Methods of modifying organic cytotoxins useful as payloads in antibody-drug conjugates and modified organic cytotoxins derived therefrom

Abstract

The present invention provides a method of modifying a cyanobacteria or mycotoxin for use as a payload in an antibody-drug conjugate, the method comprising the steps of: selecting a cyanobacteria or mycotoxin having a base structure known to have cytotoxicity to a target cell upon uptake by a transporter of the target cell, modifying the base structure with at least one structural modification, where the at least one structural modification comprises an organic inhibitory group or effector molecule that reduces intake of the modified toxin by the transporter to the target cell compared to the base structure, and an organic coupling group configured for covalently conjugating the modified toxin to an antibody. The invention further provides modified toxins derived from the method and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of European application number 22192517.5 filed on August 27, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains an electronic sequence listing having 4 sequences in xml format according to the WIPO ST.26 standard as part of the specification, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to biologically active cytotoxic anticancer payloads or drugs. In particular, the present invention relates to modified organic cytotoxins, more particularly modified cyanobacterial and fungal toxins, for use as payloads in antibody-drug conjugates. The present invention further relates to antibody-drug conjugates comprising such biologically active cytotoxic anticancer payloads coupled to antibodies that specifically target certain tumor antigens. Background Art

[0006] Today, cancer is the second leading cause of death worldwide, causing approximately 10,000,000 deaths per year worldwide. Cancer incidence has increased over the past few decades and is expected to continue to rise due to global population growth and aging.

[0007] A drawback of most anticancer drugs currently used in conventional chemotherapy is that they are often cytotoxic because they are not able to adequately discriminate between cancer cells and healthy tissue, have known severe side effects and significantly limit the quality of life of patients.

[0008] The demand for anticancer drugs with a specific spectrum of action against malignant cells has driven the development of antibody-drug conjugates.

[0009] Antibody-drug conjugates, also referred to hereinafter as "ADCs", are a class of biological drugs designed as targeted therapies for the treatment of cancer. Unlike chemotherapy, ADCs are designed to target and kill tumor cells while leaving healthy cells alone. ADCs are biological drugs that are essentially composed of a monoclonal antibody linked to a biologically active and usually cytotoxic drug (also referred to herein as a "payload"), thereby combining the target specificity of the monoclonal antibody with the cancer-killing ability of the cytotoxic drug.

[0010] Conceptually, mediated by the target specificity of monoclonal antibodies, ADCs are expected to target tumor cells and not harm healthy tissues. However, off-target effects are often seen during clinical application, such as those caused by spontaneous or enzymatic hydrolysis of the chemical linkage between the drug and antibody during transport and metabolism after systemic administration, or by the release of the payload from lysed or killed cancer cells into surrounding tissues.

[0011] Current efforts to reduce or eliminate undesirable toxic side effects on healthy cells primarily involve improving the stability of the chemical bond between the cytotoxic payload and the monoclonal antibody to avoid premature release of the drug outside the tumor cells.

[0012] For example, WO 2018 / 206715A2 and WO 2018 / 219619A1, the disclosures of which are incorporated herein by reference in their entireties, disclose modified cyanobacterial microcystins and nodularin as cytotoxic payloads, which, inter alia, incorporate non-naturally occurring amino acids, provide chemical anchoring groups for coupling the payload to a linker or antibody, respectively, resulting in a homogeneously and stably loaded ADC with limited payload release in the bloodstream.

[0013] However, the improved chemical linkage between antibodies and cytotoxic payloads can only partially overcome the above-mentioned shortcomings of current ADCs. In particular, it does not solve the problem of payload release after targeted cancer cell death. In addition, the high potency of cytotoxins creates unresolved health and safety risks in their manufacture and handling, resulting in infrastructure and production that are cost-intensive.

[0014] Against this background, it is an object of the present invention to provide an improved cytotoxin for use as a payload in antibody-drug conjugates.Another object of the present invention is to provide an improved antibody-drug conjugate.

[0015] These objects are solved according to the subject matter of the independent claims. Preferred embodiments are the subject matter of the dependent claims and the following description. Summary of the invention

[0016] In certain embodiments, the present invention provides a method for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody-drug conjugate, the method comprising the steps of: selecting a cyanobacterial or fungal toxin having a base structure known to be cytotoxic to a target cell after being taken up by a transporter protein of the target cell; modifying the base structure with at least one structural modification, wherein the at least one structural modification comprises: an organic inhibitory group or effector molecule that reduces the uptake of the modified toxin into the target cell by the transporter protein compared to the base structure, and an organic coupling group configured for covalently conjugating the modified toxin to an antibody.

[0017] In certain embodiments, the present invention provides a method for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody-drug conjugate, the method comprising the steps of selecting a cyanobacterial or fungal toxin having a base structure known to be cytotoxic to a target cell after being taken up by a transporter protein of the target cell; modifying the base structure with: (i) an organic inhibitory group or effector molecule that reduces the uptake of the modified toxin into the target cell by the transporter protein at least as compared to the base structure, and (ii) an organic coupling group configured for covalently conjugating the modified toxin to an antibody.

[0018] In certain embodiments, the present invention provides a method for making a modified cyanobacterial or fungal toxin, the method comprising the steps of: A) providing a toxin having a basic structure known to be cytotoxic to a target cell after being taken up by a transporter protein of the target cell, and, B) introducing at least one structural modification into the toxin, wherein the at least one structural modification comprises (i) an organic inhibitory group or effector molecule that reduces the uptake of the modified toxin into the target cell by the transporter protein compared to the basic structure, and (ii) an organic coupling group configured for covalently conjugating the modified toxin to an antibody.

[0019] In certain embodiments, the present invention provides a method for making a modified cyanobacterial or fungal toxin, the method comprising the steps of: A) providing a toxin having (i) a basic structure known to be cytotoxic to a target cell after being taken up by a transporter protein of the target cell and (ii) at least one functional group configured for and / or capable of covalent conjugation, B) introducing at least one structural modification into the toxin via covalent conjugation with the functional group of the toxin, wherein the at least one structural modification comprises (i) an organic inhibitory group or effector molecule that reduces the uptake of the modified toxin into the target cell by the transporter protein compared to the basic structure, and (ii) an organic coupling group configured for covalent conjugation of the modified toxin to an antibody.

[0020] In certain embodiments, the present invention provides a modified cyanobacterial or fungal toxin comprising a basic structure that is cytotoxic to a target cell after being taken up by a transporter protein of the target cell and at least one structural modification of the basic structure, wherein the at least one structural modification comprises: an organic inhibitory group or effector molecule that reduces the uptake of the modified toxin into the target cell by the transporter protein compared to the basic structure, and an organic coupling group configured to covalently conjugate the organic cytotoxin to an antibody.

[0021] In certain embodiments, the present invention provides the use of a modified toxin as defined above in the manufacture of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises covalently conjugating the modified toxin to an antibody or fragment thereof comprising an antigen-specific binding site via the organic coupling group.

[0022] In certain embodiments, the present invention provides a method for making an antibody-drug conjugate, the method comprising the steps of: a) providing a monoclonal antibody or a fragment thereof comprising an antigen-specific binding site and a toxin as defined above, b) covalently conjugating the toxin to the antibody via the organic coupling group.

[0023] In certain embodiments, the present invention provides a method for making an antibody-drug conjugate, the method comprising the steps of: a) providing a toxin having (i) a basic structure known to be cytotoxic to a target cell after being taken up by a transporter protein of the target cell, and (ii) at least a first functional group and a second functional group configured for or capable of covalent conjugation, b) introducing at least one structural modification into the toxin via covalent conjugation with the first functional group, wherein the at least one structural modification comprises an organic inhibitory group or effector molecule that reduces the uptake of the modified toxin into the target cell by the transporter compared to the basic structure, and c) covalently conjugating the toxin to an antibody via the second functional group, thereby making the antibody-drug conjugate.

[0024] In certain embodiments, the present invention provides an antibody-drug conjugate comprising a modified toxin as defined above and an antibody or fragment thereof comprising an antigen-specific binding site, wherein the antibody or fragment thereof is linked to the modified toxin via the organic coupling group.

[0025] In certain embodiments, the present invention provides a modified cyanobacterial or fungal toxin or antibody-drug conjugate as defined above for use in treating a malignant disease.

[0026] In certain embodiments, the invention provides a method for treating a human subject afflicted with a malignant disease, said method comprising administering to said subject a therapeutically effective dosing regimen of an antibody-drug conjugate as defined above.

[0027] In some embodiments according to or as applied to any of the above embodiments, the base structure comprises an oligopeptide, particularly a cyclic oligopeptide, more particularly a cyclic oligopeptide containing at least one non-proteinogenic amino acid.

[0028] In some embodiments according to or as applied to any of the above embodiments, the modified toxin or the base structure is selected from microcystin, nodularin, or amatoxin (e.g., amanitin). In specific embodiments, the modified toxin or the base structure is microcystin.

[0029] In some embodiments according to or as applied to any of the embodiments above, the base structure is a naturally occurring toxin.

[0030] In some embodiments according to or applied to any of the above embodiments, the base structure is non-naturally occurring, in particular the base structure of a naturally occurring toxin modified by at least one functional group (preferably one or two functional groups), which is configured for or capable of chemical conjugation with another organic molecule (such as an antibody or a modified molecule).

[0031] In some embodiments according to or as applied to any of the above embodiments, the base structure is microcystin having the general formula

[0032] Ring (-A 1 -X 2 -A 3 -Z 4 -A 5 -A 6 -A 7 ),

[0033] Among them, independently of each other, A 1 and A 3 Each represents a D-amino acid, A 5 Selected from Adda, DM-Adda, dm-Adda, (6Z)Adda and ADM-Adda, A 6 Selected from D-Glu and D-Glu(OCH3), A 7 is selected from the group consisting of Mdha, MdhB, Dha, L-Ser, L-MeSer, Dhb, (E)-Dhb, (Z)-Dhb, MeLan, Cys and Thr or a modified L-amino acid, and X 2 and Z 4 are independently L-amino acids, wherein A 1 , X 2 , A 3 , Z 4 and A 7 At least one of the microcystin molecules is a modified amino acid having a side chain that is not present in the natural counterpart of the microcystin, wherein the side chain comprises a functional group configured for or capable of covalently conjugating the at least one structural modification.

[0034] In some embodiments according to or as applied to any of the embodiments above, the chemical conjugation is a bioorthogonal chemical conjugation, preferably comprising a click chemistry reaction.

[0035] In some embodiments according to or applied to any of the above embodiments, the organic inhibitory group is selected from an organic group carrying at least one charge at physiological pH, a heterocycle, such as biotin, an organic carboxamide, an alcohol, or any combination thereof.

[0036] In some embodiments according to or as applied to any of the embodiments above, the transporter comprises an organic anion transporting polypeptide (OATP), particularly OATP1B1 and / or OATP1B3.

[0037] In some embodiments according to or as applied to any of the above embodiments, the target cell is a mammalian cell, particularly a cancer cell.

[0038] Preferred variants of these embodiments can be derived from the description of the invention and the detailed description of the embodiments.

[0039] Description of the Invention

[0040] A first aspect of the present invention relates to a method of modifying a cyanobacterial or fungal toxin for use as a payload in an antibody drug conjugate.

[0041] The method comprises the following steps: selecting a cyanobacterial or fungal toxin having a basic structure known to be cytotoxic to a target cell after being taken up by a transporter of the target cell; and modifying the basic structure with at least one structural modification, thereby modifying the toxin. Herein, the at least one structural modification comprises: an organic inhibitory group or effector molecule that reduces the uptake of the modified toxin into the target cell by the transporter compared to the basic structure, and an organic coupling group configured to covalently conjugate the modified toxin to an antibody.

[0042] The present invention is based on the inventor's insight that cytotoxins must usually be taken up by cells through active transport mediated by transporters or by passive diffusion before they can exert their cytotoxic function, and when coupled to antibodies, the internalization of cytotoxins becomes receptor-mediated endocytosis, such as clathrin-mediated endocytosis, caveolae-mediated endocytosis or pinocytosis, which is a process in which cells absorb ADCs by budding inwards of the plasma membrane after binding to the cell surface by antibodies, thereby transporting toxins to targeted cells. Based on this insight, the inventors recognize the potential of modified toxins, which require active transport across the cell membrane in an isolated form and cannot enter the cell by passive diffusion, with structural modifications that intentionally weaken their acceptance by the corresponding transporters, so that they contain structural "safety catches" that prevent them from being actively taken into cells and exerting their cytotoxic function. In this way, the modified toxins according to the present invention are blocked from opening non-specific cytotoxic activity as isolated compounds in the extracellular environment of the human body, such as when released prematurely from ADCs into the bloodstream or after release from dead cancer cells. In contrast, when the modified toxins of the present invention are bound to antibodies via the provided organic coupling groups, they are internalized by target cells as the payload of the antibodies via receptor-mediated endocytosis, wherein after intracellular transport and processing of the ADC, the toxins are unloaded and the cytotoxicity of the toxins is released to trigger tumor cell death. In this way, the present invention provides modified toxins used as ADC payloads with significantly enhanced safety features, which allows the dosage of the ADC to be increased to improve the therapeutic benefit without causing unacceptable side effects or toxicity. In addition, the safety of the modified toxins during production and processing is improved.

[0043] Toxins with infrastructure that requires transporters for cellular uptake of toxins are known to those skilled in the art. For example, the transport of toxins or infrastructure can be mediated by organic anion transporting polypeptides (OATPs) or their subtypes (e.g., OATP1B1, OATP1B3, and / or OATP1A2), respectively. These OATPs are mainly found in human livers. Therefore, the inventors have recognized the importance of blocking the transport of payload toxins by OATPs to avoid hepatotoxicity and improve the relative safety of ADCs.

[0044] Typically, the base structure can be a naturally occurring toxin or its base structure. The base structure can also be non-naturally occurring, such as a base structure of a naturally occurring toxin modified by at least one anchoring group, the anchoring group being configured for chemical conjugation with an antibody, as described in, for example, WO 2018 / 206715A2.

[0045] Preferably, the toxin is an oligopeptide toxin, i.e. the basic structure comprises an oligopeptide with several amino acids, such as 5-15 amino acids or 5-10 amino acids, in particular five, six, seven or eight amino acids. More preferably, the oligopeptide is cyclic. Preferably, the cyclic oligopeptide toxin comprises at least one non-protein amino acid.

[0046] Non-limiting examples of suitable cyanobacterial or fungal toxins in the sense of the present invention include microcystins, nodularins and amatoxins (eg amanitin).

[0047] In a preferred embodiment, the cyanobacterial toxin is microcystin. Microcystin is a cyanobacterial cyclic heptapeptide that effectively inhibits eukaryotic serine / threonine protein phosphatases type 1 and 2A, leading to the interruption of many essential signal transduction pathways and ultimately leading to the collapse of the cytoskeleton and cell death. Microcystin is primarily taken up by OATP1B1 and OATP1B3.

[0048] In other preferred embodiments, the cyanobacterial toxin is nodularin. Nodularin is a cyanobacterial cyclic pentapeptide that is evolutionarily related to microcystin, which also inhibits protein phosphatases type 1 and 2A as its cytotoxic mode of action and is primarily taken up by OATP1B1.

[0049] In yet other preferred embodiments, the fungal toxin is amanita nitin. Amanita nitin is a fungal bicyclic octapeptide belonging to the amanita toxin subgroup. The most prominent α-amanitin is an inhibitor of RNA polymerase II, which binds to the bridging helix of RNA polymerase II and inhibits the translocation of RNA and DNA required to empty the site for the next synthetic run.

[0050] In a preferred embodiment, the method includes incorporating at least one structural modification into the side chain of at least one amino acid or connecting at least one structural modification to the side chain of at least one amino acid. As used herein, at least one structural modification is incorporated into the side chain of amino acid, including such a situation, wherein the structural modification or its part is introduced into the basic structure in a synthetic or biosynthetic manner, for example, via an amino acid structural unit or by feeding an amino acid precursor containing the structural modification or its part, as described in WO2018 / 219619A1. At least one structural modification is connected to the side chain of amino acid, including such a situation, wherein the structural modification is chemically conjugated with the functional group of the side chain capable of such chemical conjugation. The functional group can be a functional group of an amino acid naturally present in the basic structure, such as an amino, carboxyl, hydroxyl, aldehyde, keto or thiol group. Functional groups can also be groups not present in the natural counterparts of the basic structure, such as functional groups artificially introduced into the basic structure, as described in WO 2018 / 219619A1. Preferably, the functional group is configured for biorthogonal conjugation, i.e., a chemical conjugation reaction that can occur inside a living system without interfering with natural biochemical processes. Preferably, the functional group is selected from the group consisting of an azide, an alkynyl, an alkenyl, a phosphine, a phosphonate, a tetrazine, a hydrazone, an oxime, and any combination thereof.

[0051] In preferred embodiments, at least one structural modification comprises a single organic moiety, which includes both an organic inhibitory group or an effector molecule, and an organic coupling group, respectively. In some of the following embodiments, the organic inhibitory group and the organic coupling group may be the same organic group or structurally overlapping organic groups that share one or more carbon atoms and / or heteroatoms. In particular, the organic coupling group may be part of the organic inhibitory group.

[0052] In other preferred embodiments, at least one structural modification comprises: a first structural modification comprising an organic inhibitory group or an effector molecule and a second structural modification comprising an organic coupling group. Thus, modifying the base structure may include incorporating the first structural modification into the side chain of a first amino acid of the base structure or connecting the first structural modification to the side chain of a first amino acid of the base structure, and incorporating the second structural modification into the side chain of a second amino acid of the base structure different from the first amino acid or connecting the second structural modification to the side chain of a second amino acid of the base structure different from the first amino acid. In the following preferred embodiments, the first structural modification comprises connecting an organic molecule containing an organic inhibitory group or an effector molecule or consisting of an organic inhibitory group or an effector molecule to the side chain of the first amino acid of the base structure, and incorporating the second structural modification containing an organic coupling group or consisting of an organic coupling group into the side chain of the second amino acid, the organic coupling group being configured for covalent conjugation of the toxin to the antibody, in particular a functional group configured for biorthogonal conjugation.

[0053] As used herein, selecting cyanobacteria or fungal toxins can be in principle a step in the brain or a step performed on a computer. On the other hand, modifying the basic structure with at least one structural modification may include a substep for manufacturing a modified toxin, such as (i) providing the basic structure, and (ii) modifying the basic structure with at least one structural modification. Advantageously, the step of modifying the basic structure with at least one structural modification can be carried out by semi-synthesis (also referred to as partial chemical synthesis), wherein the basic structure is separated from a natural source (such as cyanobacteria cell culture or fungal cell culture described in WO 2018 / 219619A1, respectively), and then at least one structural modification is chemically introduced into the basic structure to produce modified toxins. In this way, the present invention provides unprecedented access to improved toxins for the multiple structural changes of ADC.

[0054] In preferred embodiments, the organic inhibitory group comprises an organic group that carries at least one charge at physiological pH (i.e., pH 6-8) in aqueous medium at 20-40° C. In some embodiments, at least one charge is a positive charge. In some embodiments, at least one charge is a negative charge.

[0055] Preferably, the organic inhibitory group comprises an amphoteric group or a zwitterionic group, each having at least one cationic charge and at least one anionic charge at physiological pH. For example, the cationic charge can be based on primary, secondary and / or tertiary amines and quaternary ammonium cations and guanidine. The anionic moiety can be based, for example, on sulfonates, sulfinates, phenolic hydroxyls, thiols, carboxylates and / or thiocarboxylates. Preferably, the organic amphoteric group is an amino acid group, wherein at least one positive charge is based on an amino group and at least one negative charge is based on a carboxylic acid group. In some embodiments, the organic inhibitory group comprises a plurality of amino acids.

[0056] The inventors have found that the above-mentioned organic inhibitory group is an effective inhibitor of transporter, and surprisingly enhances the cytotoxic effect of the modified toxin compared with the basic structure without structural modification. Through the dual action of this synergistic effect, the modified toxin of the present invention provides an improved treatment window compared with conventional toxins, and realizes greater therapeutic benefits without causing unacceptable side effects or toxicity. In this regard, the detailed description of the embodiment set forth below is also referred to.

[0057] In a further preferred embodiment, the organic inhibitory group comprises a heterocycle, in particular a saturated or unsaturated, optionally substituted five-membered or six-membered ring having at least one or at least two heteroatoms, in particular having 1 to 3 heteroatoms selected from N, O and S. Preferably, the heterocycle contains at least one sulfur atom and / or at least one nitrogen atom in the ring. The organic inhibitory group may also comprise a ring system comprising or consisting of two saturated or unsaturated and optionally substituted fused heterocycles. For example, the organic inhibitory group may be biotin.

[0058] In a further preferred embodiment, the organic inhibiting group comprises an organic carboxamide and / or an alcohol. Suitable carboxamides and alcohols are exemplified in the detailed description of the embodiments below.

[0059] Some embodiments provide at least one structural modification comprising an effector molecule, which is an inhibitor of a known active transporter, or a functional portion or derivative of such an inhibitor. In some embodiments, the effector molecule comprises a competitive inhibitor ligand or an allosteric inhibitor ligand of a transporter, or a portion or derivative thereof, which at least partially has a competitive inhibition or allosteric inhibition function of a ligand. In particular, the effector molecule can be a known OATP inhibitor, which has specific inhibitory activity to OATP subtypes or has inhibitory activity to two or more OATP subtypes. Suitable inhibitors are known to those skilled in the art, including but not limited to pravastatin, erlotinib, cyclosporin A, rifampicin, nystatin, doxorubicin and ritonavir, as well as flavonoids and nostocyclopeptide or its functional portion or derivative.

[0060] As mentioned above, organic coupling group is a functional group, i.e. a substituent or part with characteristic chemical reactivity. Regardless of the rest of the toxin molecule modified, the same functional group will undergo the same or similar chemical reaction. This enables the chemical reaction and behavior of the modified toxin in the chemical conjugation with the antibody to be systematically predicted. Preferably, the organic coupling group can or is configured for bioorthogonal chemical coupling reaction, such as click chemistry reaction. Preferably, the organic coupling group is selected from amino, carboxyl, hydroxyl, azido, alkynyl, alkenyl, tetrazine, thiol, aldehyde, keto, oxime, hydrazine, hydrazone, phosphine, phosphonate and any combination thereof.

[0061] A second aspect of the present invention relates to a modified cyanobacterial or fungal toxin obtainable by the above method.

[0062] The cyanobacteria or fungal toxin of modification comprises a basic structure having cytotoxicity to the target cell after being taken up by the transporter of the target cell, and at least one structural modification of the basic structure. In this article, at least one structural modification comprises an organic inhibitory group or an effector molecule that reduces the toxin of modification taken into the target cell by the transporter compared with the basic structure, and an organic coupling group that is configured to make the toxin of modification and the antibody covalent conjugate.

[0063] In a preferred embodiment, the toxin or base structure, respectively, is microcystin having the general structure

[0064] Ring (-A 1 -X 2 -A 3 -Z 4 -A 5 -A 6 -A 7 ),

[0065] Among them, independently of each other, A 1 and A 3 Each represents a D-amino acid that can be modified, A 5 Selected from Adda, DM-Adda, dm-Adda, (6Z)Adda and ADM-Adda, A 6 Selected from D-Glu and D-Glu(OCH3), A 7 is selected from the group consisting of Mdha, MdhB, Dha, L-Ser, L-MeSer, Dhb, (E)-Dhb, (Z)-Dhb, MeLan, Cys and Thr or a modified L-amino acid, and X 2 and Z 4 Each of A and B is independently an L-amino acid that can be modified. 1 is selected from D-Ala, D-Leu and D-Ser or a modified D-amino acid. 3Selected from D-Asp and D-MeAsp or modified D-amino acids. D-MeAsp is D-erythro-β-methylaspartic acid, Mdha is N-methyldehydroalanine, Mdhb is N-methyldehydrobutyrate. Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-dec-4,6-dienoic acid, ADM-Adda is O-acetyl-9-O-demethyl-Adda, DM-Adda is 9-O-demethyl-Adda, dm-Adda is Adda demethylated at C-2, C-6 or C-8, Dha is dehydroalanine, Dhb is dehydrobutyrine, and MeLan is N-methyl-lanthionine. As used herein, a "modified amino acid" is an amino acid having a side chain that is not present in the natural counterpart or the basic structure of microcystin, respectively, and which comprises a chemical group covalently conjugated to at least one structural modification or a functional group configured for covalent conjugation to at least one structural modification. 1 , X 2 , A 3 , Z 4 and A 7 In a preferred embodiment, at least one of A is a modified amino acid. 1 , X 2 , Z 4 and A 7 In a more preferred embodiment, X 2 , Z 4 and A 7 At least one is a modified amino acid. The modified amino acid can be, for example, selected from azido-norvaline, propargyl tyrosine, azido-lysine, azido-phenylalanine, propargyl cysteine, propargyl serine, azido-alanine or any other amino acid that is not present in the corresponding natural cyanobacteria or fungal toxin or the basic structure and has a functional group that is configured for or can be covalently conjugated.

[0066] Microcystin is a potent inhibitor of type 1 and type 2A protein phosphatases. Protein phosphatases 1 (PP1) and 2A (PP2A) are two major phosphatases in eukaryotic cells that dephosphorylate serine and threonine residues. For example, the IC50 of naturally occurring microcystin-LR is 0.03 nM for type 1 protein phosphatases and 0.04 nM for type 2A protein phosphatases. 5 and A 6 The A position is essential for the inhibitory effect of microcystin on PP1 and PP2A. 1 , X 2 , A 3 , Z 4 and / or A7 Preferably, at least one structural modification of microcystin is provided at A 1 , X 2 , Z 4 and / or A 7 Position, most preferably in X 2 or Z 4 At least one structural modification is provided at a position. For example, at least one structural modification is incorporated into or covalently bound to an amino acid side chain at said position. Thus, the base structure may be 1 , X 2 , A 3 , Z 4 and / or A 7 Preferably, at least one of the A 1 , X 2 , Z 4 and / or A 7 Position, most preferably in X 2 and / or Z 4 Preferably, at least one structural modification is provided at at most two positions (eg at exactly one position).

[0067] In other preferred embodiments, the toxin or base structure, respectively, is a nodularin having the general structure

[0068] Ring [-A 1 -*A 2 -A 3 -A 4 -*A 5 ],

[0069] Among them, independently of each other, A 1 is D-Asp or D-MeAsp or a modified D-amino acid, A 2 is Arg or Har or a modified L-amino acid, A 3 Selected from Adda, DM-Adda, (6Z)Adda and MeAdda, A 4 Selected from D-Glu and D-Glu(OCH3), A 5 is Dhb or Mdhb or a modified L-amino acid, and "*" indicates an isopeptide bond.

[0070] It has been established in this field that A 3 and A 4 The A position is essential for the inhibitory effect of nodularin on PP1 and PP2A. 1 , A 2 and / or A 5At least one structural modification of the nodularin is provided at a position. For example, at least one structural modification is incorporated into or covalently bound to an amino acid side chain in said position. Thus, the basic structure may be 1 , A 2 and / or A 5 Preferably, at least one structural modification is provided at no more than two positions (e.g., at exactly one position). Preferably, at least one structural modification is provided at A 2 The bit provides at least one structural modification.

[0071] In yet other preferred embodiments, the toxin or base structure, respectively, is an amatoxin having the general structure

[0072] Ring {A 1 -Ring [A 2 -A 3 -A 4 -A 5 -A 6 ]-A 7 -A 8}(SEQ ID NO:1),

[0073] Among them, independently of each other, A 1 isoleucine (Ile), hydroxyisoleucine (Hyile) or dihydroxyisoleucine (Dihyile), A 2 is tryptophan (Trp) or hydroxytryptophan (Hytrp), A 3 is glycine (Glys), A 4 Isoleucine (Ile), A 5 is glycine (Glys), A 6 is cysteine ​​(Cys), A 7 is asparagine (Asn) or aspartic acid (Asp), and A 8 is proline (Pro) or hydroxyproline (Hypro), where A 2 and A 6 They are cross-linked via sulfoxide (S=O) bridges.

[0074] For example, the amatoxin can be one of the group consisting of α-amanitin, β-amanitin, γ-amanitin, δ-amanitin, ε-amanitin, amanullin, amanullinic acid, amanitinamide, amanitin and proamanullin. Preferably, the amatoxin is amanitin.

[0075] In a preferred embodiment, in A 2Hydroxytryptophan (Hytrp) or in A 8 At least one structural modification of amatoxin or amanitin is provided at the position hydroxyproline (Hypro). For example, at least one structural modification is incorporated into the amino acid side chain in said position or covalently bound to the amino acid side chain in said position, for example via a hydroxyl or amino group in the side chain. Preferably, at A 2 The hydroxytryptophan (Hytrp) at position 1 provides at least one structural modification.

[0076] The effectiveness of toxins is usually produced by the relationship between cellular uptake and inhibition, and the inhibition can be, for example, protein phosphatase inhibition. This relationship can be expressed, for example, in the form of an IC50 value, which corresponds to the toxin concentration required to kill 50% of the cells exposed to the toxin, hereinafter referred to as IC50_Cytotox. However, considering that the IC50_cytotox value depends not only on cellular uptake but also on the level of inhibition, a high IC50_cytotox value may be caused by poor cellular uptake or poor toxin inhibition or both, while a low IC50_cytotox value may be caused by high cellular uptake or strong inhibition or both, respectively. Therefore, the inventors recognize that the more appropriate characteristics of the cytotoxic properties of the modified toxins are expressed in the relationship between the IC50_cytotox value and the inhibition concentration of the toxin required to achieve half-maximal inhibition (e.g., inhibiting protein phosphatase 50%, hereinafter referred to as IC50_PPI). Therefore, if the IC50_cytotox / IC50_PPI ratio of the modified toxin is greater than the basic structure, then cellular uptake is impaired relative to the inhibitory function, which is a preferred feature of the modified toxin of the present invention. Therefore, preferred embodiments of the methods described herein comprise determining the IC50_cytotox / IC50_PPI ratio of the modified toxin and / or the basic structure, and optionally comparing or associating the IC50_cytotox / IC50_PPI of the modified toxin with the IC50_cytotox / IC50_PPI of the basic structure.Preferably, the IC50_cytotox / IC50_PPI ratio of the modified toxin is at least twice as high as the IC50_cytotox / IC50_PPI ratio of the basic structure, more preferably at least three times or at least four times as high, particularly preferably at least five times as high.The method for determining the IC50 value of an inhibitor is well known in the art.In this regard, also refer to the detailed description of the following embodiments.

[0077] In certain embodiments, at least one structural modification comprises a group B-In, wherein the group In represents an inhibitory group or an effector molecule that reduces the uptake of the toxin into the target cell by the transporter, and wherein the group B represents a bridging group that covalently bridges the group In to the toxin or the base structure, respectively. Preferably, the group B comprises a group selected from the group consisting of a triazole, a peptide bond, a disulfide, a C1 to C10 -alkyl, C1 to C 10 -Alkylaryl, (ethylene oxide) 1-10 groups, amide bonds, thioamide bonds, ether bonds, thioether bonds, covalent bonds containing triazoles, dihydropyridazines or higher amines such as secondary (2 0 )amine, tertiary (3 0 )amine or quaternary (4 0 ) amine, carbamate bond, thiocarbamate bond, urea bond, thiourea bond, phosphate bond, phosphoramide bond, sulfonamide bond, oxime bond or any combination thereof. In a preferred embodiment, group B comprises a triazole.

[0078] In some embodiments, the group In is selected from *-C1-C 10 Alkyl-NH3 + 、*-C1-C 10 Alkyl-CO2 - 、*-C1-C 10 Alkyl-CH(NH3 + )-CO2 - 、*-C1-C 10 Alkyl-CH(NH3 + )-C1-C4 alkyl-CO2 - 、*-C1-C 10 Alkyl-OC(O)-NH-C1-C6alkyl-CH(NH3 + )-C1-C4 alkyl-CO2 - 、*-C1-C 10 Alkyl-NH-CHO, *-C1-C 10 Alkyl-OH, *-C1-C 10 Isoalkyl -CH(NH3 + )-CO2 - 、*-C1-C4 alkyl-NH-biotin、*-C1-C4 alkyl-(O-CH2-CH2) 1-10 -NH-Biotin, *-C1-C4 Alkyl-(PEG) 2-10 -NH-biotin, *-C1-C4 alkyl-NH2 + -C1-C6 alkyl-NH2 + -C1-C6 alkyl-NH3 + 、*-phenyl-C1-C4alkyl-CH(NH3 + )-CO2 - ,

[0079]

[0080] Where "*" indicates the point of covalent attachment to the bridging group B, and "+" and "-" indicate the charge at physiological pH.

[0081] In certain embodiments, at least one structural modification may also include a label for in vivo visualization, such as a fluorescent label or a near-infrared dye, for example, which allows for deep tissue imaging in vivo by using luminescence in the near-infrared region.

[0082] As already described above, at least one structural modification may comprise a first structural modification comprising an organic inhibitory group or effector molecule that reduces the uptake of the toxin by the transporter into the target cell compared to the base structure (i.e. the toxin lacking the first structural modification) and a separate second structural modification comprising an organic coupling group configured for covalently conjugating the modified toxin to the antibody. In particular, the first and second structural modifications may be provided at different positions of microcystin, nodularin or amatoxin, respectively. For example, the base structure may be microcystin, wherein at X 2 The first structural modification is provided at position A 1 , A 3 , Z 4 or A 7 One of the positions provides the second structural modification, or vice versa. Alternatively, Z 4 The first structural modification can be provided at A 1 , X 2 , A 3 or A 7 Preferably, one of the positions X provides a second structural modification, or vice versa. 2 One of the first and second structural modifications is provided at Z 4 The first and second structural modifications are provided at the position of the amino acid. As described above, the first and second structural modifications can be independently connected to the amino acid side chain in the position or incorporated into the amino acid side chain in the position. In some embodiments, the first structural modification is covalently connected to the side chain of the amino acid, and the second structural modification is incorporated into the side chain of the amino acid, for example, by incorporating a modified amino acid with a side chain including the second structural modification, as described in WO 2018 / 206715A2. In particular, the second structural modification may include an alkynyl or an azido group, or any group configured to covalently couple the modified toxin to the above-mentioned antibody.

[0083] In some embodiments, the second structural modification can comprise a linker that incorporates an organic coupling group configured for covalent conjugation of the modified toxin to the antibody, or is disposed between the side chain of the amino acid and the organic coupling group.

[0084] According to the common understanding of the ADC field, a "linker" is a bifunctional or multifunctional part that can be used to covalently bridge a modified toxin with an antibody to form an antibody-drug conjugate. There are two types of linkers, the so-called "cleavable linker" and the "non-cleavable linker". Non-cleavable linkers are substantially stable outside the cell (i.e., extracellular). Cleaved linkers include predetermined cleavage sites that can be cleaved by enzyme activity, hydrolysis or other metabolic conditions. The linker can also include a spacer structure that separates the modified toxin or infrastructure from the antibody in space, respectively. The use of linkers is well known in the art, and the technician can easily select a suitable linker based on his knowledge and the present disclosure.

[0085] For example, the joint can include an organic coupling group, which is configured to be covalently coupled with any other modification of cysteine ​​thiol, amine (e.g., N-terminal or amino acid side chain such as lysine) or an antibody as described below. In some preferred embodiments, the joint is a non-cleavable joint. The joint can be substituted by a sulfonate substituent or other substituent, which can increase the water solubility of the reagent and promote the coupling reaction of the joint reagent with the antibody. The organic coupling group can be configured to be covalently coupled with a nucleophilic group on the antibody, the nucleophilic group includes but is not limited to N-terminal amino, side chain amino (e.g., side chain amino of lysine), side chain sulfhydryl (e.g., side chain sulfhydryl of cysteine) and hydroxyl or amino of carbohydrate in the case of glycosylated antibodies. Amino, thiol and hydroxyl are nucleophilic, and can react with, for example, an electrophilic group of a joint to form a covalent bond, the electrophilic group includes but is not limited to active esters (e.g., NHS esters, HOBt esters, haloformates) and acyl halides, alkyl and benzyl halides (e.g., haloacetamide), aldehydes, ketones, carboxyl and maleimide groups. Some antibodies have reducible interchain disulfides, i.e. cysteine ​​bridges. By treating with a reducing agent such as DTT (dithiothreitol), the antibody can be made reactive for conjugation with a linker. By reacting lysine with 2-iminothiolane (Traut's reagent), an amine is converted into a thiol, and other nucleophilic groups can be introduced into the antibody. Reactive thiol groups can be introduced into the antibody by introducing one, two, three, four or more cysteine ​​residues, for example, by preparing a mutant antibody comprising one or more non-natural cysteine ​​residues or by a commercially available chemical kit.

[0086] In some embodiments, the second structural modification (particularly the organic coupling group) is configured to be connected to the antibody via a click chemistry reaction. In the sense of the present invention, the click chemistry reaction is, for example, a copper (I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted alkyne-nitrone cycloaddition, olefin and azide [3+2] cycloaddition, olefin and tetrazine inverse-demand Diels-Alder reaction, olefin and tetrazole photoclick reaction. Therefore, the organic coupling group is preferably an azide, an alkyne, an olefin or a tetrazine.

[0087] In some specific embodiments, at least one structural modification is a single structural modification, that is, the organic inhibitory group comprises an organic coupling group or a portion thereof, or is composed of an organic coupling group or a portion thereof, or vice versa. In some preferred embodiments, the organic coupling group forms at least part of the organic inhibitor group, so that at least part of the organic inhibitor group is also configured to covalently conjugate the modified toxin to the antibody. In this way, when the modified toxin is covalently conjugated to the antibody in the ADC through the organic coupling group, the function of the organic inhibitory group can be deliberately weakened, at least partially weakened. However, after the conjugation bond is cleaved intracellularly or extracellularly, the organic inhibitor group is restored and prevents the released modified toxin from being subsequently internalized by healthy cells. In this way, both the internalization of the modified toxin when coupled to the antibody and the inhibition of the internalization of the modified toxin after release from the antibody are enhanced. In some of the following embodiments, the single structural modification comprises a functional group as an organic coupling group configured to be covalently linked to the antibody, wherein the functional group is selected from **-NH3 + 、**-COO2 - or **-OH, where "**" represents the point of attachment to the rest of the single structural modification, and "+" and "-" indicate the charge at physiological pH.

[0088] As mentioned above, the basic structure can be a naturally occurring toxin or a non-naturally occurring toxin. The latter can be, for example, a basic structure of a naturally occurring toxin modified by at least one functional group that is configured to be connected by chemical conjugation with a modifying molecule, wherein the chemical conjugation reaction between the functional group and the modifying molecule produces at least one structural modification. In a preferred embodiment, the at least one functional group is an azido or alkynyl group. However, the functional group can also be any functional group disclosed above.

[0089] The modified toxins described above can be advantageously used as cytotoxic drugs in antibody-drug conjugates to reduce the uptake of the toxin into cells by transporters compared to the base toxin without at least one structural modification.

[0090] Therefore, a third aspect of the present invention relates to the use of a modified toxin as described above in the manufacture of an antibody-drug conjugate, wherein the use comprises covalently conjugating the modified toxin to an antibody or fragment thereof comprising an antigen-specific binding site, thereby forming an antibody-drug conjugate.

[0091] Therefore, a fourth aspect of the present invention provides an antibody-drug conjugate, which comprises a modified toxin as described above as a drug and an antibody or a fragment thereof comprising an antigen-specific binding site, wherein the antibody or fragment thereof is linked to the modified toxin via an organic coupling group (i.e., covalently conjugated).

[0092] In some of the following embodiments, at least one structural modification is a single structural modification and the antibody is linked to the single structural modification via, for example, one of an amide, ester, ether, triazole, or carbamate linkage.

[0093] For the purposes of the present invention, the term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, bispecific antibodies or multispecific antibodies, and antibody fragments having an antigen-specific binding site (i.e., an antigen-binding site that immunospecifically binds to an antigen of a target cell or a portion thereof). Examples of antibody fragments include Fab, Fab', F(ab')2 and scFv fragments, single domain antibodies (also called nanobodies), diabodies, linear antibodies, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions) and epitope binding fragments of any of the above that immunospecifically bind to, for example, cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules, multispecific antibodies formed from antibody fragments. The antibody may be a murine antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antibody derived from another species. The antibody can be any type of immunoglobulin, such as IgG, IgE, IgM, IgD or IgA, of any class, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2, or a subclass of immunoglobulin.

[0094] In preferred embodiments, the antibody or fragment thereof is specific for a tumor associated antigen (TAA) (i.e., an anti-TAA antibody) or a tumor specific antigen (TSA) (i.e., an anti-TSA antibody). Tumor specific antigens are found only on cancer cells and not on healthy cells. Tumor associated antigens have elevated levels on tumor cells but are also expressed at lower levels on healthy cells. Such tumor associated antigens and tumor specific antigens are known in the art. Examples of TAA and TSA include, but are not limited to, BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession number NM-001203), E16 (LAT1, SLC7A5, Genbank accession number NM-003486), STEAP1 (six transmembrane epithelial antigen of the prostate, Genbank accession number NM-012449), 0772P (CA125, MUC16, Genbank accession number AF361486), MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM-005823), Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM-006424), Sema 5b(F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878), PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628), ETBR (endothelin B receptor, Genbank accession number AY275463), MSG783 (RNF124, hypothetical protein F1120315, Genbank accession number NM-017763), STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate six-transmembrane epithelial antigen 2, six-transmembrane prostate protein White, Genbank accession number AF455138), TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM-017636), CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor derived from teratocarcinoma, Genbank accession number NP-003203 or NM-003212), CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792, Genbank accession number M26004), CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29, Genbank accession number NM-000626 or 11038674), FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchoring protein 1a containing SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM-030764, AY358130), HER2 (Er bB2, Genbank accession number M11730), NCA (CEACAM6, Genbank accession number M18728), MDP (DPEP1, Genbank accession number BC017023), IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971), brevican (BCAN, BEHAB, Genbank accession number AF229053), EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession number NM-004442), ASLG659 (B7h, Genbank accession number AX092328), PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436), GEDA (Genbank accession number AY260763), AAP14954 lipoma HMGIC fusion-partner-like protein / pid=AAP14954.1 Homo sapiens (human)), BAFF-R (B cell activating factor receptor, BLy S receptor 3, BR3, Genbank accession number AF116456), BAFF receptor / pid=NP-443177.1-Homo sapiens, CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number AK026467), CD79a (CD79A, CD79α, immunoglobulin-related α), CXCR5 (Burkitt's lymphoma receptor 1), HLA-DOB (MHC β subunit of class II molecule), P2X5 (purinergic receptor P2X ligand-gated ion channel 5), CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP-001773.1), LY64 (lymphocyte antigen 64 (RP105)), FcRH1 (Fc receptor-like protein 1), IRTA2 (FcRH5, immunoglobulin superfamily receptor translocation-related 2), TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR), MUC1 (tumor-associated MUC1 glycopeptide epitope). .

[0095] In certain embodiments, the antibody-drug conjugate has the general formula

[0096] Ab-(LT) n ,

[0097] wherein Ab is an antibody or an antigen binding fragment thereof, T is a modified cyanobacterium or fungal toxin, L is an organic linker that provides a covalent bond between the antibody or its antigen binding fragment and the modified cyanobacterium or fungal toxin as defined above, and n is an integer of 1-16.

[0098] In some embodiments, n is an integer from 1 to 12. In some embodiments, n is an integer from 2 to 10. In some embodiments, n is an integer from 4 to 8.

[0099] The number "n" is also called the "drug-to-antibody ratio" (DAR).

[0100] In a preferred embodiment, the antibody-drug conjugate has a drug-antibody ratio (DAR) of 2-12, i.e., each antibody binds at least two and at most 12 modified toxins. Preferably, the DAR is 2-10, more preferably 2-8, and most preferably 4-8.

[0101] A fifth aspect of the present invention provides the modified cyanobacterial or fungal toxin or antibody-drug conjugate as described above for use in treating malignant diseases.

[0102] A sixth aspect of the invention provides a method for treating a human subject afflicted with a malignant disease, the method comprising administering to the subject a therapeutically effective amount or dosing regimen of an antibody-drug conjugate as defined above.

[0103] In certain embodiments, the malignant disease is cancer. Examples of cancers treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer, gastrointestinal stromal tumor (GIST)), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. For example, cancer can be characterized by the overexpression of HER2 or ErbB receptors.

[0104] The pharmaceutically effective amount may be, for example, 0.01-100 mg / kg body weight (mg / kg), more particularly 0.1-20 mg / kg or 1-10 mg / kg. A therapeutically effective dosing regimen may, for example, comprise administering a single dose or multiple doses of 0.01-100 mg / kg, more particularly 0.1-20 mg / kg or 1-10 mg / kg.

[0105] In a seventh aspect, the present invention provides a method for producing the modified cyanobacterial or fungal toxin as described above.

[0106] The method comprises the steps of: A) providing a toxin having a base structure known to be cytotoxic to a target cell after uptake by a transporter protein of the target cell, and B) introducing at least one structural modification into the toxin or the base structure, respectively, thereby forming a modified toxin.

[0107] In some embodiments, the toxin or the base structure provided in step A) comprises at least one first functional group configured for and / or capable of covalent conjugation, respectively, wherein in method step B), the at least one structural modification is introduced into the toxin or the base structure, respectively, via covalent conjugation to the at least one first functional group.

[0108] In some embodiments, method step B) comprises introducing at least a first and a second structural modification into the toxin or the base structure, respectively, wherein the first structural modification comprises an organic inhibitory group and the second structural modification comprises an organic coupling group configured for covalently conjugating the modified toxin to the antibody.

[0109] Furthermore, method step B) may comprise providing a modifying molecule, wherein the modifying molecule comprises an organic inhibitory group or effector molecule, respectively, and / or an organic coupling group, and covalently conjugating the modifying molecule to the at least one first functional group, thereby generating at least one structural modification.

[0110] The modified molecule may comprise a second functional group that is mutually reactive with the first functional group of the toxin or base structure provided in step A). ​​In particular, the first and second functional groups may be mutually reactive partners in any of the above-mentioned click chemistry reactions, such as an azide or a tetrazine and an alkyne or an alkene. However, the first and second functional groups may also be selected from any other mutually reactive functional groups mentioned above, such as an amino group and a carboxyl group, an aldehyde group or a ketone group.

[0111] In addition, it should be understood that the various embodiments described for one aspect of the present invention may also be applied to other aspects of the present invention. Therefore, the features and functions disclosed above and below related to the method for modifying cyanobacteria or mycotoxin may also relate to the purposes of modified cyanobacteria or mycotoxin, modified toxins in manufacturing antibody-drug conjugates, methods for manufacturing modified cyanobacteria or mycotoxin, antibody-drug conjugates and medical uses thereof, and vice versa. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1 Structural formulas of MC-(azidonorvaline)R (A), MC-(propargyltyrosine)R (B), and MC-L (azidolysine) (C) used as reference toxins with cytotoxic base structures;

[0113] Figure 2 According to the present invention, the structural formula of the modified toxin MC-(AznvaPrgam)R produced by modifying MC-(azidonorvaline)R with propargylamine is;

[0114] Figure 3 According to the present invention, a scheme for structural modification of MC-(propargyltyrosine) R;

[0115] Figure 4 According to the present invention, another structural modification scheme of MC-(propargyltyrosine) R;

[0116] Figure 5 According to the present invention, [AzProMDap] produced from MC-LR by Michael addition with 3-azidopropane-1-amine 7 The structural formula of MC-LR;

[0117] Figure 6 In one embodiment, as a dimer obtained by cross-linking two MC-PrgTyr-Arg monomers with a bridging molecule, the structural formula of the modified toxin according to the present invention is;

[0118] Figure 7 Depend on Figure 6 Structural formula of MC-dimer-linker conjugate composed of MC-dimer conjugated with a cleavable mc-Val-Ala-PAB-PNP linker. DETAILED DESCRIPTION

[0119] The invention will be explained in more detail below based on exemplary embodiments with reference to the accompanying drawings.Neither the examples nor the drawings should be considered limiting.

[0120] Embodiment 1:Production of MC-(azidonorvaline) R, MC-(propargyltyrosine) R and MC-L (azidolysine) and MC-LR as reference toxins with cytotoxic basic structures

[0121] Three reference toxins were produced that have a base structure known to be cytotoxic to cancer cell lines expressing OATP, namely Figure 1 Microcystin MC-(azidonorvaline)R or "MC-(Aznva)R" shown in A, Figure 1 MC-(propargyltyrosine)R or "MC-(Prtyr)R" shown in B and Figure 1 MC-L (azidolysine) or "MC-L(Azlys)" shown in C. Each base structure contains a 2 The functional groups of the modification moieties can be covalently coupled at Z sites, i.e., azide in MC-AznvaR and alkyne in MC-PrtyrR, or can be configured for cleavage at Z sites via click chemistry. 4 The functional group of the modified moiety is covalently coupled at position 1, i.e., the azide in MC-LAzlys. Therefore, the basic structure can be written as: cyclo(D-Ala-Aznva-D-MeAsp-R-Adda-D-Glu-Mdha) (SEQ ID NO: 2) for MC-(Aznva)R, cyclo(D-Ala-Prtyr-D-MeAsp-R-Adda-D-Glu-Mdha) (SEQ ID NO: 3) for MC-(Prtyr)R, and cyclo(D-Ala-Leu-D-MeAsp-Azlys-Adda-D-Glu-Mdha) (SEQ ID NO: 4) for MC-L(Azlys).

[0122] MC-(Aznva)R, MC-(Prtyr)R and MC-L(AzLys) are produced essentially as described in WO 2018 / 219619A1. Briefly, a cyanobacterial strain producing natural microcystin is cultured under conditions that allow the strain to grow. A non-limiting example of a suitable strain is PCC 7820, which is publicly available from Pasteur Culture Collection (PCC). During the culture period, the inorganic medium is supplemented with modified amino acids azidonorvaline (Aznva), propargyltyrosine (Prtyr) or azidolysine (Azlys). Those modified amino acids represent the X of the microcystin produced by the strain. 2 and Z 4 The modified structural unit at the position of X was incorporated into microcystin instead of the natural amino acid substrate during the cultivation of the strain, resulting in the biosynthesis of 2 and Z 4After cultivation, the biomass was collected and extracted, and the non-naturally occurring microcystins used as the base structure were purified by HPLC.

[0123] Except X 2 or Z 4 In addition to the modified microcystins MC-(Aznva)R, MC-(Prtyr)R, and MC-L(AzLys), the natural microcystin MC-LR was obtained by standard cultivation of the strain and separated by HPLC after biomass extraction.

[0124] Next, as described below, four base structures were modified according to the present invention with structural modifications comprising an organic inhibiting group and an organic coupling group.

[0125] Embodiment 2: Structural modification of MC-(Aznva)R, MC-(Prtyr)R, and MC-L(Azlys) using copper-catalyzed azide-alkyne-cycloaddition (CuAAC)

[0126] Several different structures were modified on X via copper-catalyzed azide-alkyne-cycloaddition (CuAAC). 2 The MC-(Aznva)R and MC-(Prtyr)R were introduced into the Z 4 The modified toxins were introduced into MC-L(Azlys) to provide various modified toxins according to the present invention.

[0127] The following modification molecules were used for the structural modification of MC-(Aznva)R:

[0128] Propargyl lysine (Prglys) is used as an example of structural modification using amphoteric organic repressing groups and amino and carboxyl groups as organic coupling groups;

[0129] Biotin alkyne (Albio) is used as an example of structural modification using a heterocyclic suppressing group and biotin as an organic coupling group;

[0130] Propargylamine (Prgam) is used as an example of structural modification with an inhibitory group carrying a positive charge at physiological pH and an amino group as an organic coupling group.

[0131] The following modification molecules were used for structural modification of MC-(Prtyr)R:

[0132] Azidoethanol (Azeth) is an example of structural modification using alcohol as the inhibiting group and hydroxyl as the organic coupling group;

[0133] Biotin-PEG-3-azide (Azbio) is used as an example of structural modification using a heterocyclic inhibitory group and biotin as an organic coupling group;

[0134] Azidohomoalanine (AzHala) is used as an example of structural modification using an amphiphilic organic inhibitory group and amino and carboxyl groups as organic coupling groups;

[0135] Azidophenylalanine (AzPhe) is another example of structural modification using an amphiphilic organic inhibitory group and amino and carboxyl groups as organic coupling groups;

[0136] N1-azidospermine (AzSpe) is another example of structural modification using an inhibitory group carrying a positive charge at physiological pH and an amino group as an organic coupling group;

[0137] Azidopropylcarboxamide (AzProam) is used as an example of structural modification using an organic carboxamide as both an inhibitory group and an organic coupling group;

[0138] Azidoacetic acid (Azacac) is another example of structural modification using an inhibitory group that carries a negative charge at physiological pH and a carboxyl group as an organic coupling group;

[0139] 2-amino-3-azidobutyric acid (AzAbu) is another example of structural modification using an amphoteric organic inhibitory group and amino and carboxyl groups as organic coupling groups;

[0140] 3-amino-4-azidobutyric acid (AzDbu) is another example of structural modification using an amphoteric organic inhibitory group and amino and carboxyl groups as organic coupling groups;

[0141] 3-Azido-D-alanine (AzDala) is another example of structural modification using an amphoteric organic inhibitory group and amino and carboxyl groups as organic coupling groups;

[0142] 3-Azido-L-alanine (AzLala) is another example of structural modification using an amphoteric organic inhibitory group and amino and carboxyl groups as organic coupling groups;

[0143] 6-Azido-L-lysine (Azlys) is another example of structural modification using an amphoteric organic inhibitory group and amino and carboxyl groups as organic coupling groups;

[0144] 3-Azido-propan-1-amine (Azpro) is used as an example of structural modification using an inhibitory group carrying a positive charge at physiological pH and an amino group as an organic coupling group;

[0145] MC-(Prtyr)R, thereby artificially generating microcystin dimers, is another example of structural modification using a zwitterionic organic inhibitory group (guanidinium) carrying a positive charge at physiological pH and a carboxyl group as an organic coupling group. The structural modification (i.e., dimerization) is achieved using the bridging molecules BM-I (1,4-diazidoacetylpiperazine) or BM-II (1-azidoacetyl-4-(2-azido-L-lysinyl)-piperazine), where BM-II carries an additional amino group as an organic coupling group.

[0146] The following modified molecules were used for structural modification of MC-L(Azlys):

[0147] Prgam is used as an example of structural modification with an inhibitory group carrying a positive charge at physiological pH and an amino group as an organic coupling group.

[0148] Propargyl lysine (Prglys) is used as an example of structural modification using amphoteric organic inhibitory groups and amino and carboxyl groups as organic coupling groups.

[0149] In each case, CuAAC was performed using 150 μL DMSO, 930 μL H2O, 1.5 μL 100 mM CuSO4 solution (0.5 equiv), 3.75 μL 200 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) solution (2.5 equiv), 75 μL 100 mM aminoguanidine (25 equiv), MC-(Prtyr)R (0.32 mg, 0.3 μmol, 1 equiv) or MC-(Aznval)R (0.31 mg, 0.3 μmol, 1 equiv) in 300 μL DMSO and 150 mL of the above-mentioned "clickable" modified molecules (10 mM, 1.5 μmol, 5 equiv).

[0150] The reaction was started by adding 37.5 μL of 100 mM sodium ascorbate solution (12.5 equiv.).The modified toxins were separated by C-18 solid phase extraction (SPE) or semi-preparative C-18 reverse phase (RP)-HPLC and lyophilized.

[0151] Figure 2 The structural formula of the modified toxin MC-(AznvaPrgam)R obtained by modifying MC-(azidonorvaline)R with propargylamine according to the present invention is exemplarily shown.

[0152] Embodiment 3: Synthesis of modified MC-(Prtyr)R compound 1

[0153] According to the present invention, MC-(Prtyr)R is modified with azidoacetyl-L-Lys(7-DCCA)-OH as an example of a heterocycle (structural modification with negative charge at physiological pH), wherein the organic coupling group is a carboxyl group. The structural modification also contains a fluorescent structure. The synthetic route is shown in Figure 3 , and are described in more detail below.

[0154] Synthesis of Boc-L-Lys(7-DCCA)-OH (1a): 7-(Diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester (7-DCCA-NHS; 10 mg, 27.9 μmol, 1 eq) and Boc-L-lysine (10.3, 41.9 μmol, 1.5 eq) were each dissolved in 300 μL of N,N-dimethylformamide (DMF), combined, and then 400 μL of 0.1 M sodium phosphate buffer (pH 8) was added. The reaction mixture was stirred at room temperature for 6 hours. The reaction product 1a was isolated by C-18 solid phase extraction (SPE) and lyophilized. The molecular structure of the reaction product is C 25 H 35 N3O7, and the monoisotopic mass is 489,2475 g / mol.

[0155] Synthesis of HL-Lys(7-DCCA)-OH (1b): Compound 1a (3.5 mg, 7.15 μmol, 1 equivalent) was dissolved in 700 μL of dichloromethane (DCM), followed by the addition of 300 μL of trifluoroacetic acid (TFA). The reaction was stirred at room temperature for 2 hours. The solvent was evaporated, and the remaining solid was triturated with ether. The precipitate was separated by centrifugation to obtain compound 1b, with a molecular structure of C 20 H 27 N3O5, and its monoisotopic mass is 389,1951 g / mol.

[0156] Synthesis of N-succinimidyl azidoacetic acid ester (1c): N-hydroxysuccinimide (NHS; 62.6 mg, 0.54 mmol, 1.1 equiv) was dissolved in 750 μL tetrahydrofuran (THF), and azidoacetic acid (37 μL, 0.49 mmol, 1 equiv) was added. N,N'-dicyclohexylcarbodiimide (DCC; 112.3 mg, 0.54 mmol, 1.1 equiv) was dissolved in 750 μL THF and added dropwise to the reaction mixture. After 17 hours, the reaction mixture was separated by suction filtration and the filtrate was evaporated. To further remove impurities, the product was dissolved in 20 mL DCM and extracted twice with 10 mL saturated NaHCO3 and 50 mL brine. The organic layer was dried over Na2SO4. The filtrate was evaporated to obtain 1c, with a molecular structure of C 22 H 28N6O6, and the monoisotopic mass is 472,2070 g / mol.

[0157] Synthesis of azidoacetyl-L-Lys(7-DCCA)-OH (1d): Compound 1b (2.8 mg, 7.2 μmol, 1 eq) and 1c (4.28 mg, 21.6 μmol, 3 eq) were dissolved in 500 μL DMF. Then, 500 μL 0.1 M sodium phosphate buffer (pH 8) was added and the reaction was stirred at room temperature for 16 hours. Compound 1d was isolated by semi-preparative C-18 RP-HPLC, and the molecular structure was C 22 H 28 N6O6, and the monoisotopic mass is 472,2070 g / mol, and is freeze-dried.

[0158] Synthesis of compound 1 by CuAAC: Copper-catalyzed azide-alkyne-cycloaddition (CuAAC) was performed with MC-(Prtyr)R (0.25 mg, 0.23 μmol, 1 eq.) and 1d (0.16 mg, 0.35 μmol, 1.5 eq.) using 350 μL DMSO, 75 μL H2O, 25 μL 100 mM CuSO4 solution (5 eq.), 25 μL 200 mM THPTA solution (10 eq.). The reaction was started by adding 25 μL 100 mM sodium ascorbate solution (5 eq.). The reaction product, compound 1, was isolated by semi-preparative C-18 RP-HPLC with the chemical structure C 77 H 102 N 16 O 19 , and the monoisotopic mass is 1554,7507 g / mol, and is freeze-dried.

[0159] Embodiment 4: Synthesis of modified MC-(Prtyr)R compound 2

[0160] According to the present invention, MC-(Prtyr)R is modified with HL-Lys(7-DCCA)-azidopropylamide as an example of a heterocycle (a structural modification with a positive charge at physiological pH), wherein the organic coupling group is an amino group. The structural modification also contains a fluorescent structure. The synthetic route is shown in Figure 3 , and are described in more detail below.

[0161] Synthesis of Boc-L-Lys(7-DCCA)-azidopropylamide (2a): Compound 1a (3 mg, 6.13 μmol, 1 eq), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HATU; 4.66 mg, 12.26 μmol, 2 eq) and N,N-diisopropylethylamine (DIPEA; 2.1 μL, 12.26 μmol, 2 eq) were dissolved in 1 mL of DMF and stirred in the dark for 15 min. Then, 3-azidopropylamine (0.7 μL, 6.75 μmol, 1.1 eq) was added and the solution was stirred in the dark for 16 h. Compound 2a was separated by C-18 SPE and the molecular structure was C 28 H 41 N7O6, and the monoisotopic mass is 571,3118 g / mol.

[0162] Synthesis of HL-Lys(7-DCCA)-azidopropylamide (2b): Compound 2a (2.9 mg, 5.1 μmol, 1 equivalent) was dissolved in 700 μL DCM, followed by the addition of 300 μL TFA. The reaction was stirred at room temperature for 2 hours. The solvent was evaporated, and the remaining solid was triturated with ether. The precipitate was separated by centrifugation to obtain compound 2b, with a molecular structure of C 23 H 33 N7O4, and its monoisotopic mass is 471,2594 g / mol.

[0163] Synthesis of compound 2 by CuAAC: Copper-catalyzed azide-alkyne-cycloaddition (CuAAC) was performed with MC-(Prtyr)R (0.25 mg, 0.23 μmol, 1 eq.) and 2b (0.16 mg, 0.35 μmol, 1.5 eq.) using 350 μL DMSO, 75 μl H2O, 25 μL 100 mM CuSO4 solution (5 eq.), and 25 μL 200 mM THPTA solution (10 eq.). The reaction was started by adding 25 μL 100 mM sodium ascorbate solution (5 eq.). The reaction product, compound 2, was isolated by semi-preparative C-18 RP-HPLC, and the molecular structure was C 78 H 107 N 17 O 17 , and the monoisotopic mass is 1553,8031 g / mol, and is freeze-dried.

[0164] Embodiment 5: Through Mdha 7 Michael addition of α,β-unsaturated carbonyl groups, structural modification of MC-LR

[0165] Several different structures were modified in A via Michael addition. 7 The Mdha residues are introduced into MC-LR to provide various modified toxins according to the present invention. 7 The reaction occurs between an α,β-unsaturated carbonyl group (Michael acceptor) and a nucleophile (Michael donor).

[0166] The following modification molecules were used for the structural modification of MC-LR:

[0167] Cysteine ​​(Cys) is an example of structural modification using amphoteric organic inhibitory groups and amino and carboxyl groups as organic coupling groups to produce [Mlan 7 ]MC-LR (Mlan = N-methyllanthionine);

[0168] Ammonia (NH3) is used as an example of structural modification using a positively charged inhibitory group at physiological pH and an amino group as an organic coupling group to produce [Mdap 7 ]MC-LR (Mdap = N-methyl-2,3-diaminopropionic acid);

[0169] N α -tert-Butyloxycarbonyl-L-lysine (Boc-Lys) is another example of structural modification using amphoteric organic inhibitory groups and amino and carboxyl groups as organic coupling groups, which after Boc-deprotection yields [LysMala 7 ]MC-LR(LysMala=N ε -lysinyl-N-methyl-alanine);

[0170] Tris(2-carboxyethyl)phosphine (TCEP) is another example of structural modification using an inhibitory group carrying a negative charge at physiological pH and a carboxyl group as an organic coupling group, yielding [TCEPMala 7 ]MC-LR (TCEPMala=tris(2-carboxyethyl)phosphino-N-methyl-alanine).

[0171] In each case, Michael addition was performed with MC-LR (1 eq.) in 50 μL DMSO and added to 150 μL of a 1% (w / v) K2CO3 aqueous solution containing the Michael donor (40-100 eq.). The reaction was stirred overnight at 40°C. The MC-Michael adduct obtained was acidified with 10% HCl and separated by semi-preparative C-18 RP-HPLC and lyophilized.

[0172] In the case of the Michael donor Boc-Lys, after the Michael reaction, the Boc protecting group was cleaved from the isolated product using 1 mL TFA / H2O / ACN (1:1:1, v / v / v) at room temperature for 1-2 hours. The final product [LysMala 7 ]MC-LR and freeze-dried.

[0173] Embodiment 6: Using copper-catalyzed azide-alkyne-cycloaddition (CuAAC), [AzProMDap 7 Structural modification of MC-LR

[0174] As described in Example 5 above, 3-azido-propan-1-amine (Azpro) was reacted with A via Michael addition. 7 The bit is introduced into MC-LR, generating [AzProMDap 7 ]MC-LR (AzProMdap = 3-N-azidopropyl-2-N-methyl-2,3-diaminopropionic acid).

[0175] Then, [AzProMDap was further modified using CuAAC with Prglys as an example of structural modification with amphoteric organic inhibitory groups and amino and carboxyl groups as organic coupling groups. 7 ]MC-LR.

[0176] As described in Example 2 above, [AzProMDap 7 ]CuAAC between MC-LR and Prglys, thus generating [AzPro(Prglys)Mlap 7 ]MC-LR.

[0177] Embodiment 7: Protocol for cell culture of cancer cell lines expressing OATPs and subsequent cytotoxicity assays

[0178] HEK293 cells stably transfected with the expression vectors pcDNA3.1(+)-OATPB1 and pcDNA3.1 / Hygro(-)-OATP1B3, and the respective empty vectors pcDNA3.1(+) and pcDNA3.1 / Hygro(-) as controls were prepared by Prof.Dr. Kindly provided by (Friedrich-Alexander-University Erlangen-Nürnberg, Germany).

[0179] All cell lines were maintained in minimal essential medium supplemented with 10% heat-inactivated fetal calf serum, a non-essential amino acid mix and 2 mM glutamine at 37°C and 5% CO2.

[0180] HEK293OATP1B1+ and corresponding control cell lines were constantly selected with 800 μg / mL geneticin (G418), while 250 μg / mL hygromycin B was used to select HEK293OATP1B3+ and its empty vector control.

[0181] To determine the cytotoxicity of the modified MC-(Aznva)R and MC-(Prtyr)R toxins described in Examples 2-4, 5×10 cells of each cell line were plated per well. 4 Cells were seeded in triplicate in 96-well plates without a selection marker. After 24 h, transporter expression was induced by the addition of 10 mM sodium butyrate. The next day, the medium was removed and the cells were incubated for 48 h with eight different concentrations of each modified toxin (0.01 nM to 3 μM) and unmodified MC-(Aznva)R and MC-(Prtyr)R as controls in medium without a selection marker.

[0182] Then, as described above, the cells were fixed, washed and stained with sulforhodamine B (SRB) (Vichai et al., Nature protocols 2006, 1, 1112-1116). In brief, 10 μL trichloroacetic acid (10%) was added directly to each well and incubated at 4°C for 1 hour. Then, the solution was removed and each well was washed three times with 200 μL H2O. Then, the wells were dried and 100 μL 0.057% SRB-solution (in 1% acetic acid) was added to each well and incubated at 4°C for 30 minutes. The solution was removed and each well was washed three times with 200 μL acetic acid (1%). After the wells were dried, 200 μL Tris buffer (10 mM, pH 10.5) was added and the plate was shaken for five minutes. The absorbance of SRB was then measured at 510 nm using a TECAN Infinity TECAN Infinity M PLEX plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). Experiments were performed at least in duplicate.

[0183] Cell viability and IC were calculated using GraphPad PRISM 6 using nonlinear regression (sigmoidal dose response). 50 Value (IC50_cytotox).

[0184] Embodiment 8:Protocol for Protein Phosphatase Inhibition (PPI) Assay

[0185] The PPI assay was performed as recently described (Heresztyn et al., Water Res 2001, 35, 3049-3056).

[0186] Before the measurement, different working solutions were prepared. The reaction buffer was freshly prepared by combining Tris buffer (250 mM, pH 8.1), magnesium chloride solution (200 mM), manganese chloride solution (10 mM) and bovine serum albumin (BSA) solution (5 mg / ml) (50:26:4:20 (v / v)) and stored on ice. For the substrate solution, the reaction buffer was mixed with p-nitrophenyl phosphate (pNPP) stock solution (60 mM) and DTT solution (20 mM) (5:4:1, (v / v)), thereby freshly preparing pNPP. Enzyme dilution buffer was obtained by combining solutions of ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA; 0.19 mM in Tris buffer (80 mM, pH 7.0)), manganese chloride (10 mM), dithiothreitol (DTT; 20 mM) and BSA (5 mg / ml) (65:20:5:10 (v / v)). For the enzyme solution, protein phosphatase-1 catalytic subunit (α-isoform from rabbit, Sigma-Aldrich) was diluted to 10 units / mL with enzyme dilution buffer and stored on ice.

[0187] The assay was performed in a 384-well plate. Diluted modified toxins and unmodified MC-(Aznva)R, MC-(Prtyr)R, MC-L(Azlys) and MC-LR (4 μL in dimethyl sulfoxide (DMSO)) were pre-incubated with enzyme solution (4 μL) at 37°C for 5 minutes. Then, 40 μL of substrate solution was added to each well and incubated at 37°C for 2 hours. The seven toxin concentrations ranged from 0.001 nM to 1 μM, while a constant substrate concentration of 20 mM was used per well. As a positive control, 4 μL of DMSO was used instead of MC solution to track substrate conversion without PPI. The negative control (blank) contained 4 μL of enzyme dilution buffer instead of the diluted enzyme to prevent any hydrolysis of the substrate. To determine the PPI, the absorbance was measured at 405 nm using a TECAN Infinity M PLEX plate reader. Experiments were performed at least in duplicate, and IC was calculated using nonlinear regression (sigmoidal dose response) with GraphPad PRISM 6. 50 Value (IC50_PPI).

[0188] Embodiment 9: Bioassay results

[0189] Tables 1-5 below summarize the results of the assays described in Examples 7 and 8 above, respectively, for the basic structures MC-(Aznva)R (Table 1), MC-(Prtyr)R (Tables 2-3), MC-L(Azlys) (Table 4) and MC-LR (Table 5) according to the invention and the corresponding modified toxins derived therefrom, for the general cytotoxicity (IC50_Cytotox) and protein phosphatase inhibition assays (IC50_PPI) for cell lines expressing OATP1B1 and cell lines expressing OATP1B3. The table also shows the ratio of the IC50_Cytotox and IC50_PPI values ​​of the modified toxins and the ratio of these IC50 values, as well as their corresponding basic structures, for evaluating whether the structural modification shows the desired effect. In this context, if the IC50_Cyototox / IC50_PPI ratio of the modified toxin is greater than the IC50_Cyototox / IC50_PPI ratio of the basic structure, it means that the therapeutic window of the modified toxin is enhanced relative to the cell line. Such enhancement of the therapeutic window occurs when the cellular uptake of the modified toxin is reduced relative to protein phosphatase inhibition and / or when the protein phosphatase inhibition of the modified toxin is increased relative to cellular uptake. In addition, if the ratio of the IC50_PPI of the modified toxin and the IC50_PPI of the basic structure is <1, it is shown that the potency of the cytotoxic mode of action of the modified toxin is enhanced compared to the basic structure, based on inhibition of the corresponding protein phosphatase.

[0190] The results of the structural modification of the base structure microcystin MC-(Aznva)R (Table 1) show that for all modified microcystins according to the invention the therapeutic window of both OATP expressing cell lines could be enhanced compared to the base structure. Furthermore, the PPI of two of the three modified microcystins was improved, as indicated by their lower IC50_PPI compared to that of the base structure, while the IC50_PPI of the third modified microcystin remained unaffected by the structural modification.

[0191] The results of the structural modifications of the basic structure MC-(Prtyr)R (Tables 2-3) show that for all modified microcystins according to the present invention, the therapeutic window of at least one OATP-expressing cell line can be enhanced compared to the basic structure. Four structural modifications increase the therapeutic window of one of the two OATP-expressing cell lines, while 14 structural modifications increase the therapeutic window of both OATP-expressing cell lines.

[0192] Furthermore, the results show that the PPI of the eight modified microcystins according to the present invention can be improved, which is indicated by their lower IC50_PPI compared to that of the base structure.

[0193] The results of the structural modifications of the base structure MC-L (Azlys) (Table 4) show that the therapeutic window of both OATP-expressing cell lines can be enhanced for the two modified microcystins according to the invention compared to the base structure.

[0194] Furthermore, the results show that the PPI of the two modified microcystins according to the present invention can be improved, which is indicated by their lower IC50_PPI compared to that of the base structure.

[0195] The results of the structural modifications of the base structure MC-LR (Table 5) show that for all modified microcystins according to the invention the therapeutic window for both OATP expressing cell lines could be enhanced compared to the base structure.

[0196] In addition, compared with the base structure, [AzPro(Prlys)Mlap 7 ]The PPI of MC-LR was improved, as indicated by a lower IC50_PPI compared to that of MC-LR.

[0197] Thus, using the modified toxins according to the invention, an increase of up to 2400-fold, ie more than three orders of magnitude, of the therapeutic window was achieved. These improvements and the advantages associated therewith could not have been anticipated by a person skilled in the art.

[0198] Embodiment 10: ADC synthesis

[0199] According to a further embodiment of the present invention, the monoclonal antibody (mAb) trastuzumab (trade name: Herceptin) targeting Her2 (human epidermal growth factor receptor 2 = ERBB2, erb-b2 receptor tyrosine kinase 2) is used as a representative benchmark mAb in ADC development, and a homodimer of MC-PrgTyr-Arg ("MC-dimer") is used as a representative example of the modified microcystin according to the present invention as a payload to synthesize ADC. The MC-dimer is conjugated to the mAb using a cleavable linker.

[0200] By combining two MC-PrgTyr-Arg monomers using their alkyne groups as 2 The coupling functionality at the 5′ position of MC-PrgTyr-Arg was cross-linked with a trifunctional bridging moiety BM-II having two terminal azide groups as complementary coupling functionalities to form a covalent link between MC-PrgTyr-Arg monomers to produce MC-dimers.

[0201]

[0202] The copper-catalyzed azide-alkyne-cycloaddition (CuAAC) reaction of two MC-PrgTyr-Arg monomers in X 2 The cross-linking between the alkyne coupling functionality at the position and the two terminal azide groups of the bridging part BM-II as complementary coupling functionality. Briefly, CuAAC was performed using a reaction mixture of 2 equivalents (eq.) of monomer, 1 equivalent of the bridging part BM-II, 10 equivalents of 100 mM CuSO4 aqueous solution and 20 equivalents of 200 mM tris((1-hydroxy-propyl-1H-1,2,3-triazol-4-yl)methyl)amine (THPTA) aqueous solution in DMSO. The reaction was started by adding 10 equivalents of 100 mM sodium ascorbate aqueous solution. The reaction products were separated by semi-preparative C-18 RP-HPLC and lyophilized. The structural formula of MC-dimer is shown in Figure 6 middle.

[0203] To produce monoclonal antibodies, genes encoding the heavy and light chain variable regions of trastuzumab are synthesized and cloned into human heavy and light chain vectors encoding human IgG1. Both constructs are verified by DNA sequencing. Vector DNA is prepared for transient transfection in CHO cells. mAbs are purified from cell culture supernatants (e.g., by protein A) with a targeted purity of >90%. Purified mAbs are quantified by absorption at 280 nm and characterized by SEC and SDS-PAGE.

[0204] MC-dimer-linker conjugates were obtained by conjugating MC-dimer with a cleavable maleimidocaproyl-Val-Ala-(p-amino-benzyl)-(p-nitro-phenyl)-carbonate (mc-Val-Ala-PAB-PNP) linker by incubating 1 equiv of MC-dimer with 1 equiv of linker and DIPEA (2 equiv) in DMF for 1-3 hours at 25°C. In this way, the amino groups incorporated into MC-dimer via the bridging moiety BM-I were coupled to the mc-Val-Ala-PAB-PNP linker by carbamate bond formation between the activated carbonate on the linker and the amino groups of MC-dimer, thereby generating mc-Val-Ala-PABC-MC-dimer (PABC = p-amino-benzyl carbamate). The produced MC-dimer-linker conjugates were separated by RP-HPLC and their identity was confirmed by mass spectrometry. The linker-MC-dimer conjugate was then purified by RP-LC, aiming for a purity of >95%. Figure 7 The structural formula of the MC-dimer-linker conjugate is shown.

[0205] To synthesize ADC, the native (non-engineered) mAb trastuzumab was dissolved in 5% tris (hydroxymethyl) aminomethane (TRIS) buffer (0.5 M, supplemented with 25 mM ethylenediaminetetraacetic acid (EDTA), pH 8.5) at a final concentration of 26.5 mg / mL. For mAb reduction, 1 equivalent of tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 10 mM) was added for a final DAR of about 2, or 2 equivalents of tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 10 mM) was added for a final DAR of about 4. The reaction was incubated at room temperature for 1.5 hours and then diluted to a final concentration of 5 mg / mL with 2 mM EDTA (pH 7.4) in PBS.

[0206] The buffer was exchanged and the solution was desalted by gel filtration on a NAP column (Zeba spin) with PBS containing 2 mM EDTA for DAR of about 2 and with an additional 3% cyclodextrin (CD) for DAR of about 4. To conjugate the MC-dimer-linker conjugate to the reduced mAb with a DAR of about 2, 4 equivalents of MC-dimer-linker from a 10 mM solution of MC-dimer-linker in dimethylacetamide (DMA) were added to the reduced mAb (5 mg / mL in PBS with 2 mM EDTA) and the reaction was incubated for 2 hours at room temperature. For a DAR of about 4, 8 equivalents of linker-MC dimer were used and added to the reduced mAb (5 mg / mL, 3% CD in PBS with 2 mM EDTA) and incubated for 3.5 hours at room temperature. To improve solubility, a final concentration of 5% DMA was used for a DAR of 2 and a final concentration of 10% DMA was used for a DAR of 4. The conjugation reaction was quenched for 30 minutes by adding an equimolar amount of N-acetyl-L-cysteine ​​(NAC) relative to the MC-dimer-linker. Then, the reaction mixture was desalted by gel filtration on a NAP column, eluted with PBS (DAR 2) or PBS with 3% CD (DAR 4). Then, activated carbon (100 mg / mL, in PBS or in PBS with 3% CD, pH 7.4) was added in an amount equal to the amount of mAb. After 1 hour, the activated carbon was removed by centrifugation (4000g, 15 minutes). The supernatant containing ADC was analyzed by SEC and HIC and sterile filtered.

[0207] Cell viability assays using cancer cell lines expressing tumor antigens (e.g., NCI-N87 expressing the Her2 antigen), such as using the CellTiter-Glo Luminescent Cell Viability Assay (Promega GmbH, Walldorf, Germany), tested the in vitro potency of the ADCs, showing that when bound to the antibody, the modified toxins of the invention were selectively internalized by target cells, where they effectively triggered tumor cell death, e.g., achieving EC50 values ​​in the picomolar range.

[0208] The results demonstrate that the present invention provides modified toxins for use as payloads in ADCs with significantly enhanced safety profiles, thereby improving the therapeutic benefit of ADCs without causing unacceptable side effects or toxicity.

[0209] The present invention is not limited by the description based on the embodiments. On the contrary, the present invention covers every new feature and every new combination of features, which particularly includes every combination of features in the patent claims and the description, even if the feature or the combination of features is not explicitly defined in the patent claims, the description or the embodiments.

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

Claims

1. A method for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody-drug conjugate, the method comprising the steps of: - selecting cyanobacterial or fungal toxins having a basic structure known to be cytotoxic to target cells after uptake by transport proteins of said target cells, - modifying said base structure with at least one structural modification, wherein the at least one structural modification comprises an organic inhibitory group or effector molecule that reduces uptake of the modified toxin into the target cell by the transporter compared to the base structure, and An organic coupling group configured to covalently conjugate the modified toxin to the antibody.

2. The method of claim 1, wherein the base structure comprises a cyclic oligopeptide having a plurality of amino acids, and Wherein modifying the base structure comprises incorporating the at least one structural modification into the side chain of at least one of the amino acids or attaching the at least one structural modification to the side chain of at least one of the amino acids.

3. The method according to any one of claims 1 or 2, wherein the base structure is a derivative of a naturally occurring toxin, wherein the derivative comprises a modification of the naturally occurring toxin having at least one functional group as described below, wherein the at least one functional group is configured for or capable of chemical conjugation to further modify the base structure with the at least one structural modification.

4. The method according to any one of claims 1 to 3, wherein the base structure is microcystin having the general formula Ring(-A 1 -X 2 -A 3 -Z 4 -A 5 -A 6 -A 7 ) in, Independently of each other, A 1 and A 3 Each represents a D-amino acid, A 5 Selected from Adda, DM-Adda, dm-Adda, (6Z)Adda and ADM-Adda, A 6 Selected from D-Glu and D-Glu(OCH3), A 7 is selected from the group consisting of Mdha, MdhB, Dha, L-Ser, L-MeSer, Dhb, (E)-Dhb, (Z)-Dhb, MeLan, Cys and Thr or a modified L-amino acid, and X 2 and Z 4 are independently L-amino acids, Among them A 1 , X 2 , A 3 , Z 4 and A 7 At least one of the microcystin molecules is a modified amino acid having a side chain that is not present in the natural counterpart of the microcystin, wherein the side chain comprises a functional group configured for or capable of covalently conjugating the at least one structural modification.

5. The method of any one of claims 1-4, wherein the at least one structural modification comprises an organic moiety comprising both the organic inhibiting group and the organic coupling group.

6. The method of claim 2, wherein the at least one structural modification comprises: a first structural modification comprising the organic inhibitory group or effector molecule and a second structural modification comprising the organic coupling group, and wherein modifying the base structure comprises incorporating the first structural modification into the side chain of a first amino acid or attaching the first structural modification to the side chain of a first amino acid, and incorporating the second structural modification into the side chain of a second amino acid different from the first amino acid or attaching the second structural modification to the side chain of a second amino acid different from the first amino acid.

7. The method according to any one of claims 1 to 6, wherein the organic inhibitory group carries at least one charge at physiological pH. The method of claim 7 , wherein the at least one charge is a positive charge.

9. The method of claim 7, wherein the at least one charge is a negative charge.

10. The method of any one of claims 1 to 9, wherein the organic inhibitory group comprises an amphoteric group carrying at least one cationic charge and at least one anionic charge at physiological pH.

11. The method of claim 10, wherein the amphiphilic group is an amino acid group.

12. The method of any one of claims 1-11, wherein the organic inhibitory group comprises a plurality of amino acids.

13. The method according to any one of claims 1 to 12, wherein the effector molecule comprises a competitive inhibitor ligand or an allosteric inhibitor ligand of the transporter or a functional part thereof.

14. The method according to any one of claims 1 to 13, wherein the organic coupling group is selected from the group consisting of amino, carboxyl, hydroxyl, azido, alkynyl, alkenyl, thiol, aldehyde, ketone, tetrazine, and any combination thereof.

15. A modified cyanobacterial or fungal toxin comprising: A base structure that is cytotoxic to a target cell after being taken up by a transporter protein of the target cell, and at least one structural modification of the base structure, wherein the at least one structural modification comprises an organic inhibitory group or effector molecule that reduces uptake of the modified toxin into the target cell by the transporter compared to the base structure, and An organic coupling group configured for covalently conjugating the toxin to the antibody.

16. The modified toxin of claim 15, wherein the at least one structural modification comprises a group B-In, wherein "In" represents the inhibitory group or the effector molecule that reduces the uptake of the toxin into the target cell by the transporter, and wherein "B" represents a bridging group that covalently bridges the inhibitory group or effector molecule to the base structure.

17. The modified toxin of claim 16, wherein the bridging group B comprises a group selected from the group consisting of a triazole, a peptide bond, a disulfide, a C1 to C 10 -alkyl, C1 to C 10 -Alkylaryl, (ethylene oxide) 1-10 groups, amide bonds, thioamide bonds, ether bonds, thioether bonds, covalent bonds containing triazoles, dihydropyridazines or higher amines such as secondary (2 0 )amine, tertiary (3 0 )amine or quaternary (4 0 ) an amine, a carbamate bond, a thiocarbamate bond, a urea bond, a thiourea bond, a phosphate bond, a phosphoramide bond, a sulfonamide bond, an oxime bond, or any combination thereof.

18. The modified toxin according to any one of claims 16 or 17, wherein the bridging group B comprises a triazole.

19. The modified toxin according to any one of claims 15-18, wherein the base structure is selected from microcystin, nodularin or amanitin.

20. The modified toxin according to any one of claims 15-19, wherein the modified toxin and the base structure are inhibitors of protein phosphatases of the target cell, and wherein the modified cytotoxin has an IC50_cytotox / IC50_PPI ratio greater than the IC50_cytotox / IC50_PPI ratio of the base structure, wherein IC50_cytotox is the IC50 value of the modified toxin or the base structure relative to the target cell, respectively, and IC50_PPI is the IC50 value of the modified toxin or the base structure relative to the protein phosphatase, respectively.

21. Use of a modified toxin according to any one of claims 15 to 20 in the manufacture of an antibody-drug conjugate, the antibody-drug conjugate comprising covalently conjugating the modified toxin to an antibody or fragment thereof comprising an antigen binding site.

22. A method for producing a modified cyanobacterial or fungal toxin according to any one of claims 15-20, comprising the steps of: A) providing a toxin having a basic structure known to be cytotoxic to target cells after being taken up by a transporter protein of said target cells and at least one functional group capable of covalent conjugation, B) introducing said at least one structural modification into said toxin via covalent conjugation to said functional group of said toxin.

23. An antibody-drug conjugate comprising a modified toxin according to any one of claims 15 to 22 and an antibody or fragment thereof comprising an antigen binding site, wherein the antibody or fragment thereof is linked to the modified toxin via the organic coupling group.

24. A method for producing an antibody-drug conjugate, the method comprising the steps of: a) providing a monoclonal antibody or a fragment thereof comprising an antigen-specific binding site and a modified cyanobacterial or fungal toxin according to any one of claims 15 to 20, b) covalently conjugating the modified toxin to the antibody via the organic coupling group.

25. A method for producing an antibody-drug conjugate, the method comprising the steps of: a) providing a toxin having (i) a base structure known to be cytotoxic to a target cell after uptake by a transporter protein of the target cell, and (ii) at least a first functional group and a second functional group configured for or capable of covalent conjugation, b) introducing at least one structural modification into the toxin via covalent conjugation to the first functional group, wherein the at least one structural modification comprises an organic inhibitory group or an effector molecule that reduces uptake of the modified toxin into the target cell by the transporter compared to the base structure, and c) covalently conjugating the toxin to the antibody via the second functional group, thereby producing the antibody-drug conjugate.

26. The modified cyanobacterial or fungal toxin according to any one of claims 15 to 20 or the antibody-drug conjugate according to claim 23 for use in the treatment of a malignant disease.

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