 2 minutes to allow the charcoal to settle at the bottom of the tube. The supernatant was removed and filtered through a 0.22 μm membrane. The resulting product was then submitted for characterization.

[0322] Conjugation method ADC-AFI-ExV1 Conjugation buffer and formulation buffer [Table 8] TIFF2026514252000046.tif25170 Experimental procedure for pilot conjugation mAbs from the original buffer (20 mM His, 150 mM NaCl, pH 6.5) were pipetteed into three 1.5 mL EP tubes and then reduced with 1.5, 2.5, and 3.5 molar equivalents of TCEP. Reaction buffer was added to each tube to bring the mAb concentration during the reaction to 4.5 mg / mL. The reaction vials were placed in an incubator shaker at 37°C with a stirring speed of 60 rpm. After 2 hours of reduction, 10 mM Target 1 in DMA was added to each of the three samples to bring the drug-to-mAb molar equivalent to 8.0. DMA solvent was added to each sample to bring the organic solvent concentration to 10%, and the samples were incubated for a further 1 hour at 4°C. After 1 hour, the samples were purified using a spin desalting column (40 K, 0.5 mL). The pilot products were characterized by SEC-HPLC, HIC-HPLC, and LC-MS.

[0323] Experimental procedure to confirm conjugation A 1 mg scale conjugation was performed to confirm the optimal conditions for a pilot conjugation with a target DAR of 4.0. The mAb in the original buffer was pipetteed into a 1.5 mL EP tube and then reduced with the optimal molar equivalent of TCEP. The reaction buffer was added to the tube to bring the mAb concentration to 4.5 mg / mL. The reaction vial was placed in an incubator shaker at 37°C with a stirring speed of 60 rpm. After 2 hours of reduction, 10 mM target 1 in DMA was added to the sample to bring the drug-to-mAb molar equivalent to 8.0. DMA was added to the sample, the organic solvent was reduced to 10%, and the mixture was incubated for a further 1 hour at 4°C. After 1 hour, the sample was purified using a spin desalting column (40 K, 0.5 mL). The product to be confirmed was characterized by SEC-HPLC and LC-MS.

[0324] Bulk conjugation experimental procedure 15 mg of mAb from the original buffer was pipetteed into a 50 mL tube and reduced to the confirmed optimal TCEP equivalent. Reaction buffer was added to the tube to adjust the mAb concentration to 4.5 mg / mL. The reaction vial was placed in an incubator shaker at 37°C with a stirring speed of 60 rpm. After 2 hours of reduction, 10 mM of Object 1 in DMA was added to the sample to adjust the drug-to-mAb molar equivalent to 8.0. DMA was added to the sample, the organic solvent was reduced to 10%, and the sample was incubated for a further 1 hour at 4°C. After 1 hour, the sample was purified using a spin desalting column (40 K, 10 mL). The product was characterized by SEC-HPLC and LC-MS. If the reduction MS-DAR meets the requirements, another 15 mg scale conjugation will be performed under the same conditions. If the reduction MS-DAR does not meet the requirements, the TCEP / mAb molar equivalent will be adjusted in the second round of the 15 mg scale conjugation. The products from both batches were pooled and dialyzed overnight. After dialyzation, the products were applied to dextran-coated charcoal to remove residual free drugs.

[0325] Efficacy study using a human gastric SNU16 subcutaneous xenograft model (CDX) This project was conducted in accordance with Wuxi AppTec's internal operational standards.

[0326] The objective of this study was to evaluate the in vivo antitumor efficacy of exatecan ADC (ADC-AFI-ExV1) in a subcutaneous xenograft model of human stomach SNU-16 in female BALB / c nude mice. Cells were injected via FACS, and target STn expression was performed before detection using mAb_V64 along with a secondary fluorescent mAb (Figure 38). The experimental design of the efficacy test relating to the inventiveness of this patent is summarized as follows: [Table 9] TIFF2026514252000048.tif46170 animal Species: House mouse Variety: BALB / c Nude Age: 6-8 weeks Weight: 17.14~23.47g Sex: Female Number of animals: 42 mice + spares Animal supplier: Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Quality Certificate Number: 20220711Abzz0619000293

[0327] Conditions for breeding enclosures The mice were housed in individual, ventilated cages maintained at a constant temperature and humidity, with 3 / 4 of the animals in each cage. Temperature: 20~26℃. Humidity 40-70%. Cage: Made of polycarbonate. Dimensions: 325mm x 210mm x 180mm. The bedding was corn cobs, which were changed twice a week.

[0328] observation All procedures for animal handling, care, and treatment in this study were carried out in accordance with the guidelines of the Laboratory Animal Care Assessment and Accreditation (AAALAC) and approved by the In-House Laboratory Animal Care Committee (IACUC) of WuXi AppTec. During routine monitoring, all effects of tumor growth and treatment on normal behavior were checked daily, including motor function, food and water intake (visual only), weight gain / loss (weight measured twice a week), eye / hairball condition, and any other abnormal effects described in the protocol. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[0329] Tumor measurement and endpoints The primary endpoint was to determine whether tumor growth could be slowed or whether the mice could be cured. Tumor size was measured twice a week using calipers, and volume was calculated using the formula: V = 0.5a × b 2 Using mm 3 This is expressed as follows. In the formula, a and b are the long and short diameters of the tumor, respectively.

[0330] The antitumor efficacy of the compounds was evaluated using TGI (%) or T / C (%). TGI (%) reflects the tumor growth inhibition rate. The TGI for each group was calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)] × 100%. T i V is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on day 0, and V i is, T i V0 is the mean tumor volume of the media control group on the same day, while V0 is the mean tumor volume of the media group on day 0.

[0331] The T / C value (in percent) is an indicator of antitumor effect, and the T / C for each group was calculated using the following formula: T / C(%)=T RTV / C RTV ×100%. T RTV This is the RTV of the treatment group, and C RTVThis is the RTV of the control group on the same day. The RTV of each tumor is given by RTV=V t It was calculated as / V0. t V0 is the average tumor volume on a given day, and V0 is the average tumor volume on the first day of treatment for the same tumor.

[0332] Tumor weight was measured at the end of the study. T / C 重量 The value (in percent) was calculated using the following formula: T / C 重量 %=T 重量 / C weight x 100%. In the formula, T 重量 and C 重量 These represent the average tumor weights for the treatment group and the media control group, respectively.

[0333] This study evaluated the therapeutic efficacy of exatecan ADC as a monotherapy in a human gastric SNU-16 xenograft model. The results for tumor size at various time points after the start of treatment for different groups are shown in Figures 4a-4b.

[0334] The additional efficacy studies reported in Appendix Figure 25a were conducted using the same animal model. The treatment schedule is reported in the legend of the same figure.

[0335] In vivo distribution The experiments conducted in this study complied with British law and included ethical review.

[0336] PET scanner Long-term images were acquired using a Siemens Inveon PET scanner. Positron emission tomography (PET) is a nuclear medicine imaging technique that generates three-dimensional images of functional processes within the body. This device detects pairs of gamma rays indirectly emitted by positron-emitting radionuclides (tracers) introduced into the body using biologically active molecules.

[0337] In preclinical studies, PET imaging can be used for non-invasive detection and investigation of diseases in small animal models.

[0338] The Siemens Inveon PET system is a state-of-the-art device for PET studies on laboratory animals, and its specifications and capabilities are described below.

[0339] LSO with 1.6 × 1.6 mm detector sensitivity Gamma counter To understand the ex vivo organ biodistribution data, a Perkin-Elmer-Wallac-Wizard gamma counter was used. The detection system consists of a thallium-activated sodium iodide crystal. It has a sample changer and a storage capacity of 100 racks (1000 samples), and provides a library of 51 radionuclides with an energy range of 15–2000 keV.

[0340] Animal models Eight-week-old female BALB / C mice were purchased from Envigo and acclimatized for seven days before transplantation of cancer cells into the right flank region. The animals were divided into three groups: two groups received parental 4T1 cell lines (WT 4T1) and one group received STn 4T1 cell lines (STn 4T1). Eight days after tumor growth, the mice were randomized and isolated as follows: half of the WT 4T1 group 89 The remaining half received intravenous injections of Zr anti-STn antibody. 89 The Zr isotype control group received treatment. The STn 4T1 group was: 89 The patient received Zr anti-STn antibody (Ab). All procedures were performed in accordance with the HO Project License PPL P15A1884A.

[0341] Radioactive tracer 89 Zr was purchased from our commercial radioactive tracer supplier (Wolfson Molecular Imaging Centre, University of Manchester) and administered intravenously to animals. Mice were imaged 2, 8, 24, 48, 72, and 96 hours after tracer injection.

[0342] A BriTec well counter was used to measure the syringe dose before and after injection, and the measurement time was recorded using a clock synchronized with the PET scanner. Then, the injection dose, calculated as the difference in syringe dose before and after injection after correcting for decay relative to the injection time, was calculated using Microsoft Excel.

[0343] Animal protocols The mice were divided into three groups. Group 1 received WT 4T1 cells and an isotype control injection. Group 2 received WT 4T1 cells and an anti-STn antibody. Group 3 received STn 4T1 cells and an anti-STn antibody. 89 A bolus injection of Zr-Ab (approximately 100 µl) was administered intravenously, and the mice were imaged with static PET at 2, 8, 24, 48, 72, and 96 hours after injection. PET scans of anesthetized mice were performed for 20 minutes. Anesthesia was induced and maintained with isoflurane (approximately 1.5% isoflurane, 3 L of oxygen) delivered in 100% oxygen. A heating pad was provided throughout, and the animals' respiration and body temperature were monitored with BioVet.

[0344] Imaging protocol In short, a 20-minute static PET scan was performed using 3D histograms and MAP / 3D reconstruction. QC measurements of the PET scanner were performed before imaging began. Immediately after the final PET scan, the mice were euthanized and tissues were collected for in vivo distribution studies. For ex vivo analysis using a gamma counter, blood, muscle, lungs, liver, spleen, kidneys, heart, pancreas, ovaries, large intestine, small intestine, stomach, TDLN, NDLN, tumors, and tail (injection site) were extracted from each animal. The tumors and ovaries were fixed in formalin and transferred to ethanol for further histological examination. Both the tumors and ovaries were sent to CellmAbs for histological evaluation. Blood samples were also collected from each animal at 2, 8, and 24 hours after the PET scan for gamma counter analysis.

[0345] Adhesive ELISA Biomolecules, including DNA, LPS, lysozyme, and cell lysates, were immobilized on ELISA plates. Various antibodies, including control IgG1 palivizumab and trastuzumab, were further incubated with novel mAb_v1 and mAb_v64 and detected in human Fc. Absorbance data (A450-A620) were reported as normalized data for the therapeutic antibody palivizumab.

[0346] Antibody stability - SEC-HPLC Stability tests were conducted under two stress conditions using various antibodies, including mAb_v1, mAb_v64, and control IgG1 (palivizumab), as follows. • Temperature stress at 45°C for 48 hours • 24 hours of pH stress at pH 3

[0347] The data is from SEC-HPLC analysis after stress testing, and as shown in Figure 6c, all tested antibodies exhibited monomer mAb form.

[0348] Internal migration assay The objective of this study was to evaluate the internal distribution dynamics of anti-STn antibodies in a breast cancer cell line (MDA-MB231-STn) that overexpresses STn, and its parental WT cell line (STn-negative), along with colorectal cancer cell lines (COLO205) and gastric cancer cell lines (SNU16), which naturally express STn. To evaluate this feature, the inventors evaluated internal distribution assays using parental L2A5, humanized Ab clone (mAb_v1), and affinity-mature mAb clones (mAb_v57, mAb_v48, mAb_v46, mAb_v53, mAb_v25, mAb_v64), as shown in Figures 7a-7d, together with a negative control (IgG1-palivizumab), an anti-STn-positive control (mAb_PC), and a benchmark mAb trastuzumab antibody control.

[0349] 1 x 10 per well sown in a 96-well plate 4Antibody internal distribution assay analysis using individual target cells. In this assay, antibodies were labeled with Zenon pH-rodo fluorophore (Invitrogen, catalog no. Z25611), which is activated only under low pH conditions found in early endosomes. Labeled antibodies (3 ug / mL) were incubated with target cells for 0, 4, or 24 hours, and pHrodo fluorescence was measured by flow cytometry. Antibody internal distribution was indirectly evaluated by measuring pHrodo fluorescence by flow cytometry. The signal-to-background ratio (S / B) was calculated by dividing the MFI data for each antibody by the MFI of a non-internal distribution mAb (palivizumab). Data normalized for palivizumab were plotted as internal distribution multipliers at each time point.

[0350] antibody binding Antibody binding validation was performed using cell lines that naturally express STn (COLO205, SNU16, OV90). All antibodies showed higher and more specific binding to target cells. Figure 8 shows the binding profiles of various antibody variants (listed in Appendix Antibody Sequences, Figures 9-13) in cell lines exhibiting various STn expression levels. Each antibody was titrated using a 7-point concentration curve, and the binding strength was measured by flow cytometry using a secondary antibody conjugated to a fluorophore (Invitrogen catalog number A-21445).

[0351] The embodiments of the present disclosure described above are intended to be illustrative only, and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to fall within the scope of the present invention as defined in any appended claims.

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody conjugated to a drug via a linker, wherein the antibody is bound to a glycan terminated with sialyl Tn (STn) or alpha-2,6-linked sialic acid, the linker comprises a linker cleavable by glucuronidase, and the drug comprises a growth inhibitor.

2. The antibody-drug conjugate (ADC) according to claim 1, wherein the linker comprises a β-glucuronide moiety.

3. Linker, equation I': 【Chemistry 1】 Equation (I') [In the formula, X is NH, N-CH] ３ , or CF ２ The β-glucuronide moiety shown in [is] The antibody-drug conjugate (ADC) according to claim 1 or 2.

4. The linker, Equation I: 【Chemistry 2】 Equation (I) An antibody-drug conjugate (ADC) according to claim 1, 2, or 3, comprising the β-glucuronide moiety shown in [reference].

5. The antibody-drug conjugate (ADC) according to any one of claims 1 to 4, wherein the linker is conjugated to or modified to conjugate a drug via a carbamate bond.

6. The antibody-drug conjugate (ADC) according to any one of claims 1 to 4, wherein the linker is conjugated to the drug via a quaternary ammonia bond, or modified to be conjugated thereto.

7. The antibody-drug conjugate (ADC) according to any one of claims 1 to 6, wherein the linker is PEG-modified with a group containing polyethylene glycol (PEG).

8. The antibody-drug conjugate (ADC) according to claim 7, wherein the linker comprises a β-glucuronide moiety that is PEG-modified with a group containing polyethylene glycol (PEG).

9. The linker has the following structure: 【Transformation 3】 An antibody-drug conjugate (ADC) according to claim 7 or 8, which is PEGylated with a group containing polyethylene glycol (PEG) based on [wherein n is 1 to 5].

10. The antibody-drug conjugate (ADC) according to claim 9, wherein n is 2 to 4.

11. The antibody-drug conjugate (ADC) according to claim 10, wherein n is 3.

12. An antibody-drug conjugate (ADC) formed by conjugating a drug to the antibody described in claim 1 using a linker, wherein the linker is a group of formula II: 【Chemistry 4】 The antibody-drug conjugate (ADC) is as described above.

13. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the linker is a base of formula IIA: 【Transformation 5】 Formula (IIA) The antibody-drug conjugate (ADC) comprising the above.

14. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the linker is of formula IIIA': 【Transformation 6】 Formula IIIA' [In the formula, X is NH, N-CH] ３ , or CF ２ The antibody-drug conjugate (ADC) is the group shown in [ ].

15. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the linker is of formula IIIA: 【Transformation 7】 Formula (IIIA) The antibody-drug conjugate (ADC) is the group shown in [reference].

16. An antibody-drug conjugate (ADC) formed by conjugating a drug to the antibody described in claim 1 using a linker, wherein the linker is of formula III: 【Transformation 8】 The antibody-drug conjugate (ADC) is as shown in [reference].

17. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the linker is of formula IVA': 【Chemistry 9】 Formula IVA' [In the formula, X is NH, N-CH] ３ , or CF ２ The antibody-drug conjugate (ADC) is as shown in [ ].

18. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the linker is of formula IVA: 【Chemistry 10】 Formula (IVA) The antibody-drug conjugate (ADC) is as shown in [reference].

19. An antibody-drug conjugate (ADC) formed by conjugating a drug to the antibody described in claim 1 using a linker, wherein the linker is of formula IV: 【Chemistry 11】 The antibody-drug conjugate (ADC) is as shown in [reference].

20. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is of formula VA': 【Chemistry 12】 Formula VA' [In the formula, X is NH, N-CH] ３ , or CF ２ The antibody-drug conjugate (ADC) is as shown in [ ].

21. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is of formula VA: 【Chemistry 13】 Formula VA The antibody-drug conjugate (ADC) is as shown in [reference].

22. An antibody-drug conjugate (ADC) formed by conjugating a drug to the antibody described in claim 1 using a linker, wherein the drug-linker portion of the ADC is of formula V: 【Chemistry 14】 The antibody-drug conjugate (ADC) is as shown in [reference].

23. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is of formula VIA': 【Chemistry 15】 Formula VIA' [wherein, X is NH, N-CH ３ , or CF ２ , as shown, the antibody-drug conjugate (ADC).

24. An antibody-drug conjugate (ADC) comprising the antibody according to claim 1, conjugated to a drug via a linker, wherein the drug-linker portion of the ADC is of formula VIA: 【Chemistry 16】 formula VIA The antibody-drug conjugate (ADC) is as shown in [reference].

25. An antibody-drug conjugate (ADC) formed by conjugating a drug to the antibody described in claim 1 using a linker, wherein the drug-linker portion of the ADC is of formula VI: 【Chemistry 17】 The antibody-drug conjugate (ADC) is as shown in [reference].

26. The antibody-drug conjugate (ADC) according to any one of claims 1 to 25, wherein the glycan terminated with sialyl Tn (STn) or alpha-2,6 linked sialic acid is a human protein or an animal protein.

27. The antibody-drug conjugate (ADC) according to any one of claims 1 to 26, wherein the drug is exatecan, deruxtecan, or a derivative thereof.

28. The antibody-drug conjugate (ADC) according to any one of claims 1 to 27, wherein the antibody is an antibody fragment.

29. The antibody-drug conjugate (ADC) according to claim 28, wherein the antibody fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabody.

30. The antibody-drug conjugate (ADC) according to any one of claims 1 to 29, wherein the antibody is a human antibody or an animal antibody.

31. Antibodies, (a) Heavy chain variable region (VH), (i) H-CDR1, H-CDR2, and H-CDR3 shown in any one of sequence numbers 1-24 or 49-88, respectively The heavy chain variable region (VH) includes a complementarity determination region (CDR) selected from the group consisting of the following: and / or, (b) Light chain variable region (VL), (i) L-CDR1, L-CDR2, and L-CDR3 shown in any one of sequence numbers 25-48 or 89-128, respectively The light chain variable region (VL) includes a complementarity determination region (CDR) selected from the group consisting of the following: The antibody-drug conjugate (ADC) according to any one of claims 1 to 30, comprising, optionally, at least one CDR comprising one or two amino acid substitutions compared to the sequence described above.

32. The antibody-drug conjugate (ADC) according to claim 31, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL).

33. The antibody pairs the following heavy chain CDRs with the light chain CDRs: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 1 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 25; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 2 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 26; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 3 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 27; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 4 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 28; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 5 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 29; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 6 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 30; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 7 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 31; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 8 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 32; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 9 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 33; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 10 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 34; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 11 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 35; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 12 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 36; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 13 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 37; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 14 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 38; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 15 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 39; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 16 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 40; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 17 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 41; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 18 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 42; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 19 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 43; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 20 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 44; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 21 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 45; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 22 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 46; Pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 23 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 47; or The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 24 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 48 It includes one of the following: The antibody-drug conjugate (ADC) according to claim 31 or 32, wherein at least one CDR may optionally contain one or two amino acid substitutions compared to the sequence described above.

34. The antibody pairs the following heavy chain CDRs with the light chain CDRs: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 1 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 25; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 3 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 27; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 2 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 26 It includes one of the following: The antibody-drug conjugate (ADC) according to claim 33, wherein at least one CDR may optionally contain one or two amino acid substitutions compared to the sequence described above.

35. The antibody pairs the following heavy chain CDRs with the light chain CDRs: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 49 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 89; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 50 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 90; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 51 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 91; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 52 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 92; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 53 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 93; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 54 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 94; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 55 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 95; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 56 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 96; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 57 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 97; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 58 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 98; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 59 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 99; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 60 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 100; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 61 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 101; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 62 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 102; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 63 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 103; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 64 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 104; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 65 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 105; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 66 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 106; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 67 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 107; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 68 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 108; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 69 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 109; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 70 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 110; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 71 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 111; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 72 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 112; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 73 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 113; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 74 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 114; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 75 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 115; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 76 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 116; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 77 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 117; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 78 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 118; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 79 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 119; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 80 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 120; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 81 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 121; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 82 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 122; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 83 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 123; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 84 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 124; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 85 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 125; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 86 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 126; Pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 87 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 127; or The pair of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 88 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 128 It includes one of the following: The antibody-drug conjugate (ADC) according to claim 31 or 32, wherein at least one CDR may optionally contain one or two amino acid substitutions compared to the sequence described above.

36. The antibody pairs the following heavy chain CDRs with the light chain CDRs: The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 49 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 89; The pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 70 and L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 110; Pairs of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 81 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 121; or The pair of H-CDR1, H-CDR2, and H-CDR3 shown in Sequence ID No. 88 with L-CDR1, L-CDR2, and L-CDR3 shown in Sequence ID No. 128 It includes one of the following: The antibody-drug conjugate (ADC) according to claim 35, wherein at least one CDR may optionally contain one or two amino acid substitutions compared to the sequence described above.

37. Antibodies, (a) Heavy chain variable region (VH) including humanized heavy chain framework region and / or, (b) Variable light chain region (VL) including the humanized light chain framework region The antibody-drug conjugate (ADC) according to any one of claims 31 to 36, comprising:

38. Antibodies, (a) Heavy chain variable region (VH), (i) The heavy chain variable region (VH) includes a humanized heavy chain framework region shown in each of the following heavy chain variable region (VH) sequences selected from the group consisting of one of sequence numbers 1 to 24 or 49 to 88, or sequences having at least 80% sequence identity with the sequences described above. and / or, (b) Light chain variable region (VL), (ii) The light chain variable region (VL) includes a humanized light chain framework region shown in one of sequence numbers 25 to 48 or 89 to 128, or a sequence selected from the group consisting of sequences having at least 80% sequence identity with the sequences described above. An antibody-drug conjugate (ADC) according to any one of claims 31 to 37, comprising:

39. An antibody is a pair of light chain framework regions and heavy chain framework regions, wherein the heavy chain framework region is as shown in the heavy chain variable region (VH) sequence shown below, and the light chain framework region is as shown in the light chain variable region (VL) sequence shown below; The pair between the heavy chain framework region of SEQ ID NO: 1 and the light chain framework region of SEQ ID NO: 25; The pair between the heavy chain framework region of SEQ ID NO: 2 and the light chain framework region of SEQ ID NO: 26; The heavy chain framework region of SEQ ID NO: 3 and the light chain framework region of SEQ ID NO: 27 Opposite of; The pair between the heavy chain framework region of SEQ ID NO: 4 and the light chain framework region of SEQ ID NO: 28; The pair between the heavy chain framework region of SEQ ID NO: 5 and the light chain framework region of SEQ ID NO: 29; The pair between the heavy chain framework region of SEQ ID NO: 6 and the light chain framework region of SEQ ID NO: 30; The pair between the heavy chain framework region of SEQ ID NO: 7 and the light chain framework region of SEQ ID NO: 31; The pair between the heavy chain framework region of SEQ ID NO: 8 and the light chain framework region of SEQ ID NO: 32; The pair between the heavy chain framework region of SEQ ID NO: 9 and the light chain framework region of SEQ ID NO: 33; The pair between the heavy chain framework region of sequence number 10 and the light chain framework region of sequence number 34; The pair between the heavy chain framework region of sequence number 11 and the light chain framework region of sequence number 35; The pair between the heavy chain framework region of sequence number 12 and the light chain framework region of sequence number 36; The pair between the heavy chain framework region of SEQ ID NO: 13 and the light chain framework region of SEQ ID NO: 37; The pair between the heavy chain framework region of sequence number 14 and the light chain framework region of sequence number 38; The pair between the heavy chain framework region of sequence number 15 and the light chain framework region of sequence number 39; The pair between the heavy chain framework region of sequence number 16 and the light chain framework region of sequence number 40; The pair between the heavy chain framework region of sequence number 17 and the light chain framework region of sequence number 41; The pair between the heavy chain framework region of sequence number 18 and the light chain framework region of sequence number 42; The pair between the heavy chain framework region of sequence number 19 and the light chain framework region of sequence number 43; The pair between the heavy chain framework region of sequence number 20 and the light chain framework region of sequence number 44; The pair between the heavy chain framework region of sequence number 21 and the light chain framework region of sequence number 45; The pair between the heavy chain framework region of sequence number 22 and the light chain framework region of sequence number 46; A pair of the heavy chain framework region of SEQ ID NO: 23 and the light chain framework region of SEQ ID NO: 47; or The pair between the heavy chain framework region of sequence number 24 and the light chain framework region of sequence number 48 It includes one of the following: The antibody-drug conjugate (ADC) according to claim 37, wherein optionally, the heavy chain framework region and / or the light chain framework region have at least 80% sequence identity with the sequence described above.

40. The antibody is a pair of light chain framework regions and heavy chain framework regions, wherein the heavy chain framework region is as shown in the heavy chain variable region (VH) sequence shown below, and the light chain framework region is as shown in the light chain variable region (VL) sequence shown below, the pair of light chain framework regions and heavy chain framework regions: The pair between the heavy chain framework region of SEQ ID NO: 49 and the light chain framework region of SEQ ID NO: 89; The pair of the heavy chain framework region of SEQ ID NO: 50 and the light chain framework region of SEQ ID NO: 90; The pair between the heavy chain framework region of sequence number 51 and the light chain framework region of sequence number 91; The pair of the heavy chain framework region of SEQ ID NO: 52 and the light chain framework region of SEQ ID NO: 92; The pair of the heavy chain framework region of SEQ ID NO: 53 and the light chain framework region of SEQ ID NO: 93; The pair between the heavy chain framework region of sequence number 54 and the light chain framework region of sequence number 94; The pair of the heavy chain framework region of SEQ ID NO: 55 and the light chain framework region of SEQ ID NO: 95; The pair of the heavy chain framework region of SEQ ID NO: 56 and the light chain framework region of SEQ ID NO: 96; The pair between the heavy chain framework region of SEQ ID NO: 57 and the light chain framework region of SEQ ID NO: 97; The pair between the heavy chain framework region of SEQ ID NO: 58 and the light chain framework region of SEQ ID NO: 98; The pair between the heavy chain framework region of SEQ ID NO: 59 and the light chain framework region of SEQ ID NO: 99; The pair between the heavy chain framework region of sequence number 60 and the light chain framework region of sequence number 100; The pair between the heavy chain framework region of sequence number 61 and the light chain framework region of sequence number 101; The pair between the heavy chain framework region of sequence number 62 and the light chain framework region of sequence number 102; The pair between the heavy chain framework region of sequence number 63 and the light chain framework region of sequence number 103; The pair between the heavy chain framework region of sequence number 64 and the light chain framework region of sequence number 104; The pair of the heavy chain framework region of sequence number 65 and the light chain framework region of sequence number 105; The pair of the heavy chain framework region of sequence number 66 and the light chain framework region of sequence number 106; The pair between the heavy chain framework region of SEQ ID NO: 67 and the light chain framework region of SEQ ID NO: 107; The pair between the heavy chain framework region of sequence number 68 and the light chain framework region of sequence number 108; The pair between the heavy chain framework region of sequence number 69 and the light chain framework region of sequence number 109; The pair between the heavy chain framework region of SEQ ID NO: 70 and the light chain framework region of SEQ ID NO: 110; The pair between the heavy chain framework region of sequence number 71 and the light chain framework region of sequence number 111; The pair between the heavy chain framework region of sequence number 72 and the light chain framework region of sequence number 112; The pair between the heavy chain framework region of sequence number 73 and the light chain framework region of sequence number 113; The pair between the heavy chain framework region of sequence number 74 and the light chain framework region of sequence number 114; The pair between the heavy chain framework region of sequence number 75 and the light chain framework region of sequence number 115; The pair between the heavy chain framework region of sequence number 76 and the light chain framework region of sequence number 116; The pair between the heavy chain framework region of SEQ ID NO: 77 and the light chain framework region of SEQ ID NO: 117; The pair between the heavy chain framework region of sequence number 78 and the light chain framework region of sequence number 118; The pair between the heavy chain framework region of SEQ ID NO: 79 and the light chain framework region of SEQ ID NO: 119; The pair of the heavy chain framework region of SEQ ID NO: 80 and the light chain framework region of SEQ ID NO: 120; The pair between the heavy chain framework region of sequence number 81 and the light chain framework region of sequence number 121; The pair between the heavy chain framework region of sequence number 82 and the light chain framework region of sequence number 122; The pair between the heavy chain framework region of SEQ ID NO: 83 and the light chain framework region of SEQ ID NO: 123; The pair between the heavy chain framework region of sequence number 84 and the light chain framework region of sequence number 124; The pair of the heavy chain framework region of sequence number 85 and the light chain framework region of sequence number 125; The pair of the heavy chain framework region of sequence number 86 and the light chain framework region of sequence number 126; A pair of the heavy chain framework region of SEQ ID NO: 87 and the light chain framework region of SEQ ID NO: 127; or The pair between the heavy chain framework region of SEQ ID NO: 88 and the light chain framework region of SEQ ID NO: 128 It includes one of the following: The antibody-drug conjugate (ADC) according to claim 37, wherein optionally, the heavy chain framework region and / or the light chain framework region have at least 80% sequence identity with the sequence described above.

41. The antibody-drug conjugate (ADC) according to any one of claims 37 to 40, wherein the heavy chain framework region and / or light chain framework region have at least 90% sequence identity with the sequence described.

42. The antibody-drug conjugate (ADC) according to claim 41, wherein the heavy chain framework region and / or light chain framework region have at least 95% or at least 99% sequence identity with the sequence described.

43. Antibodies, (a) Heavy chain variable region (VH), (i) The heavy chain variable region (VH) selected from the group consisting of one of sequence numbers 1 to 24 or 49 to 88, or sequences having at least 80% sequence identity with the sequences described above. and / or, (a) Light chain variable region (VL), (ii) The light chain variable region (VL) selected from the group consisting of one of sequence numbers 25 to 48 or 89 to 128, or sequences having at least 80% sequence identity with the sequences described above. An antibody-drug conjugate (ADC) according to any one of claims 31 to 42, comprising:

44. The antibody pairs the following heavy chain variable region (VH) and light chain variable region (VL): The pair of sequence number 1 and sequence number 25; The pair of sequence number 2 and sequence number 26; The pair of sequence number 3 and sequence number 27; The pair of sequence number 4 and sequence number 28; The pair of sequence number 5 and sequence number 29; The pair of sequence number 6 and sequence number 30; The pair of sequence number 7 and sequence number 31; The pair of sequence number 8 and sequence number 32; The pair of sequence number 9 and sequence number 33; The pair of sequence number 10 and sequence number 34; The pair of sequence number 11 and sequence number 35; The pair of sequence number 12 and sequence number 36; The pair of sequence number 13 and sequence number 37; The pair of sequence number 14 and sequence number 38; The pair of sequence number 15 and sequence number 39; The pair of sequence number 16 and sequence number 40; The pair of sequence number 17 and sequence number 41; The pair of sequence number 18 and sequence number 42; The pair of sequence number 19 and sequence number 43; The pair of sequence number 20 and sequence number 44; The pair of sequence number 21 and sequence number 45; The pair of sequence number 22 and sequence number 46; The pair of sequence number 23 and sequence number 47; or The pair of sequence number 24 and sequence number 48 It includes one of the following: The antibody-drug conjugate (ADC) according to claim 43, wherein the heavy chain variable region (VH) and / or the light chain variable region (VL) optionally have at least 80% sequence identity with the sequence described above.

45. The antibody pairs the following heavy chain variable region (VH) and light chain variable region (VL): The pair of sequence number 49 and sequence number 89; The pair of sequence number 50 and sequence number 90; The pair of sequence number 51 and sequence number 91; The pair of sequence number 52 and sequence number 92; The pair of sequence number 53 and sequence number 93; The pair of sequence number 54 and sequence number 94; The pair of sequence number 55 and sequence number 95; The pair of sequence number 56 and sequence number 96; The pair of sequence number 57 and sequence number 97; The pair of sequence number 58 and sequence number 98; The pair of sequence number 59 and sequence number 99; The pair of sequence number 60 and sequence number 100; The pair of sequence number 61 and sequence number 101; The pair of sequence number 62 and sequence number 102; The pair of sequence number 63 and sequence number 103; The pair of sequence number 64 and sequence number 104; The pair of sequence number 65 and sequence number 105; The pair of sequence number 66 and sequence number 106; The pair of sequence number 67 and sequence number 107; The pair of sequence number 68 and sequence number 108; The pair of sequence number 69 and sequence number 109; The pair of sequence number 70 and sequence number 110; The pair of sequence number 71 and sequence number 111; The pair of sequence number 72 and sequence number 112; The pair of sequence number 73 and sequence number 113; The pair of sequence number 74 and sequence number 114; The pair of sequence number 75 and sequence number 115; The pair of sequence number 76 and sequence number 116; The pair of sequence number 77 and sequence number 117; The pair of sequence number 78 and sequence number 118; The pair of sequence number 79 and sequence number 119; The pair of sequence number 80 and sequence number 120; The pair of sequence number 81 and sequence number 121; The pair of sequence number 82 and sequence number 122; The pair of sequence number 83 and sequence number 123; The pair of sequence number 84 and sequence number 124; The pair of sequence number 85 and sequence number 125; The pair of sequence number 86 and sequence number 126 The pair of sequence number 87 and sequence number 127: or The pair of sequence number 88 and sequence number 128; It includes one of the following: The antibody-drug conjugate (ADC) according to claim 43, wherein the heavy chain variable region (VH) and / or the light chain variable region (VL) optionally have at least 80% sequence identity with the sequence described above.

46. L-CDR3 undergoes further mutation, and the D (aspartic acid / aspartate) at position 93 is replaced with the following amino acid residue: A, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y An antibody-drug conjugate (ADC) according to any one of claims 31 to 45, wherein one of the following is substituted.

47. L-CDR3 undergoes further mutation, and the proline (P) at position 94 is replaced with the following amino acid residue: A, E, F, H, I, K, L, N, Q, R, T, V, W, or Y An antibody-drug conjugate (ADC) according to any one of claims 31 to 46, wherein one of the following is substituted.

48. The antibody-drug conjugate (ADC) according to claim 46, wherein one of the mutations shown for position 93 is paired with one of the mutations shown for position 94 in claim 35.

49. L-CDR3 undergoes further mutation, with DP at positions 93 and 94 corresponding to the following amino acid residue pairs: DA, DK, DN, EP, KP, NP, QP, RP, AA, EE, FF, GP, HH, II, KK, LL, NN, QQ, RR, SP, TT, VV, WW, or YY The antibody-drug conjugate (ADC) according to claim 46, 47, or 48, wherein one of the following is substituted.

50. The antibody-drug conjugate (ADC) according to any one of claims 46 to 49, wherein the light chain variable sequence VL is as shown in any one of sequence numbers 150 to 173, or is a variant having at least 80% sequence identity thereto.

51. H-CDR2 undergoes further mutation, and the D (aspartic acid / aspartate) at position 55 becomes the following amino acid residue: A, E, F, G, H, I, K, L, P, Q, R, V, W, or Y An antibody-drug conjugate (ADC) according to any one of claims 36 to 50, wherein one of the following is substituted.

52. H-CDR2 undergoes further mutation, and the G (glycine) at position 56 becomes the following amino acid residue: A, E, F, H, I, K, L, N, Q, R, T, V, W, or Y An antibody-drug conjugate (ADC) according to any one of claims 36 to 51, wherein one of the following is substituted.

53. The antibody-drug conjugate (ADC) according to claim 51, wherein one of the mutations shown for position 55 is paired with one of the mutations shown for position 56 in claim 22.

54. H-CDR2 undergoes further mutation, with DG at positions 55 and 56 becoming the following amino acid residue pair: DE, DK, DA, EG, QG, RG, AA, EE, FF, GG, HH, KK, LL, DN, PQ, QQ, RR, DT, VV, WW, or YY The antibody-drug conjugate (ADC) according to claim 51, 52, or 53, wherein one of the following is substituted.

55. The antibody-drug conjugate (ADC) according to any one of claims 51 to 54, wherein the heavy chain variable sequence VH is as shown in any one of sequence numbers 129 to 149, or is a variant having at least 80% sequence identity thereto.

56. L-CDR3 undergoes further mutation, with the "DP" at positions 93 and 94 becoming the pair of the following amino acid residues: DA, DK, DN, EP, KP, NP, QP, RP, AA, EE, FF, GP, HH, II, KK, LL, NN, QQ, RR, SP, TT, VV, WW, or YY It is replaced with one of the following: H-CDR2 undergoes further mutation, with DG at positions 55 and 56 becoming the following amino acid residue pair: DE, DK, DA, EG, QG, RG, AA, EE, FF, GG, HH, KK, LL, DN, PQ, QQ, RR, DT, VV, WW, or YY It is replaced with one of the following: The antibody-drug conjugate (ADC) according to any one of claims 46 to 55.

57. L-CDR3 undergoes further mutation, with the "DP" at positions 93 and 94 becoming the pair of the following amino acid residues: DA, DK, DN, EP, KP, NP, QP, or RP It is replaced with one of the following: H-CDR2 undergoes further mutation, with DG at positions 55 and 56 becoming the following amino acid residue pair: DE, DK, DA, EG, QG, or RG It is replaced with one of the following: The antibody-drug conjugate (ADC) according to any one of claims 46 to 56.

58. The antibody comprises a combination of a light chain variable region (VL) and a heavy chain variable region (VH), The aforementioned VL includes complementarity determination regions (CDRs) L-CDR1, L-CDR2, and L-CDR3, respectively, as shown in Sequence ID Nos. 179, 181, and 183. The aforementioned VH includes CDR H-CDR1, H-CDR2, and H-CDR3 shown in sequence numbers 185, 187, and 189, respectively. The antibody-drug conjugate (ADC) according to any one of claims 1 to 30.

59. The antibody-drug conjugate (ADC) according to claim 58, wherein VL comprises SEQ ID NOs: 178, 180, and 182, and VH comprises SEQ ID NOs: 184, 186, and 188, and optionally the heavy chain variable region (VH) and / or the light chain variable region (VL) have at least 80% sequence identity with the sequence described above.

60. The antibody-drug conjugate (ADC) according to claim 58 or 59, wherein VL comprises SEQ ID NO: 177, VH comprises SEQ ID NO: 176, and optionally the heavy chain variable region (VH) and / or the light chain variable region (VL) have at least 80% sequence identity with the sequence described above.

61. The antibody-drug conjugate (ADC) according to any one of claims 47 to 59, wherein the heavy chain variable region (VH) and / or light chain variable region (VL) have at least 90%, at least 95%, or at least 99% sequence identity with the sequence described.

62. An antibody-drug conjugate (ADC) according to any one of claims 1 to 61, wherein the glycan terminated with alpha-2,6-linked sialic acid comprises STn, 2,6-sialyl T, dicialyl T, or 2,6-sialolactosamine.

63. The antibody-drug conjugate (ADC) according to any one of claims 1 to 62, wherein the antibody undergoes glycan conversion at the glycosylation site.

64. The antibody-drug conjugate (ADC) according to any one of claims 1 to 63, wherein the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

65. The antibody-drug conjugate (ADC) according to any one of claims 1 to 64, wherein the antibody is a functional antibody fragment that binds to a glycan group terminated by STn and alpha-2,6-linked sialic acid.

66. A pharmaceutical composition comprising an antibody-drug conjugate (ADC) according to any one of claims 1 to 60, and a pharmaceutically acceptable carrier.

67. An antibody-drug conjugate (ADC) according to any one of claims 1 to 65, for use in pharmaceuticals.

68. An antibody-drug conjugate (ADC) according to any one of claims 1 to 65, for use in the treatment of a human body or an animal body.

69. An antibody-drug conjugate (ADC) according to any one of claims 1 to 65, for use in the detection and / or treatment of various types of cancer.

70. A linker that can be cleaved by glucuronidase, configured to be coupled to a drug via a quaternary ammonium salt bond, and PEGylated with a group containing polyethylene glycol (PEG).

71. A linker that can be cleaved by glucuronidase, configured to be coupled to a drug via a carbamate bond, and which does not contain an alkyne moiety.

72. A linker that can be cleaved by glucuronidase, is configured to be coupled to a drug via a carbamate bond, and is terminated at a maleimide portion.

73. A linker according to any one of claims 70 to 72, comprising a β-glucuronide moiety.

74. Equation I'; [Chemistry 18] [In the formula, X is NH, N-CH] ３ , or CF ２ A linker according to any one of claims 70 to 73, comprising the β-glucuronide moiety shown in [ ].

75. Formula I: 【Chemistry 19】 Equation (I) A linker according to any one of claims 70 to 74, comprising the β-glucuronide moiety shown in [the figure].

76. The linker according to claim 74 or 75, wherein the amide portion of formula (I') or (I) is PEGylated with a group containing polyethylene glycol (PEG).

77. The following structure: 【Chemistry 20】 A linker according to any one of claims 70 to 76, which is PEGylated with a group containing polyethylene glycol (PEG) based on the formula [wherein n is 1 to 5].

78. The linker according to claim 77, wherein n is 2 to 4.

79. The linker according to claim 78, wherein n is 3.

80. Formula II: 【Chemistry 21】 Formula (II) A linker according to any one of claims 70 to 79, comprising the base of.

81. Formula III': 【Chemistry 22】 Formula III' [In the formula, X is NH, N-CH] ３ , or CF ２ As shown in [this document], A linker according to any one of claims 70 to 80.

82. Formula III: 【Chemistry 23】 The linker according to any one of claims 70 to 81, as shown in [reference].

83. Formula IV': 【Chemistry 24】 Formula IV' [In the formula, X is NH, N-CH] ３ , or CF ２ As shown in [this document], A linker according to claim 70 or any of claims 73 to 82.

84. Formula IV: 【Chemistry 25】 The linker according to any one of claims 70 or 73 to 83, as shown in [reference].

85. Formula V': 【Chemistry 26】 Formula V' [In the formula, X is NH, N-CH] ３ , or CF ２ As shown in [this document], Drug-linker payload.

86. Formula V: 【Chemistry 27】 The drug-linker payload according to claim 85, as shown in [reference].

87. Formula VI': 【Chemistry 28】 Equation VI' [In the formula, X is NH, N-CH] ３ , or CF ２ As shown in [this document], Drug-linker payload.

88. Equation (VI): 【Chemistry 29】 The drug-linker payload according to claim 87.

89. An antibody-drug conjugate (ADC) comprising an antibody conjugated to a drug via a linker according to any one of claims 70 to 84.

90. The antibody-drug conjugate (ADC) according to claim 89, wherein the antibody is bound to a glycan terminated with sialyl Tn (STn) or alpha-2,6 linked sialic acid.

91. The antibody-drug conjugate (ADC) according to claim 89 or 90, wherein the antibody binds to a tumor biomarker.

92. The antibody-drug conjugate (ADC) according to claim 89, 90, or 91, wherein the drug comprises a growth inhibitor suitable for the treatment of cancer.

93. The antibody-drug conjugate (ADC) according to claim 89, 90, or 91, wherein the conjugate includes a labeling detection means for diagnostic purposes.

94. An antibody-drug conjugate (ADC) according to any one of claims 1 to 65 or 89 to 93, having an antibody-drug ratio (DAR) that is an integer from 1 to 10.

95. The antibody-drug conjugate (ADC) according to claim 94, having an antibody-drug ratio (DAR) that is an integer between 2 and 6.

96. The antibody-drug conjugate (ADC) according to claim 95, having an antibody-drug ratio (DAR) that is an integer between 3 and 5.

97. The antibody-drug conjugate (ADC) according to claim 96, having an antibody-drug ratio (DAR) that is an integer between 2 and 6.

98. An exatecan derivative compound having a handle portion covalently bonded to the exatecan parent molecule at a position that does not significantly affect the therapeutic or pharmacokinetic properties of the exatecan parent molecule, wherein the handle portion is a quaternary ammonium salt of the following formula: 【Transformation 30】 Formula (VII) The exatecan derivative compound comprising the above-mentioned compound.

99. The basic structure is as follows: 【Chemistry 31】 Formula (VIII) An exatecan derivative compound having the following properties.

100. The exatecan derivative compound according to claim 94 or 95, exhibiting an improved safety profile compared to the exatecan parent molecule and retaining therapeutic activity against cancer cells.

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