Amatoxin-antibody conjugate

Abstract

The present invention relates to conjugates comprising an amatoxin and an antibody, in particular an amatoxin linked to an antibody comprising a specific cysteine ​​residue.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201680027092.X, filed on March 7, 2016, entitled “Amanita Toxin-Antibody Conjugate”. Technical Field

[0002] The present invention relates to conjugates comprising amatoxin and an antibody, in particular amatoxin linked to an antibody comprising a specific cysteine ​​residue. Background Art

[0003] Amanita toxin is a cyclic peptide composed of 8 amino acids found in the mushroom Amanita phalloides (see Figure 1 ). Amanitin specifically inhibits the DNA-dependent RNA polymerase II of mammalian cells and thereby also inhibits transcription and protein biosynthesis of affected cells. Inhibition of intracellular transcription leads to cessation of growth and proliferation. Although not covalently linked, the complex between amanitin and RNA polymerase II is very tight (K D =3 nM). Dissociation of amatoxin from the enzyme is a very slow process, making it impossible for the affected cells to recover. When transcriptional inhibition persists too long, cells undergo programmed cell death (apoptosis).

[0004] Already in 1981, the use of a diazotized reaction with tryptophan (amino acid 4, see Figure 1 ) The use of amatoxin as a cytotoxic part for tumor treatment was developed by coupling anti-Thy1.2 antibodies to α-amanitin using an indole ring-linked linker (Davis & Preston, Science 1981, 213, 1385-1388). Davis & Preston determined that the position of the connection was the 7' position. Morris & Venton also demonstrated that the derivatives produced by 7'-position substitution retained cytotoxic activity (Morris & Venton, Int. J. Peptide Protein Res. 1983, 21 419-430).

[0005] Patent application EP 1 859 811 A1 (published on November 28, 2007) describes conjugates in which the γC-atom of the amino acid 1 of amatoxin of β-amanitin is directly (i.e. without a linker structure) coupled to albumin or monoclonal antibodies HEA125, OKT3 or PA-1. In addition, these conjugates show an inhibitory effect on the proliferation of breast cancer cells (MCF-7), Burkitt's lymphoma cells (Raji) and T lymphocytes (Jurkat). Although the application suggests the use of linkers, including linkers containing elements such as amides, esters, ethers, thioethers, disulfides, ureas, thioureas, hydrocarbon moieties, etc., no such structures are actually given and no further details are provided, such as the linking site on the amatoxin.

[0006] Patent applications WO 2010 / 115629 and WO 2010 / 115630 (both published on August 14, 2010) describe conjugates in which an antibody, for example an anti-EpCAM antibody, such as the humanized antibody huHEA125, is coupled to amatoxin via (i) the γ C-atom of amino acid 1 of amatoxin (ii) the 6′ C-atom of amino acid 4 of amatoxin or (iii) the δ C-atom of amino acid 3 of amatoxin, in each case directly or via a linker between the antibody and amatoxin. The proposed linkers comprise elements such as amides, esters, ethers, thioethers, disulfides, ureas, thioureas, hydrocarbon moieties, etc. In addition, these conjugates show an inhibitory effect on the proliferation of breast cancer cells (MCF-7 cell line), pancreatic cancer cells (Capan-1 cell line), colon cancer cells (Colo205 cell line) and cholangiocarcinoma cells (OZ cell line).

[0007] The disadvantage of the above method is that the coupling to lysine residues in the protein target binding domain (such as an antibody) is non-specific, resulting in conjugates with mixed components of different drug-antibody ratios (DARs), and the DAR cannot be reliably controlled. For example, there are about 70 to 100 lysine amino acid residues in a typical human IgG1 antibody. Typically, a DAR of about 4 is obtained by reacting a suitably activated amatoxin construct with lysine residues. In addition, a very heterogeneous mixture of coupling positions is observed, some resulting in higher potency of the conjugate and some with lower potency. Here, a specific degree of control cannot be obtained.

[0008] Similarly, since there are 32 cysteines in human IgG1 and 8 of them are available for conjugation after reducing the four interchain disulfide bonds, using cysteines obtained by reducing the disulfide bonds in the antibody molecule followed by conjugation with a toxin carrying a thiol-reactive group would result in the formation of a heterogeneous mixture.

[0009] It can be observed that the cytotoxic activity of the toxin-antibody conjugate increases with the increase of the obtained DAR, and therefore its therapeutic effect also increases with the increase of the obtained DAR. However, at the same time, tolerance decreases with the increase of DAR. Therefore, in order to optimize the performance of the toxin-antibody conjugate, it is very desirable to control the amount of toxin coupled to the antibody (i.e., DAR) and the specific positioning of the toxin conjugate. Only in this case, the precise adjustment of the available therapeutic window and the reproducibility of the results can be achieved.

[0010] In response to this situation, a number of methods have been developed for the specific and controlled production of drug-antibody conjugates, including, for example, site-specific conjugation with unnatural amino acids that have been introduced into the wild-type antibody sequence (see Axup et al., Proc. Natl. Acad. Sci. USA 109 (2012) 16101).

[0011] The alternative approach uses an antibody construct comprising a single cysteine ​​residue obtained by mutation of the wild-type antibody sequence. Ideally, a DAR of 2 can be achieved by mutation of a single amino acid residue in the light or heavy chain of an IgG with two copies of this mutant chain, and a DAR of 1 can be achieved by mutation of a single amino acid residue in the light or heavy chain of a monovalent antibody fragment (e.g., Fab fragment) of any such mutant chain with one copy.

[0012] WO 2006 / 034488 describes antibodies engineered by replacing one or more amino acids of a parent antibody with non-cross-linking, highly reactive cysteine ​​amino acids. The application describes various positions in the light and heavy chains where such substitutions may occur.

[0013] WO 2011 / 005481 is another application which presents a large number of positions which can be used to replace wild-type amino acid residues in an antibody sequence with reactive amino acid residues, in particular cysteine ​​residues.

[0014] WO 2008 / 070593 describes a similar approach in which amino acid residues in the Fc region of an antibody involved in Fc gamma receptor binding are replaced, including by cysteine ​​residues.

[0015] Although a lot of work has been done in this regard, there is no amatoxin conjugated in a defined manner by controlling the DAR ratio, for example by using a specific cysteine ​​for such a defined conjugation. In addition, it seems that there is no complete understanding of which amino acid positions are used for such cysteine-based conjugation. In particular, there is no available information on conjugates of amatoxin and antibodies through engineered cysteine ​​residues. However, given the high toxicity of amatoxin, it is very important to find a construct that exhibits high toxicity to the target cells of interest while exhibiting excellent stability and tolerance. So far, this goal has not been achieved.

[0016] Purpose of the Invention

[0017] Therefore, there remains a great need for a cost-effective and robust way to synthesize conjugates comprising amatoxin and antibodies, in particular amatoxin linked to antibodies comprising specific cysteine ​​residues, which are highly toxic to target cells while being stable and highly tolerated. In addition, the present invention also aims to identify positions in antibody chains that can be mutated from parental amino acid residues to cysteine ​​residues, resulting in the formation of such highly toxic, stable and highly tolerated conjugates. SUMMARY OF THE INVENTION

[0018] The present invention is based on the unexpected observation that a limited number of specific wild-type amino acid residues can be identified that can be mutated to a single unpaired cysteine ​​residue, resulting in the formation of highly toxic, stable and highly tolerated conjugates with amatoxin.

[0019] Therefore, in one aspect, the invention relates to a conjugate of the following general formula:

[0020] Ama–L–X–S–Ab,

[0021] wherein Ama is amatoxin, L is a linker, X is a moiety resulting from coupling a thiol group to a thiol-reactive group, S is a sulfur atom of a cysteine ​​amino acid residue, and Ab is an antibody sequence or a functional antibody fragment comprising said cysteine ​​residue, wherein said cysteine ​​residue (i) is located in an antibody domain selected from CL, CH1, CH2 and CH3; (ii) is located at a position where the germline sequence showing the closest sequence homology to said antibody domain contains an amino acid residue different from cysteine; and (iii) is located at a solvent exposed position.

[0022] In another aspect, the invention relates to a conjugate of the following general formula:

[0023] Ama–L–X–S–Ab,

[0024] wherein Ama is amatoxin, L is a linker, X is a moiety produced by coupling a thiol group with a thiol-reactive group, S is a sulfur atom of a cysteine ​​amino acid residue, and Ab is an antibody sequence or a functional antibody fragment comprising the cysteine ​​residue, wherein the cysteine ​​residue is selected from the following: heavy chain 118Cys, heavy chain 239Cys and heavy chain 265Cys, in particular: heavy chain 118Cys and heavy chain 265Cys.

[0025] In a third aspect, the invention relates to a method for synthesizing a conjugate of the following general formula:

[0026] Ama–L–X–S–Ab

[0027] This is achieved by reacting the compound Ama-L-X' with the antibody Ab-SH, wherein X' is a thiol-reactive group, the -SH group is a thiol of a cysteine ​​amino acid residue, and Ab is an antibody sequence comprising the cysteine ​​residue, wherein the cysteine ​​residue is selected from the following: heavy chain 118Cys, heavy chain 239Cys and heavy chain 265Cys, in particular: heavy chain 118Cys and heavy chain 265Cys.

[0028] In a fourth aspect, the present invention relates to a kit comprising (i) a compound Ama-L-X', wherein X' is a thiol-reactive group, and (ii) an antibody Ab-SH, wherein the -SH group is a thiol of a cysteine ​​amino acid residue, and Ab is an antibody sequence comprising the cysteine ​​residue, wherein the cysteine ​​residue is selected from the following list: heavy chain 118Cys, heavy chain 239Cys and heavy chain 265Cys, in particular: heavy chain 118Cys and heavy chain 265Cys.

[0029] In a fifth aspect, the present invention relates to a method for synthesizing the compound Ama-L-X', wherein X' is a maleimide group, the method comprising the step (a) reacting an amatoxin containing a nucleophilic group with a compound YLX", wherein

[0030] Y is a leaving group, and

[0031] X" is a protected maleimide group.

[0032] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising the conjugate according to the present invention.

[0033] In a seventh aspect, the present invention relates to a method for treating a disease associated with a cell presenting a target, comprising the step of contacting said cell with a conjugate according to the present invention, wherein said antibody is specific for said target. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The structural formulas of different amatoxins are shown. The standard numbering of the eight amino acids that form amatoxin is indicated. Also shown are the standard numbering of atoms in amino acids 1, 3, and 4 (Greek letters α to γ, Greek letters α to δ, and numbers from 1' to 7', respectively).

[0035] Figure 2 Schematic diagram of an IgG1 molecule and the positions of amino acid residues that have been mutated to cysteine ​​residues and used for conjugation to toxins are shown. HC-A118C; HC-S131C; HC-S239C; HC-D265C; HC-E269C; HC-N297C; HC-A327C; HC-I332C; Residues HC-D265 and HC-N297 are involved in Fcγ receptor binding; substitutions will interfere with receptor binding and subsequent uptake in receptor-positive cells.

[0036] Figure 3 A schematic diagram of the expression vectors used to produce the heavy and light chains of trastuzumab in Example 1 is shown.

[0037] Figure 4(A) shows the deconvoluted mass spectra of antibody trastuzumab HC-A118C and its conjugates: (a) deconvoluted mass spectrum of antibody trastuzumab HC-A118C; (b) deconvoluted mass spectrum of ADC trastuzumab HC-A118C-30.0880; Figure 4(B) shows the deconvoluted mass spectra of antibody trastuzumab HC-A118C light chain (a) and heavy chain (b); Figure 4(C) shows the deconvoluted mass spectra of ADC trastuzumab HC-A118C-30.0880 light chain (a) and heavy chain (b).

[0038] Figure 5(A) shows the mass spectrum of intact Her-30.0643 determined by LC / MS: drug-antibody ratio = 3.7 (amanitin / IgG); Figure 5(B) shows the mass spectrum of the Her-30.0643 light chain determined by LC / MS; Figure 5(C) shows the mass spectrum of the Her-30.0643 heavy chain determined by LC / MS.

[0039] Figure 6 Binding of four exemplary cysteine ​​mutants to the antibody target on SKOV-3 cells is shown compared to the parental antibody (two different expression products).

[0040] FIG. 7 (A) shows the analysis of different constructs on a blot stained with an anti-amanitin antibody: Lanes 1 and 3: naked antibody trastuzumab; Lanes 2 and 4: antibody trastuzumab conjugated to the amatoxin construct Her-30.0643 via lysine coupling; Lane 5: naked antibody trastuzumab with the HC-A118C mutation; Lane 6: antibody trastuzumab with the HC-A118C mutation conjugated to the amatoxin construct Her-30.1619 via cysteine ​​coupling to 118C (bromoacetamide coupling, stable linker); Lane 7: antibody trastuzumab with the HC-A118C mutation conjugated to the amatoxin construct Her-30.1704 via cysteine ​​coupling to 118C (bromoacetamide coupling, cleavable linker). Lane 8: antibody trastuzumab with HC-A118C mutation conjugated to the amanitin construct Her-30.0880 via cysteine ​​and 118C coupling (maleimide coupling, stable linker); Lane 9: antibody B conjugated to the amanitin construct Her-30.1699 via cysteine ​​and 118C coupling (maleimide coupling, cleavable linker); upper gel: denaturing, reducing conditions; exposure time: 60 s; lower gel: denaturing, non-reducing conditions; exposure time: about 5 s; Figure 7(B) shows the analysis of different constructs using (i) Coomassie blue protein staining (upper part); and (ii) anti-amanitin antibody Western blot (lower part) under denaturing, reducing conditions (left side) and denaturing, non-reducing conditions (right side).

[0041] Figures 8(A) and (B) show the results of WST-1 cytotoxicity assays using Fcγ receptor-positive THP-1 cells and different conjugates of the antibody trastuzumab with different cysteine ​​mutations: Figure 8(A): 120h incubation time; Figure 8(B): 72h incubation time trastuzumab; in both figures, the two curves on the upper right of the figures are from ADCs based on mutants HC-D265C and HC-N297C, i.e., mutants from residues known to be involved in Fcγ receptor binding; the remaining figures are from ADCs based on mutants HC-A118C; HC-S239C; HC-E269C; HC-A327C; and HC-I332C.

[0042] Fig. 9 Results of a tolerability study of the conjugate Ab trastuzumab HC-A118C (stable linker; maleimide coupled) in mice are shown. BWL: body weight loss; nd*: not determined due to insufficient coupling; nd**: not determined due to low morbidity; nd***: not determined due to lack of material; T HC-A118C-30.0880 showed no body weight loss at 37.5 mg / kg.

[0043] Fig.10 The results of tolerability studies of different amatoxin-trastuzumab conjugates in cynomolgus monkeys are shown: (A) random lysine conjugation, stable linker: (i) 0.3 mg / kg; (B) random lysine conjugation, cleavable linker: (i) 0.3 mg / kg; (C) conjugate with stable linker / maleimide coupling Ab trastuzumab HC-A118C: (i) 0.3 mg / kg; (ii) 1.0 mg / kg; (iii) 3.0 mg / kg; (iv) 10.0 mg / kg; (D) conjugate with stable linker / maleimide coupling Ab trastuzumab HC-D265C: (i) 0.3 mg / kg; (ii) 1.0 mg / kg; (iii) 3.0 mg / kg; (iv) 10.0 mg / kg.

[0044] Fig.11 Shown are the results of a BrdU incorporation experiment to determine cell viability after 72 h of incubation with different anti-HER2 ADCs and HER2-positive SKOV-3 cancer cells.

[0045] Fig.12 Shown are the results of a BrdU incorporation experiment to determine cell viability after 72 hours of incubation with different anti-HER2 ADCs and HER2-positive NCI-N87 cancer cells.

[0046] Fig.13 Shown are the results of a BrdU incorporation experiment to determine cell viability after 120 h of incubation with different anti-PSMA ADCs and PSMA-positive C4-2 prostate cancer cells.

[0047] Fig.14 Shown are the results of a subcutaneous HER2-positive SKOV-3 xenograft model treated with anti-HER2 based ADCs. DETAILED DESCRIPTION OF THE INVENTION

[0048] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, procedures and reagents described herein, as they may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood.

[0049] Preferably, the terms used herein are as in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0050] Unless the context requires otherwise, throughout the specification and the claims that follow, the words "comprise, comprises, comprising" will be understood to mean including the stated whole, components, steps, or groups of wholes or steps, and any additional wholes, components, steps, or groups of wholes, components, or steps may also be optionally present, including embodiments in which no additional wholes, components, steps, or groups of wholes, components, or steps are present. In the following embodiments, the terms "comprise" and "consisting of" are used consistently.

[0051] Several documents are quoted in this specification text. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, technical specifications, GenBank accession number sequence submissions, etc.), no matter above or below, is incorporated herein by reference in full to the extent allowed by each patent law. No content herein can be interpreted as admitting that the present invention has no right to advance the disclosure based on prior inventions.

[0052] The present invention is based on the unexpected observation that a limited number of specific amino acid residues can be identified in the parental antibody that can be mutated to a single unpaired cysteine ​​residue, resulting in the formation of highly toxic, stable and highly tolerated conjugates with amatoxin.

[0053] Therefore, in one aspect, the invention relates to a conjugate of the following general formula:

[0054] Ama–L–X–S–Ab,

[0055] wherein Ama is amatoxin, L is a linker, X is a moiety resulting from coupling a thiol group to a thiol-reactive group, S is a sulfur atom of a cysteine ​​amino acid residue, and Ab is an antibody sequence or a functional antibody fragment comprising said cysteine ​​residue, wherein said cysteine ​​residue (i) is located in an antibody domain selected from CL, CH1, CH2 and CH3; (ii) is located at a position where the germline sequence showing the closest sequence homology to said antibody domain contains an amino acid residue different from cysteine; and (iii) is located at a solvent exposed position.

[0056] In another aspect, the invention relates to a conjugate of the following general formula:

[0057] Ama–L–X–S–Ab,

[0058] Wherein, Ama is amatoxin, L is a linker, X is a portion produced by coupling a thiol group with a thiol-reactive group, S is a sulfur atom of a cysteine ​​amino acid residue, and Ab is an antibody sequence or a functional antibody fragment comprising the cysteine ​​residue, wherein the cysteine ​​residue is selected from the following: heavy chain 118Cys, heavy chain 239Cys and heavy chain 265Cys, in particular: heavy chain 118Cys and heavy chain 265Cys.

[0059] In the context of the present invention, the term "amanitin" includes all cyclic peptides consisting of 8 amino acids as isolated from Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 1978 Dec; 5(3): 185-260), and also all chemical derivatives thereof; also all semi-synthetic analogs thereof; also all synthetic analogs thereof built from building blocks according to the basic structure of the natural compound (cyclic, 8 amino acids), also all synthetic or semi-synthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids, also all synthetic or semi-synthetic analogs in which the thioether sulfoxide part is replaced by sulfide, sulfone or by an atom different from sulfur, such as the carbon atom in the carba-analogues of amanitin, in each case any such derivative or analogue functionally acting by inhibiting mammalian RNA polymerase II.

[0060] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those having a functional group (e.g., a carboxyl, amino, hydroxyl, thiol or thiol capture group) that is capable of reacting with a linker molecule or target binding moiety as defined above. Amatoxins that are particularly suitable for the conjugates of the present invention are Figure 1The α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin and amanullin acid and their salts, chemical derivatives, semi-synthetic analogs and synthetic analogs are shown. Particularly preferred amatoxins used in the present invention are α-amanitin, γ-amanitin and ε-amanitin, especially α-amanitin.

[0061] In the context of the present invention, a "linker" refers to a structure that connects two components, each of which is connected to one end of the linker. In the case where the linker is a bond, direct bonding of amatoxin to the antibody can reduce the ability of amatoxin to interact with RNA polymerase II. In specific embodiments, the linker increases the distance between the two components and alleviates the spatial interference between these components, such as between the antibody and the amatoxin in this example. In specific embodiments, there is a continuous chain of 1 to 30 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 atoms) on the linker backbone, that is, the length of the linker is defined as the shortest connection measured by the number of atoms or bonds between the amatoxin moiety and the antibody, wherein one side of the linker backbone has been reacted with the amatoxin and the other side can be reacted with the antibody or has been reacted with the antibody. In the context of the present invention, the linker is preferably C 1-20 -alkylene, C 1-20 -heteroalkylene, C 2-20- Alkenylene, C 2-20 -heteroalkenylene, C 2-20 -Alkynylidene, C 2-20-heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene or heteroaralkylene group, optionally substituted. The linker may contain one or more structural elements such as carboxamide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moiety, etc. The linker may also contain a combination of two or more of these structural elements. Each of these structural elements may be present in the linker more than once, for example twice, three times, four times, five times or six times. In certain embodiments, the linker may comprise a disulfide bond. It should be understood that the linker must be connected to the amatoxin and antibody in a single step or in two or more subsequent steps. For this purpose, the linker will carry two groups, preferably at the proximal and distal ends, which can (i) form a covalent bond with a group present in one of the components to be connected, preferably an activating group on amatoxin or a target binding peptide, or (ii) it is activated or can be activated to form a covalent bond with a group on amatoxin. Accordingly, it is preferred that chemical groups are located at the distal and proximal ends of the linker which may be the result of the coupling reaction, such as esters, ethers, carbamates, peptide bonds, and the like.

[0062] In a particular embodiment, the linker L is a linear chain of 1 to 20 atoms, particularly 2 to 18 atoms, more particularly 5 to 16 atoms, and even more particularly 6 to 15 atoms, independently selected from C, O, N and S. In a particular embodiment, at least 60% of the atoms in the linear chain are C atoms. In a particular embodiment, the atoms in the linear chain are connected by single bonds.

[0063] In particular embodiments, the linker L is an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene or heteroaralkylene group containing 1 to 4 heteroatoms selected from N, O and S, wherein the linker is optionally substituted.

[0064] The term "alkylene" refers to a divalent straight chain saturated hydrocarbon group having 1 to 20 carbon atoms, including groups having from 1 to 10 carbon atoms. In certain embodiments, the alkylene group may be a lower alkylene group. The term "lower alkylene" refers to an alkylene group having 1 to 6 carbon atoms, 1 to 5 or 1 to 4 carbon atoms in certain embodiments. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2-CH2-), n-propylene, n-butylene, n-pentylene and n-hexylene.

[0065] The term "alkenylene" refers to a divalent straight chain group having 2 to 20 carbon atoms, wherein at least one of the carbon-carbon bonds is a double bond and the other bonds may be single bonds or additional double bonds. Herein, the term "alkynylene" refers to a group having 2 to 20 carbon atoms, wherein at least one of the carbon-carbon bonds is a triple bond and the other bonds may be single bonds, double bonds or additional triple bonds. Examples of alkenylene groups include ethenylene (-CH=CH-), 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 3-butenylene and the like. Examples of alkynylene groups include ethynylene, 1-propynylene, 2-propynylene and the like.

[0066] As used herein, "cycloalkylene" means a divalent ring as part of any stable monocyclic or polycyclic system, wherein the ring has 3 to 12 carbon atoms but no heteroatoms, and wherein the ring is fully saturated, while the term "cycloalkenylene" means a divalent ring as part of any stable monocyclic or polycyclic system, wherein the ring has 3 to 12 carbon atoms but no heteroatoms, and wherein the ring is at least partially unsaturated (but excluding any arylene ring). Examples of cycloalkylene include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Examples of cycloalkenylene include, but are not limited to, cyclopentenylene and cyclohexenylene.

[0067] As used herein, the terms "heterocycloalkylene" and "heterocycloalkenylene" mean a divalent ring that is part of any stable monocyclic or polycyclic ring system, wherein the ring has 3 to about 12 carbon atoms, and wherein the ring consists of carbon atoms and at least one heteroatom, specifically at least one heteroatom independently selected from the group consisting of N, O and S, wherein heterocycloalkylene refers to a fully saturated ring and heterocycloalkenylene refers to an at least partially unsaturated ring (but excluding any arylene or heteroarylene ring).

[0068] The term "arylene" means a divalent ring or ring system that is part of any stable monocyclic or polycyclic ring system, wherein the ring or ring system has 3 to 12 carbon atoms but no heteroatoms, and the ring or ring system consists of aromatic moieties as defined by the "4n+2" π-electron rule, including phenylene.

[0069] As used herein, the term "heteroarylene" refers to a divalent ring or ring system that is part of any stable monocyclic or polycyclic ring system, wherein the ring or ring system has 3 to 20 atoms, the ring or ring system consisting of an aromatic portion as defined by the "4n+2" π-electron rule and containing carbon atoms and one or more nitrogen, sulfur and / or oxygen heteroatoms.

[0070] In the context of the present invention, the term "substituted" means that one or more hydrogens present in the backbone of the linker are replaced by a selection from the indicated group or groups, as long as the normal valence of the indicated atom or the valence of the appropriate atom of the substituted group is not exceeded and the substitution results in a stable compound. As defined herein, the term "optionally substituted" means that the linker is unsubstituted or substituted with one or more substituents as defined herein. When the substituent is a keto (or oxo, i.e., =O) group, a thio or imino group or a similar group, two hydrogens on the backbone atoms of the linker are replaced. Exemplary substituents include, for example, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, aroyl, heteroaroyl, carboxyl, alkoxy, aryloxy, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, halogen, (thio)ester, cyano, phosphoryl, amino, imino, (thio)amido, thiol, alkylthiol, acylthiol, sulfonyl, sulfate, sulfonate, sulfamoyl, sulfonamido, nitro, azido, haloalkyl (including perfluoroalkyl (e.g., trifluoromethyl)), haloalkoxy, alkylsulfanyl ... alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino, arylsulfonoamino, phosphoryl, phosphate, carbophosphate, phosphine, alkylcarboxyl, alkylcarboxamide, oxa, hydroxy, thiol, amino (optionally mono- or disubstituted by, for example, alkyl, aryl, or heteroaryl), imino, carboxamide, carbamoyl (optionally mono- or disubstituted by alkyl, aryl, or heteroaryl), amidino, aminosulfonyl, acylamino, aroylamino, (thio)urea, (arylthio)urea, alkyl(thio)urea, cycloalkyl(thio)urea, aryloxy, aralkoxy, or —O(CH2) n -OH, -O(CH2) n -NH2, -O(CH2) n COOH, -(CH2) n COOH, -C(O)O(CH2) n R, -(CH2) n N(H)C(O)OR or N(R)S(O)R, wherein n is 1-4 and R is independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, -cycloalkenyl, -(C-attached heterocycloalkyl), -(C-attached heterocycloalkenyl), -aryl and -heteroaryl, with various degrees of substitution permitted. It will be appreciated by those skilled in the art that substituents such as heterocycloalkyl, aryl, heteroaryl, alkyl, etc. or functional groups such as -OH, -NHR, etc., may themselves be substituted if appropriate. It will also be appreciated by those skilled in the art that the substituted moieties may themselves be substituted if appropriate.

[0071] In a specific embodiment, the linker L comprises a moiety selected from the group consisting of a disulfide (-SS-), an ether (-O-), a thioether (-S-), an amine (-NH-), an ester (-OC(=O)- or -C(=O)-O-), a carboxamide (-NH-C(=O)- or -C(=O)-NH-), a carbamate (-NH-C(=O)-O- or -OC(=O)-NH-) and a urea moiety (-NH-C(=O)-NH-).

[0072] In a specific embodiment of the present invention, the linker L comprises a number of m groups selected from the following: alkylene, alkenylene, alkynylene, cycloalkylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocycloalkylene, arylene, heteroarylene, aralkylene and heteroaralkylene groups, wherein each group can be optionally substituted independently, and the linker further comprises a number of n moieties independently selected from one of the following moieties: disulfide (-SS-), ether (-O-), thioether (-S-), amine (-NH-), ester (-OC(=O)- or -C(=O)-O-), carboxamide (-NH-C(=O)- or -C(=O)-NH-), carbamate (-NH-C(=O)-O- or -OC(=O)-NH-) and urea moiety (-NH-C(=O)-NH-), wherein m=n+1. In a specific embodiment, m is 2 and n is 1, or m is 3 and n is 2. In specific embodiments, the linker comprises 2 or 3 unsubstituted alkylene groups and 1 or 2 disulfide, ether, thioether, amine, ester, carboxamide, carbamate, or urea moieties, respectively, attached to the unsubstituted alkylene groups.

[0073] In a specific embodiment, the C atoms in the linear chain are independently part of an optionally substituted methylene group (-CH2-). In such a specific embodiment, the optional substituents are independently selected from halogen and C 1-6 - alkyl, especially methyl.

[0074] In certain embodiments, the linker L is a stabilizing linker.

[0075] In the context of the present invention, the term "stable linker" refers to a linker that is stable (i) in the presence of an enzyme, in particular a lysosomal peptidase such as cathepsin B, and (ii) in the reducing environment within a cell.

[0076] In a particular embodiment, the stabilizing linker does not contain (i) a substructure cleavable by an enzyme, in particular does not contain a dipeptide sequence cleavable by cathepsin B, and / or (ii) a disulfide group. In this particular embodiment, the linker has a length of up to 12 atoms, in particular 2 to 10, more particularly 4 to 9, most particularly 6 to 8 atoms.

[0077] In a particular embodiment, the LXS moiety present in the general formula of section

[0043] is selected from the group of moieties:

[0078] (amanitin side)-(CH2)2-XS-(antibody side);

[0079] (amanitin side)-(CH2)3-XS-(antibody side);

[0080] (amanitin side)-(CH2)4-XS-(antibody side);

[0081] (amanitin side)-(CH2)5X-S-(antibody side);

[0082] (amanitin side)-(CH2)6-XS-(antibody side);

[0083] (amanitin side)-(CH2)7-XS-(antibody side);

[0084] (amanitin side)-(CH2)8-XS-(antibody side);

[0085] (amanitin side)-(CH2)9-XS-(antibody side);

[0086] (Amatoxin side)-(CH2) 10 -XS-(antibody side);

[0087] (Amatoxin side)-(CH2) 11 -XS-(antibody side);

[0088] (Amatoxin side)-(CH2) 12 -XS-(antibody side);

[0089] (Amatoxin side)-(CH2) 16 -XS-(antibody side);

[0090] (amanitin side)-(CH2)2-O-(CH2)2-O-(CH2)2-XS-(antibody side);

[0091] (amanitin side)-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-XS-(antibody side); and

[0092] (Amatoxin side)-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-XS-(antibody side).

[0093] In other specific embodiments, the linker L is a cleavable linker.

[0094] In the context of the present invention, the term "cleavable linker" refers to (i) a linker that is cleavable by an enzyme, or (ii) a linker that is cleavable in a reducing environment.

[0095] In the context of the present invention, the term "enzymatically cleavable linker" refers to a linker that can be cleaved by an enzyme, in particular by a lysosomal peptidase, such as cathepsin B, leading to intracellular release of the toxin cargo conjugated to the targeting antibody after internalization (see Dubowchik et al., Bioconjug Chem. 13 (2002) 855-69). In a specific embodiment, the cleavable linker comprises a dipeptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit and Val-Cit, in particular wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and the amatoxin.

[0096] In other specific embodiments, the linker L is a reducible linker.

[0097] In the context of the present invention, the term "reducible linker" refers to a linker that can be cleaved in the intracellular reducing environment, particularly a linker containing a disulfide group, resulting in the intracellular release of the toxin cargo conjugated to the targeting antibody after internalization by the intracellular reducing environment (see Shen et al. (1985) J. Biol. Chem. 260, 10905-10908).

[0098] In other specific embodiments, the linker is a cleavable linker, in particular (i) an enzymatically cleavable linker, in particular a linker comprising a dipeptide, in particular a dipeptide cleavable by cathepsin B, or (ii) a reducible linker, in particular a linker comprising a disulfide group. In this specific embodiment, the cleavable linker has a length of up to 20 atoms, in particular 6 to 18, more particularly 8 to 16, most particularly 10 to 15 atoms.

[0099] In a specific embodiment, the linker L present in the LXS moiety in the general formula of the

[0043] segment is selected from the group of the following moieties:

[0100] (amanitin side)-(CH2)2-SS-(CH2)2-XS-(antibody side);

[0101] (amanitin side)-(CH2)3-SS-(CH2)2-XS-(antibody side);

[0102] (amanitin side)-(CH2)2-SS-(CH2)3-XS-(antibody side);

[0103] (amanitin side)-(CH2)3-SS-(CH2)3-XS-(antibody side);

[0104] (amanitin side)-(CH2)4-SS-(CH2)4-XS-(antibody side);

[0105] (amanitin side) -(CH2)2-CMe2-SS-(CH2)2-XS- (antibody side);

[0106] (amanitin side) -(CH2)2-SS-CMe2-(CH2)2-XS- (antibody side);

[0107] (amanitin side)-(CH2)3-SS-(antibody side);

[0108] (amanitin side) -CH2-C6H4-NH-Cit-Val-CO(CH2)5-XS- (antibody side);

[0109] (amanitin side) -CH2-C6H4-NH-Ala-Val-CO(CH2)5-XS- (antibody side);

[0110] (amanitin side) -CH2-C6H4-NH-Ala-Val-CO(CH2)2-XS- (antibody side);

[0111] (amanitin side) -CH2-C6H4-NH-Ala-Phe-CO(CH2)2-XS- (antibody side);

[0112] (amanitin side) -CH2-C6H4-NH-Lys-Phe-CO(CH2)2-XS- (antibody side);

[0113] (amanitin side) -CH2-C6H4-NH-Cit-Phe-CO(CH2)2-XS- (antibody side);

[0114] (amanitin side) -CH2-C6H4-NH-Val-Val-CO(CH2)2-XS- (antibody side);

[0115] (amanitin side) -CH2-C6H4-NH-Ile-Val-CO(CH2)2-XS- (antibody side);

[0116] (amanitin side) -CH2-C6H4-NH-His-Val-CO(CH2)2-XS- (antibody side);

[0117] (amanitin side) -CH2-C6H4-NH-Met-Val-CO(CH2)2-XS- (antibody side);

[0118] (amanitin side) -CH2-C6H4-NH-Asn-Lys-CO(CH2)2-XS- (antibody side); and

[0119] wherein -NH- and -CO- flanking the dipeptide sequence represent the amino and carbonyl moieties that form amide bonds with the carboxyl- and amino-termini of the dipeptide, respectively.

[0120] In the context of the present invention, the term "moiety resulting from the coupling of a thiol group with a thiol-reactive group" refers to the structure resulting from (i) nucleophilic substitution of a leaving group Y in a thiol-reactive group by a sulfur atom of a cysteine ​​residue, such as a bromoacetamide group, iodoacetamide, 4,6-dichloro-1,3,5-triazin-2-ylamino, alkylsulfone or heteroarylsulfone; (ii) addition of a HS-group of a cysteine ​​residue to an activated double bond of a thiol-reactive group, such as maleimide, or (iii) disulfide exchange of an activated disulfide or methanethiosulfonate with a sulfur atom of a cysteine ​​residue, such as with pyridine-2-thiol, 5-nitropyridine-2-thiol or methanesulfinate as a leaving group; or (iv) any other chemical reaction leading to a stable bond between a sulfur atom of a cysteine ​​residue and a reactive moiety that is part of a thiol-reactive group.

[0121] The initial moiety resulting from the coupling of the thiol group may optionally be further derivatized, for example, the succinimidyl thioether obtained from maleimide may be hydrolyzed to the succinamic acid thioether having the general structure

[0122]

[0123] In the context of the present invention, the term "thiol-reactive group" refers to a group that reacts selectively with thiol groups of free cysteines of antibodies, in particular at a pH value in the range of 6.0 to 8.0, more particularly at a pH value in the range of 6.5 to 7.5. In particular, the term "selectively" means that less than 10% of the coupling reactions of molecules comprising a thiol-reactive group with antibodies comprising at least one free cysteine ​​residue are coupling reactions with non-cysteine ​​residues (e.g. lysine residues) of the antibody, in particular less than 5%, more particularly less than 2%.

[0124] In a specific embodiment, the moiety resulting from the coupling of a thiol group to a thiol-reactive group is selected from: thiol-substituted acetamides; thiol-substituted succinimides; thiol-substituted succinamic acids; thiol-substituted heteroaryls, in particular thiol-substituted benzothiazoles, thiol-substituted phenyltetrazoles and thiol-substituted phenyloxadiazoles; and disulfide groups, in which one of the sulfur atoms is derived from a cysteine ​​residue of an antibody. In a specific embodiment, the moiety resulting from the coupling of a thiol group to a thiol-reactive group is a thiol-substituted succinimide.

[0125] The term "antibody or functional antibody fragment" as used herein refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, that is, molecules comprising an antigen binding site that immunospecifically binds to an antigen, that is, at least the antibody portion of the antigen binding fragment of the antibody. It also includes immunoglobulin-like proteins selected by technology (including, for example, phage display) to specifically bind to target molecules, such as target proteins Her-2 / neu or EpCAM. The immunoglobulin molecules of the present invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. In a specific embodiment, the antibody is IgG1. "Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (especially CDR-grafted), deimmunized or chimeric antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, nanobodies, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies of the antibodies of the present invention), FynomAb (Brack et al., Mol. Cancer Ther. 13 (2014) 2030) and epitope-binding fragments of any of the above, which contain at least one heavy chain framework position according to the present invention.

[0126] In some embodiments, the antigen-binding fragment is a human antigen-binding antibody fragment of the present invention and includes, but is not limited to, Fab, Fab' and F(ab')2, Fd and other fragments comprising at least a portion of an antibody heavy chain, comprising at least one heavy chain backbone position according to the present invention. The antigen-binding antibody fragment may comprise one or more variable domains alone or in combination with all or part of the following: hinge region, CL, CH1, CH2 and CH3 domains. Also included in the present invention are such antigen-binding fragments that further comprise any combination of one or more variable domains with hinge region, CL, CH1, CH2 and CH3 domains.

[0127] Antibodies useful in the present invention may be from any animal source, including birds and mammals. Preferably, the antibody is from humans, rodents (e.g., mice, rats, guinea pigs or rabbits), chickens, pigs, sheep, goats, camels, cattle, horses, donkeys, cats or dogs. Particularly preferably, the antibody is of human or mouse origin. As used herein, "human antibodies" include antibodies having the amino acid sequence of human immunoglobulins, and antibodies isolated from human immunoglobulin libraries or from one or more human immunoglobulin transgenics and animals that do not express endogenous immunoglobulins, such as described in U.S. Pat. No. 5,939,598 of Kucherlapati & Jakobovits.

[0128] As used herein, an antibody or a functional antibody fragment is considered to be a functional antibody if it has a specific affinity to the antigen as a target of 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM. The dissociation constant K of 50 nM or less is preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less and even more preferably 10 nM or less. D , it is said to "specifically bind" to the antigen.

[0129] In the context of the present application, the terms "target molecule" and "target epitope" refer to an antigen and an epitope of an antigen, respectively, to which an antibody or a functional antibody fragment specifically binds. Preferably, the target molecule is a tumor-associated antigen, particularly an antigen or epitope present in an increased concentration and / or a different spatial configuration on the surface of one or more tumor cell types or tumor-associated cells compared to the surface of non-tumor cells. Preferably, the antigen or epitope is present on the surface of one or more tumor or tumor stroma cell types, but not on the surface of non-tumor cells. In a specific embodiment, the antibody specifically binds to an epitope of HER-2 / neu or an epithelial cell adhesion molecule (EpCAM). In other embodiments, the antigen or epitope is preferentially expressed on cells involved in autoimmune diseases. In this specific embodiment, the antibody specifically binds to an epitope of an IL-6 receptor (IL-6R). In other embodiments, the antigen or epitope is preferentially expressed on cells involved in inflammatory diseases.

[0130] In a particular embodiment, the antibody or functional antibody fragment specifically binds an epitope present on a tumor cell, particularly wherein the antibody specifically binds an epitope of human epidermal growth factor receptor 2 (HER2).

[0131] In specific embodiments, the antibody is trastuzumab or HEA125, or an antibody fragment comprising an antigen-binding fragment of trastuzumab or HEA125.

[0132] In a specific embodiment, more than one amatoxin molecule is coupled to one antibody or one functional antibody fragment. An increase in the amount of amatoxin per conjugate will also increase toxicity. However, the increase will simultaneously reduce tolerance. Therefore, in a specific embodiment, the ratio of antibody or functional antibody fragment to amatoxin is 1 to 4 amatoxin molecules, particularly 1.5 to 3.5 amatoxin molecules, more particularly 1.8 to 2.5 amatoxin molecules, and more particularly about 2 amatoxin molecules in one antibody or one functional antibody fragment. In order to calculate the ratio in the case of antibody dimers, such as IgG, the dimer is regarded as one part.

[0133] In a specific embodiment, the antibody or antigen-binding fragment thereof is selected from a diabody, a tetrabody, a nanobody, a chimeric antibody, a deimmunized antibody, a humanized antibody or a human antibody.

[0134] In a specific embodiment, the antigen binding fragment is selected from the group consisting of Fab, F(ab')2, Fd, Fv, single chain Fv and disulfide-linked Fv (dsFv).

[0135] In the context of the present invention, the terms "heavy chain 118Cys", "heavy chain 239Cys" and "heavy chain 265Cys" refer to positions in the heavy chain of the human IgG1 antibody sequence, wherein the numbering is according to the EU numbering system of Edelman et al., Proc. Natl. Acad. Sci. USA; 63 (1969) 78-85. For example, when starting from (Trastuzumab) as the parental human IgG1 sequence, one of the following mutations will have to be made: HC-Ala118Cy; HC-Ser239Cys or HC-Asp265Cys.

[0136] In a third aspect, the invention relates to a method for synthesizing a conjugate of the general formula:

[0137] Ama–L–X–S–Ab

[0138] This is achieved by reacting the compound Ama-L-X' with the antibody Ab-SH, wherein X' is a thiol-reactive group, the -SH group is a thiol of a cysteine ​​amino acid residue, and Ab is an antibody sequence comprising the cysteine ​​residue, wherein the cysteine ​​residue is selected from the following: heavy chain 118Cys, heavy chain 239Cys and heavy chain 265Cys, in particular: heavy chain 118Cys and heavy chain 265Cys.

[0139] In a fourth aspect, the present invention relates to a kit comprising (i) a compound Ama-L-X', wherein X' is a thiol-reactive group, and (ii) an antibody Ab-SH, wherein the -SH group is a thiol of a cysteine ​​amino acid residue, and Ab is an antibody sequence comprising the cysteine ​​residue, wherein the cysteine ​​residue is selected from the following list: heavy chain 118Cys, heavy chain 239Cys and heavy chain 265Cys, in particular: heavy chain 118Cys and heavy chain 265Cys.

[0140] In a particular embodiment, the thiol-reactive group X' is selected from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazin-2-ylaminomethylsulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate and maleimide.

[0141] Thus, in a particular embodiment of the invention, X' is a maleimido substructure, wherein the nucleophilic group of the engineered cysteine ​​residue can be coupled to the double bond of the maleimide.

[0142] In a fifth aspect, the present invention relates to a method for synthesizing the compound Ama-L-X', wherein X' is a maleimide group, the method comprising the step (a) reacting an amatoxin containing a nucleophilic group with a compound YLX", wherein

[0143] Y is a leaving group, and

[0144] X" is a protected maleimide group.

[0145] In a particular embodiment, the method comprises the additional step (b) removing the protecting group from X".

[0146] In a preferred embodiment, step (a) is carried out under basic conditions, wherein the leaving group Y is selected from Br, I, tosylate or mesylate, and wherein X" is stable to basic conditions.

[0147] In a particular embodiment, X″ is a Diels-Alder-adduct obtained by reacting maleimide with 1,3-diene. In a particular embodiment, step (b) comprises removing the protecting group by a retro-Diels-Alder reaction.

[0148] In a more specific embodiment, X" is a Diels-Alder-addition product resulting from the reaction of maleimide with cyclopentadiene, furan or 2,5-dialkylfuran in step (a) and wherein the deprotection step (b) is carried out at elevated temperature in a polar aprotic solvent.

[0149] More specifically, X" is a Diels-Alder exo adduct resulting from step (a) from the reaction of maleimide with 2,5-dimethylfuran, and wherein the deprotection step (b) is carried out at a temperature between 80°C and 120°C in dimethyl sulfoxide or N-methylpyrrolidone.

[0150]

[0151] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising the conjugate according to the present invention.

[0152] In another aspect, the invention relates to a conjugate of the invention for use as a medicament.

[0153] In a seventh aspect, the present invention relates to a method for treating a disease associated with target presenting cells, comprising the step of contacting said cells with a conjugate according to the present invention, wherein said antibody or said functional antibody fragment is specific for said target.

[0154] In another aspect, the invention relates to a conjugate of the invention for use in treating a disease in a patient, in particular wherein the disease is cancer, in particular a cancer selected from the group consisting of breast cancer, pancreatic cancer, bile duct cancer, colorectal cancer, lung cancer, prostate cancer, ovarian cancer, prostate cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia and malignant lymphoma.

[0155] As used herein, "patient" refers to any mammal or bird that can benefit from the treatment of the antibody toxin conjugates described herein. Preferably, the "patient" is selected from the group consisting of experimental animals (e.g., mice or rats), livestock animals (including, e.g., guinea pigs, rabbits, chickens, pigs, sheep, goats, camels, cattle, horses, donkeys, cats or dogs) or primates (including humans). Particularly preferably, the "patient" is a human.

[0156] As used herein, "treat," "treating," or "treatment" of a disease or condition refers to accomplishing one or more of the following: (a) reducing the severity of the condition; (b) limiting or preventing the development of symptoms characteristic of the condition or conditions being treated; (c) inhibiting the worsening of symptoms characteristic of the condition or conditions being treated; (d) limiting or preventing the recurrence of the condition or conditions in a patient who previously had the condition or conditions; and (e) limiting or preventing the recurrence of symptoms in a patient who previously had symptoms of the condition or conditions.

[0157] As used herein, treatment may include administering to a patient a conjugate or pharmaceutical composition according to the invention, wherein "administering" includes in vivo administration as well as ex vivo administration directly to a tissue, such as a vein graft.

[0158] In certain embodiments, a therapeutically effective amount of a conjugate of the invention is used.

[0159] A "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal receiving the therapeutic agent, and the purpose of the administration. The effective amount for each individual case can be determined by those skilled in the art according to established methods and experience in the art.

[0160] In another aspect, the present invention relates to a pharmaceutical composition comprising an amatoxin according to the present invention, and further comprising one or more pharmaceutically acceptable diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents and / or preservatives.

[0161] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0162] In a specific embodiment, the pharmaceutical composition is used in the form of a systemically administered medicine. This includes parenteral, which particularly includes injections and infusions. The injection can be formulated in the form of an ampoule or a so-called readily available injection (e.g., a syringe readily available or a disposable syringe) and in addition formulated in a puncture bottle for multiple recovery. The administration of the injection can be in the form of subcutaneous injection (sc), intramuscular injection (im), intravenous injection (iv) or intradermal injection (ic). In particular, it is possible to produce a suitable injection formulation as a crystal suspension, solution, nanoparticle or colloidal dispersion system such as a hydrosol.

[0163] Injectable preparations can also be produced as concentrates, which can be dissolved or dispersed with aqueous isotonic diluents. Infusions can also be prepared in the form of isotonic solutions, fat emulsions, liposomal preparations and microemulsions. Similar to injections, infusion preparations can also be prepared in the form of concentrates for dilution. Injectable preparations can also be used in the form of constant infusions, such as by micropumps, both in hospitalized and ambulatory patient treatment.

[0164] To the parenteral pharmaceutical preparations, for example, albumin, plasma, bulking agents, surfactants, organic diluents, substances that influence the pH value, complexing substances or polymeric substances can be added, in particular substances that influence the adsorption of the antibody-toxin conjugate of the present invention to proteins or polymers, or they can also be added to reduce the adsorption of the antibody-toxin conjugate of the present invention to materials such as injection tools or packaging materials (such as plastic or glass).

[0165] The amatoxins of the present invention comprising antibodies can be combined with microcarriers or nanoparticles in parenteral formulations, such as, for example, finely dispersed particles based on poly(meth)acrylates, polylactates, polyglycolic acid esters, polyamino acids or polyether polyurethanes. Parenteral formulations can be modified into storage formulations, such as based on the "multiple unit principle" (if the antibody toxin conjugates of the present invention are introduced in the form of fine dispersion, dispersion or suspension or as a suspension of crystals in the drug) or based on the "single unit principle" (if the antibody toxin conjugates of the present invention are encapsulated in the formulation, such as in a tablet or rod that is subsequently implanted). Implants or storage drugs in the form of these single units and multiple unit formulations are often composed of so-called biodegradable polymers such as polyesters of lactic acid and glycolic acid, polyether polyurethanes, polyamino acids, poly(meth)acrylates or polysaccharides.

[0166] The adjuvants and carriers added during the production of the pharmaceutical composition of the invention for the preparation of parenteral preparations are preferably sterile water (sterilized water), substances that influence the pH value such as, for example, organic or inorganic acids or bases and their salts, buffer substances for adjusting the pH value, substances for isotonicity such as, for example, sodium chloride, sodium bicarbonate, glucose and fructose, tenside and surfactant and emulsifiers such as, for example, fatty acid partial esters of polyoxyethylene sorbitol (e.g. ) or fatty acid esters such as polyoxyethylene (e.g. ), fatty oils such as, for example, peanut oil, soybean oil or castor oil, synthetic esters of fatty acids such as, for example, ethyl oleate, isopropyl myristate and neutral oils (e.g. ) as well as polymeric adjuvants like, for example, gelatin, dextran, polyvinylpyrrolidone, additives that increase the solubility of organic solvents like, for example, propylene glycol, ethanol, N,N-dimethylacetamide, propylene glycol or complex-forming substances like, for example, citric acid and urea, preservatives like, for example, hydroxypropyl and methyl benzoate, benzyl alcohol, antioxidants like, for example, sodium sulfite and stabilizers like, for example, EDTA.

[0167] When the pharmaceutical composition of the invention is formulated as a suspension in a preferred embodiment, a thickener is added to prevent precipitation of the antibody toxin conjugate of the invention or a surfactant and a polyelectrolyte and / or a complex former such as EDTA to ensure resuspendibility of the precipitate. Complexes of the active ingredient with a variety of polymers can also be obtained. Examples of such polymers are polyethylene glycol, polystyrene, carboxymethyl cellulose, Or polyethylene glycol sorbitol fatty acid esters. The antibody toxin conjugates of the present invention can also be incorporated into liquid preparations in the form of inclusion compounds (e.g., using cyclodextrins). In specific embodiments, dispersants can be added as additional adjuvants. In order to produce freeze-dried preparations, scaffolding agents can be used, such as variants of mannitol, dextran, sucrose, human albumin, lactose, PVP or gelatin. Example

[0168] In the following, the present invention is explained in more detail by means of non-limiting examples:

[0169] Example 1

[0170] Engineering of cysteine ​​mutants and coupling conditions

[0171] 1.1 Antibody production

[0172] Figure 2 A schematic diagram of an IgG1 molecule and the positions of amino acid residues that have been mutated to cysteine ​​residues and used for toxin conjugation are shown. All antibodies were expressed by using expression vectors encoding the heavy and light chains ( Figure 3 ) transient transfection and production in eukaryotic Expi293 cells (Life Technologies). Gene sequences with Cys substitution mutations were synthesized by GeneArt and introduced into expression plasmids by standard molecular cloning methods based on nucleases and ligases. The results of the cloning experiments were verified by restriction enzyme analysis and sequencing (GATC Biotech, Germany). For transfection, Expi293 cells were cultured in Erlenmeyer shake flasks at 125 rpm and 8% CO2 to a density of approximately 3.0×10 per ml. 6cells. DNA and PEI reagent complexes were produced in Opti-MEM medium at a heavy:light chain ratio of 2:3. After adding the DNA:PEI complex to the medium, Expi293 cells were incubated for 24 hours. The cells were centrifuged at 460g for 15 minutes at room temperature and the medium was replaced to ensure long-term production. Cell viability was monitored and after 4 to 6 days, the cells were precipitated and the monoclonal antibodies were purified from the supernatant using a protein A column (Tosoh Biosience) through a Bio-Rad FPLC system. Aggregates and endotoxins were removed by polishing chromatography using a Superdex S-200 gel filtration column (GE Healthcare) using PBS, pH 7.4. Antibodies were identified using SDS-PAGE, UV spectroscopy, analytical SEC-HPLC, and endotoxin ELISA. The typical yield of purified antibodies was approximately 80-120 mg per liter of culture medium with <1% aggregates.

[0173] 1.2 Maleimide-amanitin conjugation

[0174] For the conjugation of maleimide-amanitin derivatives (e.g., HDP 30.0880 and HDP 30.1699), the cysteine-substituted antibodies were adjusted to 5.0 mg / ml in 1 mM EDTA in PBS (pH 7.4) and reduced with 40 equivalents of TCEP at 37°C for 3 hours. The reduced antibodies were purified by two consecutive dialysis steps in 1 mM EDTA in PBS (pH 7.4), and then the interchain disulfide bonds were oxidized by 20 equivalents of dehydroascorbic acid (dhAA) for 4 hours at room temperature. The coupling of the toxin to the substituted cysteine ​​was performed by adding 8 to 15 equivalents of maleimide-amanitin derivatives for 1 hour at room temperature, followed by quenching the reaction with 25 equivalents of N-acetyl-L-cysteine. PD-10 columns or G-25 Amanita toxin-ADC was purified by gel filtration chromatography (GE Healthcare). The drug-antibody ratio (DAR) of the ADC was determined by UV spectroscopy at 280 nm and 310 nm using the extinction coefficients of the antibody and α-amanitin. In addition, the DAR of the ADC was determined by native LC-MS ( FIG. 4(A) ) and heavy / light chain LC-MS analysis ( Figure 4(B) , 4(C)) was used to determine the DAR. According to LC-MS, the DAR ranged from 1.8 to 2.2 amanitins per IgG, and the drug was located only in the heavy chain. The quality of the ADC was checked by SDS-PAGE, Western Blotting using anti-amanitin antiserum, analytical SEC-HPLC, HIC-HPLC, and RP-HPLC. The ADC was adjusted to 3.0 to 5.0 mg / ml and stored in PBS (pH 7.4) at 4°C until further use in cell culture and in vivo models.

[0175] Example 2

[0176] 6'(6-N-maleimido-hexyl)-α-amanitin (HDP 30.0880)

[0177] Step 1: 1,7-dimethyl-10-oxa-4-azatricyclo[5.2.1.0 2,6 ]Deca-8-ene-3,5-dione, exo-isomer (HDP 30.0891)

[0178]

[0179] 4.00 g (41.2 mmol) 2,5-dimethylfuran and 5.93 g (61.7 mmol, 1.5 equivalents) maleimide were dissolved in 30 ml diethyl ether and heated to 90° C. for 12 hours in a Parr reactor. The resulting precipitate was filtered off and recrystallized from methanol:

[0180] 6.62g (83%) of crystals, melting point 137°C.

[0181] 1 H NMR (CDCl3, 500 MHz) δ (ppm): 8.68 (broad singlet, 1H), 6.31 (singlet, J, 2H), 2.88 (singlet, 2H), 1.73 (singlet, 6H). 13 C NMR (CDCl3, 100MHz) δ (ppm): 175.04, 140.82, 87.68, 53.77, 15.76.

[0182] Step 2: 4-(6-bromohexyl)-1,7-dimethyl-10-oxa-4-azatricyclo[5.2.1.0 2,6 ]Deca-8-ene-3,5-dione, exo-isomer (HDP 30.0916)

[0183]

[0184] 386 mg (2 mmol) of HDP 30.0891 and 1.952 g (8 mmol) of 1,6-dibromohexane were dissolved in 20 ml of DMF, 276 mg (2 mmol) of potassium carbonate were added, and the suspension was heated to 50 ° C for 3 hours. DMF was then evaporated and the residue was dissolved in 100 ml of dichloromethane. Inorganic salts were removed by filtration, diatomaceous earth (3 g) was added to the filtrate, and the solvent was removed under vacuum. The residue was purified by silica gel chromatography, eluting with a gradient of n-hexane-ethyl acetate to give HDP 30.0916 (483 mg) waxy crystals in a yield of 68%.

[0185] 1 H NMR (500MHz, CDCl3) δ (ppm) 6.31 (s, 2H), 3.48 (t, J = 7.2Hz, 2H), 3.39 (t, J = 6.8Hz, 2H), 2.81 (s, 1H), 1.90– 1.77(m,2H),1.70(s,5H),1.64–1.52(m,2H),1.44(dddd,J=9.2,7.4,6.5,5.4Hz,2H),1.35–1.23(m,2H). 13 C NMR (126MHz, CDCl3) δ174.81,140.81,87.52,52.33,38.42,33.65,32.50,27.54,27.33,25.64,15.87.

[0186] Step 3: 6′-(6-(1,7-dimethyl-10-oxa-4-azatricyclo[5.2.1.0 2,6 ]Deca-8-ene-3,5-dione-4-yl-hexyl)-α-amanitin (HDP 30.0903)

[0187]

[0188] 34.5 mg (37.5 μmol) of vacuum dried α-amanitin were dissolved in 1000 μl of dry dimethyl sulfoxide (DMSO) under argon and room temperature. HDP 30.0916 (106.8 mg, 8 eq.) and 1 M sodium hydroxide (41.2 μl, 1.1 eq.) were added. After 3 hours at room temperature, the reaction mixture was acidified to pH = 5 with 41.2 μl of 1 M acetic acid solution in DMSO. The solvent was removed under vacuum and the residue was purified by preparative HPLC on a C18 column eluting with a 5-100% methanol gradient. The fractions containing the product were evaporated to 27.2 mg (59%) of HDP 30.0903 as a colorless solid.

[0189] MS (ESI + )1194.17[M+H]+ ,1216.10[M+Na] +

[0190] Step 4: 6'(6-N-maleimido-hexyl)-α-amanitin (HDP 30.0880)

[0191]

[0192] HDP 30.0903 (27.2 mg, 22.7 μmol) was dissolved in 3000 μl of dry dimethyl sulfoxide. The reaction mixture was heated to 100° C. for 1.5 hours with stirring. After cooling to 40° C., DMSO was removed under vacuum and the residue was purified by preparative HPLC using the method described above.

[0193] The fractions with retention time of 17.3-18.1 minutes were collected and the solvent was evaporated. The residue was lyophilized from 3 ml of tert-butyl alcohol to give 23.6 mg (94%) of HDP 30.0880 as an off-white powder.

[0194] MS (ESI + )1098.29[M+H] + ,1120.36[M+Na] +

[0195] The following examples were prepared using the method of Example 2 with modifications apparent to those skilled in the art:

[0196]

[0197]

[0198] Embodiment 15

[0199] 6′-O-(6-(6-(N-maleimido)-hexanoylamino)hexyl)-α-amanitin (HDP 30.1948)

[0200]

[0201] Subsequently, to 10.0 mg (8.83 μmol) of 6′-O-(-6-aminohexyl)-α-amanitin (HDP 30.0134, synthesized as disclosed in EP 2621536) dissolved in 400 μl of dry DMF, 663 μl of a 20 mM solution of 6-(maleimido)hexanoic acid N-hydroxysuccinimide ester (EMCS) in DMF and 17.7 μl of a 1 M solution of DIPEA in DMF were added. After 5 hours at room temperature, 100 μl of water were added to the reaction mixture and the volatiles were evaporated. The crude product was purified by RP18 HPLC with a water-methanol gradient and the pure fractions were lyophilized from tert-butanol / water: 9.02 mg (84%) of HDP 30.1948 colorless powder.

[0202] MS (ESI + ) Measured value: 1210.99; Calculated value: 1211.54 [MH] + (C 55 H 79 N 12 O 17 S)

[0203] Measured value: 1233.32; calculated value: 1233.52[M + Na] + (C 55 H 78 N 12 NaO 17 S)

[0204] Example 16

[0205] 6′-O-(6-(N-α-maleimido)-L-2,3-diaminopropionamido)hexyl)-α-amanitin (HDP30.1958)

[0206]

[0207] Step 1: Subsequently, to a solution of 8.22 mg (17.66 μmol = 2 eq.) Mal-L-Dap(Boc)-OH xDCHA and 17.7 μl of 1 M DIPEA in DMF in 700 μl DMF, 9.19 mg (17.66 μmol = 2 eq.) PyBop and 6.01 μl (35.33 μmol = 4 eq.) DIPEA were added. After 1 minute, the mixture was added to 10.0 mg (8.83 μmol) 6'-O-(-6-aminohexyl)-α-amanitin (HDP 30.0134) dissolved in 200 μl dry DMF. After 2 hours at room temperature, 100 μl of water were added to the reaction mixture and the volatiles were evaporated. The crude product was purified by RP18 HPLC and the pure fractions were evaporated: 5.45 mg (48%) HDP 30.1954 amorphous solid.

[0208] MS (ESI + ) Measured value: 1306.58; Calculated value: 1306.54[M + Na] + (C 57 H 81 N 13 NaO 19 S)

[0209] Step 2: The Boc-protected product of step 1 was dissolved in 1 ml of trifluoroacetic acid. After 2 minutes, the mixture was evaporated to dryness at room temperature. The residue was purified by RP18 HPLC with a gradient of 0.05% TFA: methanol and the pure fractions were evaporated: 1.72 mg (31%) HDP 30.1958 amorphous solid.

[0210] MS (ESI + ) Measured value: 1306.58; Calculated value: 1184.50[M + Na] + (C 52 H 74 N 13 O 17 S)

[0211] Embodiment 17

[0212] 6′-O-(2-bromoacetamido)hexyl)-α-amanitin (HDP 30.1619)

[0213]

[0214] Subsequently, to 5.03 mg (4.44 μmol) of 6′-O-(-6-aminohexyl)-α-amanitin (HDP 30.0134) dissolved in 400 μl of dry DMF, 66.6 μl of a 100 mM solution of N-hydroxysuccinimide bromoacetate in DMF and 88.8 μl of a 100 mM solution of DIPEA in DMF were added. After 3 hours at room temperature, 50 μl of water were added and the reaction mixture was added dropwise to 10 ml of methyl tert-butyl ether (MTBE). The precipitate was separated by centrifugation and washed with 10 ml of MTBE. The crude product was purified by RP18 HPLC with a water-methanol gradient and the pure fractions were lyophilized from tert-butanol / water: 3.70 (73%) HDP 30.1619 colorless powder.

[0215] MS (ESI + ) Found: 1139.58; Calculated: 1138.39 [M+H] + (C 47 H 69 Bn 11 O 15 S)

[0216] Measured value: 1160.42; Calculated value: 1160.37 [M+Na] + (C 47 H 68 Bn 11 NaO 15 S))

[0217] Embodiment 18

[0218] 6′-O-(2-bromoacetamido)propyl)-α-amanitin (HDP 30.1618)

[0219]

[0220] The method of Example 16 was applied to 6'-O-(-3-aminopropyl)-α-amanitin disclosed in EP 2621536 to synthesize bromoacetamide HDP 30.1618:

[0221] MS (ESI + ) Measured value: 1096.22; Calculated value: 1096.34 [MH] + (C 44 H 63 Bn 11 O 15 S)

[0222] Measured value: 1118.45; calculated value: 1118.32[M + Na] + (C 44H 62 Bn 11 NaO 15 S)

[0223] Embodiment 19

[0224] 6'-[6-(6-(4-(5-(methylsulfonyl)-1,2,4-oxadiazol-2-yl)-phenoxy)hexylaminocarbonyl)-aminohexyl)]-α-amanitin (HDP 30.1926)

[0225]

[0226] Methylsulfonyl-1,2,4-oxadiazole linkers were synthesized by a variation of the method disclosed by Toda et al. in Angew. Chem. Int. Ed. 2013, 52, 12592-112596.

[0227] Step 1: 1-Azido-6-bromohexane

[0228]

[0229] 1,6-Dibromohexane (7.32 g, 30 mmol) was stirred with sodium azide (1.95 mg, 30 mmol) in 60 ml DMF overnight. The solvent was evaporated and the residue was stirred with 100 ml ethyl acetate for 5 minutes. The inorganic salts were filtered off and the monoazide was separated from the dibromide and diazide by silica gel column chromatography using a gradient of 0 to 20% dichloromethane in hexane to give 2.26 g (37%) of the product as an oil.

[0230] 1 H NMR (500MHz, CDCl3) δ3.42(t,J=6.7Hz,2H),3.28(t,J=6.9Hz,2H),1.88(dt,J=14.6,6.8Hz,2H),1.62(dt,J=14.3,7.0Hz,2H),1.53–1.36(m,4H).

[0231] 13 C NMR (126MHz, CDCl3) δ51.43,33.80,32.67,28.82,27.81,26.03.

[0232] Step 2: Ethyl 4-[(6-azidohexyl)oxy]benzoate (HDP 30.1897)

[0233]

[0234] At room temperature, add 4-hydroxybenzoic acid ethyl ester (3.324g, 20mmol) and K in the DMF solution (40mL) of step 1 product (4.122g, 20mmol) CO (5.528g, 40mmol) lasts for 4h.Then, the reaction mixture is diluted with 200ml MTBE and 200μl water. The organic layer is separated and washed with 3×100ml water, dried (MgSO ) and evaporated to dryness. Purification by silica gel column chromatography (hexane / MTBE) obtains the title compound (5.199g, 89%) as a colorless oil.

[0235] MS (ESI + ) Measured value: 314.33; Calculated value: 314.15[M + Na] + (C 15 H 21 N3NaO3)

[0236] Step 3: 4-[(6-azidohexyl)oxy]benzohydrazide (HDP 30.1899)

[0237]

[0238] To a solution of the product of step 2 (5.19 g, 17.8 mmol) in ethanol (9.0 mL) was added hydrazine monohydrate (1459 μL, 30 mmol) at room temperature, and the mixture was stirred at reflux for 22 hours. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (gradient dichloromethane to dichloromethane / ethyl acetate / methanol 6:3:1) to give a colorless solid benzoyl hydrazide derivative HDP 30.1899 (1.08 g, 22%).

[0239] MS (ESI + ) Found: 278.27; Calculated: 278.16 [M+H] + (C 13 H 20 N5O2)

[0240] Step 4: 5-[4-((6-azidohexyl)oxy)phenyl]-1,3,4-oxadiazole-2-thiol (HDP 30.1903)

[0241]

[0242] To an ethanol solution (10.0 mL) of a benzoyl hydrazide derivative (1.08 g, 3.89 mmol) was added carbon disulfide (1552 μL, 25.68 mmol) and powdered KOH (218 mg, 3.89 mmol) at room temperature, and the solution was stirred at 85° C. for 3 hours. Ethyl acetate and 1 M HCl were added to the solution. The organic layer was washed with saturated sodium bicarbonate and brine, dried over MgSO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound HDP 30.1903 (1.18 g, 95% yield) as a colorless solid.

[0243] Step 5: 5-[4-((6-azidohexyl)oxy)phenyl]-5-(methylsulfonyl)-1,3,4-oxadiazole (HDP30.1905)

[0244]

[0245] To a THF solution (15 ml) of the thiol (1.18 g, 3.69 mmol) of step 4 was added methyl iodide (257 μL, 4.13 mmol) and triethylamine (625 μl, 4.51 mmol) at 0°C. The mixture was then stirred at room temperature for 1 hour. Water (50 ml) and ethyl acetate (100 ml) were then added, and the mixture was decolorized with a 10% sodium thiosulfate solution (10 ml). The organic layer was separated, washed with brine (50 ml), and dried over MgSO4. After filtration, the organic solvent was removed under vacuum, and the residue was purified on silica gel with hexane / MTBE to give the title compound HDP 30.1905 (1.03 mg, yield 84%) as a white solid.

[0246] MS (ESI + ) Found: 334.27; Calculated: 334.13 [M+H] + (C 15 H 20 N5O2S)

[0247] Found: 356.29; Calculated: 356.12 [M+Na] + (C 15 H 19 N5NaO2S)

[0248] Step 6: 5-[4-((6-azidohexyl)oxy)phenyl]-5-(methylsulfonyl)-1,3,4-oxadiazole (HDP30.1910)

[0249]

[0250] To a solution of the product of step 5 (1.00 g, 3.00 mmol) in dichloromethane (100 mL) was added mCPBA (68 wt%, 2.79 mg, 3.66 mmol) at 0°C, and the mixture was stirred at room temperature for 24 hours. Magnesium sulfate was added, the insoluble matter was removed by filtration, and the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (hexane / MTBE) to give the title compound HDP30.1910 (858 mg, 78%).

[0251] MS (ESI + ) Found: 366.00; Calculated: 366.12 [M+H] + (C 15 H 20 N5O4S)

[0252] Found: 388.19; Calculated: 388.11 [M+Na] + (C 15 H 19 N5NaO4S)

[0253] Step 7: 5-[4-(((6-Succinimidyloxycarbonylamino)hexyl)oxy)phenyl]-5-(methylsulfonyl)-1,3,4-oxadiazole (HDP 30.1916)

[0254]

[0255] To a solution of the product of step 6 (370 mg, 1.01 mmol) in ethyl acetate (100 ml) was added N, N'-disuccinimidyl carbonate (519 mg, 2.02 mmol). The apparatus was purged with argon and palladium (10% on activated carbon) was added, followed by stirring overnight under a hydrogen atmosphere. After filtration and evaporation, the crude product was purified on silica gel using a gradient from hexane to ethyl acetate. The pure fractions were combined and lyophilized from 1,4-dioxane to give succinimidyl carbamate HDP 30.1916 (280 mg, 58%) as a colorless powder.

[0256] MS (ESI + ) Found: 481.10; Calculated: 481.14 [M+H] + (C 20 H 25 N4O8S)

[0257] 1 H NMR (500MHz, CDC l3)d 8.08–8.01(m,2H),7.06–6.99(m,2H),5.57(t,J=5.9Hz,1H),4.05(t,J=6.4Hz,2H),3 .51(s,3H),3.28(q,J=6.8Hz,2H),2.83(s,4H),1.88–1.77(m,2H),1.67–1.39(m,6H).

[0258] 13 C NMR(126MHz,CDCl3)d 170.05,166.73,163.11,161.53,151.44,129.68,115.28,114.03,68.14,42.98,41.90,29.39,28.85,26.23,25.55,25.47.

[0259] Step 8: 6'-[6-((6-((4-(5-(methylsulfonyl)-1,2,4-oxadiazol-2-yl)phenyl)oxy)hexyl)-aminocarbonylamino)hexyl]-α-amanitin (HDP 30.1926)

[0260]

[0261] Subsequently, to 10.0 mg (8.83 μmol) of 6′-O-(-6-aminohexyl)-α-amanitin (HDP 30.0134, synthesis as disclosed in EP 2621536) dissolved in 500 μl of dry DMF, 8.49 mg (17.66 μmol) of HDP 30.1916 and 6.01 μl (35.33 μmol) of DIPEA dissolved in 500 μl of DMF were added. After 18 hours at room temperature, the volatiles were evaporated. The crude product was purified by RP18 HPLC with a water-methanol gradient and the pure fractions were lyophilized from tert-butanol / water: 7.04 mg (58%) of HDP 30.1926 as a colorless powder.

[0262] MS (ESI + ) Measured value: 1383.62; Calculated value: 1383.57 [MH] + (C 61 H 87 N 14 O 19 S2)

[0263] Measured value: 1405.54; Calculated value: 1405.55[M + Na] + (C 61 H 86 N 14 NaO 19S2)

[0264] Embodiment 20

[0265] 6'-[(3-Maleimidopropionamido)-Ahx-Val-Cit-PAB]-α-amanitin (HDP 30.1426)

[0266] Dipeptide p-aminobenzylbromide was synthesized from the corresponding benzyl alcohol by adapting the method disclosed by Jeffrey et al. in J. Med. Chem. 2005, 48, 1344-1358. The general steps are exemplified by the following scheme:

[0267]

[0268] Step 1: Boc-Ahx-Val-Cit-PAB-OH (HDP 30.1267)

[0269] Firestone et al. disclose the preparation of valine-citrulline-p-aminobenzyl alcohol (H-Val-Cit-PAB-OH) and N-Boc-aminocaproic acid N-hydroxysuccinimide ester (Boc-Ahx-NHS) in US 6,214,345. By this method, crude H-Val-Cit-PAB-OH was prepared from 4.79 g (7.96 mmol) of Fmoc-Val-Cit-PAB-OH and dissolved in 50 ml of DMF. Boc-Ahx-NHS (2.88 g, 8.76 mmol) and 1489 μl (8.76 mmol) of N-ethyldiisopropylamine were added and stirred overnight at room temperature. After evaporation of DMF, the residue was stirred with 100 ml of MTBE overnight and the solid was separated by centrifugation. The precipitate was resuspended in MTBE and centrifuged again. The product was dried in vacuo to give 4.44 g (94% yield) of light brown powder.

[0270] MS (ESI + ) Measured value: 615.19; Calculated value: 615.35[M + Na] + (C 29 H 48 N6NaO7)

[0271] Step 2: Boc-Ahx-Val-Cit-PAB-OTBDMS (HDP 30.1362)

[0272] The step 1 product (4.44g, 7.49mmol) was dissolved in 20ml DMF, 6.37μl (37.45mmol) N-ethyldiisopropylamine and 3.39g (22.47mmol) tert-butyldimethylchlorosilane (TBDMSCl) were added. After 3 hours, DMF was evaporated and the residue was distributed between 120ml ethyl acetate / methanol 5:1 and 100ml 0.2M citric acid. The organic layer was washed with water and saturated sodium bicarbonate, dried (MgSO4) and concentrated under reduced pressure. The crude product was eluted from 120g silica gel with a gradient of 0 to 10% methanol in dichloromethane. The pure fractions were combined and evaporated to 2.50g (47%) solid product.

[0273] MS (ESI + ) Measured value: 729.29; Calculated value: 729.43[M + Na] + (C 35 H 62 N6NaO7Si)

[0274] Step 3: Boc-Ahx-Val-Cit(SEM)-PAB-OTBDMS (HDP 30.1368)

[0275] To a THF solution (40 mL) of the step 2 product (2.50 mg, 3.546 mmol) was added NaH (142 mg, 60% dispersion in mineral oil, 3.546 mmol). After 15 minutes, pure 2-(trimethylsilyl)ethoxymethyl chloride (SEMCl) (703 μl, 3.546 mmol) was added and the reaction mixture was stirred for 8 hours. Celite (10 g) was added to the reaction mixture and volatiles were removed under reduced pressure. The remaining solid was applied to the top of a silica gel column and gradient eluted with 0 to 10% methanol in dichloromethane. The pure fractions were combined and evaporated to give 637 mg (21%) of amorphous title compound.

[0276] MS (ESI + ) Measured value: 859.34; Calculated value: 859.52[M + Na] + (C 41 H 76 N6NaO8Si2)

[0277] Step 4: Boc-Ahx-Val-Cit(SEM)-PAB-OH (HDP 30.1370)

[0278] To a solution of the product of step 3 (567 mg, 0.677 mmol) in THF (20 mL) was added TBAF (833 μL of a 1.0 M solution, 0.833 mmol; 1.2 equiv.). After 1 hour, diatomaceous earth (1.5 g) was added to the reaction mixture and the volatiles were removed under reduced pressure. The remaining solid was applied to the top of a silica gel column and eluted with a gradient of 0 to 10% methanol in chloroform. The pure fractions were combined and evaporated to give 279 mg (57%) of the product as a white solid.

[0279] MS (ESI + ) Measured value: 745.28; Calculated value: 745.43[M + Na] + (C 35 H 62 N6NaO8Si)

[0280] Step 5: Boc-Ahx-Cit(SEM)-PAB-Br(HDP 30.1381)

[0281] To a solution of the product of step 4 (100 mg, 139 μmol) in dichloromethane (5 mL) was added triphenylphosphine (73 mg, 2 equivalents) followed by carbon tetrabromide (92 mg, 2 equivalents). After 1 hour, diatomaceous earth (1 g) was added to the reaction mixture and the volatiles were removed under reduced pressure. A gradient elution of 0 to 10% methanol in dichloromethane from 12 g of silica gel afforded 61 mg (56%) of the title product as an oil.

[0282] MS (ESI + ) Measured value: 807.15 / 809.17; Calculated value: 807.35 / 809.34[M + Na] + (C 35 H 61 BrN6NaO7Si)

[0283] Step 6: 6'-[Boc-Ahx-Cit(SEM)-PAB]-α-amanitin (HDP 30.1383)

[0284]

[0285] 44 mg (47.9 μmol) of vacuum dried α-amanitin was dissolved in 1000 μl of dry dimethyl sulfoxide (DMSO) under argon at room temperature. The product of step 5 (61 mg, 78.1 μmol) and 1M sodium hydroxide (52.7 μl, 1.1 equiv.) were added. After 4 hours at room temperature, the reaction mixture was acidified to pH=5 with 52.7 μl of 1M acetic acid solution in DMSO. The solvent was removed under vacuum and the residue was purified by preparative HPLC on a C18 column with a 5-100% methanol gradient. The fractions containing the product were evaporated to give 25.1 mg (32%) of HDP 30.1383 as a colorless solid.

[0286] MS (ESI + ) Measured value: 1646.40; Calculated value: 1645.77[M + Na] + (C 74 H 114 N 16 NaO 21 SSi)

[0287] Step 7: 6'-[H-Ahx-Cit-PAB]-α-amanitin (HDP 30.1388)

[0288]

[0289] The Boc- and SEM-protected product of step 6 (18.4 mg, 11.3 μmol) was dissolved in 2 ml of trifluoroacetic acid. After 2 minutes, the mixture was evaporated to dryness at room temperature, redissolved in 2 ml of water and 3.2% ammonia was added dropwise to pH 10. The resulting suspension was freeze-dried, applied to RP18 HPLC, eluted with a gradient of 5-100% methanol in 0.05% TFA, and the pure fractions were evaporated and freeze-dried from 2 ml of water: 12.1 mg (71%) HDP 30.1388 colorless powder.

[0290] MS (ESI + ) Found: 1393.42; Calculated: 1393.66 [M+H] + (C 63 H 93 N 16 O 18 S)

[0291] Step 8:

[0292]

[0293] Subsequently, to 9.98 mg (6.62 μmol) of the product of step 7 dissolved in 500 μl of dry DMF, 5.29 mg (19.86 μmol) of 3-(maleimido)propionic acid N-hydroxysuccinimide ester (BMPS) and 3.38 μl (19.86 μl) of DIPEA dissolved in 500 μl of DMF were added. After 2 hours at room temperature, 100 μl of water were added to the reaction mixture and the volatiles were evaporated. The crude product was purified by RP18 HPLC with a water-methanol gradient and the pure fractions were lyophilized from tert-butanol / water: 5.43 mg (53%) of the title product 6'-[(3-maleimidopropionamido)-Ahx-Cit-PAB]-α-amanitin as a colorless powder.

[0294] MS (ESI + ) Found: 1544.25; Calculated: 1544.68 [M+H] + (C 70 H 98 N 17 O 21 S)

[0295] Measured value: 1566.44; Calculated value: 1566.67 [M+Na] + (C 70 H 97 N 17 NaO 21 S)

[0296] Embodiment 21

[0297] 6'-[H-Val-Ala-PAB]-α-amanitin (HDP 30.1702)

[0298]

[0299] Using H-Val-Ala-PAB-OH as the starting material, the method of steps 1-7 of Example 20 was repeated to obtain the title substance as a colorless powder:

[0300] MS (ESI + ) Found: 1194.58; Calculated: 1194.53 [M+H] + (C 54 H 76 N 13 O 16 S)

[0301] Measured value: 1216.75; Calculated value: 1216.51 [M+Na]+(C 54 H 75 N 13 NaO 16 S)

[0302] The starting material H-Val-Ala-PAB-OH can be prepared by general methods in peptide chemistry, such as those mentioned by Howard et al. in US 2011 / 0256157.

[0303] Embodiment 22

[0304] 6'-((3-Maleamidopropionamido)-Val-Ala-PAB)-α-amanitin (HDP 30.1699)

[0305]

[0306] By using the method of step 8 of Example 20, the title substance was obtained as a colorless powder with a yield of 81% using 6'-[H-Val-Ala-PAB]-α-amanitin of Example 21:

[0307] MS (ESI + ) Measured value: 1345.48; Calculated value: 1345.55 [MH] + (C 61 H 81 N 14 O 19 S)

[0308] Measured value: 1368.17; Calculated value: 1367.53[M + Na] + (C 61 H 80 N 14 NaO 19 S)

[0309] Embodiment 23

[0310] 6′-O-[((N-α-maleimido)-L-2,3-diaminopropionamido)-Val-Ala-PAB]-α-amanitin (HDP 30.1957)

[0311]

[0312] By using the method of Example 16 and 6'-[H-Val-Ala-PAB]-α-amanitin of Example 21, the title substance was obtained as a colorless powder in 39% yield:

[0313] MS (ESI + ) Measured value: 1345.48; Calculated value: 1360.56 (C 61 H 82 N 15 O 19 S)

[0314] Embodiment 24

[0315] 6'-((2-bromoacetylamino)-Val-Ala-PAB)-α-amanitin (HDP 30.1704)

[0316]

[0317] By using the method of Example 17 and 6'-[H-Val-Ala-PAB]-α-amanitin of Example 21, the title substance was obtained as a colorless powder in a yield of 26%:

[0318] MS (ESI + ) Found: 1314.28; Calculated: 1314.45 [M+H] + (C 56 H 77 N 13 O 17 S)

[0319] Found: 1336.39; Calculated: 1336.43 [M+Na] + (C 56 H 76 N 13 NaO 17 S)

[0320] Embodiment 25

[0321] 6'-((6-(4-(5-(methylsulfonyl)-1,2,4-oxadiazol-2-yl)-phenoxy)hexylaminocarbonyl)-Val-Ala-PAB)-α-amanitin (HDP 30.1917)

[0322]

[0323] By using the method of Example 19 and 6'-[H-Val-Ala-PAB]-α-amanitin of Example 21, the title substance was obtained as a colorless powder in 44% yield:

[0324] MS (ESI + ) Measured value: 802.63 Calculated value: 802.30 [M+2Na] 2+ (C 70 H 94 N 16 Na2O 21 S2)

[0325] Embodiment 26

[0326] 6'-((6-(4-(5-(methylsulfonyl)-1,2,4-oxadiazol-2-yl)-phenoxy)hexylaminocarbonyl)-Ahx-Val-Ala-PAB)-α-amanitin (HDP 30.1930)

[0327]

[0328] By using the method of Example 19, 6'-[H-Ahx-Val-Ala-PAB]-α-amanitin prepared by the method of Example 19, steps 1-7, wherein citrulline was replaced by alanine, the title substance was obtained as a colorless powder in a yield of 37%:

[0329] MS (ESI + ) Found: 859.02; Calculated: 858.85 [M+2Na] 2+ (C 76 H 105 N 17 Na2O 22 S2) Embodiment 27

[0330] 6′-O-[3-(5-nitro-pyridin-2-yldisulfonyl)propyl)]-α-amanitin HDP 30.0951

[0331] Step 1: 6′-O-(3-S-tritylsulfonyl-propyl)-α-amanitin HDP 30.0517

[0332]

[0333] Under argon, 46 mg (50 μmol) of vacuum-dried α-amanitin was dissolved in 2500 μl of dry dimethyl sulfoxide (DMSO). 3-(S-trityl)-mercaptopropyl-1-bromide (159 mg, 8 equivalents) was added, followed by 60 μl of 1 M sodium hydroxide solution. After 1.5 hours at room temperature, the reaction mixture was acidified to pH = 5 with 50 μl of 1 M acetic acid dissolved in DMSO, and the solvent was evaporated. The residue was dissolved in 200 μl of methanol and added dropwise to a centrifuge tube containing 10 ml of tert-butyl methyl ether (MTBE). The resulting precipitate was cooled to 0°C for 10 minutes, separated by centrifugation (4000×g), and subsequently washed with 10 ml of MTBE. The supernatant was discarded, the precipitate was dissolved in 750 μl of methanol, and the precipitate was purified by preparative HPLC on a C18 column (250×21.2 mm, Luna RP-18, 10 μm, ) was purified in 3 parts. Solvent A: water, solvent B: methanol; gradient: 0 min 5% B; 5 min 5% B 20 min 100% B; 25 min 100% B; 27 min 5% B, 35 min 5% B; flow rate 30 ml / min. The fractions with retention time of 21.1-21.8 minutes were collected and the solvent was evaporated to obtain 36.5 mg (59%) of HDP 30.0517 as a colorless solid.

[0334] MS (ESI + )1234.8[M+H] + ,1257.3[M+Na] +

[0335] Step 2: 6′-O-[3-(5-nitro-pyridin-2-yldisulfonyl)propyl)]-α-amanitin HDP 30.0951

[0336]

[0337] To the product of step 1 (5.00 mg, 4.05 μmol) was added 2,2'-dithiobis(5-nitropyridine)DTNP (6.28 mg, 5 equivalents) dissolved in 200 μl trifluoroacetic acid. After 4 minutes, the volatiles were distilled off and the residue was co-evaporated with 1000 μl methanol. The crude product was purified by HPLC similar to step 1. The fractions with retention time of 18.46-19.28 min were collected, the solvent was evaporated, and the residue was lyophilized from 2 ml tert-butanol to give 2.99 mg (64%) of HDP 30.0951 as a slightly yellow solid.

[0338] MS (ESI + )1146.97[M+H] + ,1169.17[M+Na] +

[0339] Embodiment 28

[0340] The experimental setup leading to Figures 5 to 14

[0341] Cell viability assay based on BrdU incorporation ( Fig.11 , 12 、13)

[0342] To evaluate the effects of compounds on antigen-expressing tumor cell lines, 2500 cells / well were seeded in 90 μl of culture medium. The next day, 10 μl of culture medium containing different concentrations of antibody-drug conjugates was added. After 72 or 96 hours of drug contact, BrdU incorporation assays (Cell Proliferation ELISA, BrdU, Roche) were performed. Chemiluminescence was measured using a FLUOstar Optima chemiluminometer (BMG LABtech). The EC of each compound was determined by sigmoidal dose-response curve analysis using Graphpad Prism 4.0 software. 50 value.

[0343] WST-I-based cell proliferation assay (Figure 8)

[0344] To evaluate the effects of the compounds on THP-1 cells expressing Fcg receptors, 2500 cells / well were seeded in 90 μl of culture medium. The next day, 10 μl of culture medium containing different concentrations of antibody-drug conjugates was added. 96 h after drug application, 10 μl of Cell Proliferation Agent WST-1 (Roche) was added to each well. After incubation for another 4 to 24 h, the absorbance at 440 nm / 660 nm was measured using a FLUOstar Optima chemiluminometer (BMG LABtech). The EC of each compound was determined by sigmoidal dose-response curve analysis using GraphpadPrism 4.0 software. 50 value.

[0345] Cell surface binding assay ( Figure 6 )

[0346] will contain 1×10 6 100 μl cell suspension of each antigen-expressing tumor cell in PBS was incubated at 4°C for 30 minutes in the presence or absence of 0.1-50 μg / ml antibody or antibody-drug conjugate. Detection was performed with goat anti-human IgG (H+L)-Alexa Fluor 488 F(ab')2-fragment (Dianova). Fixed stained cells were analyzed by FACS on a BD FACScan device. The mean fluorescence intensity of the samples was calculated using BD CellQuest Pro software, and the binding curve was plotted against the IgG concentration after subtracting the control sample value.

[0347] Coomassie blue stained SDS-PAGE and Western Blotting (Figure 7)

[0348] Antibodies and antibody-drug conjugates were analyzed by reducing and non-reducing SDS-PAGE followed by Coomassie blue staining or Western Blot for amanitin loading according to standard methods. For detection of polyclonals, rabbit serum was used, which allows detection of amanitin and amanitin-linker compounds conjugated to proteins.

[0349] Mouse tolerance and efficacy studies ( Fig. 9 , 14 )

[0350] The maximum tolerated dose (MTD) of a single intravenous dose of the antibody-drug conjugate was evaluated, which was defined as the dose that resulted in a 10% or less weight loss in the subject. For the xenograft model, 5×10 6 Tumor cells (early passage number) were injected subcutaneously into the right flank of 7- to 8-week-old female NMRI nude mice (Janvier). Once tumors reached an average volume of 100 to 200 mm 3 Animals were randomly divided into treatment groups of 8 animals per group. All animals were treated with a single intravenous injection. By using the Endosafe-PTS system (Charles River), it was demonstrated that the endotoxin concentration per mg of protein in all batches used for in vivo experiments was <1 EU (endotoxin unit). The application volume was 10 ml / kg body weight, and PBS was used as a vehicle control. Tumor growth was measured with a caliper and the formula Tvol = (larger diameter × (smaller diameter) 2 × 0.5) to calculate tumor volume. Mice were sacrificed by carbon dioxide inhalation at moribund or at the indicated time points. All animal studies were performed in accordance with German animal welfare standards (GV-SOLAS) and were approved by the responsible board ( Statistical analyses were performed with the approval of GraphPad Software, Inc. (Karlsruhe, Karlsruhe, Referat 35). Statistical analyses were performed using Prism software (GraphPad Software, Inc.) and one-way ANOVA.

[0351] Table 1 below shows the therapeutic index of different Her2-amanitin-ADCs:

[0352] Table 1: Therapeutic index of different Her2-amanitin-ADCs

[0353]

[0354] MTD-maximum tolerated dose; MED-minimum tolerated dose; AUC-area under the curve

[0355] *: Calculation assumes 0.1

[0356] Tolerance study in cynomolgus monkeys ( Fig.10 )

[0357] Non-human primate (NHP) toxicity studies were conducted in female cynomolgus monkeys. All study protocols were approved by the Animal Care and Use Committee of the experimental institution. Non-GLP single-dose toxicity studies were conducted using increasing doses injected intravenously (IV) at three-week intervals. Comprehensive toxicological parameters were assessed in each study, including physical examination, body weight, food consumption, behavior, clinical chemistry, urinalysis, and hematology. Blood samples were collected at multiple time points. As an example of tolerance, the time course of the parameter LDH in the blood of (A) Her2-30.0643; (B) Her2-30.1465; (C) Her2-A118C-30.0880; (D) Her2-D265C-30.0880 is shown. The doses were (i) 0.3 mg / kg; (ii) 1 mg / kg; (iii) 3 mg / kg; (iv) 10 mg / kg.

[0358] Mass spectrometry (LC-MS) (Figures 4, 5)

[0359] Before MS analysis, antibodies and antibody drug conjugates were deglycosylated and reduced using standard protocols. Therefore, after deglycosylation and dialysis with PNGase F, antibody and antibody drug conjugate solutions were precipitated by adding acetonitrile, centrifuged and the precipitate was reconstructed in 6M guanidine hydrochloride solution. To analyze light and heavy chains, the reconstructed solution was reduced at 56°C for 30 minutes with 20mM dithiothreitol. Deglycosylation and reduced antibodies and antibody drug conjugates were then analyzed by nano LC-MS using an LTQ Orbitrap Elite ion trap mass spectrometer (Thermo Fisher Scientific). Before analysis, method optimization was performed for intact antibodies and reduced antibodies. The mass spectrum obtained was finally deconvoluted by suitable software (ProMass, ThermoFisher Scientific) to interpret the data.

Claims

1. A conjugate of the following general formula: Ama–L–X–S–Ab, in, Ama is amatoxin, L is a linker, X is a moiety resulting from coupling a thiol group to a thiol-reactive group, S is a sulfur atom of a cysteine ​​amino acid residue, and Ab is an antibody comprising said cysteine ​​amino acid residue, wherein said cysteine ​​amino acid residue is 265Cys of the heavy chain according to the EU numbering system, and wherein the ratio of antibody to amatoxin is between 1.8 and 2.5 amatoxin molecules, and wherein the linker is selected from a stabilizing linker and a cleavable linker.

2. The conjugate of claim 1, wherein the cleavable linker comprises a dipeptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit, and Val-Cit.

3. The conjugate of claim 2, wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and amatoxin.

4. The conjugate according to claim 1, wherein -L-X-S- is selected from -(CH2)2-XS-; -(CH2)3-XS-; -(CH2)4-XS-; -(CH2)5-XS-; -(CH2)6-XS-; -(CH2)7-XS-; -(CH2)8-XS-; -(CH2)9-XS-; -(CH2) 10 -XS-; -(CH2) 11 -XS-; -(CH2) 12 A group consisting of -XS-; -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-XS-; and -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-XS-.

5. The conjugate according to claim 4, wherein L—X—S is -(CH2)6-XS-.

6. The conjugate of claim 1, wherein -L-X-S- is selected from -(CH2)2-SS-(CH2)2-XS-; -(CH2)3-SS-(CH2)2-XS-; -(CH2)2-SS-(CH2)3-XS-; -(CH2)3-SS-(CH2)3-XS-; -(CH2)4-SS-(CH2)4-XS-; -(CH2)2-CMe2-SS-(CH2)2-XS-; -(CH2)2-SS-CMe2-(CH2)2-XS-; -(CH2)3-SS-; -CH2-C6H4-NH-Cit-Val-CO(CH2)5-XS-; -CH2-C6H4-NH-Ala-Val-CO(CH2)5-XS-; -CH2-C6H4-NH-Ala-Val l-CO(CH2)2-XS-; -CH2-C6H4-NH-Ala-Phe-CO(CH2)2-XS-; -CH2-C6H4-NH-Lys-Phe-CO(CH2)2-XS-; -CH2-C6H4-NH-Cit-Phe-CO(CH2)2-XS-; -CH2-C6H4-NH-Val-Val-CO(CH2)2-XS-; -CH2-C6H4-NH-Ile-Val-CO(CH2)2-XS-; -CH2-C6H4-NH-His-Val-CO(CH2)2-XS-; -CH2-C6H4-NH-Met-Val-CO(CH2)2-XS-; and -CH2-C6H4-NH-Asn-Lys-CO(CH2)2-XS-.

7. The conjugate of claim 1, wherein the thiol-reactive group is selected from bromoacetamide, iodoacetamide, 4,6-dichloro-1,3,5-triazin-2-ylamino, 4-[5-(methylsulfonyl)-1H-tetrazol-1-yl]phenyl, 2-(methylsulfonyl)-benzo[d]thiazol-5-yl, 4-[5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl]-phenyl, 2-pyridyldisulfide-, 2-(5-nitro-pyridyl)disulfide-, methylthiosulfonyl and maleimide.

8. The conjugate of claim 7, wherein the thiol-reactive group is maleimide.

9. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 8.

10. Use of the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer and leukemia.

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