conjugate, pharmaceutical composition, amatoxin conjugation molecule and method for synthesizing a c

专利摘要:
CONJUGATE, PHARMACEUTICAL COMPOSITION, AMATOXIN CONJUGATION MOLECULE AND METHOD FOR SYNTHESIS OF A CONJUGATE The invention relates to a therapy against tumors. In one aspect, the present invention relates to conjugates of an amatoxin and a targeting linker group, e.g., an antibody, connected by a linker comprising a urea group, which are useful in the treatment of cancer. In a further aspect the invention relates to pharmaceutical compositions comprising such conjugates.
公开号:BR112013007577B1
申请号:R112013007577-5
申请日:2011-09-29
公开日:2021-05-04
发明作者:Jan Anderl；Werner Simon；Christoph Müller
申请人:Heidelberg Pharma Gmbh；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] The invention relates to a therapy against tumors. In one aspect, the present invention relates to conjugates of an amatoxin and a targeting linker group, e.g., an antibody, connected by a linker comprising a urea group, which are useful in the treatment of cancer. In a further aspect the invention relates to pharmaceutical compositions comprising such conjugates. STATE OF THE INVENTION TECHNIQUE
[0002] Amatoxins are cyclic peptides composed of 8 amino acids. They can be isolated from Amanita phalloides mushrooms or prepared synthetically. Amatoxins specifically inhibit DNA-dependent RNA polymerase II from mammalian cells, and thus also inhibit transcription and protein biosynthesis of affected cells. Inhibition of transcription in a cell causes its growth and proliferation to stop. Although not covalently linked, the complex between amanitin and RNA polymerase II is very close (KD = 3 nm). Dissociation of amanitin from the enzyme is a very slow process, making recovery of an affected cell very unlikely. When the inhibition of transcription lasts for a long time, the cell will undergo programmed cell death (apoptosis).
[0003] The use of amatoxins as cytotoxic groups for tumor therapy was already explored in 1981 by linking an anti-Thy 1.2 antibody to an α-amanitin using a ligand coupled to the indole ring of Trp (amino acid 4; see Figure 1) via diazotation (Davis & Preston, Science 1981, 213, 1385 1388).
[0004] Patent Application EP 1 859 811 A1 (published on November 28, 2007) describes conjugates in which the carbon atom y of amino acid 1 of the amatoxin β-amanitin has been directly linked, that is, without a linker structure, albumin or monoclonal antibodies HEA125, OKT3, or PA-1. In addition, the inhibitory effect of these conjugates on the proliferation of breast cancer cells (MCF-7), Burkitt lymphoma cells (Raji), and T lymphoma cells (Jurkat) was presented. The use of linkers has been suggested, including linkers comprising elements such as an amide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon groups and the like, but no such conjugates have been shown, and no further details have been provided such as sites binding agent in amatoxins.
[0005] It is known that amatoxins are relatively non-toxic when bound to large biomolecular carriers, such as antibody molecules, and that they exert their cytotoxic activity only after the biomolecular carrier is cleaved. In light of the toxicity of amatoxins, particularly to liver cells, it is of paramount importance that amatoxin conjugates for targeted tumor therapy remain highly stable after administration into plasma, and that amatoxin release occurs after internalization into target cells. In this context, minor improvements in conjugate stability can have drastic consequences on the therapeutic window and safety of amatoxin conjugates for therapeutic purposes. PURPOSE OF THE INVENTION
[0006] Thus, there is a great need in the state of the art for amatoxin conjugates with specific linking groups that are stable in plasma, so that the harmful side effects for non-target cells are minimized. SUMMARY OF THE INVENTION
[0007] In a first aspect, the present invention relates to a conjugate comprising a targeting linker group, linked via a linker L to an amatoxin, where the linker L is connected to the amatoxin via (i) the carbon atom y from amino acid 1 of amatoxin, particularly via an amide bond; (ii) an oxygen atom attached to the δ-carbon atom of amino acid 3 of amatoxin, particularly via an ester bond, an ether bond or a urethane bond; or (iii) the 6' carbon atom of amino acid 4 of amatoxin, particularly via an oxygen atom attached to the 6' carbon atom of amino acid 4; where in each case the ligand L is connected to the targeting ligand group via a urea group.
[0008] Under a second aspect, the present invention relates to a pharmaceutical composition comprising the conjugate according to the present invention.
[0009] In another aspect, the present invention relates to an amatoxin conjugation molecule comprising a linker L connected to an amatoxin via (i) the y carbon atom of amino acid 1 of the amatoxin, particularly via an amide bond; (ii) an oxygen atom attached to the δ-carbon atom of amino acid 3 of amatoxin, particularly via an ester bond, an ether bond or a urethane bond; or (iii) the 6' carbon atom of amino acid 4 of amatoxin, particularly via an oxygen atom attached to the 6' carbon atom of amino acid 4; where in each case the linker L comprises a carbamic acid derivative -NH-C(O)-X, where X is a leaving group which can be replaced by a primary amine of a targeting linker group.
[0010] In a further aspect, the present invention relates to a method for synthesizing a conjugate of the present invention, comprising the step of reacting an amatoxin conjugation molecule of the present invention with a targeting linker group comprising an amine group primary. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 shows the structural formulas of different amatoxins. The numbers in bold (1 to 8) designate the standard numbering of the eight amino acids forming amatoxin. The standard designations of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters α to y, Greek letters α to δ, and numbers 1' to 7', respectively).

[0012] Figure 2 shows the cytotoxic activity of different conjugates of amanitin and herceptin using different ligand groups in SKOV-3 cells in a BrdU assay after incubation for 72h.
[0013] Figure 3 shows the cytotoxic activity of different conjugates of amanitin and herceptin using different ligand groups in SK-BR-3 cells in a BrdU assay after incubation for 72h.
[0014] Figure 4 shows the cytotoxic activity of different conjugates of amanitin and herceptin using different ligand groups on NCI-N87 cells in a BrdU assay after incubation for 72h.
[0015] Figure 5 shows the cytotoxic activity of different conjugates of amanitin and herceptin using different ligand groups in MDA-MB231 cells in a BrdU assay after incubation for 72h.
[0016] Figure 6 and Figure 7 show the amount of amanitin released from different amanitin-herceptin conjugates using different ligand groups after incubation in plasma for up to 14 days.
[0017] Figure 8 shows a comparison of the cytotoxic activity of different amanitin-herceptin conjugates using different ligand groups on SKOV-3 cells in a BrdU assay before and after incubation in plasma.

[0018] Figure 9 shows a comparison of the activity of two different amanitin-herceptin conjugates using different ligand groups in vivo in a SKOV-3 xenograft model. DETAILED DESCRIPTION OF THE INVENTION
Before the present invention is described in detail below, it should be noted that this invention is not limited to the particular methodology, protocols and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the sole purpose of describing particular embodiments, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used here have the same meanings commonly understood by someone ordinarily versed in the art.
[0020] Preferably, terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta , CH-4010 Basel, Switzerland).
[0021] Throughout this report and the claims that follow, unless the context indicates otherwise, the word "understands", and variations such as "understands" and "comprising", will be understood to imply the inclusion of integral data or step or group of members or steps without excluding any other members or steps or group of members or steps.
[0022] Several documents are cited throughout the text of this report. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, GenBank sequence submission accession numbers, etc.), both above and below, are incorporated herein in their entirety by reference. to the extent possible under the respective patent law. No document herein should be regarded as an admission that the invention is not capable of preceding such description by virtue of a prior invention.
[0023] The present invention will now be further described. In the following passages, different aspects of the invention are defined in greater detail. Each aspect defined in this way may be combined with any other aspect or aspects unless clearly stated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
[0024] In a first aspect, the invention relates to a conjugate comprising a targeting linker group, linked via a linker L to an amatoxin, where the linker L is connected to the amatoxin via (i) the y carbon atom of the amino acid 1 of amatoxin, particularly via an amide bond; (ii) an oxygen atom attached to the δ-carbon atom of amino acid 3 of amatoxin, particularly via an ester bond, an ether bond or a urethane bond; or (iii) the 6' carbon atom of amino acid 4 of amatoxin, particularly via an oxygen atom attached to the 6' carbon atom of amino acid 4; of targeting via a urea group.
In the context of the present invention, the term "conjugate" refers to a molecule comprising at least two different molecules linked by a covalent bond.
The term "targeting linker group", as used herein, refers to any molecule or part of a molecule that can specifically bind to a target molecule or target epitope. Preferred targeting binding groups, in the context of the present application, are (i) antibodies or targeting binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers. "Targeting binder groups" suitable for use in the present invention typically have a molecular mass of 40,000 Da (40 kDa) or more.
[0027] As used herein, a first compound (eg an antibody) is considered to specifically bind a second compound (eg an antigen, such as a target protein) if the latter has a dissociation constant KD of the second compound itself 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 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 or less, more preferably 300 nM or less, more preferably 200 nM or less, 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.
[0028] In the context of the present application, the terms "target molecule" and "target epitope", respectively, refer to an antigen and an epitope of an antigen, respectively, that are specifically linked by a targeting linker group. Preferably, the target molecule is a tumor associated antigen, in particular an antigen or an epitope that is present on the surface of one or more types of tumor cells in a high concentration and/or in a different steric configuration when compared to the surface of cells non-tumor. Preferably, the antigen or epitope itself is present on the surface of one or more types of tumor cells, but not on the surface of non-tumor cells. In particular embodiments, the targeting linker group specifically binds to an epitope of HER-2/neu or an epithelial cell adhesion molecule (EpCAM). In other modalities, the antigen or epitope itself is preferentially expressed on cells involved in autoimmune diseases. In such particular embodiments, the targeting linker group specifically binds to an epitope of the IL-6 receptor (IL-6R).
[0029] The term "antibody or antigen-binding fragment thereof", as used herein, refers to immunoglobulin molecules and immunologically active portions of the immunoglobulin molecules, that is, molecules that contain an antigen-binding site that binds bind immunospecifically to an antigen. Also encompassed are immunoglobulin-like proteins that are selected by techniques including, for example, phages displaying specific binding to a target molecule, for example, the target protein Her-2/neu or EpCAM. The immunoglobulin molecules of the invention can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of molecules. immunoglobulin. Suitable "antibodies and antigen-binding fragments thereof" for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (in particular CDR-transplanted), deimmunized antibodies , or chimeric, single chain antibodies (e.g. scFv), Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, bivalent or tetravalent antibodies (Holliger P. et al., 1993), nanoantibodies, anti-idiotypic antibodies (anti-Id, including, for example, anti-Id antibodies to antibodies of the invention), and epitope binding fragments of any of the above antibodies.
In some embodiments the antigen-binding fragments are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, chain Fv's single (scFv), single chain antibodies, disulfide linked Fv's (dsFv) and fragments comprising a VL or VH domain. Antibody fragments for binding to antigens, including single chain antibodies, can comprise the variable domains alone, or in combination with all or a portion of the following: hinge region, CL, CH1, CH2 and CH3 domains. Also included in the invention are antigen-binding fragments further comprising any combination of variable domain(s) with a hinge region, and CL, CH1, CH2 and CH3 domains.
[0031] Antibodies usable in the invention can be from any animal origin including birds and mammals. Preferably, the antibodies originate from humans, rodents (e.g., mouse, rat, guinea pig, or rabbit), chicken, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog. It is particularly preferred that the antibodies are of human or murine origin. As used herein, "human antibodies" includes antibodies having the amino acid sequence of a human immunoglobulin and includes antibodies isolated from human immunoglobulin libraries or from animals transgenic to one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described, for example , U.S. Patent No. 5,939,598 to Kucherlapati and Jakobovits.
The term "antibody-like protein" refers to a protein that has been designed (eg, by loop mutagenesis) to specifically bind to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide loop linked at both ends to a protein base. This dual structural feature strongly increases the binding affinity of the antibody-like protein to levels comparable to those of an antibody. The variable peptide loop length typically consists of 10 to 20 amino acids. The base protein is a small globular protein. Antibody-like proteins include, without limitation, affibodies, anticalins, and engineered ankyrin repeat proteins (for a review, see: Binz et al. 2005). Antibody-like proteins can be derived from large mutant libraries, for example, synthesized from large phage display libraries, and can be isolated analogously to regular antibodies. Furthermore, antibody-like binding proteins can be obtained by combined mutagenesis of surface exposed residues in globular proteins.
The term "nucleic acid aptamer" refers to a nucleic acid molecule that has been manipulated through repeated in vitro selection steps or SELEX (systematic evolution of ligands by exponential enrichment) to bind to a target molecule (For a review see: Brody and Gold, 2000). The nucleic acid aptamer can be a DNA or RNA molecule. Aptamers can contain modifications, for example, nucleotides modified such as pyrimidines substituted with 2'-fluorine.
[0034] As used herein, a "chemical derivative" (or just "derivative") of a compound refers to a species with a chemical structure similar to the compound, but containing at least one chemical group not present in the compound and/or deficient of at least one chemical group that is present in the compound. The compound to which the derivative is compared is called the “original” compound. Typically, a "derivative" can be produced from the parent compound in one or more steps of chemical reactions.
[0035] As used herein, an "analog" of a compound is structurally related to, but not identical to, the compound, and exhibits at least one activity of the compound. The compound to which the analogue is compared is known as the "original" compound. The aforementioned activities include, without limitation, binding activity to another compound; inhibitory activity, for example, enzyme inhibitory activity; toxic effects; activation activity, for example, enzyme activation activity. The analogue is not required to exhibit such activity to the same extent as the parent compound. A compound is considered an analogue within the context of the present application if it exhibits the relevant activity to a degree of at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, most preferably at least 30%, more preferably at least 40%, and most preferably at least 50%) of the activity of the parent compound. Thus, an "anatoxin of an amatoxin", as used herein, refers to a compound that is structurally related to any of α-amanitin, β-amanitin, Y—amanitin, ε-amanitin, amanin, amaninamide, amanulin , and amanulinic acid, as shown in Figure 1, and which exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, most preferably at least at least 40%, and more preferably at least 50%) of the inhibitory activity against mammalian RNA polymerase II, when compared to at least one of α-amanitin, β-amanitin, Y—amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid. An "anatoxin of an amatoxin" suitable for use in the present invention may even exhibit an inhibitory activity against mammalian RNA polymerase II greater than that of any one of α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanulinic acid. Inhibitory activity can be measured by determining the concentration at which 50% inhibition occurs (IC50 value). The inhibitory activity against mammalian RNA polymerase II can be indirectly determined by measuring the inhibitory activity on cell proliferation. A suitable assay to measure inhibition of cell proliferation is described in the examples.
A "semi-synthetic analogue" refers to an analogue that has been obtained by chemical synthesis using compounds from natural sources (eg, plant compounds, bacterial cultures, or cell cultures) as a starting material. Typically, a "semi-synthetic analogue" of the present invention was synthesized starting from a compound isolated from a mushroom of the Amanita family. In contrast, a "synthetic analogue" refers to an analogue synthesized by a so-called total synthesis from small (typically petrochemical) building blocks. Usually, such total synthesis is carried out without the aid of biological processes.
As used herein, an "aptameric conjugate" refers to a targeting linker group toxin conjugate in which the targeting linker group is a nucleic acid aptamer according to alternative (iii) above.
[0038] A "ligand" in the context of the present invention refers to a molecule that is connecting two components, each coupled to one end of the ligand, and that increases the distance between two components and alleviates steric interference between these components, as in the present case between the targeting linker group and amatoxin. In the absence of a linker, a direct link between amatoxin and the targeting linker group may decrease amatoxin's ability to interact with RNA polymerase II. In particular embodiments, a linker has a continuous chain of 1 to 30 atoms (eg, 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) in its chain, that is, the length of the ligand is defined as the shortest measured connection by the number of atoms or bonds between the amatoxin group and the targeting linker group, where one side of the linking chain has been reacted with amatoxin, and the other side with a targeting linker group. In the context of the present invention, a linker is preferably a C1-20 alkylene, C1-20 heteroalkylene, C2-20 alkenylene, C2-20 heteroalkenylene, C2-20 alkynylene, C2-20 heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene group, aralkylene, or an optionally substituted heteroaralkylene group. The linker can contain one or more structural elements such as a carboxamide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon groups and the like. The binder can further contain combinations of two or more of these structural elements. Each of these structural elements may be present in the binder more than once, for example, twice, three times, four times, five times, or six times. In some embodiments, the linker can comprise a disulfide bond. It is understood that the linker has to be coupled in one step or in two or more subsequent steps to the amatoxin and the targeting linker group. For this purpose, the linker to be used must contain 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 linked, preferably an activated group in an amatoxin or on the targeting linker peptide or (ii) that is or can be activated to form a covalent bond with a group on an amatoxin. Thus, it is preferred that there be chemical groups at the distal and proximal ends of the linker, which are the result of such a coupling reaction, for example an ester, an ether, a urethane, a peptide bond, etc.
In the context of the present invention, the term "amatoxin" includes all cyclic peptides composed of 8 amino acids such as those isolated from the genus Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit. Rev Biochem. 1978 Dec;5(3):185-260), and additionally includes all chemical derivatives thereof; additionally, all semi-synthetic analogues thereof; additionally, all synthetic analogues thereof built from blocks according to the main structure of the natural compounds (cyclic, 8 amino acids); additionally all synthetic or semi-synthetic analogues containing non-hydroxylated amino acids instead of hydroxylated amino acids, additionally all synthetic or semi-synthetic analogues in which the thioether sulphoxide group is replaced by a sulfide, sulfone, or by atoms other than sulfur, for example, an atom of carbon as a carbo-analogue of amanitin, in each case where any of these derivatives or analogues are functionally active in inhibiting mammalian RNA polymerase II.
[0040] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those with a functional group (for example, a carboxylic group, an amino group, a hydroxy group, a thiol or thiol-capturing group) that can be reacted with linker molecules or targeting linker groups as defined above. Amatoxins that are particularly suitable for the conjugates of the present invention are α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid, as shown in Figure 1, as well as salts, chemical derivatives , semi-synthetic analogues, and synthetic analogues thereof. Particularly preferred amatoxins for use in the present invention are α-amanitin, β-amanitin, and amaninamide.
[0041] In the context of the present invention, the term "connected to the targeting ligand group via a urea group" refers to a connection between the ligand and the targeting ligand group, where the targeting ligand group is directly coupled to the ligand via an -NH-C(O)-NH- group.
[0042] In joint embodiments of the present invention, the conjugate has a structure selected from one of the following structures: amatoxin-YC(O)-NH-L-NH-C(O)-NH-targeting linker group; amatoxin-δC-O-C(O)-L-NH-C(O)-NH-targeting ligand group; amatoxin-δC-O-L-NH-C(O)-NH-targeting ligand group; amatoxin-δC-O-C(O)-NH-L-NH-C(O)-NH-targeting ligand group; and amatoxin-6’C-O-L-NH-C(O)-NH-targeting ligand group.
[0043] In particular embodiments of the present invention, the targeting linker group is connected to the linker L via an amino group present in the targeting linker group, where the amino group itself forms part of the urea group itself.
In particular embodiments of the present invention, the amatoxin is chosen from α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulinic acid, or salts or analogues thereof.
In particular embodiments of the present invention, the linker L comprises one or more groups, particularly one, two or three groups, chosen from the groups: alkylene, alkenylene, alkynylene, cycloalkylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, and heteroaralkylene, where each group may optionally be independently substituted.
The term "alkylene" refers to straight chain saturated bivalent hydrocarbon groups having 1 to 20 carbon atoms, including groups having 1 to 10 carbon atoms. In certain embodiments, the alkylene groups can be short alkylene groups. The term "short alkylene" refers to alkylene groups having 1 to 6 carbon atoms, and in certain embodiments 1 to 5 or 1 to 4 carbon atoms. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2-CH2-), n-propylene, n-butylene, n-pentylene, and n-hexylene.
[0047] The term "alkenylene" refers to straight-chain bivalent groups of 2 to 20 carbon atoms, where at least one of the carbon-carbon bonds is a double bond, while the other bonds can be single bonds or double bonds additional. The term "alkynylene" herein refers to straight-chain bivalent groups of 2 to 20 carbon atoms, where at least one of the carbon-carbon bonds is a triple bond, while the other bonds can be single bonds, double bonds, or bonds. additional triples. 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, etc.
[0048] As used herein, "cycloalkylene" is intended to refer to a bivalent ring as part of any stable monocyclic or polycyclic system, where the ring itself has between 3 and 12 carbon atoms, with no heteroatom, and where such ring is completely saturated; and the term "cycloalkenylene" is intended to refer to a bivalent ring as part of any stable monocyclic or polycyclic system, where the ring itself has between 3 and 12 carbon atoms, with no heteroatom, and where such ring is by less partially unsaturated (but excluding any arylene ring). Examples of cycloalkylenes include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Examples of cycloalkenylenes include, but are not limited to, cyclopentenylene and cyclohexenylene.
[0049] As used herein, the terms "heterocycloalkylene" and "heterocycloalkenylene" are intended to refer to a bivalent ring as part of any stable monocyclic or polycyclic system, where the ring itself has between 3 and 12 atoms, and where the ring itself consists of carbon atoms and at least one heteroatom, particularly at least one heteroatom independently chosen from N, O and S, heterocycloalkylene referring to a ring of this type which is completely saturated, and heterocycloalkenylene referring to a ring which is at least partially unsaturated (but excluding any arylene or heteroarylene ring).
[0050] The term "arylene" is intended to refer to a ring or bivalent ring system as part of any stable monocyclic or polycyclic system, where the ring itself has between 3 and 20 carbon atoms, without any heteroatoms, and where the ring or ring system consists of an aromatic group as defined by the electronic rule “4n+2” π, including phenylene.
[0051] As used herein, the term "heteroarylene" is intended to refer to a ring or bivalent ring system as part of any stable monocyclic or polycyclic system, where the ring itself has between 3 and 20 atoms, and where the ring or ring system consists of an aromatic group as defined by the electronic rule “4n+2” π and contains carbon atoms and one or more nitrogen, sulfur, and/or oxygen heteroatoms.
[0052] In the context of the present invention, the term "substituted" is intended to indicate that one or more hydrogens present in the chain of a linker is replaced with a selection of the indicated group(s), provided that the normal valence of the indicated atom, or that of the appropriate atom of the group which is substituted, is not exceeded, and that the substitution results in a stable compound. The term "optionally substituted" is intended to mean that the linker is unsubstituted or substituted, as defined herein, with one or more substituents, as defined herein. When a substituent is a keto (or oxo, ie =O) group, a thio or imino group or the like, then two hydrogens of the linker chain atom 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, ( ester, cyano, phosphoryl, amino, imino, (thio)amido, sulfhydryl, alkylthio, acylthio, sulfonyl, a sulfate, a sulfonate, a solfamoyl, a sulfonamido, nitro, azido, haloalkyl, including perfluoroalkyl (such as trifluoromethyl), haloalkoxy , alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino, arylsulfonamino, phosphoryl, phosphate, phosphonate, phosphinate, alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, mercapto, amino (optionally mono- or di-substituted, e.g., bialkyl, aryl), or heteroaryl , imino, carboxamide, carbamoyl (optionally mono- or di-substituted, e.g. bialkyl, aryl, or heteroaryl), amidino, aminosulfonyl, acylamino, aroylamino, (thio)ureido, aryl(thio)ureido, alkyl(thio) ureido, cycloalkyl(thio)ureido, aryloxy, aralkoxy, or -O(CH2)n-OH, -O(CH2)n-NH2, -O(CH2)nCOOH, -(CH2)nCOOH, -C(O)O(CH2 )nR, -(CH2)nN(H)C(O)OR, or -N(R)S(O)2R where n is 1-4 and R is independently chosen from hydrogen, -alkyl, -alkenyl, -alkynyl , -cycloalkyl, -cycloalkenyl, -(C-linker-heterocycloalkyl), -(C-linker-heterocycloalkenyl), -aryl, and -heteroaryl, with multiple degrees of substitution being allowed. 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. can themselves be substituted, if appropriate. It will also be appreciated by those skilled in the art that substituted groups by themselves may also be substituted where appropriate.
[0053] In particular embodiments of the present invention, the linker L, particularly the linker L shown on page 19 lines 23 - 20 and page 20 lines 1 - 6 or on page 36 lines 10 - 16, comprises m groups chosen from the groups: alkylene, alkenylene, alkynylene, cycloalkylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, and heteroaralkylene, where each group may optionally be independently substituted; the linker further comprises n groups independently selected from: 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 urethane (-NH-C(=O)- O- or -OC(=O)-NH-), and a urea group (-NH-C(=O)-NH-), where m = n + 1. In particular embodiments, m is 2 and n is 1, or m is 3 and n is 2. In particular embodiments, the linker comprises 2 or 3 unsubstituted alkylene groups, and 1 or 2, respectively, disulfide, ether, thioether, amis, ester, carboxamide, urethane or urea groups linking the alkylene groups not replaced.
[0054] In particular embodiments, linker L, particularly linker L as shown on page 19 lines 23 - 20 and page 20 lines 1 - 6, is a linear chain of 2 to 20 atoms independently chosen from C, O, N and S, particularly between 2 and 16 atoms, more particularly between 5 and 14 atoms, and even more particularly between 6 and 12 atoms. In particular embodiments, at least 60% of the atoms in the linear chain are C atoms. In particular embodiments, the atoms in the linear chain are linked by single bonds.
[0055] In particular embodiments, the C atoms in the straight chain are independently part of optionally substituted methylene groups (-CH2-). In such particular embodiments, optional substituents are independently chosen from halogens and C1-6 alkyl, particularly methyl.
[0056] In particular embodiments, binder L, particularly binder L as shown on page 19 lines 23 - 20 and page 20 lines 1 - 6 or on page 36 lines 10 - 16, is chosen from the following group of binders: side of amatoxin: -(CH2)2- targeting ligand side amatoxin side: -(CH2)3- targeting ligand side amatoxin side: -(CH2)4- targeting ligand side amatoxin side: -( CH2)5- side of targeting ligand side of amatoxin: -(CH2)6- side of targeting ligand side of amatoxin: -(CH2)7- side of targeting ligand side of amatoxin: -(CH2)8- side of the targeting ligand side of amatoxin: -(CH2)9- side of targeting ligand side of amatoxin: -(CH2)10- side of targeting ligand side of amatoxin: -(CH2)11- side of targeting ligand side of amatoxin: -(CH2)12- targeting ligand side amatoxin side: -(CH2)16- targeting ligand side amatoxin side: targeting -(CH2)2-SS-(CH2)2- ligand side amatoxin side: targeting -(CH2)3-SS-(CH2)2- ligand side amatoxin side: targeting -(CH2)2 -SS-(CH2)3- ligand side amatoxin side: targeting -(CH2)3-SS-(CH2)3- ligand side amatoxin side: -(CH2)4-SS-(CH2)4- targeting ligand side amatoxin side: -(CH2)2-CMe2-SS-(CH2)2- targeting ligand side amatoxin side: -(CH2)2-SS-CMe2-(CH2)2- side targeting ligand amatoxin side: -(CH2)2-O-(CH2)2- side targeting ligand amatoxin side: -(CH2)2-O-(CH2)2-O-(CH2)2- side of the targeting ligand amatoxin side: -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2- side of targeting ligand amatoxin side: -(CH2)2- O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2- side of targeting ligand
[0057] In particular embodiments of the present invention, the targeting ligand group specifically binds to an epitope that is present on a tumor cell.
[0058] In particular embodiments of the present invention, the targeting linker group specifically binds to an epitope of Her-2/neu or epithelial cell adhesion molecule (EpCAM).
[0059] In particular embodiments of the present invention, the targeting binding group is selected from the group consisting of: antibody or antigen-binding fragment thereof, antibody-like protein, and nucleic acid aptamer.
In particular embodiments of the present invention, the antibody or antigen-binding fragment thereof is chosen from a bivalent antibody, a nanometric antibody, a chimeric antibody, a deimmunized antibody, a humanized antibody, or a human antibody.
In particular embodiments of the present invention, the antigen-binding fragment is chosen from the group consisting of Fab, F(ab')2, Fd, Fv, single-chain Fv, and disulfide-linked Fv's (dsFv).
In particular embodiments, the antibody is herceptin or HEA125, or an antibody fragment comprising the antigen binding fragment of herceptin or HEA125.
[0063] In particular embodiments, more than one amatoxin molecule is coupled to a targeting linker group. An increase in the number of amatoxins per conjugate will also increase toxicity. Thus, in a particular embodiment the ratio of targeting linker groups to amatoxin is between 1 targeting linker group for between 2 and 15 amatoxin molecules, particularly 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, or 15. For the purpose of calculating the ratio in the case of antibody dimers like IgGs, the dimer is considered as a targeting linker group.
[0064] In particular embodiments of the present invention, the conjugate is for use as a medicine.
[0065] In particular embodiments of the present invention, the conjugate is for the treatment of cancer in a patient, where the cancer is chosen from the group consisting of pancreatic cancer, cholangiocarcinoma, breast cancer, colorectal cancer, lung cancer, prostate cancer , ovarian cancer, stomach cancer, liver cancer, malignant melanoma, leukemia, and malignant lymphoma.
As used herein, a "patient" means any mammal or bird that would benefit from treatment with the toxin and targeting linker group conjugates described herein. Preferably, a "patient" is chosen from the group consisting of laboratory animals (eg mouse or rat), domestic animals (including, for example, guinea pig, rabbit, chicken, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog), or primates including humans. It is particularly preferred that the “patient” be a human.
[0067] As used herein, "treatment", "treating" or "treatment" of a disease or disorder means achieving one or more of the following: (a) reduction in the severity of the disorder; (b) limiting or preventing the development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients who have previously had the disorder(s); and (e) limiting or preventing the recurrence of symptoms in patients who were previously symptomatic for the disorder(s).
[0068] As used herein, treatment may comprise administering a conjugate or a pharmaceutical composition according to the present invention to a patient, where "administration" includes in vivo administration as well as administration directly to ex vivo tissue such as grafts venous.
[0069] In particular embodiments, a therapeutically effective amount of the conjugate of the present invention is used.
[0070] 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 animals receiving the therapeutic agent, and the purpose of administration. The effective amount in each individual case can be determined empirically by a skilled artisan according to methods established in the art.
[0071] Under a second aspect, the present invention relates to a pharmaceutical composition comprising the conjugate according to the present invention, further comprising one or more diluents, carriers, excipients, fillers, binders, lubricants, suspending agents, disintegrants , adsorbents, and/or pharmaceutically acceptable preservatives.
[0072] “Pharmaceutically acceptable” means approved by a regulatory agency of the federal government or a state government, or listed in the US Pharmacopoeia or other generally recognized pharmacopeias, for use in animals, and more particularly in humans.
[0073] In particular embodiments, the pharmaceutical composition is used in the form of a systematically administered medicament. This includes parenterals, which include, among others, injectables and infusions. Injectables are formulated in the form of ampoules or as so-called “ready-to-use injectables”, eg ready-to-use syringes or single-use syringes, and furthermore in pierceable vials for multiple withdrawals. Administration of injectables can be in the form of subcutaneous (s.c.), intramuscular (i.m.), intravenous (i.v.) or intracutaneous (i.c.) application. In particular, it is possible to produce the injection formulations respectively suitable as a suspension of crystals, solutions, nanoparticles or a dispersed colloidal system such as, for example, hydrosols.
[0074] Injectable formulations can also be produced as concentrates, which can be dissolved or dispersed with aqueous isotonic diluents. The infusion can also be prepared in the form of isotonic solutions, fatty emulsions, liposomal formulations and microemulsions. Similar to injectables, infusion formulations can also be prepared as concentrates for dilution. Injectable formulations can also be applied in the form of permanent infusions in patient and outpatient therapy, for example, by means of mini-pumps.
[0075] It is possible to add to parenteral pharmaceutical formulations, for example, albumin, plasma, expander, surfactant substances, organic diluents, pH-influencing substances, complexing substances or polymeric substances, in particular as substances to influence the adsorption of toxin conjugates with targeting linking group of the invention to proteins or polymers or they can further be added for the purpose of reducing the adsorption of toxin conjugates with targeting linking group of the invention to materials such as injection instruments or packaging materials, for example, plastic or glass.
The toxin and targeting linker group conjugates of the invention can be linked to microcarriers or nanoparticles in parenterals, such as, for example, to finely dispersed particles based on poly(meth)acrylates, polylactates, polyglycolates, polyamino acids or polyether urethanes. Parenteral formulations can be further modified as depot preparations, e.g. based on the "multiple unit principle", if the toxin and targeting linker conjugates of the invention are introduced in finely dispersed, dispersed and suspended forms, respectively, or as a suspension of crystals in the drug or based on the "single unit principle", if the toxin conjugate and targeting linker group of the invention is enclosed in a formulation, for example, in a lozenge or stick which is subsequently implanted. These implants or depot drugs in single-unit and multi-unit formulations generally consist of so-called biodegradable polymers such as, for example, polyesters of lactic acid and glycolic acid, polyether urethanes, polyamino acids, poly(meth)acrylates or polysaccharides.
[0077] Adjuvants and carriers added during the production of pharmaceutical compositions of the present invention formulated as parenterals are preferably aqua sterilisata (sterilized water), pH-influencing substances such as, for example, organic or inorganic acids or bases as well as salts thereof, substances buffers for adjusting pH values, substances for isotonization such as sodium chloride, sodium hydrogen carbonate, glucose and fructose, surfactants and surfactants, respectively, and emulsifiers such as fatty partial esters of polyoxyethylene sorbitans (eg Tween®) or, for example, polyoxyethylene fatty esters (eg Cremophor®), fatty oils such as peanut oil, soybean oil or castor oil, synthetic fatty acid esters such as, for example ethyl oleate, isopropyl myristate and neutral oil (eg Miglyol®), as well as polymeric adjuvants such as for example lo, gelatin, dextran, polyvinylpyrrolidone, additives which increase the solubility of organic solvents such as propylene glycol, ethanol, N,N-dimethylacetamide, propylene glycol or complex-forming substances such as citrate and urea, preservatives such as per example, hydroxypropyl and methyl esters of benzoic acid, benzyl alcohols, antioxidants such as sodium sulphite and stabilizers such as EDTA.
[0078] When formulating the pharmaceutical compositions of the present invention as suspensions, in a preferred modality, thickening agents are added to prevent coagulation of the toxin conjugates with the targeting linker group of the invention, or surfactants and polyelectrolytes to ensure the resuspension capacity sediment and/or complex-forming agents such as EDTA. It is also possible to form complexes of the active ingredient with various polymers. Examples of such polymers are polyethylene glycol, polystyrene, carboxymethylcellulose, Pluronics® or polyethylene glycol sorbide fatty ester. The targeting linker toxin conjugates of the invention may further be incorporated into liquid formulations as inclusion compounds, e.g., with cyclodextrins. In particular embodiments, dispersing agents can be added as additional adjuvants. For the production of lyophilisates, bulking agents such as mannite, dextran, sucrose, human albumin, lactose, PVP or gelatin varieties can be used.
[0079] In a further aspect, the present invention is directed to a method of treating pancreatic cancer, cholangiocarcinoma, breast cancer, colorectal cancer, lung cancer, prostate cancer, ovarian cancer, stomach cancer, kidney cancer , malignant melanoma, leukemia, or malignant lymphoma in a patient in need thereof, comprising administering to the patient an effective amount of a conjugate or pharmaceutical composition of the present invention.
[0080] In another aspect, the present invention relates to an amatoxin conjugation molecule as an intermediate for the synthesis of the conjugates of the present invention, where the amatoxin conjugation molecule comprises a ligand L connected to an amatoxin pathway (iv ) the y carbon atom of amino acid 1 of amatoxin, particularly via an amide bond; (v) an oxygen atom attached to the δ-carbon atom of amino acid 3 of amatoxin, particularly via an ester bond, an ether bond or a urethane bond; or (vi) the 6' carbon atom of amino acid 4 of amatoxin, particularly via an oxygen atom attached to the 6' carbon atom of amino acid 4; where in each case the linker L comprises a carbamic acid derivative -NH-C(O)-X, where X is a leaving group which can be replaced by a primary amine of a targeting linker group.
[0081] In one embodiment, the amatoxin conjugation molecule has a structure selected from the structures: (i) amatoxin-yC(O)-NH-L-NH-C(O)-X; (ii) amatoxin-δC-O-C(O)-L-NH-C(O)-X; (iii) amatoxin-δC-O-L-NH-C(O)-X; (iv) amatoxin-δC-O-C(O)-NH-L-NH-C(O)-X; and (v) amatoxin-6'C-O-L-NH-C(O)-X.
In certain embodiments, the amatoxin is chosen from α-amanitin, β-amanitin, y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, or amanulinic acid, or from salts or analogues thereof.
In certain embodiments, the linker L is an optionally substituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or heteroaralkylene group.
[0084] In certain embodiments, the linker L comprises a group selected from the groups: disulfide, ether, amine, ester, carboxamide, urethane, and urea.
[0085] In certain embodiments, the functional group X is selected from: t-butyloxy, succinimidyloxy, 1-O-succinimidyloxy-3-sulfonate (Sulfo-NHS), O-(4-nitrophenyloxy), O-(3-nitrophenyloxy) ), O-(2,4-dinitrophenyloxy), O-(2,4-dichloro-6-nitrophenyloxy), pentafluorophenyloxy, pentachlorophenyloxy, O-(2,4,5-trichlorophenyloxy), O-(3,4-di -hydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-3-yl), O-(endo-1-hydroxy-5-norbornene-2,3-dicarboximide-1-yl), 1- phthalimidoyloxy, 1-benzotriazolyloxy, 1-(7-aza-benzotriazolyl)oxy), and N-imidazolyl.
[0086] In another aspect, the present invention relates to a method for the synthesis of an amatoxin conjugate of the present invention, comprising the step of reacting an amatoxin conjugation molecule of the present invention with a targeting linker group comprising a primary amino group. EXAMPLES
In the following, the invention is explained in more detail by non-limiting examples: Example 1 Synthesis of α-Amanitin and Herceptin antibody Herceptin Her-DSC-30.0134 1.1 Synthesis of 6"-NH-boc-(6-aminohexyl) -α-amanitin HDP 30.0132

[0088] Under argon atmosphere and at room temperature, 30.00 mg (32.6 μmol) of vacuum-dried α-amanitin were dissolved in 900 μL of dry dimethylsulfoxide (DMSO). 3.66 mg (32.6 µmol) of potassium tert-butylate and 73.18 (261.2 µmol, 8 eq.) of NH-Boc-aminohexyl bromide (Fluka 89171) were added. After 6 hours at room temperature, the reaction mixture was acidified to pH = 5 with 50 µL of a 0.33 M acetic acid solution in DMSO. Volatiles were evaporated in vacuo and the residue dissolved in 1000 µl of methanol and diluted with 20 ml of diethyl ether. The precipitate was collected and resuspended in 1000 µL methanol. This solution was diluted with 1000 μL of water and used for purification on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol: 0.05% trifluoroacetic acid Solvent B: 10% water: 90% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-40 min 0% A
The fraction with retention time of 19.8 min was collected and the solvents were evaporated.
15.9 mg (43% yield) of a powder were obtained. MS: 1119 (M+H+); 1141 (M+Na+) 1.2 Synthesis of 6'-(-6-aminohexyl)-α-amanitin HDP 30.0134

[0091] 9.90 mg (8.85 μmol) of 6'-NH-boc-6-aminohexyl-α-amanitin HDP 30.0132 were dissolved in 250 μL of trifluoroacetic acid. The reaction mixture was stirred under an argon atmosphere at room temperature. After 2 minutes, the acid was removed under vacuum at 20°C and the residue dried. Crude amanitin ether was purified on a LaPrep-HPLC chromatograph: column: Kromasil 100-C18, d = 10 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 6 mL/min, detection in À = 295 nm). Solvent A: 95% water: 5% methanol: 0.05% trifluoroacetic acid Solvent B: 90% water: 10% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-25 min 50% A; 25-30 min 0% A; 3035 min 0% A; 35-40 min 100% A, 40-45 min 100% A
[0092] Fractions with the same retention time (14.5 min) were collected and the solvents evaporated.
9.10 mg (99% yield) of a white powder were obtained. MS: 1019 (M+H+); 1041 (M+Na+) 1.3 Synthesis of the antibody derivative HDP 30.0134, Her-DSC-30.0134 Scheme 1

[0094] In Scheme 1 (and other schemes shown in the examples), herceptin is represented by a schematic formula showing a lysine side chain with R3 and R4 representing the remaining part of the herceptin antibody protein. 1.3.1 Synthesis of the 6'-(-6-aminohexyl-6-hydroxysuccinimidyl)-α-amanitin-Herceptin Her-DSC-30.0134 conjugate with different toxin dosages (Table 2)
[0095] 5.00 mg of 6'-(-6-aminohexyl)-α-amanitin HDP 30.0134 was dissolved in 538 µL of dry dimethylformamide (DMF). 18.6 µL of a solution of dihydroxysuccinimide carbonate (DSC) in DMF (2.56 mg in 100 µL of DMF) under argon and stirring at room temperature and 10.0 µL of triethylamine were added at once. The reaction mixture was stirred at room temperature. After 12 hours, 60 mL of ice-cold diethyl ether was added. The α-amanitin-6'-(-6-aminohexyl-6-hydroxysuccinimidyl) carbonate precipitate was collected and washed. 5.00 mg of 6'-(-6-aminohexyl)-α-amanitin HDP 30.0134 was dissolved in 538 μL of dimethylformamide (DMF) Under argon and stirring at room temperature, 18.6 μL of a solution of dihydroxysuccinimide carbonate (DSC) in DMF (2.56 mg in 100 μL of DMF) and 10.0 μL of triethylamine were added at once. The reaction mixture was stirred at room temperature. After 12 hours, 60 mL of ice-cold diethyl ether was added. The precipitate of α-amanitin-6'-(-6-aminohexyl-6-hydroxysuccinimidyl) carbonate was added. collected and washed several times with diethyl ether and dried under vacuum The remaining solid was resuspended in 750 µL of DMF = solution A.
[0096] 114.0 mg of Herceptin were dissolved in 19.0 mL of buffered saline buffer (PBS, pH = 7.4) = solution B. Table 2
[0097] 3 samples of the Herceptin solution are treated with different amounts of α-amanitin-6'-(-6-aminohexyl-6-hydroxysuccinimidyl) carbonate solutions:

[0098] The three solutions of Herceptin with binding amanitin were stirred at 4°C for 14 hours and each separated by Sephadex G-25 gel filtration chromatography (column XK-16; flow rate 2 mL/min). The G-25 column was pre-washed with 500 ml of PBS solutions, pH - 7.4. The Her-DSC-30.0134 conjugate fraction was detected by UV absorption. Protein concentration was determined by RotiQuant-Assay (Carl Roth; Germany). The content of amanitin in herceptin was determined by UV absorption at wavelengths A = 280 nm and A = 310 nm. Example 2 Synthesis of α-Amanitin Conjugate with Herceptin Her-DSC-30.0256 Antibody 2.1 Preparation of 1-Isocyanate-6-BocNH-aminohexane HDP 30.0247 HDP 30.0247

2.50 g (11.56 mmol) of NH-Boc-1,6-hexamethylenediamine (Aldrich 79229) were dissolved in 35 ml dichloromethane. 35 mL of a saturated solution of NaHCO3 in water was added. After addition of 1.143 g (3.85 mmol) of bis-trichloromethyl carbonate (triphosgene), the reaction mixture was vigorously stirred at 0°C for 30 minutes. The organic phase was separated and the aqueous phase was extracted three times with 15 ml of dichloromethane. The combined organic phases were dried over MgSO4 and evaporated. The oily residue was fractionated at 150°C and 0.59 mbar in a Kugelrohr oven. 2.23 g (80% in yield) of a clear oil were obtained. MS: 242 (M+) 2.2 Synthesis of δ-O-(NH-boc-6-aminohexylcarbamoyl)-α-amanitin HDP 30.0253
cat.: dibutyl dilaurylstannate n-Bu2Sn[OCO(CH2)10CH3]2
[0100] In an inert argon atmosphere, 13.43 mg (14.6 μmol) of vacuum-dried α-amanitin were dissolved in 1000 μL of dry dimethylformamide (DMF). 7.08 mg (29.2 µmol) of NH-Boc-6-aminohexane isocyanate and 18.46 mg (29.2 µmol) of dibutyl dilaurylstannate were added and the reaction mixture stirred at room temperature. After 23 hours, an additional 13.43 mg (14.6 µmol) of NH-Boc-6-isocyanate aminohexane was added. After 52 hours, the reaction mixture was hydrolyzed with 200 µL of methanol and evaporated to dryness. The residue was dissolved in 1200 μL of DMSO and purified on a LaPrep-HPLC chromatograph: column: Kromasil 100-C18, 10 μm, 250 mm x 20 mm, with methanol/water (0.05% TFA), flow rate: 26 mL µl, detection at À=295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25 27 min 100% A ; 27-35 min 100% A. Fractions with the same retention time were collected and the solvents evaporated.
[0101] 9.06 mg (53% yield) of a white solid were obtained. MS: 1161 (M+H+); 1183 (M+Na+) 2.3 Synthesis of δ-O-(6-aminohexylcarbamoyl)-α-amanitin HDP 30.0256

[0102] 9.06 mg (7.8 μmol) of HDP 30.0253 were dissolved in 250 μL of trifluoroacetic acid (TFA) and mixed for 2 min at room temperature. The reaction mixture was completely evaporated and the residue co-evaporated twice with 1.5 ml of acetonitrile. The solid was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with acetonitrile/water, flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% acetonitrile Solvent B: 5% water: 95% acetonitrile Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0103] The fraction with a retention time of 12-17 min was collected and evaporated to a white solid.
[0104] 8.75 mg were obtained (yield 95%). MS: (1061 M+H+); 1083 (M+Na+). 2.4 Synthesis of antibody derivative and HDP 30.0256, Her-DSC-30.0256
2.4.1 Synthesis of conjugate of HDP-30.0256 and Herceptin, Her-DSC-30.0256 [3.3]
[0105] 1.00 mg of HDP 30.0256 was dissolved in 108 µL of dry dimethylformamide (DMF). Under argon and stirring, 10.0 μL of a solution of dihydroxysuccinimide carbonate (DSC) in DMF (2.56 mg in 100 μL of DMF) and 2.0 μL of triethylamine were added at once. The reaction mixture was stirred at room temperature. After overnight incubation, 30 ml of ice-cold diethyl ether was added. The precipitate was collected and washed several times with diethyl ether and dried under vacuum. The remaining solid was resuspended in 143 µL of DMF = solution A. 12.0 mg of Herceptin were dissolved in 6.0 mL of phosphate-buffered brine (PBS, pH = 7.4) = solution B. Solution A and solution B were combined. The Herceptin-amanitin linker solution was stirred at 4°C for 14 h and separated by Sephadex G-25 gel filtration chromatography (XK-16 column; 2 ml/min). The G-25 column was pre-washed with 500 ml of PBS solution, pH = 7.4. The Her-DSC-30.0256 conjugate fraction was detected by UV absorption. Protein concentration was determined by RotiQuant assay (Carl Roth; Germany). Amanitin and herceptin content was determined by UV absorption at wavelengths A = 280 nm and A = 310 nm. A content of 3.3 amanitin molecules was calculated for each molecule of Herceptin. Example 3 Synthesis of Herceptin - β-Amanitin Her-DSC-30.0304 Antibody Conjugate 3.1 BocNH-hexamethylenediamino-β-amanitin-amide HDP 30.0299

[0106] In an inert argon atmosphere, 4.65 mg (5.05 μmol) of vacuum-dried β-amanitin were dissolved in 1000 μL of dry dimethylformamide (DMF). 100 µL of a 0.15 M solution of NH-Boc-hexamethylenediamine in DMF and 100 µL of a 0.15 M solution of diisopropylethylamine (DIPEA) in DMF were added at room temperature. After the final addition of 100 μL of a 0.30 M solution of benzotriazol-1-yl-oxytripyrrolidinephosphonium hexafluorophosphate (PyBOP) in DMF, the reaction mixture was stirred for 20 h and hydrolyzed with 100 μL of water. The reaction mixture was completely evaporated in vacuo and the residue dissolved in 1000 µL of dimethylsulfoxide (DMSO). Purification was performed on a LaPrep-HPLC chromatograph: column: Kromasil 100-C18, 10 μm, 250 mm x 20 mm, with methanol/water (0.05% TFA), flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A; 27-35 min 100% A.
[0107] The fractions with the same retention time were collected and the solvents evaporated.
[0108] 4.45 mg (yield 80%) of a white solid were obtained. MS: 1119 M+H+; 1141 M+Na+. 3.2 6'-hexamethylenediamino-β-amanitin-amide HDP 30.0304

[0109] 4.14 mg (3.70 μmol) of BocNH-hexamethylenediamino-β-amanitin-amide HDP 30.0299 were dissolved in 500 μL of trifluoroacetic acid (TFA) and mixed for 2 min. Excess TFA was evaporated in vacuo and the residue co-evaporated with 2 1000 µL portions of acetonitrile. The remaining solid was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 26 mL/min, detection in À = 295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0110] Fractions with the same retention time of 13.43-14.02 min were collected and the solvents evaporated. The residue was resuspended in 2000 μL of water and the solution frozen with liquid nitrogen and freeze-dried overnight.
[0111] 4.0 mg (95% yield) of a white foam were obtained. MS: 1018 (M+H+); 1041 (M+Na+). 3.3 Synthesis of antibody derivative and HDP 30.0304, Her-DSC-30.0304 Scheme 3
conjugate of Herceptin and HDP-30.0304, Her-DSC-30.0304 [4.7]
[0112] 1.33 mg of HDP 30.0304 was dissolved in 144 µL of dry dimethylformamide (DMF). Under argon and stirring at room temperature, 13.4 µL of a solution of dihydroxysuccinimide carbonate (DSC) in DMF (2.56 mg in 100 µL of DMF) and 2.6 µL of triethylamine were added at once. The reaction mixture was stirred at room temperature. After 12 h, 30 mL of ice-cold diethyl ether was added. The precipitate was collected and washed several times with diethyl ether and dried under vacuum. The remaining solid was resuspended in 200 µL of DMF = solution A. 12.0 mg of Herceptin were dissolved in 4.0 mL of phosphate-buffered brine (PBS, pH = 7.4) = solution B. Solution A and solution B were combined. The Herceptin-amanitin linker solution was stirred at 4°C for 14 h and separated by Sephadex G-25 gel filtration chromatography (XK-16 column; 2 ml/min). The G-25 column was pre-washed with 500 ml of PBS solution, pH = 7.4. The Her-DSC-30.0304 conjugate fraction was detected by UV absorption. Protein concentration was determined by RotiQuant assay (Carl Roth; Germany). Amanitin and herceptin content was determined by UV absorption at wavelengths A = 280 nm and A = 310 nm. A content of 4.7 amanitin molecules was calculated for each Herceptin molecule. Example 4 Other Structures of Amanitin-Herceptin Conjugates



Example 5 Cytotoxicity of Amanitin-Herceptin conjugates on different HER2-positive and HER2-negative tumor cell lines in vitro
[0113] The cytotoxic activity of Her-DSC-30.0134, Her-DSC-30.0256, Her-Ester-30.0001, Her-DCC-30.0252 and Her-DCC-30.0127 was evaluated with SKOV-3 HER2-positive tumor cell lines ( from ovary), SK-BR-3 (mammary), NCI-N87 (from stomach) and the HER2-negative tumor cell line MDA-MB231 (mammary) and with a chemiluminescent BrdU incorporation assay (Roche Diagnostics) in vitro. Cell viability was determined after incubation for 72h to 96h with different concentrations of Herceptin-Amanitin conjugates at 37°C and 5% CO2 by measuring cells fixed and permeabilized with an anti-BrdU-HRP antibody in a BMG Labtech microslide reader Great. EC50 values of the dose-response curves were calculated using the Graphpad Prism 4.0 software (see Figures 2 - 5). Stability of Herceptin-Amanitin Conjugates in In Vivo Plasma 5.1. Release of amanitin after incubation in plasma
[0114] 35 μM of Herceptin-Amanitin conjugates were incubated for up to 14 days in mouse plasma in a water bath at 37°C. Samples were collected at different times and analyzed for released small molecule amanitin compounds by an ELISA method. Thus, the amanitin and the released amanitin metabolites were extracted at different times with 80% of EtOH. The solutions were clarified by centrifugation at 10,000 g for 5 minutes and the supernatants were stored at -70°C. A white Lumitrac microwell plate (Greiner) was coated with rabbit anti-amanitin antiserum overnight at 4°C. The plate was blocked with 3% BSA in PBS for 1 h at 37°C and washed three times with 0.05% Tween/PBS. Amanitin samples and amanitin solutions with defined concentrations were mixed with 1 nM biotinyl-amanitin in 1% BSA/PBS and incubated in coated wells for 1 h at 37°C. Wells were washed three times with 0.05% Tween/PBS. Streptavidin-HRP solution (Sigma-Aldrich), 1 mg/ml in PBS, was diluted 1:1000 in 3% BSA/PBS and 50 µl was added to each well. After incubation for 1 h at 37°C, the wells were washed three times with 0.05% Tween/PBS. 50 µL of luminol solution (Applichem) was added to each well and the luminescence signal was measured by a BMG Labtech Optima reader. The amounts of amanitin compounds released were calculated by linear regression (see Figures 6 - 7). 5.2. Cytotoxic activity after incubation in plasma
[0115] Herceptin-Amanitin conjugates were incubated for up to 11 days in human plasma in a water bath at 37°C. Samples were collected at different times and analyzed for remaining cytotoxic potency in SKOV-3 HER2-positive cells by a chemiluminescent BrdU incorporation assay (Roche Diagnostics) in vitro. Cell viability was determined after incubation for 72h with different concentrations of Herceptin-Amanitin conjugates at 37°C and 5% CO2 by measuring cells fixed and permeabilized with an anti-BrdU-HRP antibody in a BMG Labtech Optima microslide reader. The EC50 values of the dose-response curves were calculated using the Graphpad Prism 4.0 software (see Figure 8). Anti-tumor activity of Herceptin-Amanitin conjugates in mouse xenotransplant models with HER2-positive cancer cells
[0116] Six-week-old intact BALB/c nu/nu athymic female mice were purchased (Janvier) and randomly divided into three groups of eight mice each. 2.5 x 106 SKOV-3 cells were injected subcutaneously into the flank of each mouse. Herceptin-Amanitin conjugates were injected once intravenously at a dose of 50 μg/kg on day 19 after inoculation, while the negative control group was injected with vehicle (buffer NaCl). Parameters such as tumor survival, weight and size were recorded (see Figure 9). Example 6: Preparation of additional α-amanitin-binding compounds HDP 30.0353, HDP 30.0354, HDP 30.0355, HDP 30.0409, HDP 30.0410, HDP 30.0411 and HDP 30.0412 6.1. Amanitin-Binder HDP 30.0353 6.1.1. Synthesis of 6'O-(NH-boc-6-amino-3,4-dithiahexyl)-α-amanitin HDP 30.0341

[0117] 6.78 mg (7.38 μmol) of vacuum-dried α-amanitin were dissolved in 500 μL of dimethylsulfoxide (DMSO). 18.67 mg (59.02 µmol, 8 eq.) of NH-boc-amino-3,4-dithiahexyl bromide and 73.8 µL (7.38 µmol, 1 eq.) of LiOH (0, 1 M) in water/DMSO (1:1) were added under argon atmosphere. After 1 h, 3.5 h, 4.5 h, 6.5 h and 8 h, additional equivalents of the 0.1 M LiOH solution were added. The crude reaction mixture was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 10 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 6.5 mL/min , detection at À=295 nm). Solvent A: 95% water: 5% methanol: 0.05% trifluoroacetic acid Solvent B: 10% water: 90% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A; 27-35 min 100% A.
[0118] The fraction with a retention time of 20.8-21.4 was collected and the solvents evaporated under vacuum.
[0119] 1.29 mg (15% yield) of a white solid were obtained. MS: 1154 M+H+. 6.1.2. Synthesis of 6'-O-(-6-amino-3,4-dithiahexyl)-α-amanitin HDP 30.0353
amanitin HDP 30.0353
[0120] 1.28 mg (1.12 μmol) of 6'O-(NH-boc-6-amino-3,4-dithiahexyl)-α-amanitin HDP 30.0341 was dissolved in 200 μL of trifluoroacetic acid ( TFA). The reaction mixture was mixed under argon atmosphere at room temperature. After 1 min the trifluoroacetic acid was diluted with 1000 µL of toluene and evaporated to dryness. The temperature should not exceed 20°C. This process was repeated with 1000 μL of toluene and 1000 μL of acetonitrile (2x). The crude α-amanitin ether was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 10 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 6.5 mL/min, detection at À=295 nm). trifluoroacetic 90% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-25 min 50% A; 25-30 min 0% A; 30-35 min 0% A, 35-40 min 100% A, 40-45 min 100% A.
[0121] The fraction with a retention time of 16.1-17.0 was collected and the solvents evaporated. The residue was freeze-dried in water.
[0122] 0.19 mg (30% yield, TFA salt) of a yellow solid was obtained. MS: 1054 M+H+. 6.2. Amanitin-Binder 30.0354 6.2.1. Synthesis of 6'O-(NH-boc-7-amino-4,5-dithia-heptyl)-α-amanitin HDP 30.0349

[0123] 6.78 mg (7.38 μmol) of vacuum-dried α-amanitin were dissolved in 250 μL of dimethylsulfoxide (DMSO). 19 mg (58 µmol, 9.3 eq.) of NH-boc-7-amino-4,5-dithiaheptyl HDP 30.0345 bromide were added at room temperature under an argon atmosphere. 61.7 µL (6.10 µmol, 1 eq.) of 0.1 M LiOH in water/DMSO (1:1) were added in one go. The reaction mixture was mixed for an additional 3.5 h and an additional 10 µL (13.00 mg; 39.7 µmol; 6.3 eq.) of NH-boc-7-amino-4,5-dithia-heptyl HDP bromide 30.0345 and 61.7 µL of 0.1 M LiOH in water/DMSO (1:1) were added. After 8 h the crude reaction mixture was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 26 mL/min , detection at À=295 nm). Solvent A: 95% water: 5% methanol: 0.05% trifluoroacetic acid Solvent B: 10% water: 90% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-30 min 0% A; 30-35 min 100% A; 35-40 min 100% A.
[0124] The fraction with a retention time of 19.4 - 21.0 min was collected and the solvents evaporated completely at room temperature.
[0125] 4.83 mg (67% yield) of a white solid were obtained. MS: 1168 M+H+. 6.2.2. Synthesis of 6'-O-(-7-amino-4,5-dithia-heptyl)-α-amanitin HDP 30.0354

[0126] 4.83 mg (4.13 μmol) of 6'O-(NH-boc-7-amino-4,5-dithiaheptyl)-α-amanitin HDP 30.0349 were dissolved in 200 μL of trifluoroacetic acid ( TFA). The reaction mixture was mixed for 1 h under argon atmosphere and evaporated at room temperature. The residue was co-evaporated with 1000 μL of toluene and 1000 μL of acetonitrile. The remaining solid was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 10 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 6.5 mL/min, detection at À=295 nm). Solvent A: 95% water: 5% methanol: 0.05% trifluoroacetic acid Solvent B: 10% water: 90% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0127] The fraction with a retention time of 17.1-17.5 min was collected and evaporated. The residue was freeze-dried in water.
[0128] 0.36 mg (7.0% yield, TFA salt) of a yellow solid was obtained. MS: 1068 M+H+. 6.3. Amanitin-Binder 30.0355 6.3.1. Synthesis of 6'O-(NH-boc-7-amino-3,3-dimethyl-4,5-dithiaheptyl)-α-amanitin HDP 30.0350

[0129] 5.67 mg (6.17 μmol) of vacuum-dried α-amanitin were dissolved in 250 μL of dimethylsulfoxide (DMSO). Under argon atmosphere, 18.00 mg (51.62 µmol, 9.8 eq.) of NH-boc-7-amino-3,3-dimethyl-4,5-dithiaheptyl HDP bromide 30.0348 and 61, 7 µL (6.10 µmol, 1 eq.) of 0.1 M LiOH in water/DMSO (1:1) was added. After 2 h the reaction mixture was repeatedly treated with 10 µL (12.00 mg; 34.4 µmol; 5.6 eq.) of NH-boc-7-amino-3,3-dimethyl-4,5-dithia bromide -heptyl HDP 30.0348 and 61.7 µL of 0.1 M LiOH. After 8 h the mixture was diluted with DMSO and purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 µm, with methanol/water (0.05% TFA), flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol: 0.05% trifluoroacetic acid Solvent B: 10% water: 90% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-30 min 0% A; 30-35 min 100% A; 35-40 min 100% A.
[0130] The fraction with a retention time of 20.5-21.0 min was collected and the solvents evaporated.
[0131] 0.51 mg was obtained (7% yield; 48% based on converted α-amanitin). MS: 1196 M+H+. 6.3.2. Synthesis of 6'-O-(-7-amino-3,3-dimethyl-4,5-dithiaheptyl)-α-amanitin HDP 30.0355

[0132] 0.51 mg (0.43 μmol) of HDP 30.0350 were dissolved in 200 μL of trifluoroacetic acid (TFA) and mixed for 1 h at room temperature. Trifluoracetic acid was diluted with 1000 µL of toluene and completely evaporated at 20°C. This process was repeated with 1000 μL of toluene and 1000 μL of acetonitrile (2x). The reaction mixture was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 10 mm, 10 µm, with methanol/water (0.05% TFA), = 295 nm). Solvent A: 95% water: 5% methanol: 0.05% trifluoroacetic acid Solvent B: 10% water: 90% methanol: 0.05% trifluoroacetic acid Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0133] The fraction with a retention time of 18.2-18.6 min was collected, and the solvents evaporated. The residue was freeze-dried in water.
[0134] 0.15 mg (27% yield, TFA salt) of a yellow solid was obtained. MS: 1096 M+H+. 6.4. Amanitin-Binder 30.0355 6.4.1. Synthesis of 6'O-(NH-boc-12-amino-dodecyl)-α-amanitin HDP 30.0404

[0135] 6.67 mg (7.26 μmol) of vacuum-dried α-amanitin were dissolved in 250 μL of dimethylsulfoxide (DMSO). 21.00 mg (58.10 µmol, 8 eq.) of NH-boc-12-amino-dodecyl bromide HDP 30.0383 and 72.6 µL (7.26 µmol, 1 eq.) of 0.1 M LiOH in water/DMSO (1:1) were added. After 6 h, 36.5 µL of 0.1 M LiOH was added and the mixture disintegrated with 72.6 µL of a 0.1 M solution of acetic acid in DMSO 2 h later. The crude product of the reaction was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 26 mL/min, detection at À=295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A; 27-35 min 100% A.
[0136] The fraction with a retention time of 22.0-22.7 min was collected and the solvents evaporated.
[0137] 5.96 mg (yield 68%) of a white solid were obtained. MS: 1202 M+H+. 6.4.2. Synthesis of 6'-O-(12-amino-dodecyl)-α-amanitin HDP 30.0409

[0138] 5.96 mg (4.96 μmol) of HDP 30.0404 were dissolved in 200 μL of trifluoroacetic acid (TFA) and mixed for 1 min at room temperature. The reaction mixture was co-evaporated with 1000 μL of toluene and acetonitrile and the remaining solid was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with acetonitrile/water, flow rate: 26 mL/min, detection at À=295 nm). Solvent A: 95% water: 5% acetonitrile Solvent B: 5% water: 95% acetonitrile Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0139] The fraction with a retention time of 18.6-19.2 min was collected and evaporated to a white solid.
[0140] 6.03 mg were obtained (yield 99%, TFA salt). MS: 1102 M+H+. 6.5. Amanitin-Binder HDP 30.0410 6.5.1. Synthesis of 6'O-(NH-boc-11-amino-3,6,9-trioxa-undecyl)-α-amanitin HDP 30.0405

[0141] 6.67 mg (7.26 μmol) of vacuum-dried α-amanitin were dissolved in 250 μL of dimethylsulfoxide (DMSO). 20.51 mg (58.10 µmol, 8 eq.) of NH-boc-11-amino-3,6,9-trioxa-undecyl HDP bromide 30.0391 and 72.6 µL (7.26 µmol, 1 eq. ) of 0.1 M LiOH in water/DMSO (1:1) were added. The reaction was carried out at room temperature under an argon atmosphere. After 6 h, additional LiOH base (0.5 eq.) was added. The mixture was disintegrated after 8 h with 72.6 µL of a 0.1 M acetic acid solution in DMSO and purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 µm, with methanol/water (0.05% TFA), flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A; 27-35 min 100% A.
[0142] The fraction with a retention time of 18.1-18.6 min was collected and the solvents evaporated.
[0143] 4.68 mg (54% yield) of solid was obtained. MS: 1194 M+H+. 6.5.2. Synthesis of 6'-O-(11-amino-3,6,9-trioxa-undecyl)-α-amanitin HDP 30.0410

[0144] 4.68 mg (3.92 μmol) of HDP 30.0405 were dissolved in 200 μL of trifluoroacetic acid (TFA) and mixed for 1 min at room temperature. The reaction mixture was co-evaporated with toluene and acetonitrile and the crude solid was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 µm, with methanol/water (0.05% TFA) , flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0145] The fraction with a retention time of 18.6-19.2 min was collected and evaporated. The remaining solid was freeze-dried in water.
[0146] 2.44 mg (52% yield, TFA salt) of a white solid were obtained. MS: 1102 M+H+. 6.6. Amanitin-Binder HDP 30.0411 6.6.1. Synthesis of 6'O-(NH-boc-16-aminohexadecyl)-α-amanitin HDP 30.0406

[0147] 6.67 mg (7.26 μmol) of vacuum-dried α-amanitin were dissolved in 750 μL of dimethylsulfoxide (DMSO). 24.00 mg (58.10 µmol, 8 eq.) of NH-boc-16-aminohexadecyl HDP 30.0398 bromide were added at room temperature under an argon atmosphere. 72.6 µL (7.26 µmol, 1 eq.) of 0.1 M LiOH in water/DMSO (1:1) was added and the reaction mixture was heated to 50°C. After 6 h, additional LiOH base (36.5 µL) was added and the reaction mixture was disintegrated after 8 h with 72.6 µL of a 0.1 M acetic acid solution in DMSO. The solidified reaction mixture was diluted with DMSO and purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with methanol/water (0.05% TFA), flow rate: 26 mL /min, detection at À=295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A; 27-35 min 100% A.
[0148] The fraction with a retention time of 23.4-24.1 min was collected and the solvents evaporated.
[0149] 4.41 mg (yield 54%) of a white solid was obtained. MS: 1258 M+H+. 6.6.2. Synthesis of 6'-O-(16-aminohexadecyl)-α-amanitin HDP 30.0410

[0150] 4.41 mg (3.50 μmol) of HDP 30.0406 were dissolved in 200 μL of trifluoroacetic acid (TFA) and mixed for 2 min at room temperature. The reaction mixture was co-evaporated twice with 1000 μL of toluene and 1000 μL of acetonitrile and the solid residue was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with methanol/ water (0.05% TFA), flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0151] The fraction with a retention time of 20.5-21.2 min was collected and evaporated. The residue dried under freezing in water.
[0152] 2.44 mg (52% yield, TFA salt) of a white solid were obtained. MS: 1102 M+H+. 6.7. Amanitin-Binder HDP 30.0411 6.7.1. Synthesis of 6'O-(NH-boc-2-amino-ethyl)-α-amanitin HDP 30.0317

[0153] 20.00 mg (21.8 μmol) of vacuum-dried α-amanitin were dissolved in 900 μL of dimethylsulfoxide (DMSO). 100 µL (21.8 µmol, 1 eq.) of a 0.218 M solution of potassium t-butylate in DMSO and 39.00 mg (174.1 µmol, 8 eq.) of NH-boc-2-amino bromide -ethyl (obtained from Fluka) were added at room temperature. After 4 and 6 h, 1 and 2 additional equivalents of potassium t-butylate and NH-boc-2-amino-ethyl bromide were added. The mixture was disintegrated after 23 h with 72.6 µL of a 0.1 M acetic acid solution in DMSO and purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 µm, with methanol/water (0.05% TFA), flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A; 27-35 min 100% A.
[0154] The fraction with a retention time of 13.2-14.5 min was collected and the solvents evaporated.
[0155] 3.86 mg (16% yield) of a white solid were obtained. MS: 1062 M+H+. 6.7.2. Synthesis of 6'-O-(2-amino-ethyl)-α-amanitin HDP 30.0412

[0156] 3.86 mg (3.36 μmol) of HDP 30.3017 were dissolved in 200 μL of trifluoroacetic acid (TFA) and mixed for 2 min at room temperature. The reaction mixture was co-evaporated with 1000 μL of toluene and 1000 μL of acetonitrile and the solid was purified on a LaPrep-HPLC chromatograph: (column: Kromasil 100-C18, 250 mm x 20 mm, 10 μm, with methanol/water (0 .05% TFA), flow rate: 26 mL/min, detection at À = 295 nm). Solvent A: 95% water: 5% methanol Solvent B: 5% water: 95% methanol Gradient: 0-5 min 100% A; 5-20 min 0% A; 20-25 min 0% A; 25-27 min 100% A, 27-35 min 100% A.
[0157] The fraction with a retention time of 20.5-21.2 min was collected and evaporated. The residue dried under freezing in water.
[0158] 1.54 mg (44% yield, TFA salt) of a white solid was obtained. MS: 962 M+H+.

权利要求:
Claims (15)
[0001]
1. Conjugate comprising a targeting linker group linked via a linker L to an amatoxin, characterized in that linker L is connected to the amatoxin via (i) the y carbon atom of amino acid 1 of the amatoxin, particularly via an amide linkage; (ii) an oxygen atom attached to the δ-carbon atom of amino acid 3 of amatoxin, particularly via an ester bond, an ether bond or a urethane bond; or (iii) the 6' carbon atom of amino acid 4 of amatoxin, particularly via an oxygen atom attached to the 6' carbon atom of amino acid 4; where in each case the ligand L is connected to the targeting ligand group via a urea group.
[0002]
2. Conjugate according to claim 1, characterized in that the conjugate has a structure chosen from the following structures: (i) amatoxin-yC(O)-NH-L-NH-C(O)-NH-linker group of targeting; (ii) amatoxin-δC-O-C(O)-L-NH-C(O)-NH-targeting ligand group; (iii) amatoxin-δC-O-L-NH-C(O)-NH-targeting linker group; (iv) amatoxin-δC-O-C(O)-NH-L-NH-C(O)-NH-targeting ligand group; and (v) amatoxin-6’C-O-L-NH-C(O)-NH-targeting linker group.
[0003]
Conjugate according to any one of claims 1 or 2, characterized in that the targeting linker group is connected to the linker L via an amino group present in the targeting linker group, where the amino group forms part of the urea group.
[0004]
Conjugate according to any one of claims 1 to 3, characterized in that the amatoxin is chosen from α-amanitin, β-amanitin, Y-amanitin, ε-amanitin, amanin, amaninamide, amanulin, or amanulinic acid, or between salts or analogues thereof.
[0005]
Conjugate according to any one of claims 1 to 4, characterized in that the linker L is an optionally substituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or heteroaralkylene.
[0006]
Conjugate according to any one of claims 1 to 5, characterized in that the linker L comprises a group selected from one or more of the following groups: disulfide, ether, amine, ester, carboxamide, urethane, or urea.
[0007]
Conjugate according to any one of claims 1 to 6, characterized in that the targeting ligand group specifically binds to an epitope that is present on a tumor cell, particularly where the targeting ligand group specifically binds to an epitope of epithelial cell adhesion molecule (EpCAM).
[0008]
8. Conjugate according to any one of claims 1 to 7, characterized in that the targeting binding group is selected from the group consisting of: (i) antibody or antigen binding fragment thereof; (ii) antibody-like protein; and (iii) nucleic acid aptamer.
[0009]
Conjugate according to claim 8, characterized in that the antibody or antigen-binding fragment thereof is selected from a bivalent antibody, a tetravalent antibody, a nanometric antibody, a chimeric antibody, a deimmunized antibody, a humanized antibody or a human antibody.
[0010]
Toxin conjugate and targeting binding group, according to claims 8 or 9, characterized in that the antigen binding fragment is chosen from the group consisting of Fab, F(ab')2, Fd, Fv, single-chain Fv , and disulfide linked Fv's (dsFv).
[0011]
11. Conjugate according to any one of claims 1 to 10, characterized in that it is for use as a medicine for use in the treatment of cancer in a patient, where the cancer is selected from the group consisting of pancreatic cancer, cholangiocarcinoma, breast cancer , colorectal cancer, lung cancer, prostate cancer, ovarian cancer, stomach cancer, kidney cancer, malignant melanoma, leukemia, or malignant lymphoma.
[0012]
12. Pharmaceutical composition, characterized in that it comprises the conjugate as defined in any one of claims 1 to 10 and additionally comprises one or more diluents, carriers, excipients, fillers, binders, lubricants, surfactants, disintegrants, adsorbents; and/or pharmaceutically acceptable preservatives.
[0013]
13. Amatoxin conjugation molecule, characterized in that it comprises a linker L connected to an amatoxin via (i) the y carbon atom of amino acid 1 of the amatoxin, particularly via an amide bond; (ii) an oxygen atom attached to the δ-carbon atom of amino acid 3 of amatoxin, particularly via an ester bond, an ether bond or a urethane bond; or (iii) the 6' carbon atom of amino acid 4 of amatoxin, particularly via an oxygen atom attached to the 6' carbon atom of amino acid 4; where in each case the linker L comprises a carbamic acid derivative -NH-C(O)-X, where X is a leaving group which can be replaced by a primary amine of a targeting linker group.
[0014]
A conjugation molecule with amatoxin, according to claim 13, characterized in that X is chosen from: t-butyloxy, succinimidyloxy, 1-O-succinimidyloxy-3-sulfonate (Sulfo-NHS), O-(4-nitrophenyloxy) , O-(3-nitrophenyloxy), O-(2,4-dinitrophenyloxy), O-(2,4-dichloro-6-nitrophenyloxy), pentafluorophenyloxy, pentachlorophenyloxy, O-(2,4,5-trichlorophenyloxy), O -(3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-3-yl), O-(endo-1-hydroxy-5-norbornene-2,3-dicarboximide- 1-yl), 1-phthalimidoyloxy, 1-benzotriazolyloxy, 1-(7-aza-benzotriazolyl)oxy), and N-imidazolyl.
[0015]
15. Method for the synthesis of a conjugate as defined in any one of claims 1 to 11, characterized in that it comprises the step of reacting a conjugation molecule with amatoxin as defined in any one of claims 13 or 14 with a targeting linker group comprising a primary amine group.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07K 16/30 (2006.01), A61K 47/68 (2017.01), A61P 3 |

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2019-05-21| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-22| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-04| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/09/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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GB10012573.1|2010-09-30|

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EP10012573A|EP2436398B1|2010-09-30|2010-09-30|Amatoxin-conjugates with improved linkers|

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PCT/EP2011/004875|WO2012041504A1|2010-09-30|2011-09-29|Amatoxin-conjugates with improved linkers|

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