Anti-GUCY2C antibodies and their use

Abstract

In a first aspect, the present application relates to a corresponding antibody-drug conjugate comprising an anti-GUCY2C antibody, amatoxin, for use in the treatment of gastrointestinal cancers such as colorectal cancer and pancreatic cancer. In a second aspect, the present invention relates to a pharmaceutical composition comprising the above antibody-drug conjugate of the present invention for use in the treatment of gastrointestinal cancer. The present invention also relates to a method for treating gastrointestinal cancer using the above antibody-drug conjugate of the present invention.

Description

[Technical Field]

[0001] This application relates to an anti-GUCY2C antibody and a corresponding antibody-drug conjugate comprising amatoxin. In a further embodiment, the present invention relates to a pharmaceutical composition comprising such a conjugate for use in the treatment of gastrointestinal cancers such as colorectal cancer (CRC) or metastatic colorectal cancer (mCRC). [Background technology]

[0002] Gastrointestinal cancer accounts for approximately 26% of all cancer diagnoses and about 35% of cancer deaths worldwide. In 2018, it is estimated that there were 4.8 million new cases of gastrointestinal (GI) cancer and 3.4 million related deaths worldwide.

[0003] Gastrointestinal cancers include tumors of the esophagus, stomach, colorectal cancer, liver, and pancreas. Colorectal cancer (CRC) is the most common, accounting for approximately 10.2% of all new cases, followed by gastric cancer (5.7%), liver cancer (4.7%), esophageal cancer (3.2%), and pancreatic cancer (2.5%). Of the approximately 3.4 million GI cancer deaths worldwide in 2018, CRC accounted for about 9% of all cancer-related deaths, followed by gastric cancer (8.2%), liver cancer (8.2%), esophageal cancer (5.3%), and pancreatic cancer (4.5%) (Arnold et al. (2020), Gastroenterology 159:335-349). Therefore, colorectal cancer (CRC) is the third most common cancer in the world after lung cancer and breast cancer, but it is the second leading cause of cancer death.

[0004] CRC is the second most common cancer in adult women and the third most common in men. It is also the fourth leading cause of cancer death.

[0005] The 5-year and 10-year survival rates are 65% and 58%, respectively, with incidence and mortality rates 25% higher in men than in women. From 1975 to 2013, the incidence of CRC increased from 10 to 15 cases per 100,000 Americans aged 20 to 49. This is comparable to an approximately 20% increase in CRC cases in developing countries such as Argentina, Brazil, and China. Furthermore, 30% of all CRC patients experience metastasis, and the prognosis remains poor, with a median 5-year survival rate of only 18.5% in the United States and 27.7% in Europe.

[0006] Despite the fact that incidence rates have declined over the past 30 years due to improvements in screening and treatment paradigms, surprisingly, incidence rates among those under 50 have steadily increased, with an annual increase of 2.2% in overall incidence rates from 2012 to 2016. The exact cause of this paradigm shift is unclear, but it appears to be due to changes in dietary and other lifestyle-related risk factors, or to an increase in unsystematic screening among young adults.

[0007] Ten percent to twenty percent of all CRC patients have a positive family history, and approximately five percent of all CRC cases are related to known hereditary CRC syndromes detectable by germline. In some cases, an increase in sporadic CRC occurrences is associated with long-term inflammatory bowel disease and various lifestyle factors such as physical inactivity, unhealthy diet, smoking, obesity, and heavy alcohol consumption.

[0008] Differences in clinical outcomes and drug responses to CRC treatment depend on the location of the cancer along the colorectal and rectal tissue. Involved contributing factors include different physiological functions, gut microbiota, types of locally present immune cells, dietary carcinogens, and the timing of disease detection. Individual developmental factors may also contribute to disease severity and treatment outcomes.

[0009] The proximal (right) colon (cecum, ascending colon, and transverse colon) originates from the fetal midgut, while the distal (left) colon (splenic flexure, descending colon, sigmoid colon, and rectum) originates from the fetal hindgut. These ontogenetic differences are associated with different gene expression patterns along the proximal-distal axis. In women and African Americans, sporadic proximal colon tumors are the most frequently diagnosed, with an incidence of 51%–62%. These cases also show higher TNM stages at initial diagnosis, patterns with high levels of promoter hypermethylation across the genome (known as CpG island methylation phenotype (CIMP)), microsatellite instability (MSI) due to DNA mismatch repair deficiency (dMMR), more frequent KRAS and BRAF mutations, and a worse prognosis in terms of survival. Distal colorectal tumors tend to show chromosomal instability (CIN) and a more favorable prognosis.

[0010] Genomic profiling of CRCs revealed significant intratumoral and intertumoral heterogeneity due to the accumulation of genomic mutations and chromosomal abnormalities during disease onset and progression. Genomic instability in CRCs manifests as one of two main forms: CIN and MSI. CRCs without CIN or MSI are classified as genomically stable (GS) CRCs. In GS CRCs, DNA repair genes and tumor suppressor factors are more readily transcriptionally silenced by CIMP, while the majority of MSI CRCs and a small population of CIN CRCs are also CIMP-positive, and about 10% of CRCs are negative for CIN, MSI, or CIMP.

[0011] Chromosomal instability (CIN) is characterized by changes in chromosome number (aneuploidy) and structural changes (changes in somatic copy number, deletions, insertions, amplifications, or loss of heterozygosity) and occurs in 65%–70% of sporadic CRCs. Almost all CIN tumors show activated Wnt signaling, and 80% have inactivation due to mutations in APC, a negative regulator of the Wnt pathway. Inactivation / deletion due to TP53 mutations occurs in 60% of CIN tumors, and loss of p53 function directly drives CIN, creating a pessimistic context for the genomic instability mechanism.

[0012] Regarding genomic instability, the combination of telomer dysfunction and p53 deficiency is the major CIN mechanism, as evidenced by the appearance of late bridges in early-stage cancers of human CRCs and the spontaneous occurrence of CRCs in telomerase-deficient p53 mutant mice.

[0013] Microsatellite-instability (MSI) CRCs are characterized by the presence of microsatellites, which are DNA sequences containing repetitive motifs that tend to accumulate mutations at a higher rate than other genomic regions. MSI is the expression of dMMR phenotypes resulting from mutation-induced inactivation of MMR genes, including MLH1, MSH2, MSH3, MSH6, PMS2, and Exo1 (De' Angelis et al. Acta Biomed 2018 Dec 17;89(9-S):97-101).

[0014] Other important factors contributing to the pathogenesis of CRC are somatic gene alterations that activate major signaling pathways that promote the proliferative state of CRC cancer cells. In CRC, the main proliferative signaling pathways are the EGFR-RAS and WNT-β-catenin pathways.

[0015] EGFR signaling: EGFR activation triggers downstream RAS / RAF / MEK / ERK and PI3K / AKT signaling cascades, ultimately leading to proliferation. Mutations in EGFR itself are rare in CRC, with 1% of CRC cases attributable to mutations in the CRC (Barber et al N Engl J Med 351:2883); instead, it is overexpressed in approximately 80% of CRCs. Enhancement of EGFR activation can occur through post-translational modifications of the protein, including methylation of R198 and R200 by arginine methyltransferase 1 (PRMT1). PRMT1 enhances binding to EGF and the resulting signaling activation, even in the presence of EGFR inhibitors.

[0016] Recently, it has been shown that hepatocyte growth factor (HGF), a ligand for the MET receptor, can completely replace EGF in the growth of a single Lgr5+ mouse intestinal stem cell (ISC) into intestinal organoids, including all differentiated intestinal cell lineages. Furthermore, while HGF and EGF are equally effective in promoting organoid proliferation in Apc mutant mice, in vivo Met deletion in ISCs weakened stem cell adaptability and Apc-driven intestinal adenoma formation. These findings suggest that EGFR and MET signaling have overlapping and partially redundant functions in the mouse intestinal mucosa. More recent results also show that HGF / MET signaling can completely overcome EGFR inhibition in human ISC and CRC stem cells, enabling the proliferation of a single normal ISC and APC mutant cell into organoids even in the presence of EGFR inhibition (Joosten et al Gastroenterology 2019 Oct;157(4):1153-1155).

[0017] Other factors crucial to the clinical efficacy of EGFR blockade are the mutational status of its downstream signaling components, specifically, gain-of-function mutations in RAS, RAF, MEK, or ERK that can maintain cancer cell proliferation and survival upon EGFR inhibition. Activating mutations in KRAS, NRAS, or HRAS together are present in approximately 50% of CRC cases, with these mutations involving codons 12 or 13, and less frequently, codons 61, 117, or 146. Regarding the RAS pathway in CRC, the RAS effector BRAF has a hotspot codon 600 (V600E) mutation in 10-15% of early CRC and approximately 5% of stage IV CRC. Clinically, EGFR-mediated reactivation of MAPK signaling, which has led to the approval of EGFR inhibitors in combination with BRAF and MEK inhibitors—now the standard of treatment for patients with metastatic BRAF-mutated tumors—shows limited activity in metastatic BRAFV600E CRC.

[0018] CRC screening can be performed by direct visualization methods such as colonoscopy, colon CT, flexible sigmoidoscopy, or flexible sigmoidoscopy with fecal immunochemistry (FIT), or by stool-based tests such as fecal occult blood testing based on the guaiac method, FIT, or multi-target fecal DNA testing, or by serological tests such as SEPT9 DNA testing.

[0019] Once a CRC diagnosis is made, staging is a crucial factor in determining the patient's treatment options. Regarding tumor staging, the combined American Joint Committee on Cancer (AJCC) / Union for International Cancer Control (UICC) Tumor, Narrow, and Metastasis (TNM) system is a commonly used staging system to define the following cancer stages. Standard conventional treatments for CRC include surgery, chemotherapy, and radiotherapy. Depending on the localization and progression of the disease, a combination of these treatments may be used. Treatment options include, for example, surgical resection alone in Stage I. In Stage III, the standard treatment is adjuvant chemotherapy after curative surgery. Total mesorectal resection (TME) using laparoscopic and transanal methods is often an option in cases of localized cancer or whenever access to the tumor site is easy. However, complete removal of all cancer cells is often not possible. Approximately 66% of colorectal cancer patients in Stage II and 61% in Stage III require further treatment with adjuvant chemotherapy and / or radiotherapy. These treatments have many side effects due to their nonspecificity and cytotoxicity to any growing and dividing cells. Despite recent improvements in diagnostic and treatment options, 54% of patients still experience recurrence even after neoadjuvant treatment. Therefore, it is important to have more effective alternative treatments for treating CRC patients.

[0020] Current chemotherapy for CRC includes both monotherapy based primarily on fluoropyrimidine (5-FU) and multi-agent regimens containing one or more drugs (including oxaliplatin (OX), irinotecan (IRI), and capecitabine (CAP or XELODA or XEL)). Therefore, combination therapy regimens FOLFOX (5-FU+OX), FOXFIRI (5-FU+IRI), XELOX or CAPOX (CAP+OX), and CAPIRI (CAP+OX) represent major approaches in first-line treatment. However, chemotherapy comes with certain limitations, such as systemic toxicity, unsatisfactory response rates, unpredictable spontaneous and acquired resistance, and low tumor-specific selectivity.

[0021] Patients with metastatic colorectal cancer (mCRC) have a 5-year overall survival rate of <10%. Currently, the main chemotherapy agents used to treat these patients are fluoropyrimidines (intravenous or oral), either as monotherapy with intravenous 5-fluorouracil (5-FU) and oral capecitabine (CAP), or as multi-agent regimens including FOLFOX (5-FU and oxaliplatin), FOLFIRI (5-FU and irinotecan), XELOX / CAPOX (CAP and oxaliplatin), and CAPIRI (CAP and irinotecan). In addition to traditional chemotherapy, the EGFR-targeted drugs cetuximab and panitumumab are approved as first-line treatments for mCRC. The combination of FOLFIRI and cetuximab, or panitumumab and FOLFOX, has significantly improved treatment efficacy. However, given that BRAF or KRAS mutations dramatically worsen prognosis, as seen in clinical trials, treatment decisions for early mCRC do not take these mutations into account. Although survival rates for mCRC patients have improved in recent years, the response and prognosis of patients with the above mutations remain poor.

[0022] Given the current lack of effective treatments and limitations due to nonspecific cytotoxicity, the need for promising therapies for patients with BRAF or KRAS mutations in mCRC remains largely unmet. As a result, researchers are shifting towards developing predictive, preventive, and personalized pharmaceutical strategies to improve the treatment of CRC and mCRC.

[0023] Gastric cancer is the second leading cause of death from malignant disease worldwide, with particularly high mortality rates in East Asia, South Asia, Central Asia, Central Europe, Eastern Europe, and South America. Treatment options for gastric cancer vary depending on the stage of the cancer, with very early-stage cancers typically treatable surgically. Potentially resectable cancers may typically be treated surgically, typically with subtotal or total gastrectomy as the first course of treatment. Nearby lymph nodes and potentially adjacent organs may also be removed. Patients may also receive chemotherapy alone or chemotherapy and radiotherapy (chemoradiation). For example, patients may receive a combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) (FP treatment). Other possible treatment regimens may include epirubicin, cisplatin, and fluorouracil (ECF treatment), or regimens in which fluorouracil is replaced with capecitabine (ECX treatment), or ECF treatment in which cisplatin is replaced with oxaliplatin (EOF treatment). Alternatively, both cisplatin and fluorouracil can be replaced with oxaliplatin and capecitabine (EOX treatment).

[0024] In metastatic gastric cancer, treatment aims to control cancer growth and may include chemotherapy alone, chemotherapy and immunotherapy, or chemotherapy and radiotherapy. The combination of platinum and fluoropyrimidine (5-FU) is the world standard first-line chemotherapy regimen within the non-curative setting. After platinum and 5-FU failure, paclitaxel and ramucirumab have been established as the standard second-line treatment. However, due to treatment-related neuropathy, progression during the perioperative FLOT regimen (fluorouracil, oxaliplatin, docetaxel), or rapid recurrence thereafter, there is a demand for taxane-free second-line treatments. In recent years, trifluridine / tipiracil has been approved for patients with metastatic gastric cancer. In addition to chemotherapy, patients may be treated with immune checkpoint inhibitors such as ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), or atezolizumab (anti-PD-L1).

[0025] For diagnosis, gastrointestinal endoscopy is essential for diagnosing gastric cancer, as is diagnostic laparoscopy (SL), a minimally invasive and short procedure requiring only a small incision. Advantages of SL include providing accurate diagnosis of peritoneal dissemination and extraserosal invasion, and allowing for peritoneal lavage for cytology. In patients with advanced gastric cancer, imaging alone may not provide a diagnosis, making pre-treatment peritoneal lavage cytology crucial for treatment planning. Peritoneal lavage cytology obtained by SL for evaluation of peritoneal dissemination is considered useful for assessing the effectiveness of neoadjuvant chemotherapy and / or immunotherapy.

[0026] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for 75%–85% of all cases. Because HCC is usually diagnosed at an advanced stage, effective treatment options remain limited. Until 2007, there were no effective treatment options for patients diagnosed with advanced-stage disease, or those who progressed to advanced stages after other treatments failed. Sorafenib was the first systemic drug approved by the U.S. Food and Drug Administration (FDA) as a standard treatment for advanced HCC between 2007 and 2016. In recent years, other small molecule drugs, including lenvatinib, regorafenib, and cabozantinib, have been developed and are either approved or still in clinical trials. These drugs are also currently being tested in combination with immune checkpoint inhibitors such as nivolumab or pembrolizumab. Combinations of immune checkpoint inhibitors with angiogenesis inhibitors (such as atezolizumab and bevacizumab) are also being investigated.

[0027] Esophageal cancer, as a form of GI cancer, is treated according to its stage and may include endoscopic treatment, as well as chemotherapy and radiotherapy. Common drugs and drug combinations used to treat esophageal cancer, but not usually administered with radiation, include ECF: epirubicin (Ellence), cisplatin, and 5-FU (especially in the case of tumors at the gastroesophageal junction), DCF: docetaxel (Taxotere), cisplatin, and 5-FU, or trifluridine and tipiracil (Lonsurf), and combination drugs in pill form.

[0028] For pancreatic cancer, several chemotherapy regimens are approved, with gemcitabine being the most widely used and researched drug. Gemcitabine is often administered in combination with albumin-conjugated (Nab) paclitaxel, improving survival compared to gemcitabine monotherapy. Alternative treatment options include the multi-drug regimen FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin). In addition, combination therapies using immune checkpoint inhibitors such as pemprolizumab or nivolumab in combination with gemcitabine, nab-paclitaxel, or capecitabine are clinically evaluated for pancreatic cancer.

[0029] Guanylyl cyclase 2C (GUCY2C, EC:4.6.1.2) is a member of the receptor-enzyme protein family that synthesizes cyclic guanosine 3',5'-monophosphate (cyclic GMP; cGMP). GUCY2C is a transmembrane receptor for the endogenous hormone ligands guanyline and uroguaniline. Ligand binding to the extracellular receptor catalyzes the conversion of GTP to cyclic GMP (cGMP), initiating downstream cGMP-related signaling pathways involved in regulating intestinal homeostatic processes such as epithelial cell proliferation, differentiation, and apoptosis (Lisby et al. Expert Rev Precis Med Drug Dev. 2021;6(2):117-129).

[0030] GUCY2C is specifically expressed by intestinal epithelial cells. GUCY2C regulates the dynamic progression of cells along the crypt-villous axis and the crypt-surface axis, integrating homeostatic processes including proliferation, DNA repair, metabolic programming, lineage-specific cell fate, and epithelial-mesenchymal interactions that organize these axes. Given GUCY2C's major role in maintaining epithelial regeneration, dysregulation of the GUCY2C-cGMP axis promotes disease conditions including inflammatory bowel disease and bowel transit disorders, in addition to colorectal cancer. Importantly, silencing of the GUCY2C signaling axis is associated with colorectal tumorigenesis due to loss of ligand binding.

[0031] The GUCY2C protein can be detected in almost without exception (>95%) all primary and metastatic human colorectal tumors, as well as subsets of gastroesophageal and pancreatic tumors, regardless of anatomical site or grade, but is not detected in tumors occurring outside the GI duct. GUCY2C has also been shown to be expressed in esophageal, gastric, and pancreatic tumors (Danaee et al (2017) PLoS One.2017;12(12):e0189953).

[0032] Given the unique expression patterns in CRCs and mCRCs, various methods have been employed to utilize GUCY2C in the treatment of these diseases. These include GUCY2C-based cancer vaccines, their use as targets in CAR-T cell therapy (Magee et al. Cancer Immunol Res. 6(5), 509-516 (2018)), and the use of therapeutic antibodies and antibody-drug conjugates targeting GUCY2C. WO2013 / 163633A1 discloses anti-GUCY2C ADCs and anti-GUCY2C antibodies, including auristatin E (MMAE) and auristatin F MMAE (MMAF), which inhibit mitosis by inhibiting tubulin polymerization. Clinical trials of the anti-GUCY2C ADC TAK264 (MLN0264, 5F9vcMMAE) using MMAE as a payload have been completed. WO2021 / 205325A1 discloses the use of anti-CD3-GUCY2C bispecific antibodies and their respective applications in cancer therapy to induce cytolytic T cell responses in GUCY2C-positive target cells.

[0033] These efforts suggest that while GUCY2C-targeted therapy may be effective, given the lack of approved GUCY2C-targeted therapies, there remains a considerable need for a treatment mode with high efficacy and a favorable efficacy profile. [Overview of the Initiative] [Means for solving the problem]

[0034] Surprisingly, the inventors have found that the amatoxin-based complex according to the present invention, comprising an anti-GUCY2C antibody or an antigen-binding antibody fragment, in combination with a non-cleavable or cleavable linker that binds the anti-GUCY2C antibody or antibody fragment to amatoxin, as disclosed herein, is characterized by high potency and efficacy in in vitro and in vivo models of GUCY2C-positive cancer.

[0035] Therefore, considering the prior art, one object of the present invention is to provide a complex comprising an antibody that specifically binds to GUCY2C, at least one amatoxin that mediates a cytotoxic effect in target cells as described in this application, and the anti-GUCY2C antibody comprising at least one linker that binds the anti-GUCY2C antibody to the at least one amatoxin.

[0036] A further object of the present invention is to provide a pharmaceutical composition containing such a complex.

[0037] A further object of the present invention is to provide compounds used in methods for treating cancer.

[0038] In a further object, the present invention relates to providing compositions comprising the complex of the present invention disclosed herein and at least one immune checkpoint inhibitor for use in the treatment of cancer, particularly colorectal cancer. [Brief explanation of the drawing]

[0039] [Figure 1] Figure 1 shows the Markush structures of various amatoxins. The bold numbers (1-8) indicate the standard numbering of the eight amino acids that make up the amatoxin. The standard notation for the atoms in amino acids 1, 3, and 4 is also shown (Greek letters α-γ, α-δ, and 1'-7', respectively). [Figure 2] Figure 2 shows the in vitro cytotoxicity of the anti-GUCY2C-ADC of the present invention, including the complex (XV), assayed against HEK293 cells that stably express human GUCY2C. [Figure 3]Figure 3 shows the in vitro cytotoxicity of anti-GUCY2C-ADC against HEK cells that stably express human GUCY2C. (A) shows the cytotoxicity of anti-GUCY2C-ADC bound to complex (XII), and (B) shows the cytotoxicity of anti-GUCY2C-ADC bound to complex (XIV). The cytotoxicity assay shows that the tested anti-GUCY2C-ADC exhibits similar cytotoxic activity in the picomolar range against HEK293-GUCY2C cells. [Figure 4] Figure 4 shows the efficacy of anti-GUCY2C complexes mAb1-LALA-D265C-(XIV) and mAb8-LALA-D265C-(XIV) in a subcutaneous tumor model using HEK293-GUCY2C-(HDP)-2B3 tumor cells in female NOD Scid mice after single-dose intravenous administration of ADC, as shown. The results indicate that single-dose administration of ADC at concentrations of 1.25 mg / kg (intravenous injection) and 2.5 mg / kg (intravenous injection) effectively suppresses tumor growth. [Figure 5] Figure 5 is a Kaplan-Meier plot for survival in the subcutaneous GUCY2C tumor model shown in Figure 4, indicating increased survival in animals in groups 2, 3, 5, and 6. [Figure 6-1] Figure 6 shows the hepatotoxicity of the anti-GUCY2C complex of the present invention in cynomolgus monkeys. It shows the effects of administration of the anti-GUCY2C-ADC of the present invention, containing heavy and light chain amino acid sequences (SEQ ID NOs. 85 and 87), on the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The results show that liver enzymes ALT, AST, and LDH transiently increase upon administration of the complex of the present invention and reach baseline levels within 2-3 weeks of complex administration. Furthermore, the results show that the maximum tolerated dose of the complex containing linker payload (XIV) is 2 mg / kg, and the maximum tolerated dose of the complex containing linker payload (XII) is 5 mg / kg. [Figure 6-2]Figure 6 shows the hepatotoxicity of the anti-GUCY2C complex of the present invention in cynomolgus monkeys. It shows the effects of administration of the anti-GUCY2C-ADC of the present invention, containing heavy and light chain amino acid sequences (SEQ ID NOs. 85 and 87), on the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The results show that liver enzymes ALT, AST, and LDH transiently increase upon administration of the complex of the present invention and reach baseline levels within 2-3 weeks of complex administration. Furthermore, the results show that the maximum tolerated dose of the complex containing linker payload (XIV) is 2 mg / kg, and the maximum tolerated dose of the complex containing linker payload (XII) is 5 mg / kg. [Figure 6-3] Figure 6 shows the hepatotoxicity of the anti-GUCY2C complex of the present invention in cynomolgus monkeys. It shows the effects of administration of the anti-GUCY2C-ADC of the present invention, containing heavy and light chain amino acid sequences (SEQ ID NOs. 85 and 87), on the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The results show that liver enzymes ALT, AST, and LDH transiently increase upon administration of the complex of the present invention and reach baseline levels within 2-3 weeks of complex administration. Furthermore, the results show that the maximum tolerated dose of the complex containing linker payload (XIV) is 2 mg / kg, and the maximum tolerated dose of the complex containing linker payload (XII) is 5 mg / kg. [Figure 7] Figure 7 shows the treatment of xenograft (PDX) models derived from mouse patients with colorectal cancer using the complex of the present invention. (A) A PDX model of primary KRAS variant (G13D) adenocarcinoma stage T3N0Mx treated with the complex of the present invention, including the linker-payload complexes (XII) and (XIV) as shown. (B) A PDX model of primary adenocarcinoma stage T3N0M0 treated with the complex of the present invention, including the linker-payload complexes (XII) and (XIV) as shown. The complexes (A) and (B) were administered as a single dose or repeatedly once a week for 4 weeks ("q7dx4"). [Figure 8-1] Figure 8 shows the sequence alignment of the (A) heavy chain variable region and (B) light chain variable region of the antibody of the present invention. Alignment was performed using the Clustal W algorithm (Thompson et al, Nucleic Acids Res. 1994 Nov 11;22(22):4673-80). The CRDs of both the VH and VL sequences are indicated by boxes. [Figure 8-2] Figure 8 shows the sequence alignment of the (A) heavy chain variable region and (B) light chain variable region of the antibody of the present invention. Alignment was performed using the Clustal W algorithm (Thompson et al, Nucleic Acids Res. 1994 Nov 11;22(22):4673-80). The CRDs of both the VH and VL sequences are indicated by boxes. [Modes for carrying out the invention]

[0040] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific components of the apparatus or the specific process steps of the method described, and that the apparatus and method may vary. It should also be understood that the technical terms used herein are merely for the purpose of describing specific embodiments and are not intended to limit them. Furthermore, as used in the specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include singular and / or plural nouns. It should also be understood that when a parameter range is given separated by numerical values, this range is considered to include these limiting values. The enumeration of value ranges herein is intended to serve simply as a concise way of referring to each individual value within that range. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were individually enumerated herein.

[0041] Throughout this specification and the subsequent claims, unless otherwise required in the context, the terms “contains,” and variations thereof such as “contains,” “includes,” and “includes,” should be understood to mean that they include the stated component, integer, or process, but not that they exclude other unmentioned components, integers, or processes. The term “consists of” is a specific embodiment of the term “contains,” excluding other components, integers, or processes.

[0042] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate, unrelated embodiments. Features discussed in one embodiment are disclosed in relation to other embodiments shown herein. In some cases, if a specific feature is not disclosed in one embodiment but is disclosed in another, a person skilled in the art will understand that this does not necessarily mean that the feature is not disclosed in the other embodiments. A person skilled in the art will understand that while it is the intent of this application to disclose the feature for other embodiments, this has not been done simply for clarity and to make this specification manageable.

[0043] Furthermore, the contents of prior art documents referenced herein are incorporated by reference, particularly those disclosing standard or common methods. In this case, the incorporation by reference is primarily to provide a sufficiently feasible disclosure and to avoid lengthy repetition. The definitions of chemical groups used herein have the meanings and definitions set forth in the "Gold Book" version 2.3.3 published by the International Union of Pure and Applied Chemistry (IUPAC) (goldbook.iupac.org, ISBN: 0-9678550-9-8) (the contents of which are incorporated herein by reference).

[0044] Throughout this application, the term "approximately" is used to refer to a + / - 10% range of the numerical value used with it.

[0045] Furthermore, the content of prior art documents referenced herein is incorporated by reference, particularly those disclosing standard or common methods. In this case, the incorporation by reference is primarily to provide a sufficiently feasible disclosure and to avoid lengthy repetition.

[0046] According to a first aspect of the present invention, an isolated antibody or antibody fragment that specifically binds to guanylyl cyclase C (GUCY2C), wherein the framework region 1 (FR H 1) Complementarity Determination Area 1 (CDR) H 1) Framework domain (FR H 2) Complementarity Determination Area 2 (CDR) H 2) The heavy chain variable region (V) including framework region 3 (FRH3), complementarity determination region 3 (CDRH3), and framework region 4 (FRH4). H ) including, - FRH1 contains an amino acid sequence selected from SEQ ID NOs: 33, 40, 45, and 51. - CDRH1 contains an amino acid sequence selected from SEQ ID NOs: 34, 41, 46, and 52. - FRH2 contains the amino acid sequence corresponding to SEQ ID NO: 35;55, - CDRH2 contains an amino acid sequence selected from SEQ ID NOs: 36, 42, 47; 120. - FRH3 contains an amino acid sequence selected from SEQ ID NOs: 37, 43, 48, and 53. - CDRH3 contains an amino acid sequence selected from SEQ ID NOs: 38, 44, 49; 54. - FRH4 contains an amino acid sequence selected from SEQ ID NOs: 39 and 50. An antibody or antibody fragment is provided.

[0047] As used herein, the term "antibody" refers to a protein consisting of one or more polypeptide chains encoded by an immunoglobulin gene, or a fragment of an immunoglobulin gene, or cDNA derived therefrom. The immunoglobulin genes include any of the light chain κ and λ constant region genes, the heavy chain α, δ, ε, γ, and μ constant region genes, and many different variable region genes.

[0048] The basic immunoglobulin (antibody) unit is typically a tetramer consisting of two pairs of identical polypeptide chains, namely, a light chain (L, having a molecular weight of about 25 kDa) and a heavy chain (H, having a molecular weight of about 50 - 70 kDa). Each heavy chain consists of a heavy chain variable region (VH or V H abbreviated as) and a heavy chain constant region (CH or C H abbreviated as). The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL or V L abbreviated as) and a light chain constant region (CL or C L abbreviated as). The VH region and the VL region can be further divided into hypervariable regions, which are also called complementarity-determining regions (CDRs), and more conserved regions called framework regions (FRs) are interspersed. The VH region and the VL region each consist of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains form a binding domain that interacts with the antigen. The constant region (Fc region) is not directly involved in the binding of the antibody to the antigen, but shows various effector functions such as involvement in antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis by binding to Fcγ receptors, half-life / clearance rate by neonatal Fc receptor (FcRn), and complement activation by C1q component, resulting in chemotactic action of complement, opsonization, and cytolytic action (potentially in the case of viable cell antigen targets). Human antibodies of the IgG1 class are most potent in activating the complement system and are thus a desirable isotype for the therapeutic applications of the antibodies of the present invention.

[0049] Human Fcγ receptors include FcγR(I), FcγRIIa, FcγRIIb, FcVRIIIa, and neonatal FcRn. Here, a common set of IgG1 residues is involved in the binding of all FcγRs, but FcγRII and FcγRIII have been shown to utilize different sites outside this common set (Shields et al. (2001) J. Biol. Chem 276:6591-6604). When one group of the IgG1 residue was changed to alanine: Pro-238, Asp-265, Asp-270, Asn-297, and Pro-239 (numbered according to the EU numbering system), binding to all FcγRs decreased. All are located in the IgG CH2 domain and clustered near the hinges that bind CH1 and CH2. FcγR1 utilizes only a common set of IgG1 residues for binding, while FcγRII and FcγRIII interact with different residues in addition to this common set. Changes in several residues reduced binding only to FcγRII (e.g., Arg-292) or FcγRIII (e.g., Glu-293). Several mutants showed improved binding to FcγRII or FcγRIII, but no effect on binding to other receptors. Neonatal FcRn receptors are thought to be involved in both antibody clearance and tissue-to-tissue transcytosis (see Junghans (1997) Immunol. Res16:29-57; and Ghetie et al. (2000) Annu. Rev. Immunol. 18:739-766). Human IgG1 residues that have been confirmed to directly interact with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435.

[0050] The terms "CDR" and "CDR" as used herein L 1", CDR L 2", CDR L 3", CDR H 1", CDR H 2", CDR H"3" follows the Kabat numbering rules (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USD Department of Health and Human Services, National Institutes of Health (1987)). However, although the Kabat numbering rules are used throughout this specification for amino acid residues in variable region sequences and full-length antibody sequences, it will be apparent to those skilled in the art that there are other numbering rules for amino acid residues in variable region sequences and full-length antibody sequences. There are also other numbering rules for CDR sequences, for example, described in Chothia et al. (1989), Nature 342:877-883. The structure of the antibody and the folding of the protein may mean that other residues based on the numbering system used are considered part of the CDR sequence and may be understood as such by those skilled in the art, but these differences do not functionally mean that the antigen binding of each antibody is altered or different. Other numbering rules for CDR sequences available to those skilled in the art include the "AbM" method (University of Bath) and the "contact" method (University College London).

[0051] The CDR is crucial for the binding of the antibody or its antigen-binding site. The FR can be substituted with other sequences, as long as the three-dimensional structure necessary for antigen binding is maintained. Structural changes in the construct almost always lead to a loss of sufficient binding to the antigen.

[0052] Typically, antibodies have a dissociation constant (KD) of 10. -5 ~10 -11 It specifically binds to the same antigen with high affinity, as reflected by M or less. Generally, about 10 -4 K is greater than M D This is considered to show nonspecific binding. As used herein, an antibody that "specifically binds" to an antigen is defined as 10 -7 M or less, preferably 10 -8M or less, more convenient 5×10 -9 M or less, most preferably about 10 -8 , 10 -9 M~about 10 -10 M, 10 -11 Below, or for example, about 10 -10 M~about 10 -11 K below M D This refers to an antibody that binds to an antigen and substantially identical antigens with high affinity, meaning it possesses a specific affinity for that antigen, but does not bind to unrelated antigens (e.g., structurally unrelated or sequence-unrelated) with an affinity equivalent to that of the specific target.

[0053] For example, the antibodies of the present invention, sometimes also called immunoglobulins, may be derived from any of the generally known isotypes, such as IgA, secretory IgA, IgG, and IgM, but are not limited to these. IgG isotypes are divided into subclasses in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice.

[0054] As used herein, the terms “antibody fragment” or “antigen-binding fragment” refer to an antibody fragment or antibody analogue that retains the binding specificity of the parental anti-GUCY2C antibody as disclosed herein and includes a portion of the antigen-binding region (e.g., one or more CDRs) or variable region of the parental antibody. For example, an antibody fragment is a Fab, Fab', F(ab')2, Fv fragment, sc-Fv, unibody, bispecific antibody, linear antibody, nanobody, domain antibody, or a polyspecific antibody fragment formed from an antibody fragment. A Fab fragment consists of CH1 and a variable region of one light chain and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A Fab' fragment includes a light chain and a portion of the heavy chain including a VH domain, a CH1 domain, and a region between the CH1 domain and the CH2 domain. The F(ab')2 fragment contains two light chains and two heavy chains that include a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. The F(ab')2 fragment consists of two Fab' fragments linked by a disulfide bond between the two heavy chains. The "Fv fragment" contains variable regions derived from both the heavy and light chains, but does not contain the constant region. The term "single-chain antibody" is a single-chain recombinant protein formed by linking the heavy chain variable region VH and the light chain variable region VL of an antibody via a binding peptide. This is the smallest antibody fragment with a complete antigen-binding site. The term "domain antibody fragment" is an immunoglobulin fragment with immune function that contains only the heavy chain variable region or the light chain variable region chain. In some cases, two or more VH regions are covalently bound to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same antigen or different antigens.

[0055] According to one embodiment, the anti-GUCY2C antibody or antibody fragment of the present invention disclosed herein is located in framework region 1 (FR L 1) Complementarity Determination Area (CDR) L 1), FR L 2. CDR L 2. FR L 3. CDRL 3, and FR L Light chain variable region including 4 (V L ) including, - FR L 1 contains an amino acid sequence selected from sequence numbers 1, 13, 20, and 27. - CDR L 1 contains an amino acid sequence selected from sequence numbers 2, 14, 21, and 28. - FR L 2 contains an amino acid sequence selected from sequence numbers 3, 15, and 29. - CDR L 2 contains an amino acid sequence selected from sequence numbers 4, 16, 23, and 30. - FR L 3 contains an amino acid sequence selected from sequence numbers 5, 8, 17, 24, and 31. - CDR L 3 contains an amino acid sequence selected from sequence numbers 6, 9, 11, 18, 25, and 32. - FR L Sequence 4 contains an amino acid sequence selected from sequence numbers 7, 10, 12, 19, and 26.

[0056] According to one embodiment, the anti-GUCY2C antibody of the present invention has a framework region 1 (FR H 1) Complementarity Determination Area 1 (CDR) H 1), FR H 2. CDR H 2. FR H 3. CDR H 3, and FR H Heavy chain variable region including 4 (V H ) including, - FRH1 contains the amino acid sequence related to SEQ ID NO: 33, - CDRH1 contains the amino acid sequence corresponding to SEQ ID NO: 34, - FRH2 contains the amino acid sequence related to SEQ ID NO: 35, - CDRH2 contains the amino acid sequence corresponding to SEQ ID NO: 36, - FRH3 contains the amino acid sequence corresponding to SEQ ID NO: 37, - CDRH3 contains the amino acid sequence corresponding to SEQ ID NO: 38, - FRH4 contains the amino acid sequence corresponding to SEQ ID NO: 39, The above antibody is in framework region 1 (FR L 1) Complementarity Determination Area (CDR) L 1), FR L 2. CDR L 2. FR L 3. CDR L 3, and FR L Light chain variable region including 4 (V L ) including, - FR L 1 contains the amino acid related to SEQ ID NO: 1;121, - CDR L 1 contains the amino acid related to SEQ ID NO: 2, - FR L 2 contains the amino acids related to SEQ ID NOs: 3, 15, and 29. - CDR L 2 contains an amino acid sequence selected from SEQ ID NO: 4, - FR L 3 contains an amino acid sequence selected from SEQ ID NOs. 5 and 8. - CDR L 3 contains an amino acid sequence selected from sequence numbers 6, 9, and 11. - FR L Sequence 4 contains an amino acid sequence selected from sequence numbers 7, 10, and 12.

[0057] According to one embodiment, the anti-GUCY2C antibody or antibody fragment of the present invention has a framework region 1 (FR H 1) Complementarity Determination Area (CDR) H 1), FR H 2. CDR H 2, heavy chain variable region including FRH3, CDRH3, and FRH4 (V H ) including, - FRH1 contains the amino acid sequences related to SEQ ID NOs. 40, 45, and 51. - CDRH1 contains the amino acid sequences related to SEQ ID NOs. 41, 46, and 52. - FRH2 contains the amino acid sequence corresponding to SEQ ID NO: 35;55, - CDRH2 contains the amino acid sequences according to SEQ ID NO: 42 and 47, - FRH3 contains the amino acid sequences according to SEQ ID NO: 43, 48, and 53, - CDRH3 contains the amino acid sequences according to SEQ ID NO: 49, 44, and 54, - FRH4 contains the amino acid sequences according to SEQ ID NO: 50 and 39, The above antibody contains a light chain variable region (V L 1), a complementarity-determining region (CDR L 1), FR L 2, CDR L 2, FR L 3, CDR L 3, and FR L 4, L - FR L 1 contains the amino acids according to SEQ ID NO: 13, 20, and 27, - CDR L 1 contains the amino acids according to SEQ ID NO: 14, 21, and 28, - FR L 2 contains the amino acids according to SEQ ID NO: 15, 22, and 29, - CDR L 2 contains the amino acid sequence selected from SEQ ID NO: 16, 23, and 30, - FR L 3 contains the amino acid sequence selected from SEQ ID NO: 17, 24, and 31, - CDR L 3 contains the amino acid sequence selected from SEQ ID NO: 18, 25, and​​​​​​​​​​​​​​​​​​​H 2. CDRs according to SEQ ID NO: 38 H 3 and a heavy chain variable region (V H ).

[0059] According to one embodiment, the anti-GUCY2C antibody of the present invention disclosed herein comprises CDRs according to SEQ ID NO: 2 L 1. CDRs according to SEQ ID NO: 4 L 2. CDRs according to SEQ ID NO: 6 L 3 and a light chain variable region (V L ), and CDRs according to SEQ ID NO: 34 H 1. CDRs according to SEQ ID NO: 36 H 2. CDRs according to SEQ ID NO: 38 H 3 and a heavy chain variable region (V H ).

[0060] According to one embodiment, the anti-GUCY2C antibody of the present invention disclosed herein comprises CDRs according to SEQ ID NO: 2 L 1. CDRs according to SEQ ID NO: 4 L 2. CDRs according to SEQ ID NO: 11 L 3 and a light chain variable region (V L ), and CDRs according to SEQ ID NO: 34 H 1. CDRs according to SEQ ID NO: 36 H 2. CDRs according to SEQ ID NO: 38 H 3 and a heavy chain variable region (V H ).

[0061] According to one embodiment, the anti-GUCY2C antibody of the present invention disclosed herein comprises CDRs according to SEQ ID NO: 28 L 1. CDRs according to SEQ ID NO: 30 L 2. CDRs according to SEQ ID NO: 32 L 3 and a light chain variable region (V L ), and CDRs according to SEQ ID NO: 52 H 1. CDRs according to SEQ ID NO: 120 H 2. CDRs according to SEQ ID NO: 54 H 3 and a heavy chain variable region (V H ).

[0062] According to one embodiment, the anti-GUCY2C antibody of the present invention disclosed herein is a CDR related to SEQ ID NO: 21. L 1. CDR related to Sequence ID No. 23 L 2. CDR related to Sequence ID No. 25 L Light chain variable region including 3 (V L ) and the CDR related to Sequence ID No. 46 H 1. CDR related to Sequence ID No. 47 H 2. CDR related to Sequence ID No. 49 H Heavy chain variable region including 3 (V H ) and include.

[0063] According to one embodiment, the anti-GUCY2C antibody of the present invention disclosed herein is a CDR related to SEQ ID NO: 14. L 1. CDR related to Sequence ID No. 16 L 2. CDR related to Sequence ID No. 18 L Light chain variable region including 3 (V L ) and the CDR related to Sequence ID No. 41 H 1. CDR related to Sequence ID No. 42 H 2. CDR related to Sequence ID No. 44 H Heavy chain variable region including 3 (V H ) and include.

[0064] In one embodiment, the light chain variable region (V) of the anti-GUCY2C antibody of the present invention L ) includes the amino acid sequences related to SEQ ID NOs: 2;3;4, 121 and 8;9;10, and includes a heavy chain variable region (VH) that includes the amino acid sequences related to SEQ ID NOs: 33;34;35;36;37;38;39 (e.g., mAb#1).

[0065] In one embodiment, the light chain variable region (V) of the anti-GUCY2C antibody of the present invention L ) contains the amino acid sequences corresponding to SEQ ID NOs. 1-7 and includes a heavy chain variable region (VH) containing the amino acid sequences corresponding to SEQ ID NOs. 33;34;35;36;37;38;39 (e.g., mAb#8).

[0066] In one embodiment, the light chain variable region (V) of the anti-GUCY2C antibody of the present invention L) contains the amino acid sequences related to SEQ ID NOs: 2;3;4;5, 11, 12, 121, and the heavy chain variable region (V) containing the amino acid sequences related to SEQ ID NOs: 33;34;35;36;37;38;39. H ) including (for example, mAb#24).

[0067] In one embodiment, the light chain variable region (V) of the anti-GUCY2C antibody of the present invention L ) contains the amino acid sequence related to SEQ ID NOs. 20;21;22;23;24;25;26, and the heavy chain variable region (V) contains the amino acid sequence related to SEQ ID NOs. 45, 46, 48, 49, 50, and 55. H ) including (for example, mAb#28).

[0068] In one embodiment, the light chain variable region (V) of the anti-GUCY2C antibody of the present invention L ) contains the amino acid sequences related to SEQ ID NOs. 40, 41, 55, 42, 43, 44, and 39, and the heavy chain variable region (V) contains the amino acid sequences related to SEQ ID NOs. 13; 14; 15; 16; 17; 18; 19. H ) including (for example, mAb#40).

[0069] In one embodiment, the light chain variable region (V) of the anti-GUCY2C antibody of the present invention L ) contains the amino acid sequence related to SEQ ID NOs. 27;28;29;30;32;19, and the heavy chain variable region (V) contains the amino acid sequence related to SEQ ID NOs. 51:52;55;120;53;54; and 39. H ) including (for example, mAb#41).

[0070] According to some embodiments, the anti-GUCY2C antibody or antibody fragment according to the present invention comprises a heavy chain variable region (V) containing the following amino acid sequence. H ) and light chain variable region (V L This includes combinations of ). - Sequence ID 62, Sequence ID 56 - Sequence ID 63, Sequence ID 57 - Sequence ID 64, Sequence ID 58 - Sequence ID 65, Sequence ID 59 - Sequence ID 66, Sequence ID 60 - Sequence ID 67, Sequence ID 61

[0071] Furthermore, as long as the corresponding anti-GUCY2C antibody or antibody fragment specifically binds to the same epitope of human or cynomolgus monkey GUCY2C, the V disclosed above applies. L and V H Each combination, for example, has at least 90% or 95% sequence identity with sequence numbers 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67, either independently of the VL sequence or both of the VL and VH sequences. L , or V H , or V L and V H The present invention may include both of the above. Epitope mapping of the antibodies of the present invention may be performed by hydrogen-deuterium exchange (HDX) similar to, for example, the method disclosed in Huang et al. MAbs. 2018 Jan;10(1):95-103, or the method reviewed in, for example, Jethva and Gross, Front. Anal. Sci., 18 May 2023.

[0072] According to one embodiment, the antibody or antibody fragment according to the present invention may be a monoclonal antibody. As used herein, the term “monoclonal antibody” (“mAb”) refers to a formulation of an antibody molecule having a single binding specificity and affinity for a particular epitope, representing a homogeneous antibody population, i.e., a homogeneous population consisting of an entire immunoglobulin or a fragment thereof. Preferably, the monoclonal anti-GUCY2C antibody of the present invention disclosed herein is an IgG isotype, for example, IgG1 or IgG4, more preferably an IgG1 isotype.

[0073] Monoclonal antibodies (mAbs), such as those derived from mice, can cause undesirable immunological side effects when administered to humans due to the presence of proteins from other species that can induce antibody production. To overcome this problem, antibody humanization and maturation methods are designed to produce antibody molecules with minimal immunogenicity when applied to humans, ideally while still retaining the specificity and affinity of the non-human parent antibody (see Almagro and Fransson 2008 for review).

[0074] Therefore, according to a preferred embodiment, the anti-GUCY2C antibody of the present invention is a humanized antibody or a human antibody. As used herein, the term “humanized” antibody refers to an antibody that contains the smallest sequence derived from a non-human immunoglobulin. Thus, the “humanized” form of a non-human (e.g., mouse) antibody is a chimeric antibody that contains the smallest sequence derived from a non-human antibody. All or substantially all framework regions may also be human immunoglobulin sequences. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art and are described, for example, in Riechmann et al., Nature 332:323-7, 1988; U.S. Patents 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370. Human antibodies, such as the human anti-GUCY2C antibody of the present invention, can be produced by hybridoma using human myeloma or mouse-human heterozygous myeloma cell lines (see Kozbor (1984) J. Immunol 133, 3001). Alternative methods include the use of phage libraries or transgenic mice using human variable region repertoires (see Winter (1994) Annu. Rev. Immunol 12:433-455; Green (1999) J. Immunol. Methods 231:11-23). Several lines of transgenic mice in which mouse immunoglobulin loci are replaced with human immunoglobulin gene segments are now available (see Tomizuka (2000) PNAS 97:722-727; Fishwild (1996) Nature Biotechnol. 14:845-851; Mendez (1997) Nature Genetics, 15:146-156). During antigen challenge, such mice can produce a repertoire of human antibodies from which the desired antibody can be selected.

[0075] According to several embodiments, the constant region of the IgG heavy chain of the anti-GUCY2C IgG1 antibody of the present invention can be selected from different allotypes. In the context of the anti-GUCY2C antibody of the present invention, the term "allotype" refers to a heritable allelic variant resulting from genetic differences between individuals that can be recognized as an antigen by members of the same species. For example, the antibody of the present invention may have allotypes G1m3, G1m17,1, or G1m17,1,2, or one of G1m(f), G1m(z,a), or G1m(z,a,x).

[0076] For example, a human antibody such as the human anti-GUCY2C antibody of the present invention may contain a kappa (κ) chain encoded by the immunoglobulin kappa locus (IGK) on chromosome 2 (locus: 2p11.2) in humans, or a lambda (λ) chain encoded by the immunoglobulin lambda locus (IGL) on chromosome 22 (locus: 2q11.22) in humans. Accordingly, the anti-GUCY2C antibody of the present invention may contain two kappa light chains or two lambda light chains containing the VL sequence related to SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61.

[0077] According to one embodiment, the anti-GUCY2C antibody of the present invention or its antigen-binding fragment may be glycosylated or deglycosylated. Preferably, the antibody of the present invention is glycosylated, and the corresponding glycan may be an N-linked oligosaccharide chain at asparagine 297 of the heavy chain (numbered according to the EU numbering system, Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85). As most glycoproteins, antibodies are typically produced as a mixture of glycoforms. This mixture is particularly evident when antibodies are produced in eukaryotic cells, especially mammalian cells. For example, each glycan moiety attached to Fc-glycans such as fucose, bisected GlcNAc, galactose, and sialic acid is involved in regulating antibody sensor affinity and / or antibody function. Therefore, it is desirable to produce monoclonal antibodies characterized by a predetermined set of glycans in order to obtain antibodies with a predetermined effector function. Various methods have been developed for producing a given glycoform (see Zhang et al. (2004) Science 303:371; Sears et al. (2001) Science 291:2344; Wacker et al. (2002) Science 298:1790; Davis et al. (2002) Chem.Rev.102:579; Hang et al. (2001) Acc.Chem.Res 34:727). Preferably, the anti-GUCY2C antibody of the present invention as described herein comprises a homogeneous or predetermined number of glycoforms, for example, about 1, 2, 3, 4 to about 5, 6, 7, 8, or about 5, 6, 7 to about 8, preferably 6 or less, for example, 5, 4, 3 or less, more preferably 2 or 1 glycoform.

[0078] According to one embodiment, the anti-GUCY2C antibody or its antigen-binding fragment is a recombinant antibody or its antigen-binding fragment. As used herein, the terms “recombinant” or “recombinantly produced” refer to proteins such as the anti-GUCY2C antibody or antigen-binding fragment of the present invention disclosed herein, expressed in heterogeneous cells. For example, the anti-GUCY2C antibody or antigen-binding fragment of the present invention may be expressed in prokaryotic or eukaryotic cells. Examples of eukaryotic cells for expressing the antibody of the present invention include Gram-negative bacteria such as Escherichia coli, or Gram-positive bacteria such as Bacillus subtilis. The use of heterogeneous prokaryotic expression systems may be particularly useful for expressing the antigen-binding fragment of the anti-GUCY2C antibody of the present invention, for example, the expression of the Fab fragment may be carried out by established protocols known in the art, such as Kwong and Rader, Curr. Protoc. Protein Sci. 55:6.10.1-6.10.14.

[0079] However, the use of eukaryotic cells to express the antibody or antigen-binding fragment of the present invention is preferred due to the glycosylation of the heterologously expressed antibody. It is even more preferable to use mammalian cells to express the antibody or antigen-binding fragment of the present invention. For example, Chinese hamster ovary (CHO) cells, or any of its genetically distinct offspring such as the K1-cell line, DukX B11-cell line, or DG44-cell line, may be used to express the antibody of the present invention. Other mammalian cell lines that may be used to produce the antibody or antigen-binding fragment of the present invention include, for example, NS0 cells, fetal kidney (HEK) 293 cells, PER.C6 cells, and MCF7 cells. Cells can be transfected with an expression vector containing, for example, the coding sequence of the anti-GUCY2C antibody or antigen-binding fragment according to the present invention. The expression vector or recombinant plasmid is produced by placing the coding antibody sequence under the control of suitable regulatory genetic elements, including promoter and enhancer sequences (e.g., CMV promoter). The heavy and light chain sequences can be expressed, for example, from co-transfected individual expression vectors or from a dual expression vector. The above transfection may be transient or stable. The transfected cells are then cultured to produce transfected antibody constructs. Once stable transfection has been performed, stable clones secreting antibodies with properly bound heavy and light chains are selected by screening with a suitable assay, such as ELISA, subcloned, and propagated for future production. Corresponding methods are disclosed, for example, in WO03 / 018771.

[0080] According to one embodiment, the anti-GUCY2C antibody of the present invention includes an Fc region containing the amino acid sequence related to SEQ ID NO: 68. The antibody of the present invention, which includes an Fc region containing the amino acid sequence related to SEQ ID NO: 68, can induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0081] However, it may be desirable to reduce or eliminate the effector function of the antibodies of the present invention disclosed herein, for example, by preventing targeted cell death, undesirable cytokine secretion, or death of Fcγ receptor-expressing cells such as macrophages.

[0082] Accordingly, the anti-GUCY2C antibody or antigen-binding fragment of the present invention described herein may also include modifications and / or mutations that alter the properties of the antibody and / or fragment, for example, modifications and / or mutations that reduce ADCC, ADCP, or complement-dependent cell-mediated cytotoxicity CDC, as known in the art. Preferably, ADCC, ADCP, and CDC are reduced by at least 90%, more preferably at least 95%, more preferably at least 97.5%, and even more preferably at least 98% or 99% compared to an antibody containing a wild-type Fc region, such as the amino acid sequence relating to SEQ ID NO: 68.

[0083] The binding of IgG1 to the first component of the activated Fcγ receptor and the inhibitory Fcγ receptor (FcγR) or complement (C1q) depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are important for FcγR and complement C1q binding and have unique sequences. Substitutions of human IgG1 and IgG2 residues at positions 233–236, and IgG4 residues at positions 327, 330, and 331 significantly reduced ADCC and CDC (Armour, et al., Eur. J. Immunol. 29(8) (1999) 2613–2624; Shields, et al., J. Biol. Chem. 276(9) (2001) 6591–6604, WO2021 / 234402A2).

[0084] Accordingly, in one embodiment, the anti-GUCY2C antibody or its antigen-binding fragment disclosed herein includes a mutant Fc region, the mutant Fc region comprising at least one amino acid modification to the wild-type Fc region such that the molecule has reduced affinity for the IgG1 Fc receptors FcγRI, FcγRII, and FcγRIII, and for complement component C1q, compared to the wild-type Fc region (e.g., the amino acid sequence relating to SEQ ID NO: 68).

[0085] The affinity of IgG1 to the Fc region, such as binding to FcγR, can be determined using various techniques known in the field. These include, but are not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol.373:52-60, 2008, or radioimmunoassay (RIA)), surface plasmon resonance assays disclosed in Wilkinson et al. PLoS One. 2021;16(12):e0260954, or other kinetic assay mechanisms (e.g., BIACORE™ analysis or Octet™ analysis (forteBIO)), as well as other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration).

[0086] Therefore, the anti-GUCY2C antibodies described herein are genetically modified to include a mutant Fc region containing a modification of at least one amino acid residue that is in direct contact with FcγR, based on structural and crystallographic analysis. The terms “genetically modified” or “genetically altered” as used herein refer to the modification of the amino acid sequence or part thereof of a given or native polypeptide or protein, such as the Fc region of an antibody, by genetic technology methods such as site-directed mutagenesis, as described in Carter, Biochem. J. (1986) Vol. 237: 1-7, meaning substitution, insertion, deletion, or restoration of nucleotides and / or amino acids, or any combination thereof. The terms “amino acid substitution” or “mutation” as used herein refer to the modification of the amino acid sequence of a protein in which one or more amino acids are substituted with the same number of different amino acids, resulting in a protein with a different amino acid sequence from the original protein. Conservative amino acid substitutions are understood to refer to substitutions that do not significantly affect the structure and function of the protein due to similar size, charge, polarity, and / or higher-order structure. In this sense, the conserved amino acid group includes, for example, the nonpolar amino acids Gly, Ala, Val, Ile, and Leu; the aromatic amino acids Phe, Trp, and Tyr; the positively charged amino acids Lys, Arg, and His; and the negatively charged amino acids Asp and Glu.

[0087] Depending on the intended use of the antibody, such as in the manufacture of antibody-drug conjugates, the Fc region of the antibody may further contain at least one cysteine ​​amino acid substitution at a site where the modified cysteine ​​can be used for binding but does not disrupt the folding and assembly of the immunoglobulin. Corresponding cysteine-substituted or cysteine-modified antibodies are disclosed in WO2016040856A2, or Junutula, et al., 2008 Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; US7521541; US7723485; WO2009 / 052249, and WO2016 / 142049. A preferred cysteine ​​substitution in the Fc region of the antibodies of the present invention disclosed herein is D265C (according to the EU numbering system), as disclosed in WO2016142049A1.

[0088] In one embodiment, the anti-GUCY2C antibody of the present invention comprises an Fc region or fragment thereof containing at least one amino acid substitution selected from L234A, L234S, L234G, L235A, L235G, L235S, L235T, G236R, and D265C. Here, the amino acid numbering is according to the EU numbering system. The EU numbering system is also sometimes referred to as the "Kabat-like EU index" and refers to the numbering of human IgG1 EU antibodies, which refers to the EU antibody numbering in Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85.

[0089] Depending on the embodiment, the anti-GUCY2C antibody of the present invention includes an Fc region comprising one or a combination of the above amino acid substitutions, for example, two or more of the above amino acid substitutions (e.g., 2, 3, or 4) as shown below.

[0090] [Table 1]

[0091] In a preferred embodiment, the anti-GUCY2C antibody of the present invention comprises an Fc region comprising at least three amino acid substitutions disclosed herein, preferably the Fc region comprising at least the amino acid substitutions L234A, L235A, and D265C (SEQ ID NO: 71). The use of the Fc region comprising at least the substitutions L234A, L235A, and D265C in the anti-GUCY2C antibody of the present invention is particularly advantageous in enabling site-specific thiol-based binding of linker-payloads, such as those disclosed herein, while simultaneously reducing the interaction between the Fc region comprising the above mutations and FcγR by at least 95%, 97.5%, and 99% compared to a wild-type Fc region. Corresponding uses of such cysteine-substituted Fc regions comprising such mutations and antibodies for linker-payload binding are disclosed, for example, in WO2016142049A1.

[0092] In some embodiments, the anti-GUCY2C antibody of the present invention includes an Fc region comprising or consisting of the amino acid sequence relating to any of SEQ ID NOs: 72, 73, 74, or 75. The use of the above Fc region containing four mutations may be advantageous, for example, in eliminating residual interactions between the Fc region and FcγR, thereby obtaining the Fc-silencing antibody of the present invention.

[0093] Therefore, depending on the embodiment, the anti-GUCY2C antibody of the present invention may include the following pairs of light chain (LC) and heavy chain (HC) disclosed herein, which include at least one, preferably at least three, of the mutations disclosed herein.

[0094] [Table 2]

[0095] According to a preferred embodiment, the anti-GUCY2C antibody of the present invention comprises a light chain and a heavy chain selected from the following: LC relating to Sequence ID No. 78, HC relating to Sequence ID No. 80, or LC relating to Sequence ID No. 85, HC relating to Sequence ID No. 87, or LC relating to sequence number 92, HC relating to sequence number 94, or LC relating to SEQ ID NO: 99, HC relating to SEQ ID NO: 101, or LC relating to SEQ ID NO: 106, HC relating to SEQ ID NO: 108, or LC related to sequence number 113, HC related to sequence number 115.

[0096] According to a more preferred embodiment, the anti-GUCY2C antibody of the present invention comprises a light chain and a heavy chain selected from the following: LC relating to Sequence ID No. 78, HC relating to Sequence ID No. 80, or LC relating to Sequence ID No. 85, HC relating to Sequence ID No. 87, or LC related to sequence number 92, HC related to sequence number 94.

[0097] According to one embodiment, the anti-GUCY2C antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the antibody is a CDR selected from SEQ ID NOs: 34, 41, 46, 52; 36, 42, 47; 38, 44, or 49. H 1. CDR H 2, and CDR H Includes 3, and the framework region FR of the above antibody. H 1. FR H 2. FR H 3. FR H Sequence 4 shares at least 80%, 85%, preferably 90%, or 95% sequence similarity with any of sequence numbers 33, 40, 45, 51; 35; 55; 37, 43, 48, 53; 39, or 50.

[0098] The terms “sequence similarity,” “sequence identity,” or “sequence similar” are used interchangeably throughout this invention and refer to the similarity or identity of two or more amino acid or polynucleotide sequences to each other or to a reference sequence. Sequence identity according to this invention may be determined, for example, over the entire length of each sequence compared to each reference sequence (so-called “global alignment”), which is particularly preferred for sequences of the same or similar length. Alternatively, it may be determined over a shorter predetermined length (so-called “local alignment”), which is more preferred for sequences of different lengths. In the above context, an amino acid sequence having “sequence identity,” such as at least 95%, to a query amino acid sequence is intended to mean that the sequence of the target amino acid sequence is identical to the query sequence, except that the target amino acid sequence may contain up to 5 amino acid changes per 100 amino acids of the query amino acid sequence. For example, to obtain an amino acid sequence having at least 95% identity with the query amino acid sequence, up to 5% (5 out of 100) of the target sequence may be inserted, substituted, or deleted by other amino acids.

[0099] Methods for comparing the identity and similarity of two or more sequences are well known in the field. The percentage of identical sequences can be determined, for example, using mathematical algorithms. A preferred example of a mathematical algorithm that can be used is, but is not limited to, the algorithm described in Karlin et al. (1993), PNAS USA, 90:5873-5877. Such algorithms are incorporated into the BLAST family of programs (see also Altschul et al., 1990, J.Mol.Biol.215, 403-410, or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402, available from the NCBI homepage on the worldwide website ncbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol.83, 63-98; Pearson and Lipman (1988), Proc.Natl.Acad.Sci.USA 85, 2444-2448). Sequences that are somewhat identical to other sequences can be identified by these programs. Furthermore, the percentage of identity between two polypeptide sequences may be determined using programs available in the Wisconsin Sequence Analysis Package (Devereux et al, 1984, Nucleic Acids Res., 387-395; Womble Methods Mol Biol. 2000; 132:3-22), such as the programs BESTFIT and GAP. BESTFIT uses the "local sequence similarity" algorithm from Smith and Waterman (1981), J. Mol. Biol. 147, 195-197, to find one best-similar region between the two sequences. For example, "Gap BLAST" may be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast may be used to perform iterative searches to detect distant relationships between molecules.When using either the BLAST or GapBLAST program described above, you may use the default parameters for each program (e.g., XBLAST and NBLAST).

[0100] According to one embodiment, the present invention is (i) The anti-GUCY2C antibody of the present invention as disclosed herein, (ii) at least one toxin, and (iii) A complex comprising the antibody or antibody portion and at least one linker that binds the antibody or antibody portion to the at least one toxin, wherein the antibody specifically binds to human GUCY2C and the at least one toxin is an amatoxin.

[0101] The two terms “antibody” and “antibody portion” as used herein with respect to the complex of the present invention refer to the anti-GUCY2C antibody of the present invention disclosed herein and may be used interchangeably. The term “portion” may be used to indicate that the anti-GUCY2C antibody of the present invention is included in the complex of the present invention.

[0102] Accordingly, in one embodiment, the complex of the present invention comprises an anti-GUCY2C antibody disclosed herein. Preferably, the anti-GUCY2C antibody of the present invention comprises the light chain and heavy chain as shown in the following table.

[0103] [Table 3]

[0104] More preferably, the complex of the present invention comprises an anti-GUCY2C antibody moiety comprising a light chain amino acid sequence and a heavy chain amino acid sequence selected from the light chain amino acid sequence of SEQ ID NO: 78 and the heavy chain amino acid sequence of SEQ ID NO: 80; the light chain amino acid sequence of SEQ ID NO: 85 and the heavy chain amino acid sequence of SEQ ID NO: 87; the light chain amino acid sequence of SEQ ID NO: 92 and the heavy chain amino acid sequence of SEQ ID NO: 94; the light chain amino acid sequence of SEQ ID NO: 99 and the heavy chain amino acid sequence of SEQ ID NO: 101; the light chain amino acid sequence of SEQ ID NO: 106 and the heavy chain amino acid sequence of SEQ ID NO: 108; and the light chain amino acid sequence of SEQ ID NO: 113 and the heavy chain amino acid sequence of SEQ ID NO: 115. More preferably, the complex of the present invention comprises an anti-GUCY2C antibody moiety comprising a light chain amino acid sequence and a heavy chain amino acid sequence selected from SEQ ID NO: 78 and SEQ ID NO: 80, SEQ ID NO: 85 and SEQ ID NO: 87, or SEQ ID NO: 92 and SEQ ID NO: 94.

[0105] According to one embodiment, the complex of the present invention comprises at least one, for example, one, two, three, or four toxin molecules, wherein the toxin is an amatoxin conjugated to the anti-GUCY2C antibody of the present invention via at least one, for example, one or two linkers.

[0106] As used herein, the term "amatoxin" or "amatoxins" refers to the eight-amino acid bicyclic peptide found in Amanita phalloides (see Figure 1). Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby inhibiting transcription and protein biosynthesis in affected cells. When transcription is inhibited in cells, growth and proliferation are halted. Although not covalently bonded, the complex between amanitin and RNA polymerase II is very tightly bonded (K D (=3nM). The dissociation of amanitin from the enzyme is a very slow process, and therefore, recovery of affected cells is unlikely. If the inhibition of transcription continues for a sufficiently long time, the cells will undergo programmed cell death (apoptosis).

[0107] In the context of the present invention, the term "amatoxin" includes any bicyclic peptide consisting of eight amino acids isolated from the genus Amanita, as described by Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260), as well as any chemical derivative thereof, as well as any semi-synthetic analog thereof, as well as any synthetic analog thereof composed of components relating to the master structure (cyclic, eight amino acids) of the natural compound, as well as any synthetic or semi-synthetic analog containing non-hydroxylated amino acids instead of hydroxylated amino acids, as well as any synthetic or semi-synthetic analog in which the sulfoxide portion is substituted with an atom other than sulfone, thioether, or sulfur (for example, a carbon atom, as in the carbon analog of amanitin).

[0108] As used herein, a “derivative” of a compound refers to a species having a chemical structure similar to the compound but containing at least one chemical group not present in the originating compound and / or lacking at least one chemical group present in the originating compound. The compound compared to a derivative is known as the “parent” compound. Typically, the “derivative” may be produced from the parent compound in one or more chemical reaction steps.

[0109] As used herein, an “analog” of a compound is a structurally related compound that is not identical to it and exhibits at least one of its activities. The compound compared to the analog is known as the “parent” compound. Examples of such activities include, but are not limited to, binding activity to another compound, inhibitory activity such as enzyme inhibitory activity, toxic effects, and activating activity such as enzyme activation activity. The analog is not required to exhibit such activity to the same degree as the parent compound. A compound is considered an analog within the context of this application if it exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%) of the activity of the parent compound. Therefore, as used in the present invention, "amatoxin analogue" refers to a compound that is structurally related to any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid, and exhibits inhibitory activity against mammalian RNA polymerase II at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, 50%, 60%, more preferably at least 70%, 80%, 90%) compared to at least one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid. A suitable "amatoxin analog" for use in the present invention may even exhibit higher inhibitory activity against mammalian RNA polymerase II than any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, or amanulinic acid. The inhibitory activity is measured at a concentration (IC) at which 50% inhibition occurs. 50The inhibitory activity against mammalian RNA polymerase II can be determined indirectly by measuring the inhibitory activity against cell proliferation, or the inhibitory activity of the amatoxins and their derivatives disclosed herein may be evaluated, for example, using the RNA polymerase II activity assay disclosed in Voss et al. BMC Molecular Biology 2014, 15:7.

[0110] A "semi-synthetic analog" refers to an analog obtained by chemical synthesis using a compound derived from a natural source (e.g., plant material, bacterial culture, fungal culture, or cell culture) as an initial material. Typically, the "semi-synthetic analogs" of this invention are synthesized starting from a compound isolated from a mushroom of the Amanita family. In contrast, a "synthetic analog" refers to an analog synthesized by so-called total synthesis from small (typically petrochemical) components. This total synthesis is usually carried out without the assistance of biological processes.

[0111] According to some embodiments of the present invention, amatoxin can be selected from the group consisting of α-amanitin, β-amanitin, amanin, amaninamide, analogs thereof, derivatives thereof, and salts thereof.

[0112] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins have functional groups (e.g., carboxyl groups, amino groups, hydroxyl groups, thiol groups, or thiol-scavenging groups) that can react with the linker molecule or target binding moiety as defined below.

[0113] In the context of the present invention, the term "amanitin" particularly refers to a bicyclic structure based on an aspartic acid or asparagine residue at position 1, a proline residue at position 2, particularly a hydroxyproline residue, isoleucine, hydroxyisoleucine, or dihydroxyisoleucine (or aspartic acid in the case of amanulinic acid) at position 3, a tryptophan or hydroxytryptophan residue at position 4 (or proline in the case of proamanulin), glycine residues at positions 5 and 7 (or isoleucine residues in the case of amanulinic acid and proamanulin), an isoleucine residue at position 6, and a cysteine ​​residue at position 8, particularly when oxidized. This refers to cysteine ​​derivatives that have been converted into hydroxylated or sulfonated derivatives (see Figure 1 for amanitin numbering and representative examples), and further includes all of these chemical derivatives, all of these semi-synthetic analogs, all of these synthetic analogs composed of components related to the master structure of the natural compound (cyclic, 8 amino acids), all of these synthetic or semi-synthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids, and all of these synthetic or semi-synthetic analogs, but in any case, any of these derivatives or analogs are functionally active by inhibiting mammalian RNA polymerase II. Throughout this application, the term "amino acid 1" in relation to amatoxin refers to the asparagine residue of each amatoxin shown in Figure 1. Therefore, "amino acid 2" refers to the hydroxyproline residue, "amino acid 3" refers to the dihydroxyisoleucine residue, "amino acid 4" refers to the hydroxytryptophan residue, "amino acid 5" and "amino acid 7" refer to the glycine residue, "amino acid 6" refers to the isoleucine residue, and "amino acid 8" refers to the cysteinyl residue.

[0114] According to some embodiments of the present invention, the linker is bound to the antibody of the present invention via any of the natural Cys residues of the antibody, preferably via a disulfide bond. Preferred cysteine ​​residues for binding the linker to the anti-GUCY2C antibody of the present invention are cysteine ​​residues that form interchain disulfide crosslinks. For example, if the antibody is not a cysteine-substituted or cysteine-modified antibody, the binding of the linker to the cysteine ​​residue that forms an interchain disulfide crosslink of the IgG1 antibody of the present invention may be used in such a case. Throughout this application, the term “cysteine-substituted antibody” may be used interchangeably with “cysteine-modified antibody,” and both terms refer to an antibody in which at least one naturally occurring amino acid has been mutated to cysteine, as disclosed, for example, in WO2016040856A2 or WO2016142049A1. The use of a cysteine-modified antibody for the production of the complex of the present invention is preferred because the coupling reaction of the amatoxin-linker construct with the anti-GUCY2C antibody of the present invention yields a complex with a drug-antibody ratio (DAR) of approximately 2. Furthermore, the use of a cysteine-modified antibody does not damage the interchain disulfide bonds of the anti-GUCY2C antibody of the present invention, resulting in a more stable complex compared to when interchain disulfide bonds are used for the linker-amatoxin construct coupling. In addition, the resulting complex of the present invention with a DAR of 2 is characterized by very similar or identical pharmacokinetics as a result of the nearly homogeneous (e.g., >90%, 95% homogeneous) distribution of the complex species compared to interchain complexes where the DAR varies, for example, from about 1 to about 4. As used herein, the term “homogeneous” means that a single type of antibody-drug complex is present in a given sample, and for example, nearly homogeneous means that >90%, >95% of the ADCs or complex species have the same DAR, e.g., DAR=2.

[0115] According to a preferred embodiment, the linker is conjugated via a disulfide bond between Cys265 of the anti-GUCY2C antibody of the present invention and the linker (numbered according to the EU numbering system). Accordingly, the anti-GUCY2C antibody of the present invention comprises a genetically modified Fc region containing at least the amino acid substitution D265C (according to the EU numbering system) (sometimes referred to as Cys265).

[0116] According to some embodiments, the linker of the complex of the present invention is either a non-cleavable linker or a cleavable linker. As used herein, the term “linker” means a divalent chemical moiety comprising a covalent bond or atomic chain that covalently binds the antibody (e.g., the anti-GUCY2C antibody or a fragment thereof of the present invention) to the amatoxin, as described herein.

[0117] A “non-cleavable linker” is understood to be one that has not undergone enzymatic cleavage by cathepsin B or the like, and is released from the complex of the present invention during degradation from the antibody portion of the complex of the present invention within the target cell (e.g., lysosomal degradation). Non-cleavable linkers suitable for use according to the present invention may include, for example, one or more groups selected from binding, -(C=O)-, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkylylene, C2-C6 heteroalkylylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be substituted and / or may contain one or more heteroatoms (e.g., S, N, or O) instead of one or more carbon atoms. Non-limiting examples of such groups include (CH2) p (C=O)(CH2) p , and polyethylene glycol (PEG; (CH2CH2O) p The units are listed below. Here, p is an integer from 1 to 6, and is selected independently according to each case.

[0118] Depending on the embodiment, the non-cleavable linker according to the present invention may include a bond, -(C=O)-, -C(O)NH- group, -OC(O)NH- group, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkylylene, C2-C6 heteroalkylylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, -(CH2CH2O) p - comprising one or more groups (p is an integer from 1 to 6), where each C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkylylene, C2-C6 heteroalkylylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may be independently substituted with 1 to 5 substituents selected in each case from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0119] For example, the non-cleavable linkers disclosed herein, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkylylene, C2-C6 heteroalkylylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, may each be interrupted by one or more heteroatoms selected from O, S, and N, and may be independently substituted with 1 to 5 substituents selected in each case from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0120] According to a preferred embodiment, the non-cleavable linker of the composite of the present invention is -(CH2) n -Includes the unit, where n is an integer between 2 and 12, for example, 4 to 6, 8, 10, or 2 to 6, for example, n is 1, 2, 3, 4, 5, or 6.

[0121] In one preferred embodiment, the non-cleavable linker of the composite of the present invention is -(CH2) n - includes, where n is 6, and the above linker is expressed by the following formula: [ka]

[0122] Depending on the embodiment, the cleavable linker of the present invention disclosed herein further comprises a thiol reactive group. The thiol reactive group of the above-disclosed cleavable linker may be selected from, for example, bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazine-2-ylamino group methylsulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate, or maleimide.

[0123] According to one preferred embodiment, the thiol reactive group is the maleimide (merymidyl moiety) disclosed above. For example, the non-cleavable linker containing the maleimide may have, for example, the following structure, where the wavy line at the linker end indicates the binding site with amatoxin. [ka]

[0124] Following binding to a reactive sulfhydryl such as an anti-GUCY2C antibody disclosed herein, the meleymidyl moiety, such as a cleavable or non-cleavable linker disclosed herein, comprises the following structure. [ka] Here, the wavy lines represent binding sites of cleavable or incleavable linkers disclosed herein, and the sulfur atoms are part of the reactive cysteine, preferably Cys265, contained in the antibody of the present invention. The above structure may also be referred to as "X" or "Z".

[0125] In a preferred embodiment, the complex of the present invention, comprising a cleavable or incleavable linker disclosed herein and further comprising a thiol reactive group, may be bound to a naturally occurring sulfhydryl moiety in the antibody of the complex, or the cleavable or incleavable linker of the complex of the present invention comprising a thiol reactive group may be bound to a sulfhydryl moiety introduced into the antibody by genetic modification as described in Junutula Nat Biotechnol. 2008 Aug;26(8):925-32, etc. Preferably, the cleavable or incleavable linker disclosed herein comprising a thio reactive group is bound to a sulfhydryl moiety introduced into the Fc region of the anti-GUCY2C antibody of the complex of the present invention, for example, D265C (according to EU numbering), by genetic modification as disclosed herein.

[0126] A “cleavable linker” is understood to include at least one cleavage site. As used herein, the term “cleavage site” refers to a region that is susceptible to specific cleavage at a given location under specific conditions. These conditions include, for example, a specific enzyme or a reducing environment in a specific body or cellular compartment.

[0127] According to some embodiments, the cleavage site may be cleaved by at least one protease selected from the group consisting of cysteine ​​protease, metalloprotease, serine protease, threonine protease, and aspartate protease.

[0128] Cysteine ​​proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism involving nucleophilic cysteinethiols in three or two catalytic residues.

[0129] Metalloproteinases are proteases whose catalytic mechanism involves metals. Most metalloproteinases require zinc, but some use cobalt. The metal ion coordinates to the protein via three ligands. The ligands that coordinate to the metal ion can vary depending on whether it is histidine, glutamate, aspartate, lysine, or arginine. A fourth coordination site is occupied by an unstable water molecule.

[0130] Serine proteases are enzymes that cleave peptide bonds in proteins, with serine acting as a nucleophilic amino acid in the enzyme's active site. Based on their structure, serine proteases are broadly classified into two categories: chymotrypsin-like (trypsin-like) or subtilisin-like.

[0131] Threonine proteases are a family of proteolytic enzymes that contain a threonine (Thr) residue in their active site. The prototype member of this class of enzymes is the catalytic subunit of the proteasome, but acyltransferases have convergently evolved the same active site shape and mechanism.

[0132] Aspartate proteases are catalytic protease enzymes that use activated water molecules bound to one or more aspartate residues to catalyze the reaction of their peptide substrates. Generally, they have two highly conserved aspartate residues in their active site and are optimally active at acidic pH. Almost all known aspartyl proteases are inhibited by pepstatin.

[0133] In some embodiments of the present invention, the cleavable site can be cleaved by at least one agent selected from the group consisting of cathepsin A or B, matrix metalloproteinase (MMP), elastase, β-glucuronidase, and β-galactosidase, preferably cathepsin B.

[0134] In some embodiments of the present invention, the cleavage site is a disulfide bond, and specific cleavage is performed by a reducing environment, such as an intracellular reducing environment or acidic pH conditions. For example, the corresponding linker may have the following structure. (Amatoxin)-(CH2)2-SS-(CH2)2-XS-(Antibody) (Amatoxin)-(CH2)3-SS-(CH2)2-XS-(Antibody) (Amatoxin)-(CH2)2-SS-(CH2)3-XS-(Antibody) (Amatoxin)-(CH2)3-SS-(CH2)3-XS-(Antibody) Here, X is as disclosed above.

[0135] In some embodiments, the linker is a pH-sensitive linker, susceptible to hydrolysis at a certain pH value. Typically, a pH-sensitive linker is cleavable under acidic conditions. This cleavage strategy generally takes advantage of the lower pH of the intracellular compartments of endosomes (pH approximately 5-6) and lysosomes (pH approximately 4.8) compared to the cytosol (pH approximately 7.4) to cause hydrolysis of acid-unstable groups in the linker, such as hydrazones (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-unstable and / or hydrolyzable linker. For example, an acid-unstable linker containing acid-unstable groups (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. See, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123; Neville et al. (1989) Biol. Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, which approximates the pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether bound to the therapeutic agent via an acylhydrazone linkage). See, for example, U.S. Patent No. 5,622,929.

[0136] According to some embodiments of the present invention, the cleavable linker of the present invention is an enzymatically cleavable linker. The enzymatically cleavable linker includes a cleavage site which is an enzymatically cleavable portion containing two or more amino acids. Preferably, the enzymatically cleavable portion is phenylalanine-lysine (Phe-Lys), valine-lysine (Val-Lys), phenylalanine-alanine (Phe-Ala), valine-alanine (Val-Ala), phenylalanine-citrulline (Phe-Cit), or valine-citrulline (Val-Cit) dipeptide, or, for example, valine-alanine-valine (Val-Ala-Val), leucine-alanine-leucine (Leu-Ala-Leu), glycine-phenylalanine-lysine (Gly-Phe-Lys), isoleucine-alanine-leucine (Ile-Ala-Leu) tripeptide, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe-Lys-Gly-Pro-Leu-Gly), or alanine-alanine-proline-valine (Ala-Ala-Pro- Contains Val) peptide, or β-glucuronide or β-galactoside.

[0137] In a preferred embodiment, the cleavable linker of the complex of the present invention disclosed above is a self-destructing linker. The term “self-destructing linker” or “self-destructing spacer” refers to a bifunctional chemical moiety that can covalently bond two chemical moieties to form a normally stable tripolecular. When the bond with the first moiety is cleaved, the self-destructing spacer can spontaneously separate from the second moiety. Corresponding self-destructing linkers are disclosed, for example, in WO03026577 or WO2005 / 112919, which disclose a linker containing p-amide benzyl ether, and these may also be used, for example, in the complex of the present invention.

[0138] In a particularly preferred embodiment, the enzymatically cleavable linker according to the present invention comprises a dipeptide selected from Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit, and Val-Cit, and in particular, the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and amatoxin. [ka] [ka] [ka] [ka] [ka] [ka]

[0139] Accordingly, the complex of the present invention as disclosed herein may include an enzymatically cleavable moiety comprising any one of the dipeptide-PAB moieties disclosed above: Phe-Lys-PAB, Val-LysPAB, Phe-Ala-PAB, Val-Ala-PAB, Phe-Cit-PAB, or Val-Cit-PAB. Preferably, the cleavable moiety of the complex of the present invention comprises the dipeptide-PAB moiety: Val-Ala-PAB. [ka] Here, the PAB portion is bound to amatoxin.

[0140] According to some embodiments, the cleavable portion or cleavable linker of the present invention disclosed above comprises a thiol reactive group selected from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazine-2-ylamino group methylsulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate, or maleimide.

[0141] In one preferred embodiment, the thiol reactive group is the maleimide (merymidyl moiety) shown below. [ka]

[0142] Linkers containing the above-mentioned thiol reactive group (e.g., cleavable and / or non-cleavable linkers) are particularly useful for the covalent coupling of the linker-amatoxin complex disclosed herein to antibodies containing reactive thiols (e.g., cysteine-modified antibodies containing at least one reactive cysteine ​​residue for coupling). For example, the above-mentioned linker is particularly useful for the coupling of the linker-amatoxin complex disclosed herein to the cysteine-modified anti-GUCY2C antibody of the present invention containing the amino acid Cys265 (D265C, according to the EU numbering system).

[0143] According to one particularly preferred embodiment, the linker of the present invention comprises (i) a structure prior to coupling to an antibody disclosed herein, or (ii) a structure after said coupling. [ka] [ka]

[0144] In some embodiments of the present invention, the complex described comprises an amatoxin containing (i) an amino acid 4 having a 6'-deoxy position and (ii) an amino acid 8 having an S-deoxy position.

[0145] In some embodiments, the linker of the complex of the present invention is bound to amatoxin via (i) the γC-atom of amatoxin amino acid 1, (ii) the δC-atom of amatoxin amino acid 3, or (iii) the 6'-C-atom of amatoxin amino acid 4. The corresponding binding sites where the linker is bound to amatoxin are shown in Figure 1, where the binding site to the γC-atom of amatoxin amino acid 1 is indicated as "R3", the binding site to the δC-atom of amatoxin amino acid 3 is indicated as "R1", and the binding site to the 6'-C-atom of amatoxin amino acid 4 is indicated as "R4".

[0146] According to preferred embodiments of the present invention, the complexes of the present invention disclosed herein each contain, as linker-amatoxin moieties, any of the compounds of the following formulas (I) to (XI). [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

Chem.

Chem.

[0147] According to a preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody disclosed herein that is linked to an amatoxin linker moiety via a thioether bond according to any one of the following formulas XII to XXII.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0148] According to a preferred embodiment, the number n of amatoxin-linker complexes covalently bound to the anti-GUCY2C antibody of the present invention is about 1 to about 2, 3, 4, 6, 7, 8, or about 1, 2 to about 3, 4, or about 1.5, 1.6, 1.7, 1.8 to about 1.9, 2.0, 2.1, 2.2, 2.5, 3, or about 1.9, 2.0 to about 2.1, 2.2, and preferably n is about 2 (e.g., 1.8 to about 2.2).

[0149] According to a preferred embodiment, the composite of the present invention is selected from the group consisting of the following: - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XXIII) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XXIV) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, forming a complex (XXVI). - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXVII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXVIII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, forming a complex (XXIX). - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XIX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, forming a complex (XXX). - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 62, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 56, wherein the complex (XXXI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, forming a complex (XXXII). - A complex (XXXIII) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Here, n is approximately 1 to approximately 2.

[0150] According to a preferred embodiment, the composite of the present invention is selected from the group consisting of the following: - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XXXIV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XXXV) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XXXVI) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XXXVII) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XXXVIII) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XXXIX) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XL) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XLI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XLII) a complex comprising the antibody, - An antibody consisting of two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID NO: 57, wherein at least one amatoxin-linker moiety of formula (XXI) is linked via a thioether bond of the linker to the sulfhydryl group of the heavy chain 265Cys residue according to the EU numbering system of the said antibody, a complex (XLIII) comprising the antibody, - An antibody consisting of two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID NO: 57, wherein at least one amatoxin-linker moiety of formula (XXII) is linked via a thioether bond of the linker to the sulfhydryl group of the heavy chain 265Cys residue according to the EU numbering system of the said antibody, a complex (XLIV) comprising the antibody. Here, n is from about 1 to about 2.

[0151] According to a preferred embodiment, the complex of the present invention is selected from the group consisting of: - An antibody consisting of two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID NO: 58, wherein at least one amatoxin-linker moiety of formula (XII) is linked via a thioether bond of the linker to the sulfhydryl group of the heavy chain 265Cys residue according to the EU numbering system of the said antibody, a complex (XLV) comprising the antibody, - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (XLVI) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein the complex (XLVII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 64, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 58, wherein the complex (XLVIII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein the complex (XLIX) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, and the antibody is further conjugated to the linker. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein the complex (LI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein the complex (LII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 64, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, forming a complex (LIII). - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LIV) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein the antibody is conjugated to at least one amatoxin-linker portion of formula (XXII) via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LV). Here, n is approximately 1 to approximately 2.

[0152] According to a preferred embodiment, the composite of the present invention is selected from the group consisting of the following: - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 65, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LVI) - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 65, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 59, wherein the complex (LVII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein the complex (LVIII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein the complex (LIX) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 65, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 59, wherein the complex (LX) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LXI) - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 65, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 59, wherein the complex (LXII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 65, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 59, wherein the complex (LXIII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 65, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 59, wherein the complex (LXIV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein the complex (LXV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - A complex (LXVI) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Here, n is approximately 1 to approximately 2.

[0153] According to a preferred embodiment, the composite of the present invention is selected from the group consisting of the following: - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXVII), - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXVIII), - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein the complex (LXIX) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LXX) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein the complex (LXXI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, forming a complex (LXXII). - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 66, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 60, wherein the complex (LXXIII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein the complex (LXXIV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 66, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXV), - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein the complex (LXXVI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, - A complex (LXXVII) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Here, n is approximately 1 to approximately 2.

[0154] According to a preferred embodiment, the composite of the present invention is selected from the group consisting of the following: - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXVIII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXIX), - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LXXX) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXXI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LXXXII) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LXXXIII) - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXXIV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising an amino acid sequence corresponding to or containing the VH domain of SEQ ID NO: 67, and two light chains each comprising an amino acid sequence corresponding to or containing the VL domain of SEQ ID NO: 61, wherein the complex (LXXXV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXXVI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXXVII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. - An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the antibody is conjugated to at least one amatoxin-linker portion of formula (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXXVIII). Here, n is approximately 1 to approximately 2.

[0155] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0156] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0157] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO. 78, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0158] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0159] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0160] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0161] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0162] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XIX) contains an anti-GUCY2C antibody conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0163] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0164] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0165] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0166] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0167] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0168] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0169] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0170] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0171] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0172] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0173] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XIX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0174] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0175] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0176] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0177] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0178] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0179] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0180] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0181] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0182] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0183] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0184] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XIX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0185] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0186] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0187] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0188] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0189] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0190] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0191] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0192] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0193] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0194] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0195] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XIX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0196] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0197] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0198] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0199] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0200] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0201] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0202] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0203] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0204] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0205] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0206] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0207] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XIX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0208] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0209] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0210] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0211] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0212] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0213] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0214] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0215] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0216] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0217] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0218] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XIX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0219] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising, each, two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0220] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0221] According to some embodiments, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether linkage of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0222] According to a more preferred embodiment, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each containing the amino acid sequence of SEQ ID NO: 62 and two light chains each containing the amino acid sequence of SEQ ID NO: 56, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0223] According to a more preferred embodiment, the complex of the present invention is an anti-GUCY2C antibody comprising two heavy chains each containing the amino acid sequence of SEQ ID NO: 63 and two light chains each containing the amino acid sequence of SEQ ID NO: 57, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0224] According to a more preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains comprising or containing the amino acid sequence of SEQ ID NO: 80, and two light chains comprising or containing the amino acid sequence of SEQ ID NO: 78, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0225] According to a more preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains comprising or containing the amino acid sequence according to SEQ ID NO: 87, and two light chains comprising or containing the amino acid sequence according to SEQ ID NO: 85, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0226] According to a more preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains comprising or containing the amino acid sequence of SEQ ID NO: 94 and two light chains comprising or containing the amino acid sequence of SEQ ID NO: 92, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0227] In a more preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains each comprising or containing the amino acid sequence relating to SEQ ID NO: 101, and two light chains each comprising or containing the amino acid sequence relating to SEQ ID NO: 99, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0228] According to a more preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains comprising or containing the amino acid sequence of SEQ ID NO: 108 and two light chains comprising or containing the amino acid sequence of SEQ ID NO: 106, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0229] According to a more preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains each comprising or containing the amino acid sequence relating to SEQ ID NO: 115, and two light chains each comprising or containing the amino acid sequence relating to SEQ ID NO: 113, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0230] In one particularly preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains each consisting of or containing the amino acid sequence of SEQ ID NO: 80 and two light chains each consisting of or containing the amino acid sequence of SEQ ID NO: 78, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

[0231] In one particularly preferred embodiment, the complex of the present invention comprises an anti-GUCY2C antibody comprising two heavy chains each comprising or containing the amino acid sequence corresponding to SEQ ID NO: 87, and two light chains each comprising or containing the amino acid sequence corresponding to SEQ ID NO: 85, wherein the anti-GUCY2C antibody is conjugated to at least one amatoxin-linker moiety selected from formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, and more preferably, the amatoxin-linker moiety is selected from one of formulas (XII), (XIII), or (XIV).

[0232] Depending on the embodiment, the composite according to the present invention disclosed above is an IC 50 However, 10x10 -9 M, 9×10 -9 M, 8×10 -9 M, 7×10 -9 M, 6×10 -9 M, 5×10 -9 M, 4×10 -9 M, 3×10 -9 M, or 2×10 -9 Higher than M, preferably 10 × 10 -10 M, 9×10 -10 M, 8×10 -10 M, 7×10 -10 M, 6×10 -10 M, 5×10 -10 M, 4×10 -10 M, 3×10 -10 M, or 2×10 -10 Higher than M, more comfortable, 9x10 -11 M, 8×10 -11 M, 7×10 -11 M, 6×10 -11 M, 5×10 -11 M, 4×10 -11 M, 3×10 -11 M, 2×10 -11 M, or 1 × 10-11 It can have higher cytotoxic activity than M.

[0233] In one embodiment, the present invention provides a polynucleotide encoding an amino acid sequence including any of SEQ ID NOs: 62, 63, 64, 65, 66, or 67.

[0234] According to one embodiment, the present invention provides a polynucleotide encoding an amino acid sequence including any of SEQ ID NOs: 56, 57, 58, 59, 60, or 61.

[0235] According to one embodiment, the polynucleotide of the present invention is the following V H Array and V L Array combinations: - Sequence IDs 62, 56 - Sequence IDs 63, 57 - Sequence IDs 64, 58 - Sequence IDs 65, 59 - Sequence IDs 66, 60 - Sequence IDs 67, 61, Or V above L Array and V H Amino acid sequences that are at least 90% or 95% similar to any of the sequences. The more selected amino acid sequences are encoded, each containing one or a combination of them as disclosed above.

[0236] As used herein, the term "polynucleotide" refers to a nucleic acid sequence. The nucleic acid sequence may be a DNA sequence or an RNA sequence, and preferably, the nucleic acid sequence is a DNA sequence. The polynucleotides of the present invention are preferably provided as isolated (i.e., isolated from their natural context) polynucleotides or in a genetically modified form. Isolated polynucleotides as used herein may also include, for example, polynucleotides present in cellular contexts other than their natural cellular context, i.e., heterologous polynucleotides. The term polynucleotide includes single-stranded polynucleotides and double-stranded polynucleotides. It also includes chemically modified polynucleotides, including naturally derived modified polynucleotides (e.g., glycosylated or methylated polynucleotides) or artificially modified polynucleotides (e.g., biotinylated polynucleotides).

[0237] According to one embodiment, the present invention provides an expression vector comprising the polynucleotide of the present invention. The expression vector according to the present invention may comprise, for example, at least one polynucleotide encoding an amino acid sequence including an amino acid sequence selected from SEQ ID NOs. 62, 63, 64, 65, 66, or 67, or at least one polynucleotide encoding an amino acid sequence including an amino acid sequence selected from SEQ ID NOs. 56, 57, 58, 59, 60, or 61. Alternatively, the expression vector may comprise, for example, one polynucleotide encoding an amino acid sequence including an amino acid sequence selected from SEQ ID NOs. 62, 63, 64, 65, 66, or 67, and one polynucleotide encoding an amino acid sequence including an amino acid sequence selected from SEQ ID NOs. 56, 57, 58, 59, 60, or 61. For example, the expression vector may comprise a polynucleotide encoding an amino acid sequence including an amino acid sequence relating to SEQ ID NOs. 62, 56; SEQ ID NOs. 63, 57; SEQ ID NOs. 64, 58; SEQ ID NOs. 65, 59; SEQ ID NOs. 66, 60; or SEQ ID NOs. 67, 61. The term “expression vector” or “vector” preferably includes plasmids, phages, viruses, or retroviral vectors, or artificial chromosomes such as bacterial or yeast artificial chromosomes. Preferably, the expression vector comprising the polynucleotide of the present invention further comprises a selectable marker for proliferation and / or selection in a suitable host cell. The polynucleotide according to the present invention contained in the expression vector is operably bound to an expression regulatory sequence that enables expression or proliferation in prokaryotic or eukaryotic cells, particularly eukaryotic cells or isolated fractions thereof. The expression of the polynucleotide comprises transcription of the polynucleotide, preferably to translatable mRNA. Furthermore, the expression vector of the present invention comprises a regulatory sequence that ensures the initiation of transcription and optionally a polyA signal that ensures the termination of transcription and stabilization of the transcript. Further regulatory elements may include transcription and translation enhancers.Regulatory elements that may allow expression in prokaryotic host cells include, for example, the lac, trp, or tac promoters in E. coli, while examples of regulatory elements that allow expression in eukaryotic host cells include the AOX1 or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Roussarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells. Suitable expression regulatory sequences are well known in the art. In addition to elements involved in transcription initiation, such regulatory elements may also include transcription termination signals such as the SV40-polyA site or tk-polyA site downstream of the polynucleotide. Expression vectors are known in the art and include, for example, pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogene), or pSPORT1 (GIBCO BRL). The expression vectors of the present invention may be incorporated into host cells, for example, by various techniques known in the art. For example, plasmid vectors can be introduced into precipitates such as calcium phosphate precipitate or rubidium chloride precipitate, or into complexes with charged lipids, or into carbon-based clusters such as fullerenes. Alternatively, plasmid vectors may be introduced by heat shock or electroporation. If the expression vector is a virus, it may be packaged in vitro using an appropriate packaging cell line before being applied to host cells. Retroviral vectors may be replicable or non-replicable. In the latter case, viral replication will generally occur only in complementary hosts / cells.Available transformation and transfect methods have been reviewed in Fus-Kujawa A. et al (2021) An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro. Front. Bioeng. Biotechnol. 9:701031, or Chong ZX, et al. (2021) Transfection types, methods and strategies: a technical review. PeerJ 9:e11165, and transfect protocols are, for example, “Molecular Cloning - A laboratory manual”, 4. th This information is found in the 2001 edition, ISBN 978-1-936113-42-2, chapter 15, pages 1131-1203.

[0238] According to one embodiment, the present invention relates to a host cell comprising the expression vector or polynucleotide of the present invention disclosed herein. As used herein, “host cell” means a prokaryotic or eukaryotic cell comprising the expression vector or polynucleotide of the present invention. The host cell of the present invention is preferably a eukaryotic cell such as yeast cells (e.g., Saccharomyces cerevisiae, Hansenula polymorphia, Schizosaccharomyces pombe, Schwaniomyces occidentalis, Kluiveromyces lactis, Yarowia liporitica, and Pichia pastris), insect cells (e.g., Sf9, Sf21, S2, Hi5, or BTI-TN-5B1-4). More preferably, the host cells of the present invention are HEK293, HEK293T, HEK293E, HEK293F, NS0, per.C6, MCF-7, HeLa, Cos-1, Cos-7, PC-12, 3T3, Vero, vero-76, PC3, U87, SAOS-2, LNCAP, DU145, A431, A549, B35, H1299, HUVEC, Jurkat, MDA-MB-231, MDA-MB-468, MDA-MB-435, Caco-2, CHO, CH The host cells are mammalian cells selected from O-K1, CHO-B11, CHO-DG44, BHK, AGE1.HN, Namalwa, WI-38, MRC-5, HepG2, L-929, RAB-9, SIRC, RK13, 11B11, 1D3, 2.4G2, A-10, B-35, C-6, F4 / 80, IEC-18, L2, MH1C1, NRK, NRK-49F, NRK-52E, RMC, CV-1, BT, MDBK, CPAE, MDCK.1, MDCK.2, and D-17. More preferably, the host cells are selected from CHO, CHO-K1, CHO-B11, CHO-DG44, HEK293, HEK293T, HEK293E, HEK293F, NS0, or per.C6 cells.

[0239] In one embodiment, the present invention relates to the use of the anti-GUCY2C antibody of the present invention in the production of the complex of the present invention disclosed herein.

[0240] According to a second aspect, an object of the present invention is to provide a pharmaceutical composition comprising the complex of the present invention disclosed herein.

[0241] The above pharmaceutical composition may further contain, for example, one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, flow promoters, disintegrants, adsorbents, and / or preservatives.

[0242] The above-mentioned pharmaceutical preparations may be prepared for administration in aqueous form, or in lyophilized form, they may be converted to a liquid form by adding sterile water for injection before administration. This sterile water for injection may or may not contain preservatives. Examples of preservatives include, but are not limited to, benzyl alcohol, antioxidants (such as vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), amino acids such as cysteine ​​and methionine, citric acid and sodium citrate, and synthetic preservatives such as parabens such as methylparaben and propylparaben.

[0243] The above pharmaceutical formulation may further contain one or more stabilizers, for example, an amino acid, a sugar polyol, a disaccharide, and / or a polysaccharide. The above pharmaceutical formulation may further contain one or more surfactants, one or more isotonic agents, and / or one or more metal ion chelating agents, and / or one or more preservatives.

[0244] The pharmaceutical formulations described herein may be suitable for at least intravenous, intramuscular, or subcutaneous administration. Alternatively, the complex according to the present invention may be provided in a depot formulation that enables sustained release of a biological activator over a period of time.

[0245] In yet another embodiment of the present invention, a primary package containing the above-described formulation according to the above-described embodiment of the present invention is provided (for example, a drug-filled syringe or pen, vial, or infusion bag).

[0246] The drug-filled syringe or pen may contain the formulation in a lyophilized form (which must then be solubilized with sterile water for injection before administration) or in an aqueous form. The syringe or pen is often a single-use, disposable item and may have a volume between 0.1 ml and 20 ml. However, the syringe or pen may also be for multiple uses or multiple doses.

[0247] The vials described above may also contain formulations in lyophilized or aqueous form and may function as single-use or multi-use devices.

[0248] According to one embodiment, the present invention is - At least one immune checkpoint inhibitor, and - At least one composite according to the present invention as disclosed above herein The present invention provides a composition containing the following:

[0249] In the context of the present invention, the term “immune checkpoint inhibitor,” or simply “checkpoint inhibitor” or “ICI,” means any agent or compound that directly or indirectly reduces the amount or inhibits the function of an immune checkpoint receptor protein or molecule found on the surface of immune cells (e.g., T cells), or any agent or compound that directly or indirectly reduces the amount or inhibits the function of a ligand that binds to the immune checkpoint receptor protein or molecule, either as a soluble compound or on the surface of an immune cell inhibitory cell. Such inhibitory cells may be, for example, cancer cells, regulatory T cells, tolerogenic antigen-presenting cells, bone marrow-derived suppressor cells, tumor-associated macrophages, or cancer-associated fibroblasts. The ligand can typically bind to an immune checkpoint receptor protein or molecule on immune cells. Non-limiting examples of immune checkpoint receptor protein-ligand pairs are PD-1 and PD-L1. PD-1 is an immune checkpoint receptor protein found on T cells. PD-L1, which can be overexpressed by cancer cells, binds to PD-1 and helps cancer cells evade attack by the host immune system. Therefore, immune checkpoint inhibitors prevent PD-1 / PD-L1 interactions by blocking PD-1 on T cells (i.e., acting as PD-I inhibitors) or PD-L1 on cancer cells (i.e., acting as PD-L1 inhibitors), thereby maintaining or restoring antitumor T cell activity or blocking inhibitory cancer cell activity. Other non-exclusive examples of immune checkpoint inhibitors include CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, OX40, GITR, ICOS, CD276(B7-H3), B7-H4(VTCN1), IDO, KIR, CD122, CD137, CD94 / NKG2A, CD80, CD86, Galectin-3, LSECtin, CD112, Ceacam-1, Gal-9, PtdSer, HMGB1, HVEM, CD155, and BTLA(CD272). The corresponding compositions and their respective uses are disclosed in WO2022 / 096604.

[0250] According to one embodiment, the immune checkpoint inhibitor according to the present invention is an antibody selected from the group consisting of nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, semiprimab, ipilimumab, PD-1, PD-2, PD-3, PD-4, or PD-5, tremelimumab, or antigen-binding fragments thereof, or antigen-binding derivatives thereof. Preferably, the antibody is avelumab, pembrolizumab, nivolumab, or ipilimumab, or an antigen-binding fragment thereof, or an antigen-binding derivative of the antibody.

[0251] Nivolumab (CAS Registry No.: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 and lacks detectable antibody-dependent cytotoxicity (ADCC). Nivolumab is disclosed, for example, in US8,008,449 and WO2006 / 121168. Nivolumab has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0252] Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab is disclosed, for example, in WO2009 / 101611.

[0253] Pembrolizumab (formerly known as lambrolizumab; trade name Keytruda; also known as MK-3475), disclosed by Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, is a humanized IgG4 monoclonal antibody that binds to PD-1 and contains a mutation at C228P designed to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed, for example, in US8,354,509 and WO2009 / 114335. Pembrolizumab is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0254] Atezolizumab is a fully humanized, modified monoclonal antibody against PD-L using an IgG1 isotype, disclosed in WO2010 / 077634.

[0255] Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody disclosed in WO2013 / 079174A1.

[0256] Durvalumab is a human immunoglobulin G1 kappa (IgG1κ) anti-PD-L1 monoclonal antibody disclosed in WO2011 / 066389A1.

[0257] Semiprimab is a human IgG4 monoclonal antibody that binds to PD-1 and thereby blocks its interaction with PD-L1 and PD-L2. Semiprimab is disclosed as H4H7798N in WO15112800A1.

[0258] Ipilimumab (CAS registry number: 477202-00-9, sometimes also known as 10D1, MDX010, or MDX-101) is a human IgG1 antibody that binds to cytotoxic T-lymphocyte antigen-4 (CTLA4). CTLA-4 is an inhibitory molecule that competes with stimulating CD28 for binding to B7 on antigen-presenting cells. Both CTLA-4 and CD28 are presented on the surface of T cells. Ipilimumab is a human IgG1 that binds to CTLA-4 to prevent the inhibition of the T-cell-mediated immune response against tumors. Ipilimumab is disclosed, for example, as the antibody "10D1" in WO01 / 14424.

[0259] PD1-1 to PD1-5 refer to the anti-PD-1 antibodies disclosed in WO2018 / 220169.

[0260] Tremelimumab (sometimes called tisilimmab, CP-675, CP-675,206, CAS registry number 745013-59-6) is a fully human anti-CTLA4 antibody.

[0261] As used herein, INN includes all biosimilar antibodies of the corresponding founding antibody disclosed herein, including, but not limited to, biosimilar antibodies permitted under, for example, 42 United States Code § 262(k) in the United States and equivalent rules in other jurisdictions.

[0262] In one embodiment, the present invention relates to a complex, a pharmaceutical composition, or a composition of the present invention disclosed herein for use in the treatment of cancer, wherein the cancer is selected from gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), or liver cancer. As used herein, the term “treatment” or phrase “for treating” means any type of treatment that benefits a patient suffering from a disease such as cancer, particularly gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), or pancreatic cancer, including improvement of the patient’s condition (e.g., one or more symptoms) and / or delay of the progression of said condition.

[0263] According to one embodiment, gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), or liver cancer disclosed herein is characterized by a deletion or translocation of chromosome 17p13.1, the deletion of which may be, for example, a hemizygous deletion of chromosome 17p13.1. Accordingly, gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), or liver cancer disclosed herein, treated with the complex, pharmaceutical composition, or composition of the present invention disclosed herein, is characterized by a deletion or hemizygous deletion of chromosome 17p13.1.

[0264] According to one embodiment, gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), or liver cancer disclosed herein, which may be treated with the complex, pharmaceutical composition, or composition of the present invention disclosed herein, are characterized by a POLR2A gene, or a hemizygous deletion of the TP53 gene and the POLR2A gene. The term “hemizygous” as used herein refers to an individual or cell that has only one complete allele of a gene or chromosomal segment, rather than the usual two. A hemizygous cell or organism is one in which the genome contains only one complete allele at a given locus, whether the allele is wild-type or mutant. For example, the cells of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, and pancreatic cancer disclosed herein are hemizygous with respect to chromosomal locus 17p13. Preferably, the cells of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, and pancreatic cancer disclosed above are hemizygotes for the genes TP53 and POLR2A. As used herein, “TP53” refers to the “tumor protein 53” gene which encodes a tumor suppressor protein (P53) comprising a transcriptional activation domain, a DNA-binding domain, and an oligomerization domain. The encoded protein modulates the expression of target genes in response to various cellular stresses, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or metabolic changes. Mutations in this gene are associated with various human cancers, including hereditary cancers such as Leif Raumeni syndrome.

[0265] The tumor suppressor gene TP53 is often inactivated by mutation or deletion in most human tumors. As used herein, "POLR2A" refers to the POLR2A gene, which encodes the largest subunit of the human RNA polymerase II complex and is essential for polymerase activity in mRNA synthesis. Hemizygous deletions of chromosome 17p13, such as del(17p13.1), may be detected by fluorescence in situ hybridization (FISH) as disclosed in Merz et al. Am J Hematol. 2016 Nov;91(11):E473-E477.

[0266] The cells of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, and pancreatic cancer disclosed herein may not be homogeneous in terms of, for example, the loss of TP53 and / or POLR2A. For example, about 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% to about 70%, 75%, 80%, 85%, 90%, 95%, 100% of the cancer cells disclosed above may be about 70%, 75%, 80%, 85%, 90%, 95%, 100%, or about 70%, 75%, 80%, 85% to about 90%, 92.5%, 95%, 97.5%, 100% of the cancer cells may be about TP53 and / or POLR2A. Alternatively, they may be hemizygous with respect to POLR2A, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, and 95% of the cancer cells disclosed herein are hemizygous with respect to del(17p13) or TP53 and / or POLR2A.

[0267] For example, the complexes, pharmaceutical compositions, or compositions of the present invention may be particularly advantageous for use in the treatment of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, and pancreatic cancer characterized by hemizygous deletions of chromosome 17p13.1, or TP53 and / or POLR2A. This is because cells characterized by hemizygous deletions of chromosome 17p13.1, TP53, and / or POLR2A are at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, and 1000-fold more sensitive to the complexes, pharmaceutical compositions, or compositions of the present invention disclosed herein. Therefore, for example, it may be beneficial to determine whether the cancer cells disclosed herein include or consist of cells that are hemizygous for the loss of TP53 and / or POLR2A. This is because the desired therapeutic effect can be achieved using at least 10, 25, 50, 100, 250, 500, or 1000 times fewer of the complexes, pharmaceutical compositions, or compositions of the present invention disclosed herein. Assays for evaluating the sensitivity of cancer cells to the complexes, pharmaceutical compositions, or compositions of the present invention disclosed herein can be performed, for example, as described in Liu et al., Nature. 2015 April 30;520(7549):697-701.

[0268] According to one embodiment, the complex, pharmaceutical composition, or composition comprising the complex and an immune checkpoint inhibitor according to the present invention is for use in the treatment of a solid tumor in a patient, the solid tumor in the patient being characterized by the expression of GUCY2C on the surface of the cancer cells of the solid tumor. Accordingly, the tumor cells express, for example, amino acids 24-430 or any fragment of SEQ ID NO: 76 on their surface, which are recognized and specifically bound by the antibody, antibody fragment, or complex of the present invention disclosed herein. As used herein, “specifically bind” or “specifically bind” means that the binding of the antibody or complex of the present invention disclosed herein is such that the Kd for its antigen (e.g., an epitope of human GUCY2C) is at least about 10 -6 M, 10-7 M, 10 -8 M, or about 10 -8 M~about 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or approximately 5 x 10 -9 M, 5×10 -10 M ~ approx. 2.5×10 -11 M, 5×10 -11 M, 2.5 x 10 -12 M, 5×10 -12 This refers to being M. As used herein, the term “epitope” refers to a part of a macromolecule, preferably a polypeptide such as human GUCY2C that is recognized by an antigen-binding molecule, such as the antibody of the present invention disclosed herein, or its antigen-binding fragment, or its antigen-binding derivative, particularly by the antigen-binding site of the above molecule. An epitope defines the smallest binding site for an antibody molecule and thus represents the target of the antibody molecule’s specificity. Epitopes can be further defined as structural epitopes or functional epitopes. A “structural epitope” consists of amino acids or other molecules in a region that is in close contact with the antibody, usually revealed by its structure. A “functional epitope” is defined as such a part of a molecule that energetically contributes to binding such that changes would reduce the binding affinity. Therefore, whether proximal or paratope, the residues that come into contact with the paratope and which residues contribute to the affinity are important considerations when defining an epitope. Structural epitopes may be, for example, linear continuous sequences of approximately 5 to 50 or 100 amino acids in length, or higher-order structural epitopes formed by the three-dimensional structure of a polypeptide, which may also contain discontinuous amino acids of the polypeptide. The affinity of the anti-GUCY2C antibody or complex of the present invention can be measured using well-known methods such as in vitro assays using plasmon resonance (BIAcore, GE-Healthcare, Uppsala, Sweden).

[0269] The solid tumors disclosed above may also be characterized by, for example, a deletion of the POLR2A gene, or a hemizygous deletion of the TP53 gene and the POLR2A gene. For example, about 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% to about 70%, 75%, 80%, 85%, 90%, 95%, 100%, or about 70%, 75%, 80%, 85% to about 90%, 92.5%, 95%, 97.5%, 100% of the cells of the solid tumor may be characterized by, for example, a deletion of the POLR2A gene, or a hemizygous deletion of the TP53 gene and / or POLR2A gene. LR2A may be hemizygous, or, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, 95% of the cells of the solid tumor disclosed above are hemizygous for del(17p13), or TP53 and / or POLR2A.

[0270] According to one embodiment, the present invention also relates to an antibody that competes with the antibody of the present invention for binding to human GUCY2C. The antibody that competes with the antibody of the present invention for binding to human GUCY2C may be determined using an Octet HTX biosensor (Pall ForteBio) in a manner similar to that disclosed, for example, in WO2015 / 112800, or in a manner similar to that disclosed, for example, in Syedbasha et al., J Vis Exp. 2016;(109):53575. The entire contents of those documents are incorporated herein by reference.

[0271] According to one embodiment, the complex or pharmaceutical composition according to the present invention disclosed herein is for use in the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer in patients who have not responded to treatment (e.g., first-line or second-line treatment) including cetuximab or panitumumab alone, or in combination with 5-fluorouracil (5-FU) and orally administered capecitabine (CAP), FOLFOX (5-FU and oxaliplatin), FOLFIRI (5-FU and irinotecan), XELOX / CAPOX (CAP and oxaliplatin), CAPIRI (CAP and irinotecan), or FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin). As used herein, the terms “first-line therapy,” “first-line treatment,” or “first-line therapy” mean a therapy or treatment whose label does not include a requirement or recommendation that it should only be used after other therapies or treatments have been shown to be inadequate or unsuccessful. The terms may also include therapies and / or treatments that are not administered to individual subjects who require other active ingredients (other than the primary active ingredient). The term “second-line treatment” or “second-line therapy” means treatment of a disease or condition after initial treatment (first-line treatment) has failed, ceased to work, or shown unacceptable adverse effects due to serious side effects and associated loss of quality of life.

[0272] According to some embodiments, the complexes or pharmaceutical compositions of the present invention, either alone or optionally in combination with the immune checkpoint inhibitors disclosed above, are for use in the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer in patients who have failed standard treatment options, such as first-line and / or second-line treatments, or even third-line treatments. For example, the complexes or pharmaceutical compositions of the present invention may be for use in patients with CRC who have failed the above treatment options, or in patients with CRC who have failed third-line treatment with the combination drug trifluridine / tipiracil (TAS-102) after treatment with chemotherapy based on fluoropyrimidine, oxaliplatin, and irinotecan, anti-VEGF therapy, and, in the case of RAS-wild-type tumors, anti-EGFR therapy.

[0273] According to one embodiment, the complex (anti-GUCY2C complex) or pharmaceutical composition of the present invention is for use in the treatment of colorectal cancer or metastatic colorectal cancer (mCRC), which is characterized by KRAS mutations and / or BRAF mutations. Examples of KRAS mutations according to the present invention include, but are not limited to, KRAS mutations G13D, G12C, G12V, and G12D. Examples of BRAF mutations according to the present invention include, but are not limited to, BRAF mutations V600E, V600K, or V600R. Accordingly, the complex or pharmaceutical composition of the present invention is for use in the treatment of CRC or mCRC characterized by having one of the following mutations: KRAS G12C, KRAS G12V, KRAS G12D, G13D, and / or BRAF V600E, BRAF V600K, or BRAF V600R.

[0274] According to some embodiments, the complexes or pharmaceutical compositions of the present invention disclosed above for use in the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer include one or more immune checkpoint inhibitors disclosed above (e.g., anti-PD-1 checkpoint inhibitors (e.g., nivolumab, PD-1 to PD-5, pidilizumab, or pemprolizumab) or anti-PD-L1 checkpoint inhibitors (e.g., avelumab), anti-CTLA-4 checkpoint inhibitors (e.g., ipilimumab or tremelimumab), etc.) or KRAS inhibitors (e.g., BI It may also be used in combination with checkpoint inhibitors such as 1823911, sotrasib, adagrasib, LY3537982, JNJ-74699157), or KRAS-SOS1 inhibitors (e.g., BI1701963), or BRAF inhibitors (e.g., vemurafenib, dabrafenib, or encorafenib), or combinations of dabrafenib and trametinib, vemurafenib and cobimetinib, or encorafenib and binimetinib, or combinations of the above checkpoint inhibitors with KRAS inhibitors or BRAF inhibitors.

[0275] In one embodiment, the present invention relates to the use of the complex or pharmaceutical composition of the present invention in the manufacture of pharmaceuticals for the treatment of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), liver cancer, or GUCY2C-positive or GUCY2C-expressing solid tumors.

[0276] In other embodiments, the present invention relates to a method for treating a patient suffering from gastrointestinal cancer, the method comprising administering to the patient a therapeutically effective amount of the complex or pharmaceutical composition of the present invention alone or in combination with further pharmacologically active compounds. Further pharmacologically active compounds include, but are not limited to, one or more immune checkpoint inhibitors disclosed above (e.g., anti-PD-1 checkpoint inhibitors (e.g., nivolumab, PD-1 to PD-5, pidilizumab, or pemprolizumab), or anti-PD-L1 checkpoint inhibitors (e.g., avelumab), anti-CTLA-4 checkpoint inhibitors (e.g., ipilimumab, tremelimumab), or KRAS inhibitors (e.g., BI Examples include 1823911, sotrasib, adagrasib, LY3537982, JNJ-74699157), or KRAS-SOS1 inhibitors (e.g., BI1701963), or BRAF inhibitors (e.g., vemurafenib, dabrafenib, or encorafenib), or combinations of dabrafenib and trametinib, vemurafenib and cobimetinib, or encorafenib and binimetinib, or combinations of the above checkpoint inhibitors with KRAS inhibitors or BRAF inhibitors.

[0277] In one embodiment, the present invention relates to a method for treating a patient suffering from gastrointestinal cancer, the method comprising administering to the patient a therapeutically effective dose of a complex or pharmaceutical composition disclosed, the patient receiving cetuximab or panitumumab alone, or 5-fluorouracil (5-FU) and orally administered capecitabine (CAP), FOLFOX (5-FU and oxaliplatin), FOLFIRI (5-FU and irinotecan), XELOX / CAPOX (CAP and oxaliplatin), CAPIRI (CAP and irinotecan) This treatment is for patients who have not responded to therapy (e.g., first-line or second-line treatment) including tetrauridine (Trifluridine), oxaliplatin, and folfirinox (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin), or for example, patients who have failed third-line treatment with the combination drug trifluridine / tipiracil (TAS-102) after treatment with chemotherapy based on fluoropyrimidine, oxaliplatin, and irinotecan, anti-VEGF therapy, and, in the case of RAS-wild-type tumors, anti-EGFR therapy. Therefore, this treatment method may be used, for example, to treat patients with refractory colorectal cancer or refractory metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer.

[0278] In some embodiments, the colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer treated in the therapeutic method of the present invention is characterized by a hemizygous deletion of TP53, POLR2A, or del(17p13).

[0279] Depending on the embodiment, the colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer treated in the therapeutic method of the present invention is characterized by KRAS mutations and / or BRAF mutations disclosed herein, for example, the KRAS mutations include mutations G13D, G12C, G12V, and G12D, or the BRAF mutations include BRAF mutations V600E, V600K, or V600R.

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[0281] Patent Documents US5,122,368 US5,824,805 US5,622,929 US5,622,929 WO2016040856A2 US7521541 US7723485 WO2009 / 052249 WO2016 / 142049 WO03 / 018771 WO2013 / 163633 WO2021 / 205325A1 WO2021 / 234402A2 WO2016142049A1 WO03026577 WO2005 / 112919 WO2022 / 096604 US8,008,449 WO2006 / 121168 US8,354,509 WO2009 / 114335 WO2009 / 101611. US8,354,509 WO2009 / 114335 WO2010 / 077634 WO2013 / 079174A1 WO2011 / 066389A1. WO15112800A1 WO15112800A1 WO01 / 14424 WO2018 / 220169 WO2015 / 112800 US5,530,101 US5,585,089 US5,693,761 US5,693,762 US6,180,370

[0282] array [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15] [Table 4-16] [Examples]

[0283] Although the present invention is illustrated and described in the drawings and the above description, these illustrations and descriptions are illustrative and not limiting, and the present invention is not limited to the disclosed embodiments. Other modifications to the disclosed embodiments can be understood and achieved by those skilled in the art in practicing the claimed invention, based on a review of the drawings, this disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or processes, and the indefinite articles “a” or “an” do not exclude plurals. The mere fact that certain measurements are listed in different dependent claims does not imply that combinations of these measurements cannot be conveniently used. None of the reference numerals in the claims should be construed as limiting the scope of the present invention.

[0284] All amino acid sequences disclosed herein are shown from the N-terminus to the C-terminus. All nucleic acid sequences disclosed herein are shown 5'->3'.

[0285] Example 1: Synthesis of amatoxin Various amanitin derivatives were used for binding with the antibody of the present invention. These amanitin derivatives were produced according to the methods disclosed in WO2016 / 142049, WO2017 / 149077, WO2018 / 115466, WO2019 / 197654, WO2019 / 030173, WO20216947A1, and WO2022 / 096604 (the entirety of each disclosure is incorporated herein). Amanitin derivative (IX) of the present invention can be obtained by a method similar to the synthesis of compound XVIa disclosed in WO2022 / 096604 using β-amanitin.

[0286] Synthesis of the amatoxin-linker payload for complex (XXII) [ka]

[0287] Natural amanine (9.04 mg, 10 μmol) and maleimide-VAL-ALA-PAB linker (13.3 mg, 3 eq.) were dissolved in 300 μl of DMF. 300 μl of 0.1 M TBTU solution in DMF (30 μmol, 3 eq) and 0.2 M DIPEA solution in DMF (60 μmol, 6.0 eq) were added at room temperature. The reaction was monitored by RP-HPLC. After 2 hours, the reaction was quenched with 100 μl of water, and the crude product was purified by preparative RP-HPLC. The product, including the fraction, was evaporated and lyophilized from 2 ml of tert-butanol / water (4:1). Yield: 5.39 mg (41%) of colorless freeze-dried material MS(ESI+) [MH] + found: 1329.85;calc.:1329.56(C 61 H 81 N 14 O 18 S + )

[0288] The obtained amatoxin-linker complex (XXIIa) can be reacted as disclosed in Example 2 below to obtain the complex (XXII) of the present invention.

[0289] Example 2: Synthesis of amatoxin-linker structure The antibody was conjugated to the amatoxin-linker complex using the so-called thiomab method. In this method, conjugation occurs through the coupling of a maleimide residue in the toxin linker construct with a free SH group on a cysteine ​​residue in the antibody, as shown in the reaction scheme below. [ka]

[0290] The principle of this bonding method is disclosed in Junutula et al. (2008), which is incorporated herein by reference.

[0291] The antibody used in this experiment contains a D265C substitution in both Fc domains to provide a cysteine ​​residue with a free SH group. Each technique is disclosed in WO2016 / 142049A1 (the contents of which are incorporated herein by reference), and this yields a homogeneous product with a constant drug-to-antibody ratio ("DAR") of approximately 2 and site-specific binding.

[0292] Example 3: In vitro cytotoxicity of the anti-GUCY2C complex of the present invention In vitro cytotoxicity tests, as shown in Figures 2 and 3, were performed on human GUCY2C-positive HEK293-GUCY2C-Mono2 cells (Creative Biogen) or HEK293-GUCY2C-HDP-2B3 cells overexpressing human GUCY2C. Cell viability was quantitatively measured 96 hours after adding the complex to the cell growth medium using a commercially available BrdU kit.

[0293] HEK293-GUCY2C-HDP-2B3, which expresses human GUCY2C on its cell surface, was obtained by transient transfection of HEK293 wild-type cells with an overexpression plasmid encoding human guanylate cyclase 2C (GUCY2C or GCC, Uniprot number P25092), which has the amino acid sequence corresponding to SEQ ID NO. 76 and is resistant to genetisin (G418). After 4 days, cells were placed in a culture medium containing G418 to generate a stable cell pool expressing GCC and G418 resistance, thereby selecting stably transfected cells. Stable single-cell clones were isolated from this cell pool using limiting dilution. GCC surface expression was confirmed for the stable cell clone HEK293-GUCY2C-HDP-2B3 by flow cytometry and cytotoxicity assay (BrdU ELISA), respectively.

[0294] Example 4: Efficacy of anti-GUCY2C complex in a subcutaneous human fetal kidney model after a single intravenous administration. In this study, nine experimental groups were used, each consisting of 8 to 10 animals (female NOD SCID mice, n=126, 7 weeks old at the start of the experiment) with subcutaneous HEK293-GUCY2C-(HDP)-2B3 tumors (see Table 1). -16 days (16 days before the start of the experiment), 5 × 10 6 126 mice were subcutaneously disseminated in the right flank with 200 μL of RPMI (phenol red-free, containing 50% Matrigel(R)) added to 200 μL of RPMI. The average tumor volume was approximately 150 mm². 3 Once the tumor size was reached, the animals were randomized into nine groups according to tumor size. Treatment was started on day 1 after group assignment. Mice were treated with PBS, mAb1-LALA-D265C-(XIV), mAb8-LALA-D265C-(XIV), mAb1-LALA-D265C, or mAb8-LALA-D265C (see Table 1 for details). Tumor volume was measured twice a week with calipers (see below), and body weight was measured simultaneously. If the tumor volume was >1600 mm³ 3In such cases, or when it was necessary to slaughter the mice for ethical reasons, the animals were slaughtered and their corpses were dissected.

[0295] Tumor growth was monitored by caliper measurement. Tumor size was given by formula W 2 The tumor volume was calculated using the formula: ×L / 2 (L = tumor length, W = tumor vertical width, L > W). If abnormal growth was observed, the tumor volume was calculated using the formula: [(L × W × D) / 6000] × π.

[0296] Both the mAb1-LALA-D265C-(XIV) and mAb8-LALA-D265C-(XIV) composites of the present invention had a DAR of 2.

[0297] [Table 5] "mAb1-LALA-D265C" and "mAb8-LALA-D265C" refer to antibodies containing the heavy chain amino acid sequence and light chain amino acid sequence: SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO: 85, SEQ ID NO: 87, respectively.

[0298] Example 5: Toxicity and serum pharmacokinetics of the present invention complex in cynomolgus monkeys The purpose of this study was to measure the toxicity and serum pharmacokinetics of the mAb8-LALA-D265C-(XIV) and mAb8-LALA-D265C-(XII) complexes of the present invention after a single intravenous (IV) infusion to untreated male and female cynomolgus monkeys.

[0299] Test design Eight untreated cynomolgus monkeys (8 monkeys, 4 of each sex) were divided into two groups of four monkeys each. Group 1 included three phases, and Group 2 included two phases. Animals in Group 1, Phases 1, 2, and 3 (days 1, 22, and 43) were administered mAb8-LALA-D265C-(XIV) by single intravenous infusion at doses of 1 mg / kg, 2 mg / kg, and 3 mg / kg, respectively. Animals in Group 2, Phases 1 and 2 (days 1 and 22) were administered mAb8-LALA-D265C-(XII) by single intravenous infusion at doses of 5 mg / kg and 10 mg / kg, respectively. Blood / serum samples were collected before administration and on days 3, 8, 15, and 22 after each administration to measure the levels of liver enzymes ALT, AST, and LDH in the serum.

[0300] Dosage administration of the complex The animal's body weight was measured before dose administration on each administration day to calculate the actual volume administered. All animals in groups 1 and 2 received a single intravenous infusion of mAb8-LALA-D265C-(XIV) and mAb8-LALA-D265C-(XII) via peripheral venous access.

[0301] Blood collection and plasma preparation Approximately 0.8 mL of blood was collected from the peripheral blood vessels of each test animal at each time point. The actual time of each sample collection was recorded. The deviation in collection time was less than 1 minute from before administration to 1 hour after administration, and less than 5% of the nominal time after 1 hour after administration.

[0302] All blood samples were collected in commercially available BD tubes containing polymeric silica activator. After blood collection, the tubes containing the blood samples were left to stand at room temperature for at least 30 minutes. Then, within 1 hour of collection, the tubes were centrifuged at 4°C and 3200×g for 10 minutes. After centrifugation, clarified serum was collected. Typically, the serum was divided into two parts, one (approximately 150 μL) for PK analysis and the other as a backup. Liver enzymes ALT, AST, and LDH were analyzed using commercially available kits.

[0303] Example 6: In vivo efficacy study evaluating two antibody-drug conjugate colon cancer PDX models grown subcutaneously in NMRI nude mice. A patient-derived xenograft (PDX) colon cancer model was developed in NMRI nude mice at Charles River Discovery Research Services Germany GmbH, in accordance with local animal welfare guidelines.

[0304] In this study, 10 NMRI nude mice, aged 4–6 weeks, were assigned to each group, with randomized tumor volumes ranging from 50 to 250 mm². 3 Preferably 80-200mm 3 The tumor volume was measured twice a week using calipers in mm². 3 I evaluated it using that method.

[0305] As shown in Figure 7, the complexes mAb8-LALA-D265C-(XII) and mAb8-LALA-D265C-(XIV) of the present invention were administered as a single dose ("single dose," i.e., once every 7 days for 4 weeks ("q7dx4")). Sequencing of tumor cDNA revealed a G13D mutation at codon 38 of KRAS in the PDX model V887_CXF1034_TV (Figure 7(A)).

Claims

1. An isolated antibody or antibody fragment that specifically binds to guanylyl cyclase C (GUCY2C), wherein the framework region 1 (FR Ｈ 1) Complementarity Determination Region 1 (CDR) Ｈ 1) FR Ｈ 2. CDR Ｈ 2. Heavy chain variable region (V) including FRH3, CDRH3, and FRH4 Ｈ ) including, FRH1 contains an amino acid sequence selected from SEQ ID NOs: 33, 40, 45, and 51. CDRH1 contains an amino acid sequence selected from SEQ ID NOs: 34, 41, 46, and 52. FRH2 contains an amino acid sequence selected from SEQ ID NOs: 35; 55. CDRH2 contains an amino acid sequence selected from SEQ ID NOs: 36, 42, 47; 120. FRH3 contains an amino acid sequence selected from SEQ ID NOs: 37, 43, 48, and 53. CDRH3 contains an amino acid sequence selected from SEQ ID NOs: 38, 44, 49; 54. FRH4 contains an amino acid sequence selected from SEQ ID NOs: 39 and 50. Antibody or antibody fragment.

2. The above antibody comprises a light chain variable region (V Ｌ ) containing framework region 1 (FR Ｌ 1), complementarity determining region (CDR Ｌ 1), FR Ｌ 2, CDR Ｌ 2, FR Ｌ 3, CDR Ｌ 3, and FR Ｌ 4, FR Ｌ 1 contains an amino acid sequence selected from SEQ ID NOs: 1, 13, 20, and 27. CD-R Ｌ 1 contains an amino acid sequence selected from SEQ ID NOs: 2, 14, 21, and 28. FR Ｌ 2 contains an amino acid sequence selected from sequence numbers 3, 15, and 29. CD-R Ｌ 2 contains an amino acid sequence selected from sequence numbers 4, 16, 23, and 30. FR Ｌ 3 contains an amino acid sequence selected from sequence numbers 5, 8, 17, 24, and 31. CD-R Ｌ 3 contains an amino acid sequence selected from sequence numbers 6, 9, 11, 18, 25, and 32. FR Ｌ 4 contains an amino acid sequence selected from sequence numbers 7, 10, 12, 19, and 26. The antibody or antibody fragment according to claim 1.

3. The above antibody or antibody fragment is in framework region 1 (FR Ｈ 1) Complementarity Determination Region 1 (CDR) Ｈ 1) FR Ｈ 2. CDR Ｈ 2. Heavy chain variable region (V) including FRH3, CDRH3, and FRH4 Ｈ ) including, FRH1 contains the amino acid sequence related to SEQ ID NO: 33, CDRH1 contains the amino acid sequence related to SEQ ID NO: 34, FRH2 contains the amino acid sequence related to SEQ ID NO: 35, CDRH2 contains the amino acid sequence related to SEQ ID NO: 36, FRH3 contains the amino acid sequence corresponding to SEQ ID NO: 37, CDRH3 contains the amino acid sequence corresponding to SEQ ID NO: 38, FRH4 contains the amino acid sequence related to SEQ ID NO: 39, The above antibody or antibody fragment is in framework region 1 (FR Ｌ 1) Complementarity Determination Region (CDR) Ｌ 1) FR Ｌ 2. CDR Ｌ 2. FR Ｌ 3. CDR Ｌ 3, and FR Ｌ Light chain variable region including 4 (V Ｌ ) including, FR Ｌ 1 contains the amino acid related to SEQ ID NO: 1, CD-R Ｌ 1 contains the amino acid related to SEQ ID NO: 2, FR Ｌ 2 contains amino acids selected from SEQ ID NOs: 3, 15, and 29. CD-R Ｌ 2 includes the amino acid sequence related to SEQ ID NO: 4, FR Ｌ 3 contains an amino acid sequence selected from SEQ ID NOs. 5 and 8. CD-R Ｌ 3 contains an amino acid sequence selected from sequence numbers 6, 9, and 11. FR Ｌ 4 contains an amino acid sequence selected from sequence numbers 7, 10, and 12. The antibody or antibody fragment according to claims 1 to 2.

4. The above antibody or antibody fragment is in framework region 1 (FR Ｈ 1) Complementarity Determination Region (CDR) Ｈ 1) FR Ｈ 2. CDR Ｈ 2. Heavy chain variable region (V) including FRH3, CDRH3, and FRH4 Ｈ ) including, FRH1 contains an amino acid sequence selected from SEQ ID NOs: 40, 45, and 51. CDRH1 contains an amino acid sequence selected from SEQ ID NOs: 41, 46, and 52. FRH2 contains an amino acid sequence selected from SEQ ID NOs: 35; 55. CDRH2 contains an amino acid sequence selected from SEQ ID NOs: 42 and 47. FRH3 contains an amino acid sequence selected from SEQ ID NOs: 43, 48, and 53. CDRH3 contains an amino acid sequence selected from sequence numbers 49, 44, and 54. FRH4 contains the amino acid sequence related to SEQ ID NO: 50, The above antibody or antibody fragment is in framework region 1 (FR Ｌ 1) Complementarity Determination Region (CDR) Ｌ 1) FR Ｌ 2. CDR Ｌ 2. FR Ｌ 3. CDR Ｌ 3, and FR Ｌ Light chain variable region including 4 (V Ｌ ) including, FR Ｌ 1 contains an amino acid selected from SEQ ID NOs: 13, 20, and 27. CD-R Ｌ 1 contains amino acids selected from SEQ ID NOs: 14, 21, and 28. FR Ｌ 2 contains amino acids selected from SEQ ID NOs: 15, 22, and 29. CD-R Ｌ 2 contains an amino acid sequence selected from SEQ ID NOs: 16, 23, and 30. FR Ｌ 3 contains an amino acid sequence selected from SEQ ID NOs: 17, 24, and 31. CD-R Ｌ 3 contains an amino acid sequence selected from SEQ ID NOs: 18 and 25. FR Ｌ 4 contains an amino acid sequence selected from SEQ ID NOs: 19 and 26. The antibody or antibody fragment according to claims 1 to 2.

5. The above antibody or antibody fragment has a heavy chain variable region (V) related to SEQ ID NO 62. Ｈ ) and the light chain variable region related to Sequence ID No. 56 (V Ｌ The antibody or antibody fragment according to any one of claims 1 to 3, comprising )

6. The above antibody or antibody fragment has a heavy chain variable region (V) related to SEQ ID NO.

63. Ｈ ) and the light chain variable region related to Sequence ID 57 (V Ｌ The antibody or antibody fragment according to any one of claims 1 to 3, comprising )

7. The above antibody is related to the heavy chain variable region (V) of SEQ ID NO:

64. Ｈ ) and the light chain variable region related to Sequence ID 58 (V Ｌ The antibody or antibody fragment according to any one of claims 1 to 3, comprising )

8. The above antibody or antibody fragment has a heavy chain variable region (V) related to SEQ ID NO:

65. Ｈ ) and the light chain variable region related to Sequence ID 59 (V Ｌ The antibody or antibody fragment according to claim 4, comprising )

9. The above antibody or antibody fragment has a heavy chain variable region (V) related to SEQ ID NO.

66. Ｈ ) and the light chain variable region (V) related to Sequence ID 60 Ｌ The antibody or antibody fragment according to claim 4, comprising )

10. The antibody or antibody fragment according to claim 4, wherein the antibody or antibody fragment comprises a heavy chain variable region (VH) related to SEQ ID NO: 67 and a light chain variable region (VL) related to SEQ ID NO:

61.

11. The antibody according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody.

12. The antibody according to any one of claims 1 to 11, wherein the antibody is a humanized antibody or a human antibody.

13. The antibody according to any one of claims 1 to 12, wherein the antibody is an IgG type antibody, preferably an IgG1 antibody.

14. The antibody according to any one of claims 1 to 13, wherein the antibody is a recombinant antibody.

15. The antibody according to claim 14, wherein the antibody includes a wild-type heavy chain constant (Fc) region.

16. The antibody according to claim 15, wherein the above-mentioned Fc region includes the amino acid sequence relating to SEQ ID NO:

68.

17. The antibody according to claim 14, wherein the antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cell-mediated cytotoxicity (CDC).

18. The antibody according to claim 14, wherein the antibody comprises a heavy chain constant (Fc) region having at least one amino acid substitution selected from L234A, L234S, L234G, L235A, L235G, L235S, L235T, G236R, D265C (according to the EU numbering system).

19. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region containing the amino acid substitution D265C (according to EU numbering).

20. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region containing amino acid substitutions L234A, L235A, and D265C (according to the EU numbering system).

21. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region including amino acid substitutions L234A, L235A, G236R, and D265C (according to the EU numbering system).

22. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region including amino acid substitutions L234G, L235S, G236R, and D265C (according to the EU numbering system).

23. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region including amino acid substitutions L234S, L235G, G236R, and D265C (according to the EU numbering system).

24. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region including amino acid substitutions L234S, L235T, G236R, and D265C (according to the EU numbering system).

25. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region including mutants L234S, L235G, G236R, and D265C (according to the EU numbering system).

26. The antibody according to claim 18, wherein the antibody comprises a light chain (LC) amino acid sequence related to SEQ ID NO: 78 and a heavy chain (HC) amino acid sequence related to one of SEQ ID NOs: 79, 80, 81, 82, 83, or 84.

27. The antibody according to claim 18, wherein the antibody comprises a light chain (LC) amino acid sequence related to SEQ ID NO: 85 and a heavy chain (HC) amino acid sequence related to one of SEQ ID NOs: 86, 87, 88, 89, 90, or 91.

28. The antibody according to claim 18, wherein the antibody comprises a light chain (LC) amino acid sequence relating to SEQ ID NO: 92 and a heavy chain (HC) amino acid sequence relating to one of SEQ ID NOs: 93, 94, 95, 96, 97, or 98.

29. The antibody according to claim 18, wherein the antibody comprises a light chain (LC) amino acid sequence relating to SEQ ID NO: 99 and a heavy chain (HC) amino acid sequence relating to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105.

30. The antibody according to claim 18, wherein the antibody comprises a light chain (LC) amino acid sequence relating to SEQ ID NO: 106 and a heavy chain (HC) amino acid sequence relating to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112.

31. The antibody according to claim 18, wherein the antibody comprises a light chain (LC) amino acid sequence relating to SEQ ID NO: 113 and a heavy chain (HC) amino acid sequence relating to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119.

32. The antibody according to any one of claims 1 to 31, wherein the antibody specifically binds to an epitope contained in amino acids 24 to 430 of SEQ ID NO: 76 or amino acids 23 to 430 of SEQ ID NO:

77.

33. An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the antibody is selected from SEQ ID NOs: 34, 41, 46, 52; 36, 42, 47; 38, 44, or 49. Ｈ 1. CD-R Ｈ 2, and CDR Ｈ 3 includes the framework region FR of the above antibody Ｈ 1. FR Ｈ 2. FR Ｈ 3. FR Ｈ Antibody 4 shares at least 80%, 85%, preferably 90%, or 95% sequence similarity with any of sequence numbers 33, 40, 45, 51; 35; 55; 37, 43, 48, 53; 39, or 50.

34. A complex comprising (i) an antibody according to any one of claims 1 to 33, (ii) at least one toxin, and (iii) at least one linker which binds the antibody portion to the at least one toxin, wherein the antibody specifically binds to human GUCY2C and the at least one toxin is an amatoxin.

35. The complex according to claim 34, wherein the linker is bound to the antibody via any of the naturally derived cysteine ​​residues of the antibody, preferably via any of the naturally derived cysteine ​​residues that form an interchain disulfide bond of the antibody, and / or via a disulfide bond.

36. The complex according to claim 34, wherein the linker is linked via a disulfide bond between Cys265 of the antibody and the linker (numbered according to the EU numbering system).

37. The composite according to claim 34, wherein the linker is a non-cleavable linker or a cleavable linker.

38. The complex according to claim 37, wherein the above-mentioned cleavable linker is selected from the group consisting of an enzymatically cleavable linker, preferably a linker cleavable by a protease, and a chemically cleavable linker, preferably a linker containing a disulfide bridge.

39. The complex according to claim 38, wherein the cleavable linker is cleaved by cathepsin A or B, matrix metalloproteinase (MMP), elastase, β-glucuronidase, and β-galactosidase, preferably cathepsin B.

40. The composite according to claim 39, wherein the above-mentioned cleavable linker is a linker that can be cleaved by cathepsin B.

41. The complex according to claim 40, wherein the linker cleavable by the cathepsin B comprises a dipeptide selected from Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit.

42. The complex according to any one of claims 34 to 41, wherein the linker is bound to the amatoxin via (i) the γC- atom of amatoxin amino acid 1, or (ii) the δC- atom of amatoxin amino acid 3, or (iii) the 6'-C- atom of amatoxin amino acid 4.

43. The amatoxin is a complex according to any one of claims 34 to 42, comprising (i) an amino acid 4 having a 6'-deoxy position and (ii) an amino acid 8 having an S-deoxy position.

44. The above-mentioned complex is the complex according to any one of claims 34 to 43, comprising any of the following amatoxin-linker compounds of formula (I) to (XI). 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】

45. The complex according to any one of claims 34 to 44, comprising the antibody bound to an amatoxin linker moiety via a thioether bond relating to any one of the following formulas (XII) to (XXII), wherein the amatoxin linker moiety is bound to a thiol group of a cysteine ​​residue of the antibody, and n is preferably about 1, 2, 3 to about 4, 5, 6, 7, 8, preferably about 1, 1.5, 2, 2.5 to about 3.5, 4.5, 5.5, and more preferably about 1.5 to about 3.5, or about 1.8 to about 2.

5. 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】

46. The complex according to claim 45, wherein the amatoxin linker portion is bound to the thiol group of the cysteine ​​residue of the antibody, and n is about 1 to about 2, preferably about 2.

47. The above complex is (i) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXIII) includes an antibody to which at least one amatoxin-linker portion of formula (XII) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (ii) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXIV) includes an antibody to which at least one amatoxin-linker portion of formula (XIII) is linked via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (iii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XIV) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (XXV) (iv) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXVI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (v) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the antibody is linked to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system (XXVII), (vi) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXVIII) includes an antibody to which at least one amatoxin-linker portion of formula (XVII) is linked via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (vii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XVIII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (XXIX), (viiii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XIX) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (XXX) (ix) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXXI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (x) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XXI) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (XXXII) (xi) A complex (XXXIII) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein at least one amatoxin-linker portion of formula (XXII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Selected from the group consisting of, The composite according to any one of claims 34 to 46, wherein n is approximately 1 to approximately 2.

48. The above complex is (i) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (XXXIV), (ii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XXXV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (iii) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XIV) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (XXXVI), (iv) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XXXVII) includes an antibody to which at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (v) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 62, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 56, wherein the complex (XXXVIII) includes an antibody to which at least one amatoxin-linker portion of formula (XVI) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (vi) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XVII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (XXXIX), (vii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XVIII) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (XL) (viiii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XLI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (ix) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XLII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (x) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein the complex (XLIII) includes an antibody to which at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (xi) A complex (XLIV) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 63, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 57, wherein at least one amatoxin-linker portion of formula (XXII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Selected from the group consisting of, The composite according to any one of claims 34 to 46, wherein n is approximately 1 to approximately 2.

49. The above complex is (i) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (XLV), (ii) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XIII) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (XLVI) (iii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XIV) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (XLVII) (iv) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XV) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (XLVIII), (v) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein the complex (XLIX) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (vi) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XVII) is linked to the linker via a thioether bond between the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system, and the above antibody, a complex (L), (vii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XVIII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LI) (viiii) A complex (LII) comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XIX) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. (ix) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XX) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (LIII) (x) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XXI) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (LIV) (xi) A complex (LV) comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 64, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 58, wherein at least one amatoxin-linker portion of formula (XXII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Selected from the group consisting of, The composite according to any one of claims 34 to 46, wherein n is approximately 1 to approximately 2.

50. The above complex is (i) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LVI), (ii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XIII) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (LVII) (iii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XIV) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LVIII), (iv) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XV) is linked to the above-mentioned antibody via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above-mentioned antibody, (iv) an antibody comprising at least one amatoxin-linker portion of formula (XV), (v) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XVI) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody, (LX) (vi) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XVII) is linked to the linker via a thioether bond between the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system (LXI), (vii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XVIII) is linked to the above linker via a thioether bond between the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (LXII) (viiii) A complex (LXIII) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XIX) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. (ix) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XX) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXIV), (x) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XXI) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXV), (xi) A complex (LXVI) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 65, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 59, wherein at least one amatoxin-linker portion of formula (XXII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Selected from the group consisting of, The composite according to any one of claims 34 to 46, wherein n is approximately 1 to approximately 2.

51. The above complex is (i) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXVII), (ii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XIII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXVIII), (iii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XIV) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXIX), (iv) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XV) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXX), (v) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XVI) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXI), (vi) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XVII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXII), (vii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein the antibody is linked to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system (LXXIII), (viiii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein the antibody is linked to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXIV), (ix) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XX) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (LXXV) (x) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein the complex (LXXVI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (xi) A complex (LXXVII) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 66, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 60, wherein at least one amatoxin-linker portion of formula (XXII) is linked via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. Selected from the group consisting of, The composite according to any one of claims 34 to 46, wherein n is approximately 1 to approximately 2.

52. The above complex is (i) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXVIII), (ii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XIII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXIX), (iii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XIV) is linked to the above linker via a thioether bond between the above linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the above antibody, (LXXX) (iv) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XV) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXXI), (v) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the antibody is linked to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system (LXXXII), (vi) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the antibody is linked to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system (LXXXIII), (vii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XVIII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody (LXXXIV), (viiii) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXXV) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (ix) an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXXVI) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (x) An antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein the complex (LXXXVII) includes an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system. (xi) A complex (LXXXVIII) comprising an antibody comprising two heavy chains each comprising a VH domain consisting of or containing the amino acid sequence of SEQ ID NO: 67, and two light chains each comprising a VL domain consisting of or containing the amino acid sequence of SEQ ID NO: 61, wherein at least one amatoxin-linker portion of formula (XXII) is linked to the linker via a thioether bond between the linker and the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody. Selected from the group consisting of, The composite according to any one of claims 34 to 46, wherein n is approximately 1 to approximately 2.

53. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

54. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

55. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

56. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XV) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

57. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

58. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

59. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

60. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

61. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

62. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, and 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

63. The complex according to claim 47, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 79, 80, 81, 82, 83, or 84, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 78, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

64. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

65. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

66. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

67. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

68. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

69. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

70. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

71. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

72. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

73. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

74. The complex according to claim 48, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 86, 87, 88, 89, 90, or 91, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 85, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

75. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

76. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

77. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

78. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

79. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

80. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

81. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

82. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

83. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

84. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

85. The complex according to claim 49, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs. 93, 94, 95, 96, 97, or 98, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs. 92, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

86. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

87. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NOs: 99, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

88. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XIV) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

89. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

90. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NOs: 99, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

91. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XVII) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

92. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NOs: 99, wherein at least one amatoxin-linker portion of formula (XVIII) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

93. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs. 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NOs. 99, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

94. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NOs: 99, wherein at least one amatoxin-linker portion of formula (XX) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

95. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 99, wherein at least one amatoxin-linker portion of formula (XXI) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

96. The complex according to claim 50, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 100, 101, 102, 103, 104, or 105, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NOs: 99, wherein at least one amatoxin-linker portion of formula (XXII) is conjugated via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

97. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

98. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

99. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

100. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

101. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

102. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

103. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

104. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

105. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

106. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

107. The complex according to claim 51, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 107, 108, 109, 110, 111, or 112, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 106, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

108. The complex according to claim 52, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

109. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

110. The complex according to claim 52, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

111. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XV) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

112. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

113. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

114. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XVIII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

115. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XIX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

116. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NOs: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XX) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain according to the EU numbering system of the antibody.

117. The complex according to claim 52, wherein the complex is an antibody comprising two heavy chains each comprising or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each comprising or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXI) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

118. The complex according to claim 52, wherein the complex comprises an antibody comprising two heavy chains each consisting of or containing an amino acid sequence corresponding to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119, and two light chains each consisting of or containing an amino acid sequence corresponding to SEQ ID NO: 113, wherein the complex comprises an antibody conjugated to at least one amatoxin-linker portion of formula (XXII) via a thioether bond of the linker to the sulfhydryl group of the 265Cys residue of the heavy chain of the antibody according to the EU numbering system.

119. A polynucleotide encoding a polypeptide containing any of the amino acid sequences in sequence numbers 62, 63, 64, 65, 66, or 67.

120. A polynucleotide encoding a polypeptide containing any of the amino acid sequences of sequence numbers 56, 57, 58, 59, 60, or 61.

121. An expression vector comprising the polynucleotide according to claim 119 and / or claim 120.

122. A host cell comprising the expression vector according to claim 121, or the polynucleotide according to claim 119 and / or claim 120.

123. The host cell according to claim 122, wherein the host cell is a eukaryotic cell, preferably a mammalian cell.

124. An antibody according to any one of claims 1 to 33 for use in the manufacture of a complex.

125. A pharmaceutical composition comprising the complex described in any one of claims 47 to 118.

126. The pharmaceutical composition according to claim 125, further comprising one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, flow promoters, disintegrants, adsorbents, and / or preservatives.

127. At least one immune checkpoint inhibitor, and at least one composite according to any one of claims 44 to 118 A composition containing the following:

128. The composition according to claim 127, wherein the above-mentioned immune checkpoint inhibitor is an antibody selected from the group consisting of nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, semiprimab, ipilimumab, PD-1, PD-2, PD-3, PD-4, or PD-5, or an antigen-binding fragment thereof, or an antigen-binding derivative thereof, and preferably the above antibody is avelumab, pembrolizumab, nivolumab, or ipilimumab, or an antigen-binding fragment thereof, or an antigen-binding derivative of the above antibody.

129. A complex according to any one of claims 47 to 118, a pharmaceutical composition according to claim 125, or a composition according to claim 127, for use in the treatment of a cancer selected from colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or gastrointestinal cancer such as liver cancer.

130. The above-mentioned colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer is characterized by a hemizygous deletion of TP53, POLR2A, or del(17p13), using the complex according to any one of claims 44 to 118, the pharmaceutical composition according to claim 125 or 126, or the composition according to claim 127.

131. A complex according to any one of claims 44 to 118, or a pharmaceutical composition according to claim 125, for use in the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer in patients who have not responded to first-line treatment comprising cetuximab or panitumumab alone, or in combination with 5-fluorouracil (5-FU) and orally administered capecitabine (CAP), FOLFOX (5-FU and oxaliplatin), FOLFIRI (5-FU and irinotecan), XELOX / CAPOX (CAP and oxaliplatin), CAPIRI (CAP and irinotecan), or FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin).

132. A complex according to any one of claims 44 to 118, a pharmaceutical composition according to claim 125 or 126, or a composition according to claim 127, for use in the treatment of a refractory cancer selected from colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer.

133. A complex according to any one of claims 44 to 118, or a pharmaceutical composition according to claim 125, for use in the treatment of colorectal cancer or metastatic colorectal cancer (mCRC) characterized by a KRAS mutation and / or a BRAF mutation.

134. Use of the complex according to any one of claims 44 to 118, or the pharmaceutical composition according to any one of claims 125 to 126, in the manufacture of a pharmaceutical for the treatment of colorectal cancer including metastatic colorectal cancer (mCRC), gastric cancer, and gastrointestinal cancer including pancreatic cancer and liver cancer.

135. A method for treating a patient suffering from a solid tumor expressing amino acids 24-430 or fragments of SEQ ID NO: 76 on its surface, comprising administering to the patient, either alone or in combination with an immune checkpoint inhibitor, a therapeutically effective amount of the complex according to any one of claims 44-118 or the pharmaceutical composition according to claim 125.

136. A method for treating a patient suffering from gastrointestinal cancer, comprising administering to the patient, either alone or in combination with an immune checkpoint inhibitor, a therapeutically effective amount of the complex described in any one of claims 44 to 118 or the pharmaceutical composition described in claim 125.

137. The method according to claim 136, wherein the gastrointestinal cancer is one of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer.

138. The method according to claim 137, wherein the patient is a patient who has not responded to first-line or second-line treatment comprising cetuximab or panitumumab alone, or in combination with 5-fluorouracil (5-FU) and orally administered capecitabine (CAP), FOLFOX (5-FU and oxaliplatin), FOLFIRI (5-FU and irinotecan), XELOX / CAPOX (CAP and oxaliplatin), CAPIRI (CAP and irinotecan), or FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin).

139. The method according to claim 137, wherein the patient has refractory colorectal cancer, refractory metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer.

140. A method for treating a patient according to any one of claims 137 to 139, wherein the above-mentioned colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer is characterized by a hemizygous deletion of TP53, POLR2A, or del(17p13).

141. A method for treating a patient according to any one of claims 136 to 140, wherein the above-mentioned colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer is characterized by a KRAS mutation and / or a BRAF mutation.

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