Anti-GUCY2C antibodies and uses thereof

Abstract

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

Description

Technical Field

[0001] The present application relates to anti-GUCY2C antibodies and corresponding antibody-drug conjugates comprising amanitin. In another aspect, the present invention relates to pharmaceutical compositions comprising such conjugates for use in the treatment of gastrointestinal cancers such as colorectal cancer (CRC) or metastatic colorectal cancer (mCRC). Background Art

[0002] Gastrointestinal cancers account for approximately 26% of all cancer diagnoses and cause approximately 35% of cancer deaths globally. In 2018, there were an estimated 4.8 million new gastrointestinal (GI) cancer cases and 3.4 million related deaths worldwide.

[0003] Gastrointestinal cancers include esophageal cancer, gastric cancer, colorectal cancer, liver cancer, esophageal cancer, and pancreatic cancer, among which 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%). CRC accounts for approximately 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%). Among the approximately 3.4 million GI cancer deaths globally in 2018 (Arnold et al. (2020) Gastroenterology 159:335-349). Thus, 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] In women, CRC is the second most common adult cancer, while in men it is the third most common cancer, and it is the fourth leading cause of cancer death.

[0005] The 5-year and 10-year survival rates are 65% and 58% respectively, and the incidence and mortality rates are 25% higher in men than in women. Among Americans aged 20-49 from 1975 to 2013, the CRC incidence increased from 10 cases per 100,000 people to 15 cases. At the same time, in developing countries such as Argentina, Brazil, and China, the number of CRC cases increased by approximately 20%. In addition, 30% of all patients with CRC 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 the incidence rate has decreased over the past 30 years due to improvements in screening and treatment paradigms, shockingly, between 2012 - 2016, the incidence rate among individuals less than 50 years old has been steadily increasing, and the annual total incidence rate has risen by 2.2%. The exact etiology of this paradigm shift is unclear; however, it may be attributed to changes in diet and other lifestyle risk factors, or it may be attributable to the increase in unsystematic screening among the young.

[0007] 10% - 20% of all patients with CRC have a positive family history, and approximately 5% of all CRC cases are associated with known hereditary CRC syndromes that can be detected through germline testing. In some cases, the increased incidence of sporadic CRC is associated with long-term inflammatory bowel disease and variable lifestyle factors such as lack of physical activity, unhealthy diet, smoking, obesity, and heavy alcohol consumption.

[0008] Differences in the clinical outcomes and drug responsiveness of CRC treatment depend on the location of the cancer along the colon and rectum. Related contributing factors include their unique physiological functions, gut microbiome, regional resident immune cell types, dietary carcinogens, and the timing of disease detection. Additionally, ontogenetic factors can affect disease severity and treatment outcomes:

[0009] The proximal (right) large intestine (cecum, ascending colon, and transverse colon) is derived from the embryonic midgut, while the distal (left) large intestine (splenic flexure, descending colon, sigmoid colon, and rectum) is derived from the embryonic hindgut. These ontogenetic differences are associated with differential gene expression patterns along the proximal - distal axis. In women and African Americans, proximal sporadic colon tumors are more frequently diagnosed, accounting for 51% - 62% of cases, and also show a higher TNM stage at first diagnosis, exhibit a high level of genome-wide promoter hypermethylation pattern, i.e., CpG island methylation phenotype (CIMP), display microsatellite instability (MSI) due to DNA mismatch repair mechanism deficiency (dMMR), are more frequently mutated in KRAS and BRAF, and have a worse prognosis in terms of survival. Distal colorectal tumors are more likely to exhibit chromosomal instability (CIN) and show a more favorable prognosis.

[0010] Genomic profiling of CRC has revealed significant intra - and inter - tumor heterogeneity resulting from the accumulation of gene mutations and chromosomal aberrations during disease initiation and progression. Genomic instability in CRC manifests as one of two main forms (CIN and MSI). CRCs lacking CIN or MSI are classified as genomically stable (GS) CRCs.

[0011] In GS CRC, DNA repair genes and tumor suppressors may be transcriptionally silenced via CIMP, but a large proportion of MSI CRCs and a small subset of CIN CRCs are also CIMP-positive, and approximately 10% of CRCs are negative for CIN, MSI, or CIMP.

[0012] Chromosomal instability (CIN) CIN is characterized by alterations in chromosome number (aneuploidy) and structure (somatic copy number alterations, 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 mutant inactivation of APC (a negative regulator of the Wnt pathway). Mutant inactivation / deletion of TP53 occurs in 60% of CIN tumors, and loss of p53 function directly drives CIN and provides a permissive background for genomic instability mechanisms.

[0013] Regarding genomic instability, the combination of telomere dysfunction and p53 deficiency is a major CIN mechanism, as revealed by the occurrence of anaphase bridges in early-stage human CRC carcinomas and the development of spontaneous CRCs in p53-mutant mice lacking telomerase.

[0014] Microsatellite instability (MSI) CRC is characterized by the presence of microsatellites (i.e., DNA sequences containing repetitive motifs that tend to accumulate a higher mutation rate than other genomic regions). MSI is the phenotypic manifestation of dMMR caused by mutant inactivation of MMR genes (including MLH1, MSH2, MSH3, MSH6, PMS2, and Exo1) (De'Angelis et al. Acta Biomed December 17, 2018; 89(9-S):97-101).

[0015] Other important factors contributing to CRC etiology are somatic gene alterations that activate key signal transduction pathways promoting the proliferative state of CRC cancer cells. In CRC, the major proliferative signaling pathways are the EGFR-RAS and WNT-β-catenin pathways.

[0016] EGFR signaling EGFR activation triggers downstream RAS / RAF / MEK / ERK and PI3K / AKT signaling cascades, which ultimately lead to proliferation. In CRC, mutations in EGFR itself are rare, with 1% of CRC cases attributable to mutations (Barber et al. N Engl J Med 351:2883), and instead overexpression is shown in approximately 80% of CRCs. Enhanced EGFR activation can occur via post-translational modifications that involve methylation of R198 and R200 by protein arginine methyltransferase 1 (PRMT1), which enhances EGFR binding to EGF and subsequent signaling activation, even in the presence of EGFR inhibitors.

[0017] It has recently been demonstrated that hepatocyte growth factor (HGF), the ligand of the MET-receptor, can completely substitute for EGF in the growth of single Lgr5+ murine intestinal stem cells (ISCs) into intestinal organoids (including all differentiated intestinal cell lineages). In addition, HGF and EGF are equally effective in promoting the expansion of Apc-mutant mouse organoids, while deletion of Met in ISCs in vivo attenuates stem cell fitness and the formation of Apc-driven intestinal adenomas. These findings suggest that EGFR and MET signaling have overlapping and partially redundant functions in the murine intestinal mucosa. Recent results also indicate that in human ISCs and CRC stem cells, HGF / MET signaling can completely overcome EGFR inhibition, allowing single normal ISCs as well as APC mutant cells to expand into organoids even in the presence of EGFR inhibition (Joosten et al. Gastroenterology October 2019;157(4):1153-1155).

[0018] Another factor that is decisive for the clinical efficacy of EGFR blockade is the mutational status of its downstream signaling components, specifically, gain-of-function mutations in RAS, RAF, MEK, or ERK, which can maintain cancer cell proliferation and survival after EGFR inhibition. Activating mutations in KRAS, NRAS, or HRAS are present together in approximately 50% of CRC cases; these mutations involve codons 12 or 13 and less frequently codons 61, 117, or 146. Regarding the RAS pathway in CRC, in the hot spot codon 600 (V600E), the RAS effector (BRAF) is mutated in 10%-15% of early CRCs and in approximately 5% of stage IV CRCs. Clinically, due to EGFR-mediated reactivation of MAPK signaling, single-agent inhibition of BRAF shows limited activity in metastatic BRAFV600E CRC, leading to the approval of combinations of EGFR inhibitors with BRAF and MEK inhibitors, which have now become the standard of care for patients with metastatic BRAF-mutated tumors.

[0019] Screening for CRC can be accomplished via direct visualization methods (such as colonoscopy, CT colonography, flexible sigmoidoscopy, flexible sigmoidoscopy combined with fecal immunochemical testing (FIT)), or fecal-based tests (such as guaiac-based fecal occult blood testing, FIT, or multitargeted fecal DNA testing), or serological tests (such as SEPT9 DNA testing).

[0020] Once a diagnosis of CRC is made, staging is an important factor as it determines the treatment options for the patient. For tumor staging, the Tumor, Node, Metastasis (TNM) system jointly developed by the American Joint Committee on Cancer (AJCC) / Union for International Cancer Control (UICC) is a commonly used staging system that defines the following cancer stages: The standard conventional treatments for CRC are surgery, chemotherapy, and radiotherapy. These treatments can be used in combination depending on the location and progression of the disease. Treatment options include, for example, for stage I, surgical resection alone. For stage III, the standard of care is adjuvant chemotherapy after curative surgery. Total mesorectal excision (TME) by laparoscopic and transanal surgical approaches is usually the option for localized cancer and when the tumor location is easily accessible. However, it is usually impossible to completely remove all cancer cells. Approximately 66% of stage II and 61% of stage III colon and rectal patients must undergo further treatment with adjuvant chemotherapy and / or radiotherapy. These treatments have many side effects due to their non-specific and cytotoxic nature to any cells that are growing and dividing. Despite recent improvements in diagnostic and treatment options, 54% of patients will still relapse even after neoadjuvant treatment. Therefore, it is crucial to have more alternative and effective treatments for CRC patients.

[0021] Current chemotherapy for CRC includes monotherapy mainly based on fluoropyrimidine (5-FU) and multi-drug regimens containing one or several drugs including oxaliplatin (OX), irinotecan (IRI), and capecitabine (CAP or XELODA or XEL), where the combination therapy regimens FOLFOX (5-FU + OX), FOXFIRI (5-FU + IRI), XELOX or CAPOX (CAP + OX), and CAPIRI (CAP + IRI) represent the main approaches for first-line treatment. However, chemotherapy is associated with certain limitations such as systemic toxicity, unsatisfactory response rates, and unpredictable innate and acquired drug resistance as well as low tumor-specific selectivity.

[0022] The 5-year overall survival rate of patients with metastatic colorectal cancer (mCRC) is <10%. Currently, the main chemotherapeutic agents used to treat affected patients are fluoropyrimidine-based (intravenous or oral), either as monotherapy, such as intravenous 5-fluorouracil (5-FU) and oral capecitabine (CAP); or as multi-drug regimens, including the combinations 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, EGFR-targeted drugs (cetuximab and panitumumab) have been approved for first-line treatment of mCRC. The combination of cetuximab with FOLFIRI or panitumumab with FOLFOX has significantly enhanced therapeutic efficacy. However, treatment decisions for early-stage mCRC do not take into account BRAF or KRAS mutations, as these mutations lead to extremely poor prognoses, as seen in clinical trials. Although the survival rate of patients with mCRC has improved in recent years, the response and prognosis of patients with the above mutations remain poor.

[0023] The current lack of effective therapies, the presence of non-specific cytotoxicity limitations, and the large unmet need for prospective therapies in patients with BRAF- or KRAS-mutated mCRC have prompted researchers to turn to the development of predictive, preventive, and personalized medical strategies to improve the treatment of CRC and mCRC.

[0024] Gastric cancer is the second leading cause of death from malignant diseases globally, with particularly high mortality rates in East Asia, South Asia, and Central Asia; Central and Eastern Europe; and South America. Treatment options for gastric cancer vary depending on the cancer stage: very early-stage cancers can usually be treated surgically. Potentially resectable cancers can usually be treated surgically (using subtotal gastrectomy or total gastrectomy) as the first treatment. Nearby lymph nodes and parts of nearby organs may also be removed. In addition, patients can receive chemotherapy alone or chemotherapy plus radiotherapy (chemoradiotherapy), for example, patients can receive a combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) (FP therapy). Other possible treatment regimens include epirubicin, cisplatin, and fluorouracil (ECF therapy), or a regimen in which fluorouracil is replaced by capecitabine (ECX therapy) or an ECF therapy in which cisplatin is replaced by oxaliplatin (EOF therapy). Alternatively, both cisplatin and fluorouracil can be replaced by oxaliplatin and capecitabine (EOX therapy).

[0025] In metastatic gastric cancer, treatment aims to control the growth of the cancer and may include chemotherapy alone, chemotherapy plus immunotherapy, or chemotherapy together with radiotherapy. The combination of platinum and fluoropyrimidine (5-FU) is the global standard first-line chemotherapy regimen in non-curative settings. After failure of platinum and 5-FU, paclitaxel plus ramucirumab has been established as the standard second-line therapy. However, treatment-related neuropathy, progression, or rapid recurrence after the perioperative FLOT regimen (fluorouracil, oxaliplatin, docetaxel) has increased the need for a taxane-free second-line therapy. Trifluridine / tipiracil has recently been approved for patients with metastatic gastric cancer. In addition to chemotherapy, patients can 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).

[0026] Diagnostically, gastrointestinal endoscopy and staging laparoscopy (SL), which is a minimally invasive, simple procedure requiring only small incisions, are indispensable for the diagnosis of gastric cancer. The advantages of SL include providing an accurate diagnosis of peritoneal dissemination and serosal invasion, and the ability to perform peritoneal lavage for cytological examination. In patients with advanced gastric cancer in whom imaging has not yielded a diagnosis, peritoneal lavage cytology obtained prior to treatment can be very important for treatment planning. Peritoneal lavage cytology obtained by SL for the evaluation of peritoneal dissemination is considered useful for assessing the efficacy of neoadjuvant chemotherapy and / or immunotherapy.

[0027] The most common liver tumor among all primary liver cancers is hepatocellular carcinoma (HCC), accounting for 75%-85% of cases. HCC is usually diagnosed at an advanced stage, for which effective treatment options remain limited. Until 2007, there were no effective treatment options for patients diagnosed with advanced disease or those who progressed to advanced disease after failure of other treatments. Between 2007 and 2016, sorafenib was the first systemic drug approved by the US Food and Drug Administration (FDA) as the standard treatment for advanced HCC. In recent years, other small molecule drugs have been developed, which include lenvatinib, regorafenib, or cabozantinib, which have been approved or are still undergoing clinical trials. These drugs are also currently being tested in combination with immune checkpoint inhibitors such as nivolumab or pembrolizumab. In addition, combinations of immune checkpoint inhibitors and angiogenesis inhibitors (such as atezolizumab and bevacizumab) are being tested.

[0028] Esophageal cancer, as a form of GI cancer, is treated according to its stage and can include endoscopic treatment as well as chemotherapy and radiotherapy. Common drugs and drug combinations used to treat esophageal cancer but not usually given with radiation include ECF: epirubicin (Ellence), cisplatin, and 5-FU (especially for tumors at the gastroesophageal junction), DCF: docetaxel (Taxotere), cisplatin, and 5-FU, or trifluridine and tipiracil (Lonsurf) (a combination drug in pill form).

[0029] For pancreatic cancer, several chemotherapy regimens have been approved, and the most widely used and best studied agent is gemcitabine. Gemcitabine is usually administered in combination with albumin-bound (Nab) paclitaxel, which improves survival time compared to gemcitabine monotherapy. Alternative treatment options include the multi-drug regimen FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin). In addition, for pancreatic cancer, combination therapies using immune checkpoint inhibitors (such as pembrolizumab or nivolumab) in combination with gemcitabine, nab-paclitaxel, or capecitabine are under clinical evaluation.

[0030] Guanylyl Cyclase 2C (GUCY2C, EC: 4.6.1.2) is a member of the receptor enzyme protein family that synthesizes guanosine 3’,5’-cyclic monophosphate (cyclic GMP; cGMP). GUCY2C is a transmembrane receptor for the endogenous hormone ligands: guanylin and uroguanylin. The ligand binding to the extracellular receptor catalyzes the conversion of GTP to cyclic GMP (cGMP) and initiates the downstream cGMP-related signaling pathway, which is related to the regulation of intestinal homeostasis processes such as epithelial cell proliferation, differentiation, and apoptosis (Lisby et al. Expert Rev Precis Med Drug Dev. 2021; 6(2): 117-129.).

[0031] GUCY2C is specifically expressed by intestinal epithelial cells. GUCY2C regulates the dynamic progression of cells along the crypt-villus and crypt-surface axes, thus coordinating homeostasis processes including proliferation, DNA repair, metabolic programming, lineage-specific cell fate, and epithelial-mesenchymal interactions that organize this axis. Given the major role of GUCY2C in maintaining epithelial regeneration, the dysregulation of the GUCY2C-cGMP axis promotes pathologies including inflammatory bowel disease and intestinal transport disorders in addition to colorectal cancer. Importantly, the silencing of the GUCY2C signaling axis is associated with colorectal tumorigenesis through the loss of ligand binding.

[0032] GUCY2C protein can be detected almost universally (>95%) in all subsets of primary and metastatic human colorectal tumors, as well as gastroesophageal and pancreatic tumors, regardless of anatomical location or grade, but not in tumors arising outside the GI tract. GUCY2C has also been shown to be expressed in esophageal tumors, gastric tumors, and pancreatic tumors (Danaee et al. (2017) PLoS One. 2017;12(12):e0189953).

[0033] Given its unique expression pattern in CRC and mCRC, multiple approaches have been taken in the treatment of these diseases to exploit GUCY2C, including: cancer vaccines using GUCY2C, using it as a target for CAR-T cell therapy (Magee et al. Cancer Immunol Res. 6(5), 509-516 (2018)), or using therapeutic antibodies and antibody-drug conjugates targeting GUCY2C. WO 2013 / 163633 A1 discloses anti-GUCY2C antibodies and anti-GUCY2C ADCs comprising auristatin E (MMAE) and auristatin F MMAE (MMAF), which inhibit mitosis by inhibiting tubulin polymerization. The clinical trial of anti-GUCY2C ADC TAK264 (MLN0264, 5F9vcMMAE) using MMAE as the payload has been terminated. WO 2021 / 205325 A1 discloses anti-CD3-GUCY2C bispecific antibodies and their respective uses in inducing cytolytic T cell responses in GUCY2C-positive target cells in cancer therapy.

[0034] These efforts suggest that therapies targeting GUCY2C may be effective; however, given the lack of approved therapies targeting GUCY2C, there is still a significant need for treatment modalities with high potency and favorable efficacy profiles. SUMMARY OF THE INVENTION

[0035] The inventors unexpectedly found that the amatoxin-based conjugates according to the present invention are characterized by high potency and efficacy in in vitro and in vivo models of GUCY2C-positive cancers, said conjugates comprising a combination of an anti-GUCY2C antibody or an antigen-binding antibody fragment thereof and a non-cleavable or cleavable linker that links the anti-GUCY2C antibody or antibody fragment to an amatoxin as disclosed herein.

[0036] In view of the prior art, it is therefore an object of the present invention to provide an antibody that specifically binds to GUCY2C, and a conjugate comprising said anti-GUCY2C antibody, said conjugate comprising at least one amatoxin and at least one linker that links the anti-GUCY2C antibody to the at least one amatoxin, mediating a cytotoxic effect in target cells, as described in the present application.

[0037] Another object of the present invention is to provide a pharmaceutical composition comprising such a conjugate.

[0038] Another object of the present invention is to provide a compound for use in a method of treating cancer.

[0039] In another object, the present invention relates to providing a composition comprising a conjugate of the present invention as disclosed herein and at least one immune checkpoint inhibitor for use in treating cancer, particularly for use in treating colorectal cancer. Description of the Drawings

[0040] Figure 1 . Markush structure of various amanitins. The bold numbers (1 to 8) designate the standard numbering of the eight amino acids that form the amanitin. The standard names of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters α to γ, Greek letters α to δ, and numbering 1' to 7', respectively).

[0041] Figure 2 . In vitro cytotoxicity of the anti-GUCY2C-ADC of the present invention comprising the conjugate (XV) against HEK293 cells stably expressing human GUCY2C.

[0042] Figure 3 In vitro cytotoxicity of anti-GUCY2C-ADC against HEK cells stably expressing human GUCY2C. (A) Cytotoxicity of anti-GUCY2C-ADC conjugated with conjugate (XII), (B) Cytotoxicity of anti-GUCY2C-ADC conjugated with conjugate (XIV). The cytotoxicity assay shows that the tested anti-GUCY2C ADC exhibits similar cytotoxic potential against HEK293-GUCY2C cells in the picomolar range.

[0043] Figure 4 . Efficacy of anti-GUCY2C conjugates mAb1-LALA-D265C-(XIV) and mAb8-LALA-D265C-(XIV) in a subcutaneous tumor model of HEK293-GUCY2C-(HDP)-2B3 tumor cells in female NOD Scid mice after a single-dose intravenous administration of ADC as indicated. The results show that a single-dose administration of ADC at concentrations of 1.25 mg / kg intravenous and 2.5 mg / kg intravenous results in effective inhibition of tumor growth.

[0044] Figure 5 . Figure 4The Kaplan - Meyer plot of the survival rate of the subcutaneous GUCY2C - tumor model shown indicates an increase in the survival rate of the animals in groups 2, 3, 5, and 6.

[0045] Figure 6 . The hepatotoxicity of the anti - GUCY2C conjugate of the present invention in cynomolgus monkeys. The effects of the anti - GUCY2C - ADC of the present invention, which comprises the heavy - chain and light - chain amino acid sequences SEQ ID NO:85 and SEQ ID NO:87, on the liver enzymes alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH). The results show that after administration of the conjugate of the present invention, the liver enzymes ALT, AST, and LDH transiently increase and reach baseline levels within 2 - 3 weeks after administration of the conjugate. The results further show that for the conjugate comprising linker - payload (XIV), the maximum tolerated dose is 2 mg / kg, and for the conjugate comprising linker - payload (XII), the maximum tolerated dose is 5 mg / kg.

[0046] Figure 7 . Mouse patient - derived xenograft (PDX) models of colorectal cancer treated with the conjugate of the present invention. (A) PDX models of primary KRAS mutant (G13D) adenocarcinoma at stage T3N0Mx treated with the conjugate of the present invention comprising linker - payload conjugates (XII), (XIV) as indicated. (B) PDX models of primary adenocarcinoma at stage T3N0M0 treated with the conjugate of the present invention comprising linker - payload conjugates (XII), (XIV) as indicated. The conjugates in (A) and (B) are administered as a single dose or repeated weekly for 4 weeks (“q7dx4”).

[0047] Figure 8 . Sequence alignment of (A) the heavy - chain variable region and (B) the light - chain variable region of the antibody of the present invention. The alignment was completed using the Clustal W algorithm (Thompson et al., Nucleic Acids Res. November 11, 1994; 22(22):4673 - 80). The CRDs of both the V H and V L sequences are indicated by boxes. Detailed Description

[0048] Before describing the invention in detail, it should be understood that the invention is not limited to the specific component parts of the described apparatus or the process steps of the described method, as such apparatus and methods may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. Additionally, it should be understood that, given a range of parameters defined by numerical values, the range is considered to include those defined values. The recitation of a range of values herein is merely intended to serve as a shorthand for individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0049] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" should be understood to mean including the stated member, integer, or step, but not excluding any other unstated member, integer, or step. The term "consisting of" is a specific embodiment of the term "comprise" where any other unstated member, integer, or step is excluded.

[0050] It should also be understood that the embodiments disclosed herein are not to be construed as separate embodiments that are unrelated to each other. Features discussed in one embodiment are meant to be disclosed in combination with other embodiments shown herein as well. If in one instance a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, one skilled in the art will understand that this does not necessarily mean that the feature is not intended to be disclosed in that other embodiment. One skilled in the art will understand that the gist of this application is that the feature is also disclosed for other embodiments, but this is not done only for the sake of clarity and to keep the specification within a manageable scope.

[0051] In addition, the content of the prior art documents mentioned herein is incorporated by reference. This particularly refers to prior art documents that disclose standard or conventional methods. In such cases, the main purpose of incorporation by reference is to provide sufficient enabling disclosure and to avoid lengthy repetition. The definitions of chemical groups as used herein shall have the meanings provided in the "Compendium of Chemical Terminology" ("Gold Book") (version 2.3.3, goldbook.iupac.org, ISBN: 0-9678550-9-8) published by the International Union of Pure and Applied Chemistry (IUPAC) and as defined therein, and the content of said document is hereby incorporated by reference.

[0052] Throughout this application, the term "about" is used, which shall mean + / - 10% of the value used with it.

[0053] In addition, the content of the prior art documents mentioned herein is incorporated by reference. This particularly refers to prior art documents that disclose standard or conventional methods. In such cases, the main purpose of incorporation by reference is to provide sufficient enabling disclosure and to avoid lengthy repetition.

[0054] According to a first aspect of the invention, there is provided an antibody or antibody fragment that specifically binds to guanylate cyclase C (GUCY2C), wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ), and the heavy chain variable region comprises framework region 1 (FR H 1), complementarity determining region 1 (CDR H 1), framework region (FR H 2), complementarity determining region 2 (CDR H 2), framework region 3 (FRH3), complementarity determining region 3 (CDRH3) and framework region 4 (FRH4), wherein

[0055] - FRH1 comprises an amino acid sequence selected from SEQ ID NO: 33, 40, 45, 51;

[0056] - CDRH1 comprises an amino acid sequence selected from SEQ ID NO: 34, 41, 46, 52;

[0057] - FRH2 comprises an amino acid sequence according to SEQ ID NO: 35; 55;

[0058] - CDRH2 comprises an amino acid sequence selected from SEQ ID No: 36, 42, 47; 120

[0059] - FRH3 comprises an amino acid sequence selected from SEQ ID NO:37, 43, 48, 53;

[0060] - CDRH3 comprises an amino acid sequence selected from SEQ ID No:38, 44, 49; 54

[0061] - FRH4 comprises an amino acid sequence selected from SEQ ID NO:39, 50.

[0062] As used herein, the term "antibody" shall refer 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 the light chain κ, λ and heavy chain α, δ, ε, γ and μ constant region genes and any of a number of different variable region genes.

[0063] The basic immunoglobulin (antibody) structural unit is generally a tetramer composed of two pairs of identical polypeptide chains (light chains (L, having a molecular weight of about 25 kDa) and heavy chains (H, having a molecular weight of about 50 - 70 kDa)). Each heavy chain consists of a heavy chain variable region (abbreviated as VH or V H ) and a heavy chain constant region (abbreviated as CH or C H ). The heavy chain constant region consists of three domains (i.e., CH1, CH2 and CH3). Each light chain contains a light chain variable region (abbreviated as VL or V L ) and a light chain constant region (abbreviated as CL or C L ). The VH and VL regions can be further subdivided into hypervariable regions (which are also called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains form the binding domain that interacts with the antigen. The constant region (Fc region) does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis via binding to Fcγ receptors; participating in the half-life / clearance rate via the neonatal Fc receptor (FcRn); and participating in complement activation via the C1q component, causing chemotaxis, opsonization of the complement, and potential cytolysis in the case of viable cellular antigen targets. Human antibodies of the IgG1 class are the most potent in activating the complement system and are therefore the ideal isotype for the therapeutic applications of the antibodies of the present invention.

[0064] Human Fcγ receptors include FcγR(I), FcγRIIa, FcγRIIb, FcγRIIIa, and the neonatal FcRn. A common set of IgG1 residues has been shown to be involved in binding to all FcγRs, while FcγRII and FcγRIII use distinct sites outside of this common set (Shields et al. (2001) J. Biol. Chem 276:6591-6604). A set of IgG1 residues that, when mutated to alanine, reduces binding to all FcγRs: Pro-238, Asp-265, Asp-270, Asn-297, and Pro-239 (numbered according to the EU numbering system). All are in the IgG CH2 domain and cluster near the hinge connecting CH1 and CH2. While FcγR1 uses only the common set of IgG1 residues for binding, FcγRII and FcγRIII interact with distinct residues in addition to interacting with the common set. Alteration of some residues reduces binding only to FcγRII (e.g., Arg-292) or FcγRIII (e.g., Glu-293). Some variants show improved binding to FcγRII or FcγRIII without affecting binding to other receptors. The neonatal FcRn receptor is thought to be involved in antibody clearance and transcytotic transport across tissues (see: Junghans (1997) Immunol. Res 16:29-57; and Ghetie et al. (2000) Annu. Rev. Immunol. 18:739-766). Human IgG1 residues that have been determined to directly interact with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435.

[0065] As used herein, the terms "CDR", "CDR L 1", "CDR L 2", "CDR L 3", "CDR H 1", "CDR H 2", "CDR H3" follows the Kabat numbering convention (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)). However, although the Kabat numbering convention for amino acid residues in variable domain sequences and full-length antibody sequences is used throughout the specification, it will be clear to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those listed in Chothia et al. (1989) Nature 342:877-883. The structure and protein folding of antibodies may mean that other residues based on the numbering system used are considered part of the CDR sequence, and the skilled person will understand that this is so, however, these differences do not functionally imply altered or different antigen binding of the corresponding antibodies. Other numbering conventions for CDR sequences available to the skilled person include the "AbM" (University of Bath) and "contact" (University College London) methods.

[0066] The CDRs are most important for the binding of an antibody or its antigen-binding portion. The FRs can be replaced by other sequences, provided that the three-dimensional structure required for antigen binding is retained. Structural changes in the constructs most often result in the loss of adequate binding to the antigen.

[0067] Antibodies usually bind specifically to their cognate antigens with high affinity. -5 Up to 10 -11 A dissociation constant (KD) of M or less reflects a dissociation constant of 10. -4 M of K D Indicates non-specific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and substantially the same antigen, meaning that it has a 10 -7 M or less, preferably 10 -8 M or less, even more preferably 5×10 -9 M or less, and most preferably about 10 -8 , 10 -9 M to about 10 -10 M, 10 -11 or less, or for example about 10 -10 M to about 10 -11 M or smaller K D , but does not bind to an unrelated (e.g., structurally or sequence unrelated) antigen with an affinity equal to that for the specific target.

[0068] The antibodies of the present invention, which may also be referred to as immunoglobulins for example, can be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into the following subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice.

[0069] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to such antibody fragments or antibody analogs that retain the binding specificity of the parental anti-GUCY2C antibody as disclosed herein and contain a part (e.g., one or more CDRs) or the variable region of the antigen-binding region of the parental antibody. Antibody fragments are, for example, Fab, Fab’, F(ab’)2, Fv fragments, sc-Fv, unibody, diabody, linear antibody, nanobody, domain antibody, or multispecific antibody fragments formed from antibody fragments. The Fab fragment consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab’ fragment contains a part of the heavy chain and the light chain, and the part of the heavy chain contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains. The F(ab’)2 fragment contains two light chains and two heavy chains, and the two heavy chains contain a part 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 is composed of two Fab’ fragments bound together by a disulfide bond between the two heavy chains. The "Fv fragment" contains the variable regions from both the heavy chain and the light chain but lacks the constant region. The term "single-chain antibody" is a single-chain recombinant protein formed by linking the variable region VH of the heavy chain of an antibody and the variable region VL of the light chain by a linker peptide. It is the smallest antibody fragment with a complete antigen-binding site. The term "domain antibody fragment" is an immunoglobulin fragment with immunological function that contains only the variable region chain of the heavy chain or the light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.

[0070] According to one embodiment, the anti-GUCY2C antibody or antibody fragment of the present invention as disclosed herein comprises a light chain variable region (V L ), the light chain variable region comprising 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 L4, wherein

[0071] -FR L 1 comprises an amino acid sequence selected from SEQ ID NO: 1, 13, 20, 27;

[0072] -CDR L 1 comprises an amino acid sequence selected from SEQ ID NO: 2, 14, 21, 28;

[0073] -FR L 2 comprises an amino acid sequence selected from SEQ ID NO: 3, 15, 29

[0074] -CDR L 2 comprises an amino acid sequence selected from SEQ ID NO: 4, 16, 23, 30;

[0075] -FR L 3 comprises an amino acid sequence selected from SEQ ID No: 5, 8, 17, 24, 31;

[0076] -CDR L 3 comprises an amino acid sequence selected from SEQ ID NO: 6, 9, 11, 18, 25, 32;

[0077] -FR L 4 comprises an amino acid sequence selected from SEQ ID NO: 7, 10, 12, 19, 26.

[0078] According to one embodiment, the anti-GUCY2C antibody of the present invention comprises a heavy chain variable region (V H ), said heavy chain variable region comprising framework region 1 (FR H 1), complementarity determining region 1 (CDR H 1), FR H 2, CDR H 2, FRH3, CDRH3 and FRH4, wherein

[0079] -FRH1 comprises the amino acid sequence according to SEQ ID NO: 33

[0080] -CDRH1 comprises the amino acid sequence according to SEQ ID NO: 34,

[0081] -FRH2 comprises the amino acid sequence according to SEQ ID NO: 35;

[0082] -CDRH2 comprises the amino acid sequence according to SEQ ID NO: 36;

[0083] -FRH3 comprises the amino acid sequence according to SEQ ID NO: 37;

[0084] - The CDRH3 comprises the amino acid sequence according to SEQ ID NO: 38;

[0085] - The FRH4 comprises the amino acid sequence according to SEQ ID NO: 39, and

[0086] wherein the antibody comprises a light chain variable region (V L ), and the light chain variable region comprises 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, wherein

[0087] - FR L 1 comprises the amino acid according to SEQ ID NO: 1; 121

[0088] - CDR L 1 comprises the amino acid according to SEQ ID NO: 2;

[0089] - FR L 2 comprises the amino acids according to SEQ ID NO: 3, 15, 29

[0090] - CDR L 2 comprises the amino acid sequence selected from SEQ ID NO: 4;

[0091] - FR L 3 comprises the amino acid sequences selected from SEQ ID No: 5, 8;

[0092] - CDR L 3 comprises the amino acid sequences selected from SEQ ID NO: 6, 9, 11;

[0093] - FR L 4 comprises the amino acid sequences selected from SEQ ID NO: 7, 10, 12.

[0094] According to one embodiment, the anti-GUCY2C antibody or antibody fragment of the present invention comprises a heavy chain variable region (V H ), and the heavy chain variable region comprises framework region 1 (FR H 1), complementarity determining region (CDR H 1), FR H 2, CDR H 2, FRH3, CDRH3 and FRH4, wherein

[0095] -FRH1 contains the amino acid sequences according to SEQ ID NO:40, 45, 51;

[0096] -CDRH1 contains the amino acid sequences according to SEQ ID NO:41, 46, 52,

[0097] -FRH2 contains the amino acid sequences according to SEQ ID NO:35; 55

[0098] -CDRH2 contains the amino acid sequences according to SEQ ID NO:42, 47;

[0099] -FRH3 contains the amino acid sequences according to SEQ ID NO:43, 48, 53;

[0100] -CDRH3 contains the amino acid sequences according to SEQ ID NO:49, 44, 54

[0101] -FRH4 contains the amino acid sequences according to SEQ ID NO:50, 39, and

[0102] wherein said antibody comprises a light chain variable region (V L ), said light chain variable region comprising 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, wherein

[0103] -FR L 1 contains the amino acids according to SEQ ID NO:13, 20, 27;

[0104] -CDR L 1 contains the amino acids according to SEQ ID NO:14, 21, 28;

[0105] -FR L 2 contains the amino acids according to SEQ ID NO:15, 22, 29;

[0106] -CDR L 2 contains the amino acid sequences selected from SEQ ID NO:16, 23, 30;

[0107] -FR L 3 contains the amino acid sequences selected from SEQ ID No:17, 24, 31;

[0108] -CDR L3 comprises an amino acid sequence selected from SEQ ID NO:18, 25; 32

[0109] -FR L 4 comprises an amino acid sequence selected from SEQ ID NO:19, 26.

[0110] According to one embodiment, an anti-GUCY2C antibody of the invention as disclosed herein comprises a light chain variable region (V L ) and a heavy chain variable region (V H ), the light chain variable region comprising CDR L 1 according to SEQ ID NO:2, CDR L 2 according to SEQ ID NO:4, CDR L 3 according to SEQ ID NO:9, and the heavy chain variable region comprising CDR H 1 according to SEQ ID NO:34, CDR H 2 according to SEQID NO:36, CDR H 3.

[0111] According to one embodiment, an anti-GUCY2C antibody of the invention as disclosed herein comprises a light chain variable region (V L ) and a heavy chain variable region (V H ), the light chain variable region comprising CDR L 1 according to SEQ ID NO:2, CDR L 2 according to SEQ ID NO:4, CDR L 3 according to SEQ ID NO:6, and the heavy chain variable region comprising CDR H 1 according to SEQ ID NO:34, CDR H 2 according to SEQ ID NO:36, CDR H 3.

[0112] According to one embodiment, an anti-GUCY2C antibody of the invention as disclosed herein comprises a light chain variable region (V L ) and a heavy chain variable region (V H ), the light chain variable region comprising CDR L 1 according to SEQ ID NO:2, CDR L 2 according to SEQ ID NO:4, CDR L 3 according to SEQ ID NO:11, and the heavy chain variable region comprising CDR H 1 according to SEQ ID NO:34, CDRH 2. CDRs according to SEQ ID NO:38 H 3.

[0113] According to one embodiment, the anti-GUCY2C antibody of the invention as disclosed herein comprises a light chain variable region (V L L) and a heavy chain variable region (V H H), the light chain variable region comprising 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 the heavy chain variable region comprising 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.

[0114] According to one embodiment, the anti-GUCY2C antibody of the invention as disclosed herein comprises a light chain variable region (V L L) and a heavy chain variable region (V H H), the light chain variable region comprising CDRs according to SEQ ID NO:21 L 1. CDRs according to SEQ ID NO:23 L 2. CDRs according to SEQ ID NO:25 L 3, and the heavy chain variable region comprising CDRs according to SEQ ID NO:46 H 1. CDRs according to SEQ ID NO:47 H 2. CDRs according to SEQ ID NO:49 H 3.

[0115] According to one embodiment, the anti-GUCY2C antibody of the invention as disclosed herein comprises a light chain variable region (V L L) and a heavy chain variable region (V H H), the light chain variable region comprising CDRs according to SEQ ID NO:14 L 1. CDRs according to SEQ ID NO:16 L 2. CDRs according to SEQ ID NO:18 L 3, and the heavy chain variable region comprising CDRs according to SEQ ID NO:41 H 1. CDRs according to SEQ ID NO:42 H 2. CDRs according to SEQ ID NO:44 H 3.

[0116] In one embodiment, the light chain variable region (V L ) of the anti-GUCY2C antibody of the present invention comprises the amino acid sequences according to SEQ ID NO.2; 3; 4, 121 and SEQ ID NO:8; 9; 10, and the heavy chain variable region (V H ) comprises the amino acid sequences according to SEQ ID NO:33; 34; 35; 36; 37; 38; 39 (e.g., mAb#1).

[0117] In one embodiment, the light chain variable region (V L ) of the anti-GUCY2C antibody of the present invention comprises the amino acid sequences according to SEQ ID NO.1-7, and the heavy chain variable region (V H ) comprises the amino acid sequences according to SEQ ID NO:33; 34; 35; 36; 37; 38; 39 (e.g., mAb#8).

[0118] In one embodiment, the light chain variable region (V L ) of the anti-GUCY2C antibody of the present invention comprises the amino acid sequences according to SEQ ID NO.2; 3; 4; 5, 11, 12, 121, and the heavy chain variable region (V H ) comprises the amino acid sequences according to SEQ ID NO:33; 34; 35; 36; 37; 38; 39 (e.g., mAb#24).

[0119] In one embodiment, the light chain variable region (V L ) of the anti-GUCY2C antibody of the present invention comprises the amino acid sequences according to SEQ ID NO.20; 21; 22; 23; 24; 25; 26, and the heavy chain variable region (V H ) comprises the amino acid sequences according to SEQ ID NO:45, 46, 48, 49, 50 and 55 (e.g., mAb#28).

[0120] In one embodiment, the light chain variable region (V L ) of the anti-GUCY2C antibody of the present invention comprises the amino acid sequences according to SEQ ID NO.40, 41, 55, 42, 43, 44, 39, and the heavy chain variable region (V H ) comprises the amino acid sequences according to SEQ ID NO:13; 14; 15; 16; 17; 18; 19 (e.g., mAb#40).

[0121] In one embodiment, the light chain variable region (V L) Comprising the amino acid sequences according to SEQ ID NO.27; 28; 29; 30; 32; 19, and the heavy chain variable region (V H ) Comprising the amino acid sequences according to SEQ ID NO:51; 52; 55; 120; 53; 54; and 39 (e.g., mAb#41).

[0122] According to some embodiments, the anti-GUCY2C antibody or antibody fragment according to the present invention comprises a subsequent combination of a heavy chain variable region (V H ) and a light chain variable region (V L ), said combination comprising the amino acid sequences according to the following:

[0123] - SEQ ID NO:62, SEQ ID NO:56;

[0124] - SEQ ID NO:63, SEQ ID NO:57

[0125] - SEQ ID NO:64, SEQ ID NO:58

[0126] - SEQ ID NO:65, SEQ ID NO:59

[0127] - SEQ ID NO:66, SEQ ID NO:60

[0128] - SEQ ID NO:67, SEQ ID NO:61.

[0129] Each of the V L and V H combinations disclosed above may also, for example, comprise V L , or V H , or V L and V H both (each VL, VH sequence independently or both the VL and VH sequences together have at least 90%, 95% sequence identity with SEQ ID No:56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 67), provided that the corresponding anti-GUCY2C antibody or antibody fragment specifically binds to the same epitope on human or cynomolgus monkey GUCY2C. Epitope mapping of the antibodies of the present invention can be done, for example, by hydrogen-deuterium exchange (HDX) analogous to the method disclosed in Huang et al. MAbs. January 2018; 10(1):95 - 103 or for example as reviewed in Jethva and Gross, Front. Anal. Sci., May 18, 2023.

[0130] According to one embodiment, the antibody or antibody fragment according to the invention can be a monoclonal antibody. As used herein, the term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules having a single binding specificity and affinity for a particular epitope, representing a homogeneous population of antibodies, i.e., a homogeneous population consisting of intact immunoglobulins or fragments. Preferably, the monoclonal anti-GUCY2C antibody of the invention as disclosed herein is of the IgG isotype (e.g., IgG1 or IgG4), more preferably of the IgG1 isotype.

[0131] Monoclonal antibodies (mAbs) derived from, for example, mice can cause unwanted immune side effects when administered to humans due to the fact that they contain proteins from another species that can trigger antibodies. To overcome this problem, antibody humanization and maturation methods have been designed to produce antibody molecules with minimal immunogenicity when applied to humans, while ideally still retaining the specificity and affinity of the non-human parental antibody (reviewed in Almagro and Fransson 2008).

[0132] Thus, 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 a minimal sequence derived from a non-human immunoglobulin. Thus, a "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains a minimal sequence derived from a non-human antibody. All or substantially all of the framework regions may also be framework regions of human immunoglobulin sequences. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically a portion of the human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art and have been described, for example, in Riechmann et al., Nature 332:323-7, 1988; U.S. Patent Nos.: 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 prepared by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines, see Kozbor (1984) J. Immunol 133, 3001. Alternative methods include using phage libraries or transgenic mice, both of which use human variable region libraries (see Winter (1994) Annu. Rev. Immunol 12:433-455; Green (1999) J. Immunol. Methods 231:11-23). Several transgenic mouse strains are now available in which their murine immunoglobulin loci have been replaced by human immunoglobulin gene segments (see Tomizuka (2000) PNAS 97:722-727; Fishwild (1996) Nature Biotechnol. 14:845-851; Mendez (1997) Nature Genetics, 15:146-156). After antigen stimulation, such mice are capable of generating a human antibody repertoire from which antibodies of interest can be selected.

[0133] According to some embodiments, the constant region of the IgG heavy chain of the anti-GUCY2C IgG1 antibody of the present invention may be selected from different allotypes. In the context of the anti-GUCY2C antibody of the present invention, the term "allotype" refers to genetic allelic variants caused by genetic differences between individuals, which can be recognized as antigenic by members of the same species. For example, the antibody of the present invention may have one of the following allotypes: G1m3, G1m17,1 or G1m17,1,2 allotypes or G1m(f), G1m(z,a) or G1m(z,a,x).

[0134] Human antibodies (e.g., the human anti-GUCY2C antibodies of the present invention) can comprise the following: a kappa (κ) chain, which is encoded in humans by the immunoglobulin kappa locus (IGK) on chromosome 2 (locus: 2p11.2); or a lambda (λ) chain, which is encoded in humans by the immunoglobulin lambda locus (IGL) on chromosome 22 (locus: 2q11.22). Thus, the anti-GUCY2C antibodies of the present invention can comprise two κ light chains or two λ light chains, which light chains comprise a VL sequence according 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.

[0135] According to one embodiment, the anti-GUCY2C antibody or antigen-binding fragment thereof of the present invention can be glycosylated or deglycosylated. Preferably, the antibody of the present invention is glycosylated, and the corresponding glycan can 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). Like most glycoproteins, antibodies are generally produced as a mixture of glycoforms. This mixture is particularly evident when antibodies are produced in eukaryotic cells, especially mammalian cells. Each glycan moiety added to the Fc-glycan (e.g., fucose, bisecting GlcNAc, galactose, and sialic acid) is involved in modulating antibody effector affinity and / or antibody function. Thus, there is a need to produce monoclonal antibodies characterized by a defined set of glycans to obtain antibodies with defined effector functions. A variety of methods have been developed to produce defined glycoforms, 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. The anti-GUCY2C antibodies of the present invention as described herein preferably comprise homogeneous or a defined number of glycoforms, such as about 1, 2, 3, 4 to about 5, 6, 7, 8, or about 5, 6, 7 to about 8, preferably 6 or less, such as 5, 4, 3 or less, more preferably two or one.

[0136] According to one embodiment, the anti-GUCY2C antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof. As used herein, the term "recombinant" or "recombinant-produced" refers to a protein that has been expressed in a heterologous cell (such as the anti-GUCY2C antibody or antigen-binding fragment of the invention as disclosed herein). For example, the anti-GUCY2C antibody or antigen-binding fragment of the invention can be expressed in a prokaryotic cell or a eukaryotic cell. Prokaryotic cells for expressing the antibody of the invention include, for example, Gram-negative bacteria (such as Escherichia coli (E. coli)) or Gram-positive bacteria (such as Bacillus subtilis). The use of a heterologous prokaryotic expression system can be particularly useful for the expression of the antigen-binding fragment of the anti-GUCY2C antibody of the invention and can be accomplished according to established protocols known in the art (for the expression of Fab fragments, for example, Kwong and Rader, Curr. Protoc. Protein Sci. 55:6.10.1-6.10.14.).

[0137] However, due to the glycosylation of heterologously expressed antibodies, eukaryotic cells are preferably used for expressing the antibody or antigen-binding fragment of the invention. Even more preferably, mammalian cells are used to express the antibody or antigen-binding fragment of the invention. For example, Chinese hamster ovary (CHO) cells or any genetically distinct progeny thereof (such as the K1 cell line, DukX B11 cell line, DG44 cell line) can be used to express the antibody of the invention. Other mammalian cell lines that can be used to produce the antibody or antigen-binding fragment of the invention include, for example, NS0 cells, embryonic kidney (HEK) 293 cells, PER.C6 cells, MCF7 cells. The cells can be transfected, for example, with an expression vector containing the coding sequence of the anti-GUCY2C antibody or antigen-binding fragment according to the invention. An expression vector or recombinant plasmid is produced by placing the antibody coding sequence under the control of suitable regulatory gene elements (including promoter and enhancer sequences, such as the CMV promoter). The heavy and light chain sequences can be expressed, for example, by separate co-transfected expression vectors or by a dual expression vector. The transfection can be transient transfection or stable transfection. The transfected cells are then cultured to produce the transfected antibody construct. In the case of stable transfection, stable clones secreting antibodies with appropriately associated heavy and light chains are then selected by screening with an appropriate assay (e.g., ELISA), subcloned, and propagated for future production. Corresponding methods are disclosed, for example, in WO 03 / 018771.

[0138] According to one embodiment, the anti-GUCY2C antibody of the invention comprises an Fc region containing the amino acid sequence according to SEQ ID NO:68. The antibody of the invention containing an Fc region containing the amino acid sequence according to SEQ ID NO:68 is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0139] However, it may be desirable to reduce or eliminate the effector functions of the antibodies of the invention as disclosed herein, for example to prevent target cell death, unwanted cytokine secretion, or killing of cells expressing Fcγ receptors (such as macrophages).

[0140] Thus, the anti-GUCY2C antibodies or antigen-binding fragments of the invention as described herein may also include modifications and / or mutations that alter the properties of the antibody and / or fragment (such as those known in the art that reduce ADCC, ADCP, or complement-dependent cytotoxicity CDC). Preferably, compared to an antibody containing a wild-type Fc region containing, for example, the amino acid sequence according to SEQ ID NO:68, ADCC, ADCP, and CDC are reduced by at least 90% or more, more preferably at least 95%, more preferably at least 97.5%, even more preferably at least 98%, 99%.

[0141] The binding of IgG1 to activating and inhibitory Fcγ receptors (FcγR) or the first component of complement (C1q) depends on residues located in the hinge region and CH2 domain. Two regions of the CH2 domain are critical 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 greatly reduce 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, WO 2021 / 234402 A2).

[0142] Thus, in one embodiment, the anti-GUCY2C antibody or antigen-binding fragment thereof as disclosed herein comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region such that the molecule has a reduced affinity for IgG1 Fc receptors FcγRI, FcγRII, and FcγRIII and for complement component C1q compared to the wild-type Fc region (e.g., containing the amino acid sequence according to SEQ ID NO:68).

[0143] The affinity for the Fc region (such as the binding of IgG1 to FcγR) can be determined using a variety of techniques known in the art, such as, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Volume 373: 52-60, 2008; or radioimmunoassay (RIA)); or surface plasmon resonance assays as disclosed, for example, in Wilkinson et al. PLoS One. 2021; 16(12):e0260954; or other mechanisms for kinetic-based assays (e.g., BIACORE™ analysis or Octet™ analysis (forteBIO)); and other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration).

[0144] Accordingly, the anti-GUCY2C antibodies described herein have been genetically engineered to contain a variant Fc region that, based on structural and crystallographic analysis, contains a modification of at least one amino acid residue that directly contacts FcγR. As used herein, the term "genetically engineered" or "genetic engineering" refers to modifying the amino acid sequence or a portion thereof of a given or native polypeptide or protein (e.g., the Fc region of an antibody) in the sense of nucleotide and / or amino acid substitutions, insertions, deletions, or revertant mutations or any combination thereof by genetic technology methods (e.g., site-directed mutagenesis as described in Carter, Biochem. J. (1986) 237: 1-7). As used herein, the term "amino acid substitution" or "mutation" refers to a modification of the amino acid sequence of a protein in which one or more amino acids are replaced by the same number of different amino acids, resulting in a protein with an amino acid sequence different from the original protein. Conservative amino acid substitutions are understood to involve substitutions that do not significantly affect the structure and function of the protein due to similar size, charge, polarity, and / or conformation. The group of conservative amino acids in this sense means, for example, non-polar amino acids Gly, Ala, Val, Ile, and Leu; aromatic amino acids Phe, Trp, and Tyr; positively charged amino acids Lys, Arg, and His; and negatively charged amino acids Asp and Glu.

[0145] Depending on the intended use of the antibody (e.g., use in the manufacture of antibody-drug conjugates), the Fc region of the antibody can further comprise at least one cysteine amino acid substitution at a site where engineered cysteine can be used for conjugation without interfering with immunoglobulin folding and assembly. Antibodies with corresponding cysteine substitutions or cysteine-engineered antibodies are disclosed in WO 2016040856A2 or Junutula et al., 2008 Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO 2009 / 052249 and WO 2016 / 142049. A preferred cysteine substitution in the Fc region of the antibodies of the invention as disclosed herein is D265C (according to the EU numbering system) as disclosed in WO 2016142049A1.

[0146] In one embodiment, the anti-GUCY2C antibody of the invention comprises an Fc region or a fragment thereof, the Fc region or a fragment thereof comprising at least one amino acid substitution selected from L234A, L234S, L234G, L235A, L235G, L235S, L235T, G236R, D265C, wherein the amino acid numbering is according to the EU numbering system. The EU numbering system can also be referred to as the "EU index in Kabat" and refers to the numbering of human IgG1 EU antibodies, which refers to the numbering of the EU antibodies of Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85.

[0147] In some embodiments, the anti-GUCY2C antibody of the invention comprises an Fc region, the Fc region comprising the above amino acid substitutions alone or in combination (e.g., two or more (e.g., 2, 3 or 4) of the above amino acid substitutions), as listed below:

[0148] Fc amino acid substitutions (according to the EU numbering system) According to the following amino acid sequences D265C SEQ ID NO:70 L234A, L235A SEQ ID NO:69 L234A, L235A, D265C SEQ ID NO:71 L234A, L235A, G236R, D265C SEQ ID NO:72 L234G, L235S, G236R, D265C SEQ ID NO:73 L234S, L235T, G236R, D265C SEQ ID NO:74 L234S, L235G, G236R, D265C SEQ ID NO:75

[0149] According to a preferred embodiment, the anti-GUCY2C antibody of the present invention comprises an Fc region, which Fc region comprises at least three amino acid substitutions as disclosed above, preferably the Fc region comprises at least the amino acid substitutions L234A, L235A, D265C (SEQ ID NO: 71). 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, for example, for reducing the interaction of the Fc region comprising said mutations with FcγR (reducing by at least 95%, 97.5%, 99% compared to the wild-type Fc region), while allowing site-specific thiol-based conjugation of the linker-payload (e.g., those as disclosed herein). The use of such cysteine-substituted Fc regions and antibodies comprising such mutations for linker-payload conjugation is disclosed, for example, in WO 2016142049 A1.

[0150] In some embodiments, the anti-GUCY2C antibody of the present invention comprises an Fc region comprising or consisting of the amino acid sequence according to any one of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75. Use of the Fc region comprising four mutations can, for example, be advantageous for eliminating the residual interaction of the Fc region with FcγR, thereby yielding the Fc-silent antibodies of the present invention.

[0151] In some embodiments, the anti-GUCY2C antibody of the present invention can thus comprise the following pairings of a light chain (LC) and a heavy chain (HC) as disclosed herein, the light chain and the heavy chain comprising at least one, preferably at least three mutations as disclosed herein:

[0152] Light chain (LC) amino acid sequence Heavy chain (HC) amino acid sequence SEQ ID NO:78 SEQ ID NO:79, 80, 81, 82, 83 or 84. SEQ ID NO:85 SEQ ID NO:886, 87, 88, 89, 900 or 91 SEQ ID NO:92 SEQ ID NO:93, 94, 95, 96, 97 or 98 SEQ ID NO:99 SEQ ID NO:100, 101, 102, 103, 104 or 105 SEQ ID NO:106 SEQ ID NO:107, 108, 109, 110, 111 or 112 SEQ ID NO:113 SEQ ID NO:114, 115, 116, 117, 118 or 119

[0153] According to a preferred embodiment, the anti-GUCY2C antibody of the present invention comprises a light chain and a heavy chain selected from:

[0154] LC according to SEQ ID NO: 78, HC according to SEQ ID NO: 80, or

[0155] LC according to SEQ ID NO: 85, HC according to SEQ ID NO: 87, or

[0156] LC according to SEQ ID NO: 92, HC according to SEQ ID NO: 94, or

[0157] LC according to SEQ ID NO: 99, HC according to SEQ ID NO: 101, or

[0158] LC according to SEQ ID NO:106, HC according to SEQ ID NO:108, or

[0159] LC according to SEQ ID NO:113, HC according to SEQ ID NO:115.

[0160] According to a more preferred embodiment, the anti-GUCY2C antibody of the invention comprises a light chain and a heavy chain selected from:

[0161] LC according to SEQ ID NO:78, HC according to SEQ ID NO:80, or

[0162] LC according to SEQ ID NO:85, HC according to SEQ ID NO:87, or

[0163] LC according to SEQ ID NO:92, HC according to SEQ ID NO:94.

[0164] According to one embodiment, the anti-GUCY2C antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the antibody comprises CDRs selected from SEQ ID NO:34, 41, 46, 52; 36, 42, 47; 38, 44 or 49 H 1, CDR H 2 and CDR H 3; and wherein the framework regions FR H 1, FR H 2, FR H 3, FR H 4 have at least 80%, 85%, preferably 90% or 95% sequence similarity to any one of SEQ ID NO:33, 40, 45, 51; 35; 55; 37, 43, 48, 53; 39 or 50.

[0165] The terms "sequence similarity", "sequence identity", or "sequence similar" are used interchangeably throughout the present 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. The sequence identity according to the present invention can be determined, for example, over the full length of each sequence compared to the corresponding reference sequence (so-called "global alignment") or over a shorter, defined length (so-called "local alignment"), the global alignment being particularly suitable for sequences of the same or similar length, and the local alignment being more suitable for sequences of unequal length. In the above context, an amino acid sequence having at least, for example, 95% "sequence identity" with a query amino acid sequence is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence, except that the subject amino acid sequence may contain at most five amino acid changes per 100 amino acids of the query amino acid sequence. For example, in order to obtain an amino acid sequence having at least 95% identity with a query amino acid sequence, at most 5% (5 out of 100) of the amino acid residues in the subject sequence can be inserted or replaced by another amino acid or deleted.

[0166] Methods for comparing the identity and sequence similarity of two or more sequences are well known in the art. The percentage of identity between two sequences can be determined, for example, by using a mathematical algorithm. Preferred but non-limiting examples of mathematical algorithms that can be used are the algorithms of Karlin et al. (1993), PNAS USA, 90:5873-5877. This algorithm is 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, which can be accessed via the NCBI homepage at the World Wide Web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 83, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U.S.A 85, 2444-2448.). These programs can identify sequences that are identical to other sequences to some extent. In addition, 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) (e.g., the programs BESTFIT and GAP) can be used to determine the % identity between two polypeptide sequences. BESTFIT uses the "local sequence similarity" algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147, 195-197.) and finds the single best region of similarity between two sequences. For example, "gapped BLAST" can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used for iterative searches, which detect remote relationships between molecules. When using any of the above BLAST, GappedBLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used.

[0167] According to one embodiment, the present invention relates to a conjugate comprising

[0168] (i) an anti-GUCY2C antibody of the present invention as disclosed herein,

[0169] (ii) at least one toxin, and

[0170] (iii) At least one linker connecting the antibody or antibody portion to the at least one toxin, wherein the antibody specifically binds to human GUCY2C and wherein the at least one toxin is an amanitin.

[0171] As used herein, the terms "antibody" and "antibody portion" in connection with the conjugates of the invention both refer to one or more anti-GUCY2C antibodies of the invention as disclosed herein and may be used interchangeably. The term "portion" may be used to indicate the fact that the anti-GUCY2C antibodies of the invention are included in the conjugates of the invention.

[0172] Thus, in one embodiment, the conjugate of the invention comprises an anti-GUCY2C antibody as disclosed herein. Preferably, the anti-GUCY2C antibody of the invention comprises a light chain and a heavy chain according to the following table:

[0173] Light chain (LC) amino acid sequence Heavy chain (HC) amino acid sequence SEQ ID NO:78 SEQ ID NO:80, 81, 82, 83 or 84. SEQ ID NO:85 SEQ ID NO::887, 88, 89, 90 or 91 SEQ ID NO:92 SEQ ID NO:94, 95, 96, 97 or 98 SEQ ID NO:99 SEQ ID NO:101, 102, 103, 104 or 105 SEQ ID NO:106 SEQ ID NO:108, 109, 110, 111 or 112 SEQ ID NO:113 SEQ ID NO:115, 116, 117, 118 or 119

[0174] More preferably, the conjugate of the invention comprises an anti-GUCY2C antibody portion comprising a light chain amino acid sequence and a heavy chain amino acid sequence selected from: the light chain amino acid sequence according to SEQ ID NO:78 and the heavy chain amino acid sequence according to SEQ ID NO:80; the light chain amino acid sequence according to SEQ ID NO:85 and the heavy chain amino acid sequence according to SEQ ID NO:87; the light chain amino acid sequence according to SEQ ID NO:92 and the heavy chain amino acid sequence according to SEQ ID NO:94; the light chain amino acid sequence according to SEQ ID NO:99 and the heavy chain amino acid sequence according to SEQ ID NO:101; the light chain amino acid sequence according to SEQ ID NO:106 and the heavy chain amino acid sequence according to SEQ ID NO:108; and the light chain amino acid sequence according to SEQ ID NO:113 and the heavy chain amino acid sequence according to SEQ ID NO:115. More preferably, the conjugate of the invention comprises an anti-GUCY2C antibody portion 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; or SEQ ID NO:85 and SEQ ID NO:87; or SEQ ID NO:92 and SEQ ID NO:94.

[0175] According to one embodiment, the conjugate of the invention comprises at least one (e.g., 1, 2, 3, 4) toxin molecules, wherein the toxin is an amanitin, and the amanitin is bound to the anti-GUCY2C antibody of the invention via at least one (e.g., one or two) linker.

[0176] As used herein, the term "amatoxin or amatoxins" refers to an eight - amino - acid bicyclic peptide found in the Amanita phalloides mushroom (see Figure 1 ). Amatoxins specifically inhibit DNA - dependent RNA polymerase II in mammalian cells, thereby also inhibiting transcription and protein biosynthesis in the affected cells. Inhibition of transcription in the cells leads to a halt in growth and proliferation. Although the complex between amatine and RNA - polymerase II is not covalently bound, it is very tight (K D = 3 nM). The dissociation of amatine from the enzyme is a very slow process, and thus it is unlikely that the affected cells will recover. When the transcription inhibition persists for a long enough time, the cells will undergo programmed cell death (apoptosis).

[0177] In the context of the present invention, the term "amatoxin" includes all eight - amino - acid bicyclic peptides isolated from the genus Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5(1978)185 - 260), as well as all their chemical derivatives; all their semi - synthetic analogs; all their synthetic analogs constructed from building blocks based on the main structure (cyclic, eight amino acids) of the natural compound, as well as all synthetic or semi - synthetic analogs containing non - hydroxylated amino acids instead of hydroxylated amino acids, and all synthetic or semi - synthetic analogs in which the sulfoxide moiety is replaced by a sulfone, a thioether, or an atom different from sulfur (e.g., a carbon atom in a carbon analog of amatine).

[0178] As used herein, a "derivative" of a compound refers to a class of compounds having a chemical structure similar to that of the compound, but containing at least one chemical group that is not present in the compound from which it is derived and / or lacking at least one chemical group that is present in the compound from which it is derived. The compound to which the derivative is compared is called the "parent" compound. Generally, a "derivative" can be produced from the parent compound in one or more chemical reaction steps.

[0179] As used herein, an "analogue" of a compound is structurally related but not identical to the compound and exhibits at least one activity of the compound. The compound to which the analogue is compared is called the "parent" compound. Such activities include, but are not limited to: binding activity to another compound; inhibitory activity (e.g., enzyme inhibitory activity); toxic effects; activating activity (e.g., enzyme activating activity). It is not required that the analogue exhibit the same degree of such activity as the parent compound. In the context of the present application, a compound is considered an analogue 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%, and more preferably at least 50%) of the relevant activity of the parent compound. Thus, as used herein, an "analogue of amatoxin" refers to a compound that is structurally related to any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, and amanullinic acid and 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%, 50%, 60%, and more preferably at least 70%, 80%, 90%) of the inhibitory activity against mammalian RNA polymerase II as compared to at least one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, and amanullinic acid. An "analogue of amatoxin" suitable for use in the present invention may even exhibit stronger inhibitory activity against mammalian RNA polymerase II than any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, or amanullinic acid. The inhibitory activity can be measured by determining the concentration at which 50% inhibition occurs (IC 50 value). The inhibitory activity against mammalian RNA polymerase II can be indirectly determined by measuring the inhibitory activity on cell proliferation, or alternatively, the inhibitory activity of amatoxins and their respective derivatives as disclosed herein can be evaluated, for example, using the RNA polymerase II activity assay disclosed in Voss et al., BMC Molecular Biology 2014, 15:7.

[0180] "Semi-synthetic analogs" refer to analogs obtained by chemical synthesis using compounds from natural sources (e.g., plant material, bacterial cultures, fungal cultures, or cell cultures) as starting materials. Generally, the "semi-synthetic analogs" of the present invention have been synthesized starting from compounds isolated from mushrooms of the Amanitaceae family. In contrast, "synthetic analogs" refer to analogs synthesized from small (usually petrochemical) building blocks by so-called total synthesis. Generally, such total synthesis is carried out without the aid of biological processes.

[0181] According to some embodiments of the present invention, the amatoxins may be selected from α-amanitin, β-amanitin, amanitine, amanitine amide, and their analogs, derivatives, and salts.

[0182] Functionally, amatoxins are defined as peptides or ester peptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are amatoxins having functional groups (e.g., carboxyl, amino, hydroxyl, thiol group, or thiol trapping group) that can react with a linker molecule or a target binding moiety as defined below.

[0183] 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 (particularly a hydroxyproline residue) at position 2, an isoleucine, hydroxyisoleucine, or dihydroxyisoleucine (or aspartic acid for amanullinic acid) at position 3, a tryptophan or hydroxytryptophan residue (or proline for proamanullin) at position 4, glycine residues at positions 5 and 7 (or isoleucine residues in the case of amanullinic acid and proamanullin), an isoleucine residue at position 6, and a cysteine residue (particularly a cysteine derivative oxidized to a sulfoxide or sulfone derivative) at position 8 (for the numbering and representative examples of amanitin, see Figure 1 ), and further includes all of its chemical derivatives; all of its semi-synthetic analogs; all of its synthetic analogs constructed from building blocks according to the main structure (cyclic, 8 amino acids) of the natural compound, all synthetic or semi-synthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids, and all synthetic or semi-synthetic analogs, in each case, wherein any such derivative or analog has functional activity by inhibiting mammalian RNA polymerase II. Throughout the present application, the term "amino acid 1" with respect to amatoxins refers to as Figure 1The asparagine residue of the corresponding amanitin depicted therein, accordingly, "amino acid 2" refers to a hydroxyproline residue, "amino acid 3" refers to a dihydroxyisoleucine residue, "amino acid 4" refers to a hydroxytryptophan residue, "amino acid 5" and "amino acid 7" refer to glycine residues, "amino acid 6" refers to an isoleucine residue, and "amino acid 8" refers to a cysteine residue.

[0184] According to some embodiments of the present invention, the linker is attached to the antibody of the present invention via any native Cys residue of the antibody (preferably via a disulfide bond). The preferred cysteine residue for attaching the linker to the anti-GUCY2C antibody of the present invention is the cysteine residue that forms the interchain disulfide bridge. If the antibody is not a cysteine-substituted antibody or a cysteine-engineered antibody, then this conjugation of the linker with the cysteine residue that forms the interchain disulfide bridge of the IgG1 antibody of the present invention can be used, for example. Throughout this application, the term "cysteine-substituted antibody" may be used interchangeably with "cysteine-engineered antibody", both terms referring to an antibody in which at least one naturally occurring amino acid has been mutated to cysteine, as disclosed, for example, in WO 2016040856 A2 or WO 2016142049 A1. The use of cysteine-engineered antibodies to make the conjugates of the present invention is preferred because the conjugation reaction of the amanitin-linker construct with the anti-GUCY2C antibody of the present invention will produce a conjugate with a drug-to-antibody ratio (DAR) of approximately 2. In addition, the use of cysteine-engineered antibodies will keep the interchain disulfide bond of the anti-GUVY2C antibody of the present invention intact, resulting in a more stable conjugate compared to a conjugate that uses the interchain disulfide bond to couple the linker-amanitin construct. Furthermore, compared to an interchain conjugate with a DAR that varies, for example, between about 1 and about 4, the resulting conjugate of the present invention with a DAR of 2 is characterized by highly similar or identical pharmacokinetics because the DAR2 distribution of the conjugate species is nearly homogeneous (e.g., >90%, 95% homogeneous). As used herein, the term "homogeneous" refers to the presence of a single species of antibody-drug conjugate in a given sample, e.g., nearly homogeneous refers to a sample in which >90%, >95% of the ADC or conjugate species have the same DAR (e.g., DAR = 2).

[0185] According to a preferred embodiment, the linker is attached via a disulfide bond connection between Cys265 of the anti-GUCY2C antibody of the present invention and the linker (numbered according to the EU numbering system). Thus, the anti-GUCY2C antibody of the present invention comprises a genetically engineered Fc region that at least comprises the amino acid substitution D265C (according to the EU numbering system), which may also be referred to as Cys265.

[0186] According to some embodiments, the linker of the conjugate of the present invention is an uncleavable or cleavable linker. As used herein, the term "linker" means a divalent chemical moiety that comprises a covalent bond or chain of atoms that covalently attaches or covalently links an antibody (such as an anti-GUCY2C antibody of the present invention) or a fragment thereof to an amanitin as described herein.

[0187] An "uncleavable linker" is understood not to be subject to enzymatic cleavage, for example by cathepsin B, and to be released from the conjugate of the present invention during degradation of the antibody portion of the conjugate of the present invention in the target cell (e.g., lysosomal degradation). Uncleavable linkers suitable for use according to the present invention may include, for example, one or more groups selected from: a bond, -(C=O)-, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, where each may optionally be substituted, and / or may include one or more heteroatoms (e.g., S, N, or O) that replace one or more carbon atoms. Non-limiting examples of such groups include (CH2) p , (C=O)(CH2) p and polyethylene glycol (PEG; (CH2CH2O) p ), units, where p is an integer from 1-6 independently selected in each case.

[0188] In some embodiments, the uncleavable linker according to the present invention comprises one or more of the following: 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 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, -(CH2CH2O)p- group (where p is an integer from 1-6), where each C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene or heteroarylene may optionally be substituted with 1 to 5 substituents independently selected for each case from: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, thioalkyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0189] For example, each C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene or heteroarylene of the non-cleavable linker as disclosed herein may optionally be interrupted by one or more heteroatoms selected from O, S and N and may for example optionally be substituted by 1 to 5 substituents independently selected for each case from: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, thioalkyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto and nitro.

[0190] According to a preferred embodiment, the non-cleavable linker of the conjugate of the present invention comprises -(CH2) n - units, where n is an integer from 2 to 12, such as 4-6, 8, 10 or 2-6, for example n is 1, 2, 3, 4, 5 or 6.

[0191] In a preferred embodiment, the non-cleavable linker of the conjugate of the present invention comprises -(CH2) n -, where n is 6 and the linker is represented by the formula:

[0192]

[0193] In some embodiments, the non-cleavable linker of the present invention as disclosed herein further comprises a thiol-reactive group. The thio-reactive group of the non-cleavable linker as disclosed above may for example be selected from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazin-2-ylaminomethyl-sulfonylphenyltetrazole or methylsulfonylphenyl oxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate or maleimide.

[0194] According to a preferred embodiment, the thiol-reactive group is maleimide (maleimide moiety) as disclosed above. For example, the non-cleavable linker containing the maleimide may for example have the following structure, where the wavy line at the end of the linker indicates the attachment point to the amanitin:

[0195]

[0196] After conjugation to the reactive thiol on, for example, an anti-GUCY2C antibody as disclosed herein, the maleimide moiety of the cleavable or non-cleavable linker as disclosed herein comprises the following structure:

[0197]

[0198] The wavy line indicates the attachment site of the cleavable or non-cleavable linker as disclosed herein, and the sulfur atom is 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".

[0199] According to a preferred embodiment, the conjugate of the present invention comprising a cleavable or non-cleavable linker as disclosed herein and further comprising a thiol-reactive group can be conjugated to a naturally occurring thiol moiety in the antibody of the conjugate, or the cleavable or non-cleavable linker comprising a thiol-reactive group in the conjugate of the present invention can be conjugated to a thiol moiety introduced into the antibody by genetic engineering as described, for example, in Junutula Nat Biotechnol. August 2008; 26(8):925-32. Preferably, the cleavable or non-cleavable linker comprising a thio-reactive group as disclosed herein is conjugated to a thiol moiety that has been introduced into the Fc region of the anti-GUCY2C antibody of the conjugate of the present invention by genetic engineering (e.g., D265C (according to EU numbering)) as disclosed above.

[0200] "Cleavable linker" should be understood to comprise at least one cleavage site. As used herein, the term "cleavage site" shall refer to a moiety that is susceptible to specific cleavage at a defined position under specific conditions. The conditions are, for example, a specific enzyme or a reducing environment in a specific body or cellular compartment.

[0201] According to some embodiments, the cleavage site can be cleaved by at least one protease selected from cysteine proteases, metalloproteases, serine proteases, threonine proteases, and aspartic proteases.

[0202] Cysteine proteases (also known as thiol proteases) are proteases that have a common catalytic mechanism that involves a nucleophilic cysteine thiol group in a catalytic triad or dyad.

[0203] Metalloproteases are proteases whose catalytic mechanism involves a metal. Most metalloproteases require zinc, but some use cobalt. The metal ion coordinates with the protein via three ligands. The ligands coordinating the metal ion can vary with histidine, glutamate, aspartate, lysine, and arginine. The fourth coordination position is occupied by a labile water molecule.

[0204] Serine proteases are enzymes that cleave peptide bonds in proteins; serine serves as the nucleophilic amino acid at the enzyme active site. Serine proteases are divided into two major classes based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

[0205] Threonine proteases are a family of proteolytic enzymes that contain a threonine (Thr) residue within the active site. The prototype member of this class of enzymes is the catalytic subunit of the proteasome. However, acyltransferases have acquired the same active site geometry and mechanism through convergent evolution.

[0206] Aspartic proteases are catalytic proteases that use an activated water molecule bound to one or more aspartic acid residues to catalyze their peptide substrates. Typically, they have two highly conserved aspartic acids in the active site and exhibit optimal activity at acidic pH. Almost all known aspartyl proteases are inhibited by pepstatin.

[0207] In some embodiments of the present invention, the cleavage 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).

[0208] In some embodiments of the present invention, the cleavage site is a disulfide bond and is specifically cleaved by a reducing environment (e.g., an intracellular reducing environment, such as acidic pH conditions). For example, the corresponding linker can have the following structure:

[0209] (Amatoxin)-(CH2)2-S-S-(CH2)2-X-S-(antibody)

[0210] (Amatoxin)-(CH2)3-S-S-(CH2)2-X-S-(antibody);

[0211] (Amatoxin)-(CH2)2-S-S-(CH2)3-X-S-(antibody);

[0212] (Amatoxin)-(CH2)3-S-S-(CH2)3-X-S-(antibody),

[0213] wherein X is as disclosed above.

[0214] In some embodiments, the linker is a pH-sensitive linker and is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers are cleavable under acidic conditions. This cleavage strategy generally takes advantage of the lower pH in endosomes (pH ~5 - 6) and lysosomes (pH ~4.8) intracellular compartments compared to the cytosol (pH ~7.4) to trigger hydrolysis of acid-labile groups (such as hydrazones) in the linker (Jain et al. (2015) Pharm Res 32:3526 - 40). In some embodiments, the linker is an acid-labile and / or hydrolysable linker. For example, acid-labile linkers that are hydrolysable in lysosomes and contain acid-labile groups (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amides, orthoesters, acetals, ketals, etc.) can be used. See, e.g., 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 those in blood), but are unstable at pH values below 5.5 or 5.0 (approximate pH of lysosomes). In certain embodiments, the hydrolysable linker is a thioether linker (such as a thioether attached to a therapeutic agent via an acylhydrazone bond). See, e.g., U.S. Patent No. 5,622,929.

[0215] According to some embodiments of the invention, the cleavable linker of the invention is an enzymatically cleavable linker. The enzymatically cleavable linker contains a cleavage site, which is an enzymatically cleavable moiety comprising two or more amino acids. Preferably, the enzymatically cleavable moiety comprises a 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, a 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, a phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys GlyPro Leu Gly) or an alanine-alanine-proline-valine (Ala Ala Pro Val) peptide, or a β-glucuronide or a β-galactoside.

[0216] In a preferred embodiment, the cleavable linker of the conjugate of the invention as disclosed above is a suicide linker. The term "suicide linker" or "suicide spacer" refers to a bifunctional chemical moiety capable of covalently linking two chemical moieties into a normally stable three-component molecule. If the bond to the first moiety is cleaved, the suicide spacer is capable of spontaneously separating from the second moiety. Corresponding suicide linkers are disclosed, for example, in WO 03026577 (disclosing linkers containing p-aminobenzyl ethers of amides) or WO 2005 / 112919, and such linkers can also be used, for example, in the conjugates of the present invention.

[0217] In a particularly preferred embodiment, the enzymatically cleavable linker according to the invention comprises a dipeptide selected from Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit and Val-Cit, particularly wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and the amanitin:

[0218]

[0219] Thus, the conjugate of the invention as disclosed herein may comprise an enzymatically cleavable moiety comprising any one of the dipeptide-PAB moieties Phe-Lys-PAB, Val-LysPAB, Phe-Ala-PAB, Val-Ala-PAB, Phe-Cit-PAB or Val-Cit-PAB as disclosed above. Preferably, the cleavable moiety of the conjugate of the invention comprises the dipeptide-PAB moiety Val-Ala-PAB

[0220]

[0221] wherein the PAB moiety is linked to the amanitin.

[0222] According to some embodiments, the cleavable moiety or cleavable linker of the invention as disclosed above comprises a thiol-reactive group selected from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazin-2-ylaminomethyl-sulfonylphenyltetrazole or methylsulfonylphenyl oxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate or maleimide.

[0223] According to a preferred embodiment, the thiol-reactive group is the maleimide (maleimidyl moiety) depicted as follows:

[0224]

[0225] Linkers containing the thiol reactive group (e.g., cleavable and / or non-cleavable linkers) are particularly useful for covalently conjugating linker-amanitin conjugates as disclosed herein to antibodies containing reactive thiols (e.g., cysteine-engineered antibodies containing at least one reactive cysteine residue for conjugation). For example, the linker is particularly useful for conjugating a linker-amanitin conjugate as disclosed herein to a cysteine-engineered anti-GUCY2C antibody of the invention containing the amino acid Cys265 (D265C, according to the EU numbering system).

[0226] According to a particularly preferred embodiment, the linker of the invention contains structure (i) before conjugation to an antibody as disclosed herein or structure (ii) after conjugation.

[0227]

[0228] In some embodiments of the invention, the conjugate as described contains amanitin, which contains (i) amino acid 4 having a 6'-deoxy position and (ii) amino acid 8 having an S-deoxy position.

[0229] In some embodiments, the linker of the conjugate of the invention is attached to amanitin via: (i) the γC atom of amanitin amino acid 1, or (ii) the δC atom of amanitin amino acid 3, or (iii) the 6'-C atom of amanitin amino acid 4. The corresponding attachment sites where the linker is attached to amanitin are indicated in Figure 1 where the attachment site to the γC atom of amanitin amino acid 1 is designated "R3", the attachment site to the δC atom of amanitin amino acid 3 is designated "R1", and the attachment site to the 6'-C atom of amanitin amino acid 4 is designated "R4".

[0230] According to a preferred embodiment of the invention, the conjugate of the invention as disclosed herein contains any one of the compounds of formulas (I) to (XI) below as the linker-amanitin moiety:

[0231]

[0232]

[0233]

[0234]

[0235] According to a preferred embodiment, the conjugate of the invention contains an anti-GUCY2C antibody as disclosed herein conjugated via a thioether linkage to an amanitin linker moiety according to any one of formulas XII to XXII:

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] wherein the amanitin-linker moiety is coupled to the thiol group of a cysteine residue of the anti-GUCY2C antibody of the present invention, and wherein n is preferably from about 1, 2, 3 to about 4, 5, 6, 7, 8, preferably, wherein n is from about 1, 1.5, 2, 2.5 to about 3.5, 4.5, 5.5, more preferably, wherein n is from about 1.5 to about 3.5. The thiol group of the cysteine residue of the anti-GUCY2C antibody of the present invention can be, for example, a naturally occurring cysteine residue of the antibody, such as those that form interchain disulfide bonds after reduction of the cysteine residue for thiol-based conjugation, or the thiol group of the cysteine residue of the anti-GUCY2C antibody of the present invention can be genetically engineered, preferably at position D265 (D265C according to the EU numbering).

[0242] According to a preferred embodiment, the number n of amanitin-linker conjugates covalently bound to the anti-GUCY2C antibody of the present invention is from about 1 to about 2, 3, 4, 6, 7, 8, or from about 1, 2 to about 3, 4, or from about 1.5, 1.6, 1.7, 1.8 to about 1.9, 2.0, 2.1, 2.2, 2.5, 3, or from about 1.9, 2.0 to about 2.1, 2.2, n is preferably about 2 (e.g., from 1.8 to about 2.2).

[0243] According to a preferred embodiment, the conjugate of the present invention is selected from

[0244] - conjugate (XXIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0245] - Conjugate (XXIV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0246] - Conjugate (XXV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0247] - Conjugate (XXVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0248] - Conjugate (XXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0249] - Conjugate (XXVIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0250] - Conjugate (XXIX), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0251] - Conjugate (XXX), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0252] - Conjugate (XXXI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0253] - Conjugate (XXXII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0254] - Conjugate (XXXIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0255] wherein n is from about 1 to about 2.

[0256] According to a preferred embodiment, the conjugates of the present invention are selected from

[0257] - Conjugate (XXXIV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0258] - Conjugate (XXXV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0259] - Conjugate (XXXVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0260] - Conjugate (XXXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0261] - Conjugate (XXXVIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0262] - Conjugate (XXXIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0263] - Conjugate (XL), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0264] - Conjugate (XLI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0265] - Conjugate (XLII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0266] - Conjugate (XLIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0267] - Conjugate (XLIV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0268] where n is from about 1 to about 2.

[0269] According to a preferred embodiment, the conjugate of the invention is selected from

[0270] - Conjugate (XLV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0271] - Conjugate (XLVI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0272] - Conjugate (XLVII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0273] - Conjugate (XLVIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0274] - Conjugate (XLIX), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0275] - Conjugate (L), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0276] - Conjugate (LI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0277] - Conjugate (LII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0278] - Conjugate (LIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0279] - Conjugate (LIV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0280] - Conjugate (LV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0281] where n is from about 1 to about 2.

[0282] According to a preferred embodiment, the conjugates of the present invention are selected from

[0283] - Conjugate (LVI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0284] - Conjugate (LVII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0285] - Conjugate (LVIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0286] - Conjugate (LIX), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0287] - Conjugate (LX), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0288] - Conjugate (LXI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0289] - Conjugate (LXII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0290] - Conjugate (LXIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0291] - Conjugate (LXIV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0292] - Conjugate (LXV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0293] - Conjugate (LXVI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0294] wherein n is from about 1 to about 2.

[0295] According to a preferred embodiment, the conjugates of the invention are selected from

[0296] - Conjugate (LXVII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0297] - Conjugate (LXVIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0298] - Conjugate (LXIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0299] - Conjugate (LXX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0300] - Conjugate (LXXI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0301] - Conjugate (LXXII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0302] - Conjugate (LXXIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0303] - Conjugate (LXXIV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0304] - Conjugate (LXXV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0305] - Conjugate (LXXVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0306] - Conjugate (LXXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0307] wherein n is from about 1 to about 2.

[0308] According to a preferred embodiment, the conjugates of the invention are selected from

[0309] - Conjugate (LXXVIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0310] - Conjugate (LXXIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0311] - Conjugate (LXXX), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0312] - Conjugate (LXXXI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0313] - Conjugate (LXXXII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0314] - Conjugate (LXXXIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0315] - Conjugate (LXXXIV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0316] - Conjugate (LXXXV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0317] - Conjugate (LXXXVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0318] - Conjugate (LXXXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0319] - Conjugate (LXXXVIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system,

[0320] where n is from about 1 to about 2.

[0321] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or containing the amino acid sequence according to one of SEQ ID Nos. 79, 80, 81, 82, 83, 84, and each light chain consisting of or containing the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0322] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or containing the amino acid sequence according to one of SEQ ID Nos. 79, 80, 81, 82, 83, 84, and each light chain consisting of or containing the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0323] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or containing the amino acid sequence according to one of SEQ ID Nos. 79, 80, 81, 82, 83, 84, and each light chain consisting of or containing the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0324] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0325] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0326] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0327] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0328] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0329] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0330] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0331] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 78. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0332] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0333] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0334] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0335] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0336] According to some embodiments, the conjugate of the invention comprises an anti-GUCY2C antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0337] According to some embodiments, the conjugate of the invention comprises an anti-GUCY2C antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0338] According to some embodiments, the conjugate of the invention comprises an anti-GUCY2C antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0339] According to some embodiments, the conjugate of the invention comprises an anti-GUCY2C antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0340] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0341] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0342] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0343] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0344] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0345] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0346] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0347] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0348] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0349] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0350] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0351] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0352] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0353] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No 93, 94, 95, 96, 97 or 98, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No.92. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0354] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No.100, 101, 102, 103, 104 or 105, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No.99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0355] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No.100, 101, 102, 103, 104 or 105, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No.99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0356] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0357] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0358] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0359] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0360] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0361] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0362] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0363] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0364] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0365] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0366] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0367] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0368] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0369] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0370] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0371] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0372] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0373] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0374] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0375] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0376] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 106. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0377] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0378] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0379] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain consists of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain consists of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0380] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0381] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0382] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0383] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0384] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0385] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0386] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0387] According to some embodiments, the conjugate of the present invention comprises an anti-GUCY2C antibody, which is composed of two heavy chains and two light chains. Each heavy chain is composed of or comprises an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, and each light chain is composed of or comprises an amino acid sequence according to SEQ ID No. 113. The antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0388] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which antibody consists of two heavy chains and two light chains, each heavy chain comprising the amino acid sequence according to SEQ ID No. 62, each light chain comprising the amino acid sequence according to SEQ ID No. 56, and the antibody being conjugated to at least one amanitin-linker moiety selected from the group consisting of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0389] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which antibody consists of two heavy chains and two light chains, each heavy chain comprising the amino acid sequence according to SEQ ID No. 63, each light chain comprising the amino acid sequence according to SEQ ID No. 57, and the antibody being conjugated to at least one amanitin-linker moiety selected from the group consisting of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0390] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which antibody consists of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to SEQ ID No. 80, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety selected from the group consisting of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0391] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to SEQ ID No. 87, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 85. The antibody is conjugated to at least one amanitin-linker moiety selected from the group consisting of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0392] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to SEQ ID No. 94, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 92. The antibody is conjugated to at least one amanitin-linker moiety selected from the group consisting of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0393] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains. Each heavy chain consists of or comprises the amino acid sequence according to SEQ ID No. 101, and each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 99. The antibody is conjugated to at least one amanitin-linker moiety selected from the group consisting of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0394] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to SEQ ID No. 108, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody being conjugated to at least one amanitin-linker moiety selected from the group consisting of formulae (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0395] According to a more preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to SEQ ID No. 115, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety selected from the group consisting of formulae (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0396] According to a particularly preferred embodiment, the conjugate of the present invention comprises an anti-GUCY2C antibody, which consists of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to SEQ ID No. 80, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety selected from the group consisting of formulae (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

[0397] According to a particularly preferred embodiment, the conjugate of the invention comprises an anti-GUCY2C antibody, said antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to SEQ ID No. 87, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, said antibody being conjugated to at least one amanitin-linker moiety selected from the group consisting of formulae (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, and even more preferably, the amanitin-linker moiety is selected from one of formulae (XII), (XIII) or (XIV).

[0398] In some embodiments, the conjugate according to the invention as disclosed above may have an IC -9 of better than 10x10 -9 M, 9x10 -9 M, 8x10 -9 M, 7x10 -9 M, 6x10 -9 M, 5x10 -9 M, 4x10 -9 M, 3x10 -9 M, preferably better than 10x10 -10 M, 9x10 -10 M, 8x10 -10 M, 7x10 -10 M, 6x10 -10 M, 5x10 -10 M, 4x10 -10 M, 3x10 -10 M, 2x10 - 10 M, and more preferably better than 9x10 -11 M, 8x10 -11 M, 7x10 -11 M, 6x10 -11 M, 5x10 -11 M, 4x10 -11 M, 3x10 -11 M, 2x10 -11 M or 1x10 -11 of the cytotoxic activity of the 50

[0399] ​In one embodiment, the present invention provides a polynucleotide encoding an amino acid sequence comprising any one of SEQ ID NO:62, 63, 64, 65, 66 or 67.

[0400] According to one embodiment, the present invention provides a polynucleotide encoding an amino acid sequence comprising any one of SEQ ID NO:56, 57, 58, 59, 60 or 61.

[0401] According to one embodiment, the polynucleotide of the present invention encodes an amino acid sequence comprising an amino acid sequence selected from the following V H and V L sequences in combination:

[0402] - SEQ ID NO:62, 56

[0403] - SEQ ID NO:63, 57

[0404] - SEQ ID No:64, 58

[0405] - SEQ ID No:65, 59

[0406] - SEQ ID No:66, 60

[0407] - SEQ ID NO:67, 61,

[0408] or an amino acid sequence that is at least 90%, 95% similar to any one of the above V L and V H sequences (either alone or in the combinations disclosed above).

[0409] As used herein, the term "polynucleotide" refers to a nucleic acid sequence. The nucleic acid sequence can be a DNA or RNA sequence, preferably the nucleic acid sequence is a DNA sequence. The polynucleotide of the present invention should preferably be provided as an isolated polynucleotide (i.e., isolated from its natural environment) or in a genetically modified form. The isolated polynucleotide mentioned herein can also cover, for example, a polynucleotide present in a cellular environment other than its natural cellular environment, i.e., a heterologous polynucleotide. The term polynucleotide covers single-stranded as well as double-stranded polynucleotides. In addition, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides (such as glycosylated or methylated polynucleotides) or artificially modified polynucleotides (such as biotinylated polynucleotides).

[0410] According to one embodiment, the present invention provides an expression vector, which comprises the polynucleotide of the present invention. The expression vector according to the present invention may, for example, comprise the following: at least one polynucleotide encoding an amino acid sequence comprising an amino acid sequence selected from SEQ ID NO: 62, 63, 64, 65, 66 or 67; or at least one polynucleotide encoding an amino acid sequence comprising an amino acid sequence selected from SEQ ID NO: 56, 57, 58, 59, 60 or 61. The expression vector of the present invention may also, for example, comprise the following: a polynucleotide encoding an amino acid sequence comprising an amino acid sequence selected from SEQ ID NO: 62, 63, 64, 65, 66 or 67; and a polynucleotide encoding an amino acid sequence comprising an amino acid sequence selected from NO: 56, 57, 58, 59, 60 or 61; for example, the expression vector may comprise a polynucleotide encoding an amino acid sequence comprising an amino acid sequence according to SEQ ID NO: 62, 56; SEQ ID NO: 63, 57; SEQ ID NO: 64, 58; SEQ ID NO: 65, 59; SEQ ID NO: 66, 60; or SEQ ID NO: 67, 61. The term "expression vector" or "vector" preferably encompasses plasmid, phage, viral or retroviral vectors as well as 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 propagation and / or selection in a suitable host cell. The polynucleotide according to the present invention comprised in the expression vector is operably linked to an expression control sequence, thereby allowing expression or propagation in a prokaryotic cell or a eukaryotic cell (especially in a eukaryotic cell or a separated part thereof). The expression of the polynucleotide includes the transcription of the polynucleotide, preferably transcribed into translatable mRNA. The expression vector of the present invention further comprises a regulatory sequence ensuring transcription initiation and optionally a polyA signal ensuring transcription termination and transcript stability. Additional regulatory elements may include transcriptional and translational enhancers. Possible regulatory elements allowing expression in a prokaryotic host cell include, for example, the lac, trp or tac promoters in Escherichia coli, and examples of regulatory elements allowing expression in a eukaryotic host cell are the AOX1 or GAL1 promoters in yeast or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells. Suitable expression control sequences are well known in the art. In addition to the elements responsible for transcription initiation, such regulatory elements may also comprise 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 can be incorporated into host cells, for example, by various techniques known in the art. For example, plasmid vectors can be introduced in precipitates (such as calcium phosphate precipitates or rubidium chloride precipitates), or in complexes with charged lipids, or in carbon-based clusters (such as fullerenes). Alternatively, plasmid vectors can be introduced by heat shock or electroporation techniques. If the expression vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before being applied to host cells. Retroviral vectors can be replication-competent or replication-defective. In the latter case, viral propagation typically occurs only in complementary host / cells. Available transformation and transfection techniques are 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 transfection protocols are described, for example, in "Molecular Cloning - A laboratory manual", 4th edition (2001), ISBN 978-1-936113-42-2, Chapter 15, pages 1131-1203.

[0411] According to one embodiment, the present invention relates to a host cell comprising the expression vector of the present invention or the polynucleotide of the present invention as disclosed herein. As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell comprising the expression vector or polynucleotide of the present invention. Preferably, the host cell of the present invention is a eukaryotic cell such as a yeast cell (e.g., Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, Yarrowia lipolytica, and Pichia pastoris), an insect cell (e.g., Sf9, Sf21, S2, Hi5, or BTI-TN-5B1-4), and more preferably, the host cell of the present invention is a mammalian cell selected from the following: HEK293, HEK293T, HEK293E, HEK 293F, 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, CHO-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., and more preferably the host cell is selected from CHO, CHO-K1, CHO-B11, CHO-DG44, HEK293, HEK293T, HEK293E, HEK 293F, NS0, or per.C6 cells.

[0412] In one embodiment, the present invention relates to the use of the anti-GUCY2C antibody of the present invention in the manufacture of the conjugate of the present invention as disclosed herein.

[0413] According to a second aspect, an object of the present invention is to provide a pharmaceutical composition, which comprises the conjugate of the present invention as disclosed herein.

[0414] The pharmaceutical composition may further comprise, for example, one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives.

[0415] In an aqueous form, the pharmaceutical formulation may be ready for administration, while in a lyophilized form, the formulation may be transferred into a liquid form, for example, by adding water for injection before administration, and the water for injection may or may not contain a preservative, such as, but not limited to, benzyl alcohol, antioxidants such as vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium, amino acids cysteine and methionine, citric acid and sodium citrate, synthetic preservatives such as parabens (methylparaben and propylparaben).

[0416] The pharmaceutical formulation may further comprise one or more stabilizers, which may be, for example, amino acids, sugar polyols, disaccharides, and / or polysaccharides. The pharmaceutical formulation may further comprise one or more surfactants, one or more isotonic agents, and / or one or more metal ion chelating agents, and / or one or more preservatives.

[0417] The pharmaceutical formulation as described herein may be suitable for at least intravenous, intramuscular, or subcutaneous administration. Alternatively, the conjugate according to the present invention may be provided in a depot formulation, which allows for sustained release of the bioactive agent over a period of time.

[0418] In yet another aspect of the present invention, there is provided a primary package (such as a drug-loaded syringe or pen, vial, or infusion bag), which contains the formulation according to the previous aspect of the present invention.

[0419] The drug-loaded syringe or pen may contain the formulation in a lyophilized form (which must then be dissolved, for example, with water for injection before administration) or in an aqueous form. The syringe or pen is typically 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 a multi-use or multi-dose syringe or pen.

[0420] The vial may also contain the formulation in a lyophilized form or in an aqueous form and may serve as a single- or multi-use device.

[0421] According to one embodiment, the present invention provides a composition, which comprises - at least one immune checkpoint inhibitor, and

[0422] -At least one conjugate according to the invention as disclosed above.

[0423] In the context of the present invention, the term "immune checkpoint inhibitor" or simply "checkpoint inhibitor" or "ICI" refers to any agent or compound that directly or indirectly reduces the level of an immune checkpoint receptor protein or molecule found on the surface of immune cells (e.g., T cells) or inhibits the function of said immune checkpoint receptor protein or molecule, or refers to any agent or compound that directly or indirectly reduces the level of a ligand that binds to said immune checkpoint receptor protein or molecule as a soluble compound or on the surface of immunosuppressive cells of the immune system or inhibits the function of said ligand. Such immunosuppressive cells can be, for example, cancer cells, regulatory T cells, tolerogenic antigen-presenting cells, myeloid-derived suppressor cells, tumor-associated macrophages or cancer-associated fibroblasts. The ligand is generally capable of binding to an immune checkpoint receptor protein or molecule on an immune cell. Non-limiting examples of immune checkpoint receptor protein-ligand pairs are PD-1, 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. Thus, immune checkpoint inhibitors prevent PD-1 / PD-L1 interaction by blocking PD-1 on T cells (i.e., as a PD-I inhibitor) or PD-L1 on cancer cells (i.e., as a PD-L1 inhibitor), thereby maintaining or restoring anti-tumor T cell activity or blocking inhibitory cancer cell activity. Other non-limiting 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). Corresponding compositions and their respective uses are disclosed in WO2022 / 096604.

[0424] According to one embodiment, the immune checkpoint inhibitor according to the invention is an antibody selected from nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, ipilimumab, PD-1, PD-2, PD-3, PD-4 or PD-5, tremelimumab or an antigen-binding fragment thereof, or an antigen-binding derivative thereof, preferably, wherein the antibody is avelumab, pembrolizumab, nivolumab or ipilimumab or an antigen-binding fragment thereof, or an antigen-binding derivative of the antibody.

[0425] Nivolumab (CAS Registry Number: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 without detectable antibody-dependent cellular cytotoxicity (ADCC). Nivolumab is disclosed, for example, in US 8,008,449 and WO 2006 / 121168. It has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0426] Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab is disclosed, for example, in WO 2009 / 101611.

[0427] Pembrolizumab (previously also known as lambrolizumab; trade name Keytruda; also known as MK-3475), as disclosed, for example, in 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; it contains a mutation at C228P designed to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed, for example, in US 8,354,509 and WO 2009 / 114335. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0428] Atezolizumab is a fully humanized, engineered monoclonal antibody of the IgG1 isotype against PD-L1, as disclosed in WO 2010 / 077634.

[0429] Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody as disclosed in WO 2013 / 079174 A1.

[0430] Durvalumab is a human immunoglobulin G1κ (IgG1κ) anti-PD-L1 monoclonal antibody as disclosed in WO 2011 / 066389 A1.

[0431] Cemiplimab is a human IgG4 monoclonal antibody that binds to PD-1, thereby blocking the interaction of PD-1 with PD-L1 and PD-L2. Cemiplimab is disclosed as H4H7798N in WO 15112800 A1.

[0432] Ipilimumab (CAS Registry Number: 477202-00-9, which may also be referred to as 10D1 or MDX010, 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 the stimulatory CD28 for binding to B7 on antigen-presenting cells. Both CTLA-4 and CD28 are present on the surface of T cells. Ipilimumab is a human IgG1 that binds to CTLA-4 and thereby prevents the inhibition of T cell-mediated immune responses against tumors. Ipilimumab is disclosed as antibody "10D1" in, for example, WO 01 / 14424.

[0433] PD1-1 to PD1-5 refer to anti-PD-1 antibodies as disclosed in WO 2018 / 220169.

[0434] Tremelimumab (which may also be referred to as ticilimumab, CP-675, CP-675,206, CAS Registry Number: 745013-59-6) is a fully human anti-CTLA4 antibody.

[0435] As used herein, INN is meant to also encompass all biosimilar antibodies corresponding to the original antibodies disclosed herein, including but not limited to those biosimilar antibodies authorized under subsection (k) of 42 USC § 262 in the United States and equivalent regulations in other jurisdictions.

[0436] In one embodiment, the invention relates to the conjugates of the invention as disclosed herein, or the pharmaceutical compositions or compositions of the invention as 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 "treating" or the phrase "to treat" refers to any type of treatment that confers a benefit to a patient suffering from a disease such as cancer, more particularly gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), or pancreatic cancer, including improving the patient's condition (e.g., in terms of one or more symptoms) and / or delaying the progression of the condition.

[0437] According to one embodiment, the gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), or liver cancer as disclosed herein is characterized by a deletion or translocation of chromosome 17p13.1, whereby the deletion can be, for example, a hemizygous deletion of chromosome 17.p13.1. Thus, the gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), or liver cancer as disclosed herein to be treated with the conjugates, pharmaceutical compositions, or compositions of the invention as disclosed herein is characterized by a deletion or hemizygous deletion of chromosome 17p13.1.

[0438] According to one embodiment, gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), or liver cancer, which can be treated, for example, with the conjugates, pharmaceutical compositions, or compositions of the invention as disclosed herein, are characterized by hemizygous deletion of the POLR2A gene or the TP53 and POLR2A genes. As used herein, the term "hemizygous" refers to an individual or cell that has only one (as opposed to the usual two) complete alleles or chromosomal segments. Hemizygosity refers to a cell or organism whose genome includes only one complete allele at a given locus (whether the allele is wild-type or mutant), e.g., cells of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer as disclosed herein are hemizygous at chromosomal locus 17p13, preferably cells of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer as disclosed above are hemizygous for the genes TP53 and POLR2A. As used herein, "TP53" refers to the "tumor protein 53" gene that encodes a tumor suppressor protein (P53), which contains transcriptional activation, DNA-binding, and oligomerization domains. The encoded protein regulates the expression of target genes in response to different cellular stimuli, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or metabolic changes. Mutations in this gene are associated with a variety of human cancers, including hereditary cancers such as Li-Fraumeni syndrome.

[0439] The tumor suppressor gene TP53 is frequently inactivated in most human tumors by mutation or deletion. As used herein, "POLR2A" refers to the POLR2A gene, which encodes the largest subunit of the human RNA polymerase II complex and is indispensable for polymerase activity in mRNA synthesis. Hemizygous deletion of chromosome 17p13 (e.g., del(17p13.1)) can be detected by fluorescence in situ hybridization (FISH) as disclosed in Merz et al. Am J Hematol. November 2016;91(11):E473-E477.

[0440] As disclosed herein, the cells of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, and pancreatic cancer may not be a homogeneous cell population, for example, with respect to the deletion 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% or about 70%, 75%, 80%, 85% to about 90%, 92.5%, 95%, 97.5%, 100% of the cancer cells disclosed above may be hemizygous for del(17p13.1), TP53, and / or POLR2A, or for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, 95% of the cancer cells disclosed herein may be hemizygous for del(17p13) or for TP53 and / or POLR2A.

[0441] The conjugates, pharmaceutical compositions, or compositions of the present invention for use in treating gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (characterized by hemizygous deletion of chromosome 17p13.1 or TP53 and / or POLR2A) may be particularly advantageous, for example, because cells characterized by hemizygous deletion of chromosome 17p13.1, TP53, and / or POLR2A are at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1000-fold more sensitive to the conjugates, pharmaceutical compositions, or compositions of the present invention disclosed herein. Therefore, it may be beneficial, for example, to determine whether the cells of the cancers disclosed herein contain or consist of cells that are hemizygous for the deletion of TP53 and / or POLR2A, because the desired therapeutic effect can be achieved using at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold less of the conjugates, pharmaceutical compositions, or compositions of the present invention disclosed herein. The assay for evaluating the sensitivity of cancer cells to the conjugates, pharmaceutical compositions, or compositions of the present invention disclosed herein can be done, for example, as described in Liu et al., Nature. April 30, 2015; 520(7549):697-701.

[0442] According to one embodiment, the conjugate, pharmaceutical composition, or composition comprising the conjugate of the invention and an immune checkpoint inhibitor of the invention is for use in treating a solid tumor in a patient, wherein the solid tumor of the patient is characterized by the expression of GUCY2C on the surface of the cancer cells of the solid tumor. Thus, tumor cells express, for example, amino acids 24-430 of SEQ ID NO.:76 or any fragment thereof on their surface that is recognized and specifically bound by an antibody, antibody fragment, or conjugate of the invention as disclosed herein. As used herein, the term "specifically binds" or "specifically binding" refers to the binding of an antibody or conjugate of the invention as disclosed herein to its antigen (e.g., an epitope of human GUCY2C) having a Kd of at least about 10 -6 M, 10 -7 M, 10 -8 M, or about 10 -8 M to about 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or about 5x 10 -9 M, 5x 10 -10 M to about 2.5x 10 -11 M, 5x 10 - 11 M, 2.5x 10 -12 M, 5x 10 -12M. As used herein, the term "epitope" refers to a portion of a macromolecule (preferably a polypeptide such as human GUCY2C) that is recognized by an antigen-binding molecule (such as an antibody of the invention or an antigen-binding fragment or an antigen-binding derivative thereof disclosed herein), and more specifically recognized by the antigen-binding site of said molecule. The epitope defines the minimal binding site of the antibody molecule and thus represents the target of the specificity of the antibody molecule. An epitope can be further defined as a structural epitope or a functional epitope. A "structural epitope" consists of amino acids or other molecules in a region that is in close contact with the antibody and is usually revealed by the structure. A "functional epitope" is defined as those portions of a molecule that contribute energetically to binding such that the binding affinity decreases when they are altered. Thus, when defining an epitope, the residues in contact with the paratope and which residues contribute to the affinity and whether they are proximal are important considerations. A structural epitope can be, for example, a linear contiguous sequence of about 5 amino acids to about 50, 100 amino acids in length, or a conformational epitope formed by the three-dimensional structure of the polypeptide and can contain non-contiguous amino acids of the polypeptide. The affinity of an anti-GUCY2C antibody or conjugate of the invention can be measured using well-known methods (e.g., in an in vitro assay using surface plasmon resonance (BIAcore, GE-Healthcare Uppsala, Sweden)).

[0443] The features of the solid tumors disclosed above can also, for example, consist of hemizygous deletions of the POLR2A gene or the TP53 and POLR2A genes. 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 solid tumor cells can be hemizygous for del(17p13.1), TP53 and / or POLR2A, or for example at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, 95% of the solid tumor cells disclosed above are hemizygous for del(17p13) or for TP53 and / or POLR2A.

[0444] According to one embodiment, the invention also relates to an antibody that competes with the antibody of the invention for binding to human GUCY2C. Antibodies that compete with the antibody of the invention for binding to human GUCY2C can be determined, for example, using an Octet HTX biosensor (Pall ForteBio Corp.) in a similar manner as disclosed in WO 2015 / 112800 or, for example, in a similar manner as disclosed in Syedbasha et al., J Vis Exp. 2016;(109):53575, the content of which is hereby incorporated by reference in its entirety.

[0445] According to one embodiment, the conjugate or pharmaceutical composition according to the invention as disclosed herein is for use in the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer in a patient who is unresponsive to treatment, such as first-line or second-line treatment, the first-line or second-line treatment including cetuximab or panitumumab alone or in combination with: 5-fluorouracil (5-FU) and oral 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 term "first-line therapy" or "first-line treatment" or "first-line therapy" means a therapy or treatment the label of which does not include a requirement or recommendation that the therapy or treatment should only be used after other therapies or treatments have shown unsatisfactory or unsuccessful results. It can also include a therapy and / or treatment in which no other active agent (other than the primary active agent) is administered to an individual subject in need. The term "second-line treatment" or "second-line therapy" refers to the treatment of a disease or disorder after an initial treatment (first-line treatment) has failed, ceased to be effective or has intolerable side effects due to severe side effects and associated loss of quality of life.

[0446] According to some embodiments, the conjugate or pharmaceutical composition of the invention (alone or optionally in combination with an immune checkpoint inhibitor as disclosed above) is for use, for example, in the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer in a patient in whom standard of care treatment options have failed (e.g., first-line and / or second-line or even third-line treatment failure). For example, the conjugate or pharmaceutical composition of the invention can be for use, for example, in a patient with CRC who has experienced failure of the above treatment options or who has experienced failure of third-line treatment with the combination drug trifluridine / tipiracil (TAS-102) after treatment with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, anti-VEGF therapy and anti-EGFR therapy for RAS wild-type tumors.

[0447] According to one embodiment, the conjugate (anti-GUCY2C conjugate) or pharmaceutical composition of the present invention is for use in the treatment of colorectal cancer or metastatic colorectal cancer (mCRC), wherein the colorectal cancer or metastatic colorectal cancer (mCRC) is characterized by a KRAS mutation and / or a BRAF mutation. The KRAS mutations according to the present invention include, but are not limited to, the KRAS mutations G13D, G12C, G12V, and G12D. The BRAF mutations according to the present invention include, but are not limited to, the BRAF mutations V600E, V600K, or V600R. Thus, the conjugate 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.

[0448] According to some embodiments, the conjugate or pharmaceutical composition of the present invention as disclosed above for use in the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer can be combined with one or more of the immune checkpoint inhibitors as disclosed above, such as anti-PD-1 (e.g., nivolumab, PD-1-PD-5, pidilizumab, or pembrolizumab), or anti-PD-L1 (e.g., avelumab), anti-CTLA-4 (e.g., ipilimumab or tremelimumab) checkpoint inhibitors; or for example, combined with a KRAS inhibitor (such as BI 1823911, sotorasib, adagrasib, LY3537982, JNJ-74699157, or a KRAS-SOS1 inhibitor (e.g., BI1701963)); or combined with a BRAF inhibitor (e.g., vemurafenib, dabrafenib, or encorafenib, or a combination of dabrafenib and trametinib, a combination of vemurafenib and cobimetinib, or a combination of encorafenib and binimetinib); or combined with a combination of the above checkpoint inhibitors with a KRAS inhibitor or a BRAF inhibitor.

[0449] In one embodiment, the present invention relates to the use of the conjugate or pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of gastrointestinal cancer, colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), liver cancer, or a GUCY2C-positive or GUCY2C-expressing solid tumor.

[0450] In another aspect, the present invention relates to a method for treating a patient suffering from gastrointestinal cancer, wherein the method comprises administering to the patient a therapeutically effective amount of the conjugate or pharmaceutical composition of the present invention, either alone or in combination with another pharmacologically active compound, such as but not limited to: one or more of the immune checkpoint inhibitors disclosed above, for example, anti-PD-1 (e.g., nivolumab, PD-1-PD-5, pidilizumab or pembrolizumab), or anti-PD-L1 (e.g., avelumab), anti-CTLA-4 (e.g., ipilimumab, tremelimumab) checkpoint inhibitors; or for example, a KRAS inhibitor (such as BI 1823911, sotorasib, adagrasib, LY3537982, JNJ-74699157 or a KRAS-SOS1 inhibitor (e.g., BI1701963)); or a BRAF inhibitor (e.g., vemurafenib, dabrafenib or encorafenib, or a combination of dabrafenib and trametinib, a combination of vemurafenib and cobimetinib, or a combination of encorafenib and binimetinib); or a combination of the above checkpoint inhibitors with a KRAS inhibitor or a BRAF inhibitor.

[0451] In one embodiment, the present invention relates to a method for treating a patient suffering from gastrointestinal cancer, wherein the method comprises administering to the patient a therapeutically effective amount of the conjugate or pharmaceutical composition as disclosed above, wherein the patient has no response to first-line or second-line treatment with, for example, cetuximab or panitumumab alone or in combination with: 5-fluorouracil (5-FU) and oral capecitabine (CAP), FOLFOX (5-FU and oxaliplatin), FOLFIRI (5-FU and irinotecan), XELOX / CAPOX (CAP and oxaliplatin), CAPIRI (CAP and irinotecan), FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan and oxaliplatin), or for example, where the patient has 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 anti-EGFR therapy for RAS wild-type tumors. Thus, the above treatment method can be used, for example, to treat patients suffering from refractory colorectal cancer or refractory metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer.

[0452] In some embodiments, the colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer to be treated in the treatment method of the present invention is characterized by hemizygous deletion of TP53, POLR2A or del(17p13).

[0453] In some embodiments, the colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer, or liver cancer to be treated in the treatment methods of the present invention is characterized by a KRAS mutation and / or a BRAF mutation as disclosed herein. For example, the KRAS mutations include the mutations G13D, G12C, G12V, and G12D, or the BRAF mutations BRAF V600E, V600K, or V600R.

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[0549] Sequence

[0550] Table 1: Amino Acid Sequences of the Present Invention

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572] Example

[0573] Although the present invention has been described in detail in the drawings and the foregoing description, such description and illustration are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0574] 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'.

[0575] Example 1: Synthesis of Amanitin

[0576] A variety of amatoxin derivatives are used for conjugation with the antibodies of the present invention. The amatoxin derivatives are generated according to the methods disclosed in, for example, WO2016 / 142049, WO 2017 / 149077, WO 2018 / 115466, WO 2019 / 197654, WO 2019 / 030173, WO20216947 A1, and WO 2022 / 096604, and the corresponding disclosures of said documents are incorporated herein in their entirety. The amatoxin derivative (IX) of the present invention can be obtained using β-amanitin in a manner similar to the synthesis of compound XVIa disclosed in WO 2022 / 096604.

[0577] Synthesis of Amanitoxin-Linker Payload for Conjugate (XXII)

[0578]

[0579] Amanitin + Maleimide-VAL-ALA-PAB Linker (XXIIa)

[0580] Dissolve natural amanitin (9.04 mg, 10 μmol) and maleimide-VAL-ALA-PAB linker (13.3 mg, 3 equivalents) in 300 μl of DMF. Add a solution of 0.1 M TBTU in DMF (30 μmol, 3 equivalents) and 300 μl of 0.2 M DIPEA in DMF (60 μmol, 6.0 equivalents) at room temperature. Monitor the reaction by RP-HPLC. After 2 h, quench the reaction with 100 μl of water and purify the crude product by preparative RP-HPLC. Evaporate the fraction containing the product from 2 ml of tert-butanol / water 4:1 and lyophilize:

[0581] Yield: 5.39 mg (41%) colorless lyophilized product

[0582] MS(ESI+)[MH] + Found: 1329.85; Calculated: 1329.56 (C 61 H 81 N 14 O 18 S + )

[0583] The obtained amatoxin-linker conjugate (XXIIa) can be reacted as disclosed in Example 2 below to produce the conjugate (XXII) of the present invention.

[0584] Example 2: Synthesis of the amanitin-linker construct

[0585] The antibody is conjugated with the amanitin linker conjugate by the so-called Thiomab technology. In this method, conjugation occurs by coupling the maleimide residue of the toxin linker construct to the free SH group of a cysteine residue in the antibody, as shown in the following reaction scheme:

[0586]

[0587] The principle of this conjugation method is disclosed in Junutula et al. (2008), the content of which is incorporated herein by reference.

[0588] The antibody used in this experiment contains the D265C substitution in two Fc domains to provide a cysteine residue with a free SH group. The corresponding technology is disclosed in WO 2016 / 142049A1, the content of which is incorporated herein by reference, and it produces a homogeneous product with a fixed drug-to-antibody ratio ("DAR") of approximately 2 and site-specific conjugation.

[0589] Example 3: In vitro cytotoxicity of the anti-GUCY2C conjugate of the present invention

[0590] In vitro cytotoxicity studies as depicted in Figure 2 and Figure 3 were performed on human GUCY2C-positive HEK293-GUCY2C-Mono2 cells (Creative Biogen) or HEK293-GUCY2C-HDP-2B3 cells overexpressing human GUCY2C. Quantitative determination of cell viability was completed 96 hours after adding the conjugate to the cell growth medium using a commercially available BrdU kit.

[0591] HEK293-GUCY2C-HDP-2B3 expressing human GUCY2C on the 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) with the amino acid sequence according to SEQ ID NO:76 and resistance to geneticin (G418). Selection of stable transfected cells was completed after 4 days by placing the cells in a medium containing G418 to generate a stable cell pool expressing GCC and G418 resistance. Stable single-cell clones were isolated from this cell pool by using limited dilution. The surface expression of GCC in the stable cell clone HEK293-GUCY2C-HDP-2B3 was confirmed by flow cytometry and cytotoxicity assay (BrdU ELISA), respectively.

[0592] Example 4: Function of the anti-GUCY2C conjugate in a subcutaneous human embryonic kidney model after single-dose intravenous administration Effect

[0593] In this study, 9 experimental groups were used, with 8 - 10 animals in each group (female NOD SCID mice, n = 126, 7 - week - old at the start of the experiment), and the animals were loaded with subcutaneous HEK293 - GUCY2C - (HDP) - 2B3 tumors (see Table 1). On day - 16 (16 days before the start of the experiment), 126 mice were subcutaneously inoculated in their right flank with 5 x 10 6 HEK293 - GUCY2C - (HDP) - 2B3 tumor cells in 200 μL of RPMI without phenol red and containing 50% . Once the average tumor volume reached approximately 150 mm 3 , the animals were randomly divided into 9 groups according to tumor size. Therapy started one day after grouping. The mice were treated with PBS, mAb1 - LALA - D265C - (XIV), mAb8 - LALA - D265C - (XIV), mAb1 - LALA - D265C, or mAb8 - LALA - D265C (details see Table 1). Tumor volume was measured twice a week with calipers (see below), and body weight was determined simultaneously. If the tumor volume > 1600 mm 3 or the mice needed to be sacrificed due to ethical reasons, the animals were sacrificed and necropsy was performed.

[0594] Tumor growth was monitored by measuring with calipers. Tumor size was calculated according to the formula W 2 x L / 2 (L = length and W = the vertical width of the tumor, L > W). If abnormal growth was observed, the tumor volume was calculated according to the following formula: [(L x W x D) / 6000] x π.

[0595] The DAR of both conjugates mAb1 - LALA - D265C - (XIV) and mAb8 - LALA - D265C - (XIV) of the present invention is 2.

[0596] Table 1: Experimental groups

[0597]

[0598] "mAb1 - LALA - D265C" and "mAb8 - LALA - D265C" refer to antibodies containing the heavy - chain and light - chain amino - acid sequences SEQ ID NO:78, SEQ ID NO:80, and SEQ ID NO:85, SEQ ID NO:87, respectively.

[0599] Example 5: Toxicity and serum pharmacokinetics of the conjugate of the present invention in cynomolgus monkeys

[0600] The objective of this study was to determine the toxicity and serum pharmacokinetics of the conjugates mAb8-LALA-D265C-(XIV) and mAb8-LALA-D265C-(XII) of the present invention after single intravenous (i.v.) infusion administration to male and female naive cynomolgus monkeys.

[0601] Study design

[0602] Eight (8, 4 of each sex) naive cynomolgus monkeys were divided into two groups, with 4 animals in each group. Group 1 included three phases and Group 2 included two phases. Animals in the first, second, and third phases (Day 1, Day 22, and Day 43) of Group 1 were administered mAb8-LALA-D265C-(XIV) at 1, 2, and 3 mg / kg, respectively, by single intravenous infusion. Animals in the first and second phases (Day 1 and Day 22) of Group 2 were administered mAb8-LALA-D265C-(XII) at 5 mg / kg and 10 mg / kg, respectively, by single intravenous infusion. Blood / serum samples were collected before dosing and on Days 3, 8, 15, and 22 after each dosing to determine the hepatic enzyme levels of ALT, AST, and LDH in the serum.

[0603] Dose administration of the conjugate

[0604] The animals were weighed on each day of dosing before dose administration to calculate the actual dose volume. All animals in Group 1 and Group 2 received single intravenous infusion administration of mAb8-LALA-D265C-(XIV) and mAb8-LALA-D265C-(XII) from the peripheral vein.

[0605] Blood collection and plasma preparation

[0606] Approximately 0.8 mL of blood was collected from each study animal via the peripheral vasculature at each time point. The actual time of each sample collection was recorded. The sampling time deviation from pre-dose to 1 hour post-dose time points was less than 1 minute, and the sampling time deviation at time points after 1 hour post-dose was less than 5% of the nominal time.

[0607] All blood samples were collected into commercially available BD tubes containing polymeric silica activator. After blood collection, the tubes containing the blood samples were allowed to stand at room temperature for at least 30 minutes. Then, they were centrifuged at 3200×g for 10 minutes at 4°C within 1 hour after collection. The clear serum was then collected. Typically, the serum was divided into 2 aliquots, one aliquot (about 150 μL) for PK analysis and the other aliquot for backup. The analysis of hepatic enzymes ALT, AST, and LDH was completed using commercial kits.

[0608] Example 6:In vivo efficacy study of two antibody-drug conjugate colon cancer PDX models grown subcutaneously in NMRI nude mice In vivo efficacy study

[0609] A patient-derived xenograft (PDX) colon cancer model was established in NMRI nude mice by Charles River Discovery Research Services Germany GmbH according to local animal welfare guidelines.

[0610] For the study, 10 NMRI nude mice, 4 - 6 weeks old, were assigned to each group, and the tumor volume at randomization was 50 mm 3 to 250 mm 3 , preferably 80 - 200 mm 3 . The tumor volume was evaluated twice a week in mm 3 using calipers.

[0611] As Figure 7 indicated, the conjugates mAb8-LALA-D265C-(XII) and mAb8-LALA-D265C-(XIV) of the invention were administered as a single dose (“single dose”) or once every 7 days for 4 weeks (“q7dx4”). Sequencing of tumor cDNA revealed a G13D mutation in 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 guanylate cyclase C (GUCY2C), wherein the antibody comprises a heavy chain variable region (V H ), and the heavy chain variable region comprises framework region 1 (FR H 1), complementarity determining region 1 (CDR H 1), FR H 2, CDR H 2, FRH3, CDRH3, and FRH4, wherein - FRH1 comprises an amino acid sequence selected from SEQ ID NO: 33, 40, 45, 51; - CDRH1 comprises an amino acid sequence selected from SEQ ID NO: 34, 41, 46, 52; - FRH2 comprises an amino acid sequence selected from SEQ ID NO: 35; 55; - CDRH2 comprises an amino acid sequence selected from SEQ ID No: 36, 42, 47; 120 - FRH3 comprises an amino acid sequence selected from SEQ ID NO: 37, 43, 48, 53; - CDRH3 comprises an amino acid sequence selected from SEQ ID No: 38, 44, 49; 54 - FRH4 comprises an amino acid sequence selected from SEQ ID NO: 39, 50.

2. The antibody or antibody fragment according to claim 1, wherein the antibody comprises a light chain variable region (V L ), and the light chain variable region comprises 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, wherein - FR L 1 comprises an amino acid sequence selected from SEQ ID NO: 1, 13, 20, 27; - CDR L 1 comprises an amino acid sequence selected from SEQ ID NO: 2, 14, 21, 28; - FR L 2 comprises an amino acid sequence selected from SEQ ID NO: 3, 15, 29 - CDR L 2 comprises an amino acid sequence selected from SEQ ID NO: 4, 16, 23, 30; - FR L3 comprises an amino acid sequence selected from SEQ ID No: 5, 8, 17, 24, 31; -CDR L 3 comprises an amino acid sequence selected from SEQ ID NO: 6, 9, 11, 18, 25, 32; -FR L 4 comprises an amino acid sequence selected from SEQ ID NO: 7, 10, 12, 19, 26.

3. The antibody or antibody fragment according to claims 1-2, wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ), the heavy chain variable region comprises framework region 1 (FR H 1), complementarity determining region 1 (CDR H 1), FR H 2, CDR H 2, FRH3, CDRH3 and FRH4, wherein -FRH1 comprises the amino acid sequence according to SEQ ID NO: 33 -CDRH1 comprises the amino acid sequence according to SEQ ID NO: 34, -FRH2 comprises the amino acid sequence according to SEQ ID NO: 35; -CDRH2 comprises the amino acid sequence according to SEQ ID NO: 36; -FRH3 comprises the amino acid sequence according to SEQ ID NO: 37; -CDRH3 comprises the amino acid sequence according to SEQ ID NO: 38; -FRH4 comprises the amino acid sequence according to SEQ ID NO: 39, and wherein the antibody or antibody fragment comprises a light chain variable region (V L ), the light chain variable region comprises 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, wherein -FR L 1 comprises the amino acid according to SEQ ID NO: 1; -CDR L 1 comprises the amino acid according to SEQ ID NO: 2; -FR L 2 comprises an amino acid selected from SEQ ID NO: 3, 15, 29 -CDR L 2 comprises the amino acid sequence according to SEQ ID NO:4; -FR L 3 comprises the amino acid sequences selected from SEQ ID No:5, 8; -CDR L 3 comprises the amino acid sequences selected from SEQ ID NO:6, 9, 11; -FR L 4 comprises the amino acid sequences selected from SEQ ID NO:7, 10, 12.

4. The antibody or antibody fragment according to claims 1-2, wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ), the heavy chain variable region comprising framework region 1 (FR H 1), complementarity determining region (CDR H 1), FR H 2, CDR H 2, FRH3, CDRH3 and FRH4, wherein -FRH1 comprises the amino acid sequences selected from SEQ ID NO:40, 45, 51; -CDRH1 comprises the amino acid sequences selected from SEQ ID NO:41, 46, 52; -FRH2 comprises the amino acid sequences selected from SEQ ID NO:35; 55 -CDRH2 comprises the amino acid sequences selected from SEQ ID NO:42, 47; -FRH3 comprises the amino acid sequences selected from SEQ ID NO:43, 48, 53; -CDRH3 comprises the amino acid sequences selected from SEQ ID NO:49, 44, 54 -FRH4 comprises the amino acid sequence according to SEQ ID NO:50, and wherein the antibody or antibody fragment comprises a light chain variable region (V L ), the light chain variable region comprising 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, wherein -FR L 1 comprises the amino acids selected from SEQ ID NO:13, 20, 27; -CDR L1 comprises an amino acid selected from SEQ ID NO: 14, 21, 28; -FR L 2 comprises an amino acid selected from SEQ ID NO: 15, 22, 29; -CDR L 2 comprises an amino acid sequence selected from SEQ ID NO: 16, 23, 30; -FR L 3 comprises an amino acid sequence selected from SEQ ID No: 17, 24, 31; -CDR L 3 comprises an amino acid sequence selected from SEQ ID NO: 18, 25; -FR L 4 comprises an amino acid sequence selected from SEQ ID NO: 19, 26.

5. The antibody or antibody fragment according to any one of claims 1 - 3, wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ) according to SEQ ID NO: 62 and a light chain variable region (V L ) according to SEQ ID NO:

56.

6. The antibody or antibody fragment according to any one of claims 1 - 3, wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ) according to SEQ ID NO: 63 and a light chain variable region (V L ) according to SEQ ID NO:

57.

7. The antibody or antibody fragment according to any one of claims 1 - 3, wherein the antibody comprises a heavy chain variable region (V H ) according to SEQ ID NO: 64 and a light chain variable region (V L ) according to SEQ ID NO:

58.

8. The antibody or antibody fragment according to claim 4, wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ) according to SEQ ID NO: 65 and a light chain variable region (V L ) according to SEQ ID NO:

59.

9. The antibody or antibody fragment according to claim 4, wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ) according to SEQ ID NO: 66 and a light chain variable region (V L ) according to SEQ ID NO:

60.

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

61.

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

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

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

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

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

16. The antibody according to claim 15, wherein the Fc region comprises the amino acid sequence according 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 cytotoxicity (CDC).

18. The antibody according to claim 14, wherein the antibody comprises a heavy chain constant (Fc) region, and the heavy chain constant region comprises 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, and the heavy chain constant region comprises the amino acid substitution D265C (according to the EU numbering).

20. The antibody according to claim 18, wherein the antibody comprises a heavy chain constant (Fc) region, and the heavy chain constant region comprises the 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, and the heavy chain constant region comprises the 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, and the heavy chain constant region comprises the 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, and the heavy chain constant region comprises the 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, and the heavy chain constant region comprises the 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, and the heavy chain constant region comprises the mutations 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 according to SEQ ID NO: 78 and a heavy chain (HC) amino acid sequence according 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 according to SEQ ID NO: 85 and a heavy chain (HC) amino acid sequence according 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 according to SEQ ID NO: 92 and a heavy chain (HC) amino acid sequence according 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 according to SEQ ID NO: 99 and a heavy chain (HC) amino acid sequence according 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 according to SEQ ID NO: 106 and a heavy chain (HC) amino acid sequence according 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 according to SEQ ID NO: 113 and a heavy chain (HC) amino acid sequence according to one of SEQ ID NOs: 114, 115, 116, 117, 118, or 119.

32. The antibody according to any one of claims 1-31, wherein the antibody specifically binds to an epitope comprising amino acids 24-430 of SEQ ID NO:76 or amino acids 23-430 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 comprises CDRs selected from SEQ ID NO:34, 41, 46, 52; 36, 42, 47; 38, 44 or 49 H 1, CDR H 2 and CDR H 3; and wherein the framework regions FR H 1, FR H 2, FR H 3, FR H 4 have at least 80%, 85%, preferably 90% or 95% sequence similarity to any one of SEQ ID NO:33, 40, 45, 51; 35; 55; 37, 43, 48, 53; 39 or 50.

34. A conjugate comprising (i) an antibody according to any one of claims 1-33, (ii) at least one toxin, and (iii) at least one linker connecting the antibody moiety to the at least one toxin, wherein the antibody specifically binds to human GUCY2C and wherein the at least one toxin is amanitin.

35. The conjugate according to claim 34, wherein the linker is connected to the antibody via any naturally occurring cysteine residue of the antibody, preferably via any naturally occurring cysteine residue forming an interchain disulfide bond of the antibody and / or via a disulfide bond connection.

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

37. The conjugate according to claim 34, wherein the linker is an uncleavable linker or a cleavable linker.

38. The conjugate according to claim 37, wherein the cleavable linker is selected from enzymatically cleavable linkers, preferably protease-cleavable linkers and chemically cleavable linkers, preferably linkers comprising a disulfide bridge.

39. The conjugate 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 conjugate according to claim 39, wherein the cleavable linker is a cathepsin B-cleavable linker.

41. The conjugate according to claim 40, wherein the cathepsin B-cleavable linker 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 conjugate according to any one of claims 34-41, wherein the linker is linked to the amatoxin via: (i) the γC atom of amatoxin amino acid 1, or (ii) the δ-C atom of amatoxin amino acid 3, or (iii) the 6’-C atom of amatoxin amino acid 4.

43. The conjugate according to any one of claims 34-42, wherein the amatoxin comprises (i) amino acid 4 having a 6’-deoxy position and (ii) amino acid 8 having an S-deoxy position.

44. The conjugate according to any one of claims 34-43, wherein the conjugate comprises any one of the amatoxin-linker compounds of the following formulas (I) to (XI) 45. The conjugate according to any one of claims 34-44, wherein the conjugate comprises the antibody conjugated to an amatoxin linker moiety according to any one of formulas (XII) to (XXII) via a thioether linkage: wherein the amatoxin linker moiety is coupled to the thiol group of a cysteine residue of the antibody, and wherein n is preferably from about 1, 2, 3 to about 4, 5, 6, 7, 8, preferably, wherein n is from about 1, 1.5, 2, 2.5 to about 3.5, 4.5, 5.5, more preferably, wherein n is from about 1.5 to about 3.5, or from about 1.8 to about 2.

5.

46. The conjugate according to claim 45, wherein the amatoxin linker moiety is coupled to the thiol group of a cysteine residue of the antibody, and wherein n is from about 1 to about 2, preferably wherein n is about 2.

47. The conjugate according to any one of claims 34 - 46, wherein the conjugate is selected from (i) conjugate (XXIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ii) conjugate (XXIV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iii) conjugate (XXV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iv) conjugate (XXVI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (v) Conjugate (XXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, said antibody conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vi) Conjugate (XXVIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, said antibody conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vii) Conjugate (XXIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, said antibody conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (viii) Conjugate (XXX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, said antibody conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ix) Conjugate (XXXI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (x) Conjugate (XXXII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (xi) Conjugate (XXXIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, wherein n is from about 1 to about 2.

48. The conjugate according to any one of claims 34 - 46, wherein the conjugate is selected from (i) Conjugate (XXXIV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ii) Conjugate (XXXV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iii) Conjugate (XXXVI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iv) Conjugate (XXXVII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (v) Conjugate (XXXVIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 62, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 56, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vi) Conjugate (XXXIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, said antibody conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vii) Conjugate (XL) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, said antibody conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (viii) Conjugate (XLI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, said antibody conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ix) Conjugate (XLII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, said antibody conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (x) Conjugate (XLIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amatoxin-linker moiety of formula (XXI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (xi) Conjugate (XLIV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 63, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 57, the antibody being conjugated to at least one amatoxin-linker moiety of formula (XXII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, where n is from about 1 to about 2.

49. The conjugate according to any one of claims 34 - 46, wherein the conjugate is selected from (i) Conjugate (XLV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amatoxin-linker moiety of formula (XII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ii) Conjugate (XLVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amatoxin-linker moiety of formula (XIII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iii) Conjugate (XLVII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iv) Conjugate (XLVIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (v) Conjugate (XLIX), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vi) Conjugate (L), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vii) Conjugate (LI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (viii) Conjugate (LII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ix) Conjugate (LIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (x) Conjugate (LIV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (xi) Conjugate (LV), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 64, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 58, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, where n is from about 1 to about 2.

50. The conjugate according to any one of claims 34-46, wherein the conjugate is selected from (i) Conjugate (LVI), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ii) Conjugate (LVII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iii) Conjugate (LVIII), which comprises an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iv) Conjugate (LIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (v) Conjugate (LX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vi) Conjugate (LXI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vii) Conjugate (LXII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (viii) Conjugate (LXIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ix) Conjugate (LXIV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (x) Conjugate (LXV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (xi) Conjugate (LXVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 65, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 59, the antibody conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, where n is from about 1 to about 2.

51. The conjugate according to any one of claims 34 - 46, wherein the conjugate is selected from (i) Conjugate (LXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (ii) Conjugate (LXVIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (iii) Conjugate (LXIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (iv) Conjugate (LXX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (v) Conjugate (LXXI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (vi) Conjugate (LXXII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (vii) Conjugate (LXXIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (viii) Conjugate (LXXIV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, said antibody conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (ix) Conjugate (LXXV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (x) Conjugate (LXXVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (xi) Conjugate (LXXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 66, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 60, the antibody conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, where n is from about 1 to about 2.

52. The conjugate according to any one of claims 34 - 46, wherein the conjugate is selected from (i) Conjugate (LXXVIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ii) Conjugate (LXXIX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated via a thioether linkage of the linker to at least one amanitin-linker moiety of formula (XIII) and to the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iii) Conjugate (LXXX) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated via a thioether linkage of the linker to at least one amanitin-linker moiety of formula (XIV) and to the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (iv) Conjugate (LXXXI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated via a thioether linkage of the linker to at least one amanitin-linker moiety of formula (XV) and to the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (v) Conjugate (LXXXII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, the antibody being conjugated via a thioether linkage of the linker to at least one amanitin-linker moiety of formula (XVI) and to the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vi) Conjugate (LXXXIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, said antibody conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (vii) Conjugate (LXXXIV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, said antibody conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (viii) Conjugate (LXXXV) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, said antibody conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (ix) Conjugate (LXXXVI) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, said antibody conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system, (x) Conjugate (LXXXVII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, said antibody conjugated to at least one amatoxin-linker moiety of formula (XXI) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, (xi) Conjugate (LXXXVIII) comprising an antibody consisting of two heavy chains and two light chains, each heavy chain comprising a VH domain consisting of or containing the amino acid sequence according to SEQ ID No. 67, each light chain comprising a VL domain consisting of or containing the amino acid sequence according to SEQ ID No. 61, said antibody conjugated to at least one amatoxin-linker moiety of formula (XXII) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system, wherein n is from about 1 to about 2.

53. The conjugate according to claim 47, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or containing the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consisting of or containing the amino acid sequence according to SEQ ID No. 78, said antibody conjugated to at least one amatoxin-linker moiety of formula (XII) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system.

54. The conjugate according to claim 47, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or containing the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consisting of or containing the amino acid sequence according to SEQ ID No. 78, said antibody conjugated to at least one amatoxin-linker moiety of formula (XIII) via a thioether linkage of said linker and the sulfhydryl group of the heavy chain 265Cys residue of said antibody according to the EU numbering system.

55. The conjugate according to claim 47, wherein the conjugate comprises an antibody, the antibody consists of two heavy chains and two light chains, each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 78, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

56. The conjugate according to claim 47, wherein the conjugate comprises an antibody, the antibody consists of two heavy chains and two light chains, each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 78, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

57. The conjugate according to claim 47, wherein the conjugate comprises an antibody, the antibody consists of two heavy chains and two light chains, each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 78, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

58. The conjugate according to claim 47, wherein the conjugate comprises an antibody, the antibody consists of two heavy chains and two light chains, each heavy chain consists of or comprises the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consists of or comprises the amino acid sequence according to SEQ ID No. 78, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

59. The conjugate according to claim 47, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

60. The conjugate according to claim 47, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

61. The conjugate according to claim 47, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

62. The conjugate according to claim 47, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83, 84, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 78, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

63. The conjugate according to claim 47, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 79, 80, 81, 82, 83 or 84, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 78, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

64. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

65. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

66. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

67. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

68. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

69. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

70. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

71. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

72. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

73. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

74. The conjugate according to claim 48, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 86, 87, 88, 89, 90 or 91, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 85, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

75. The conjugate according to claim 49, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

76. The conjugate according to claim 49, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

77. The conjugate according to claim 49, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

78. The conjugate according to claim 49, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

79. The conjugate according to claim 49, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

80. The conjugate according to claim 49, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

81. The conjugate according to claim 49, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

82. The conjugate according to claim 49, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and the sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

83. The conjugate according to claim 49, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, the antibody being conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

84. The conjugate according to claim 49, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

85. The conjugate according to claim 49, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 93, 94, 95, 96, 97 or 98, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 92, the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

86. The conjugate according to claim 50, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

87. The conjugate according to claim 50, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

88. The conjugate according to claim 50, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

89. The conjugate according to claim 50, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

90. The conjugate according to claim 50, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

91. The conjugate according to claim 50, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

92. The conjugate according to claim 50, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

93. The conjugate according to claim 50, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

94. The conjugate according to claim 50, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

95. The conjugate according to claim 50, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

96. The conjugate according to claim 50, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 100, 101, 102, 103, 104 or 105, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 99, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

97. The conjugate according to claim 51, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

98. The conjugate according to claim 51, wherein the conjugate comprises an antibody, the antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

99. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

100. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

101. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

102. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

103. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

104. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

105. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

106. The conjugate according to claim 51, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 106, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and a sulfhydryl group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

107. The conjugate according to claim 51, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 107, 108, 109, 110, 111 or 112, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 106, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

108. The conjugate according to claim 52, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

109. The conjugate according to claim 52, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

110. The conjugate according to claim 52, wherein the conjugate comprises an antibody composed of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XIV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

111. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XV) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

112. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

113. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

114. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising the amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising the amino acid sequence according to SEQ ID No. 113, and the antibody being conjugated to at least one amanitin-linker moiety of formula (XVIII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

115. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 113, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XIX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

116. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 113, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XX) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

117. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 113, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XXI) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

118. The conjugate according to claim 52, wherein the conjugate comprises an antibody consisting of two heavy chains and two light chains, each heavy chain consisting of or comprising an amino acid sequence according to one of SEQ ID No. 114, 115, 116, 117, 118 or 119, each light chain consisting of or comprising an amino acid sequence according to SEQ ID No. 113, and the antibody is conjugated to at least one amanitin-linker moiety of formula (XXII) via a thioether linkage of the linker and a thiol group of the heavy chain 265Cys residue of the antibody according to the EU numbering system.

119. A polynucleotide encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 62, 63, 64, 65, 66 or 67.

120. A polynucleotide encoding a polypeptide, said polypeptide comprising the amino acid sequence of any one of SEQ ID NO:56, 57, 58, 59, 60 or 61.

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

122. A host cell, said 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. The antibody according to any one of claims 1-33, said antibody for use in the manufacture of a conjugate.

125. A pharmaceutical composition, said pharmaceutical composition comprising the conjugate according to any one of claims 47-118.

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

127. A composition, said composition comprising - at least one immune checkpoint inhibitor, and - at least one conjugate according to any one of claims 44-118.

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

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

130. The conjugate according to any one of claims 44 - 118 for the said use, or the pharmaceutical composition according to claim 125 or claim 126, or the composition according to claim 127, for the treatment of colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer, wherein the colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer is characterized by hemizygous deletion of TP53, POLR2A or del(17p13).

131. The conjugate according to any one of claims 44 - 118 or the 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 a patient who has not responded to first - line treatment, which first - line treatment comprises cetuximab or panitumumab alone or in combination with: 5 - fluorouracil (5 - FU) and oral 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. The conjugate according to any one of claims 44 - 118, or the pharmaceutical composition according to claim 125 or claim 126, or the composition according to claim 127 for use in the treatment of refractory cancer, wherein the cancer is selected from colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer.

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

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

135. A method of treating a patient having a solid tumor that expresses amino acids 24 - 430 or a fragment thereof of SEQ ID NO:76 on the surface of the solid tumor, wherein the method comprises administering to the patient a therapeutically effective amount of the conjugate according to any one of claims 44 - 118 or the pharmaceutical composition according to claim 125, alone or in combination with an immune checkpoint inhibitor.

136. A method of treating a patient suffering from gastrointestinal cancer, wherein the method comprises administering to the patient a therapeutically effective amount of the conjugate according to any one of claims 44-118 or the pharmaceutical composition according to 125, either alone or in combination with an immune checkpoint inhibitor.

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 non-responsive to first-line or second-line treatment, the first-line or second-line treatment comprising cetuximab or panitumumab alone or in combination with: 5-fluorouracil (5-FU) and oral capecitabine (CAP), FOLFOX (5-FU and oxaliplatin), FOLFIRI (5-FU and irinotecan), XELOX / CAPOX (CAP and oxaliplatin), CAPIRI (CAP and irinotecan), 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. The method of treating a patient according to any one of claims 137-139, wherein the colorectal cancer, metastatic colorectal cancer (mCRC), gastric cancer, pancreatic cancer or liver cancer is characterized by hemizygous deletion of TP53, POLR2A or del(17p13).

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

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