Methods for modifying organic cytotoxins for use as payloads in antibody drug conjugates and modified organic cytotoxins derived therefrom

Abstract

The present invention provides a method for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody-drug conjugate, comprising the steps of selecting a cyanobacterial or fungal toxin having a basic structure known to be cytotoxic to a target cell after uptake by a transport protein of the target cell, and modifying the basic structure with at least one structural modification, the at least one structural modification including an organic inhibitory group or effector molecule that reduces uptake of the modified toxin into the target cell by the transport protein compared to the basic structure, and an organic coupling group configured for covalently conjugating the modified toxin to an antibody. The present invention further provides the modified toxin obtained by this method and uses thereof.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This patent application claims the benefit of European Patent Application No. 22192517.5, filed August 27, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] (Sequence Listing) This application contains as part of the description an electronic sequence listing containing four sequences in XML format in accordance with the WIPO ST.26 standard, the contents of which are incorporated herein by reference in their entirety.

[0003] (Technical field) 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, used as payloads in antibody-drug conjugates. The present invention further relates to antibody-drug conjugates comprising a biologically active, cytotoxic anticancer drug payload bound to an antibody that specifically targets a particular tumor antigen. [Background technology]

[0004] Cancer is currently the second leading cause of death in the world, accounting for approximately 10 million deaths annually. Due to global population growth and aging, the incidence of cancer has been increasing over the past few decades and is expected to continue to rise.

[0005] Most of the anticancer drugs currently used in conventional chemotherapy have the drawback of being unable to sufficiently distinguish between cancer cells and healthy tissues, and therefore are generally cytotoxic, with known severe side effects and significant limitations on the quality of life of patients.

[0006] The need for anti-cancer drugs with specific spectra of action against malignant cells has driven the development of antibody-drug conjugates.

[0007] Antibody-drug conjugates, also referred to below as "ADCs," are a type of biopharmaceutical designed as targeted therapies for cancer treatment. Unlike chemotherapy, ADCs aim to target and kill tumor cells while sparing healthy cells. ADCs are biopharmaceuticals consisting essentially of a monoclonal antibody conjugated to a biologically active, often cytotoxic, drug, also referred to below as a "payload," thereby combining the targeting specificity of a monoclonal antibody with the cancer-killing potential of a cytotoxic drug.

[0008] Conceptually, ADCs are supposed to target tumor cells and protect healthy tissues with the targeting specificity of monoclonal antibodies. However, in clinical applications, off-target effects are often observed, for example, due to spontaneous or enzymatic hydrolysis of the chemical bond between the drug and antibody during transport and metabolism after systemic administration, or release of the payload into surrounding tissues from lysed or killed cancer cells.

[0009] Current efforts to reduce or eliminate unwanted toxic side effects on healthy cells are primarily directed at 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.

[0010] For example, Patent Publications WO 2018 / 206715 and WO 2018 / 219619, the disclosures of which are incorporated herein by reference in their entirety, disclose modified cyanobacterial microcystin and nodularin as cytotoxic payloads that incorporate, inter alia, non-naturally occurring amino acids that provide chemical anchor groups for attaching the payload to a linker or antibody, respectively, thereby resulting in homogeneously and stably loaded ADCs that limit release of the payload into the bloodstream.

[0011] However, improving the chemical linkage between antibodies and cytotoxic payloads only partially overcomes the drawbacks of current ADCs. In particular, the payload release problem persists after the death of the target cancer cells. Furthermore, the high potency of cytotoxins poses unresolved health and safety risks during manufacturing and handling, leading to costly infrastructure and production.

[0012] Against this background, it is an object of the present invention to provide improved cytotoxins for use as payloads in antibody drug conjugates. Another object of the present invention is to provide improved antibody drug conjugates.

[0013] 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 description below. Summary of the Invention

[0014] In certain embodiments, a method is provided for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody-drug conjugate, comprising: selecting a cyanobacterial or fungal toxin having a basic structure known to be cytotoxic to a target cell after uptake by a transport protein of the target cell; and modifying the basic 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 transport protein compared to the basic structure; and an organic coupling group configured for covalently conjugating the modified toxin to an antibody.

[0015] In certain embodiments, a method is provided for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody-drug conjugate, the method comprising: selecting a cyanobacterial or fungal toxin having a basic structure known to be cytotoxic to a target cell after uptake by a transport protein of the target cell; and modifying the basic structure with (i) at least one organic inhibitory group or effector molecule that reduces uptake of the modified toxin into the target cell by the transport protein compared to the basic structure, and (ii) an organic coupling group configured for covalently conjugating the modified toxin to an antibody.

[0016] In certain embodiments, the present invention provides a method for producing a modified cyanobacterial or fungal toxin, comprising the steps of: (A) providing a toxin having a basic structure known to be cytotoxic to a target cell after uptake by a transport 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 uptake of the modified toxin into the target cell by the transport protein compared to the basic structure; and (ii) 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 producing a modified cyanobacterial or fungal toxin, comprising the steps of: (A) providing a toxin having a basic structure known to be cytotoxic to a target cell after uptake by a transport protein of the target cell and (ii) at least one functional group configured for and / or capable of covalent conjugation; and (B) introducing at least one structural modification into the toxin via covalent conjugation to the functional group of the toxin, wherein the at least one structural modification comprises: (i) an organic inhibitory group or effector molecule that reduces uptake of the modified toxin into the target cell by the transport protein compared to the basic structure; and (ii) an organic coupling group configured to covalently compete the modified toxin for an antibody.

[0018] In certain embodiments, the invention provides modified cyanobacterial or fungal toxins comprising a base structure that has cytotoxicity to a target cell after uptake by a transport 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 transport protein compared to the base structure, and an organic coupling group configured for covalently conjugating the organic cytotoxin to an antibody.

[0019] In certain embodiments, the present invention provides the use of the above-described modified toxin in the manufacture of an antibody drug conjugate comprising covalently conjugating the modified toxin via an organic coupling group to an antibody or fragment thereof comprising an antigen-specific binding site.

[0020] In certain embodiments, the present invention provides a method for producing an antibody-drug conjugate, comprising the steps of: (a) providing a monoclonal antibody or fragment thereof comprising an antigen-specific binding site and a toxin as described above; and (b) covalently conjugating the toxin to the antibody via an organic coupling group.

[0021] In certain embodiments, the present invention provides a method for producing an antibody-drug conjugate, comprising: (a) providing a toxin having (i) a base structure known to be cytotoxic to a target cell after uptake by a transport 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 to 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 transport protein compared to the base structure; and (c) covalently conjugating the toxin to an antibody via the second functional group, thereby producing the antibody-drug conjugate.

[0022] In certain embodiments, the present invention provides an antibody drug conjugate comprising the modified toxin described above and an antibody or fragment thereof comprising an antigen-binding site, wherein the antibody or fragment thereof is attached to the modified toxin via an organic coupling group.

[0023] In certain embodiments, the present invention provides a modified cyanobacterial or fungal toxin, or an antibody drug conjugate as defined above, for use in the treatment of malignant disease.

[0024] In certain embodiments, the present invention provides a method of treating a human subject suffering from a malignant disease, the method comprising administering to the subject a therapeutically effective dosing regimen of an antibody drug conjugate as defined above.

[0025] In some embodiments according to or applied to any of the above embodiments, the basic structure comprises an oligopeptide, in particular a cyclic oligopeptide, more particularly a cyclic oligopeptide comprising at least one non-proteinogenic amino acid.

[0026] In some embodiments according to or as applied to any of the above embodiments, the modified toxin or base structure, respectively, is selected from the group consisting of an amatoxin, such as microcystin, nodularin, or amanitin. In particular embodiments, the modified toxin or base structure, respectively, is microcystin.

[0027] In some embodiments according to or as applied to any of the above embodiments, the base structure is a naturally occurring toxin.

[0028] In some embodiments according to or adapted from any of the above embodiments, the base structure is non-naturally occurring, in particular the base structure of a naturally occurring toxin modified with at least one functional group, preferably one or two functional groups, configured for or capable of chemical conjugation with another organic molecule, such as an antibody or modified molecule.

[0029] In some embodiments according to or applied to any of the above embodiments, the base structure has the general formula cyclo(-A 1 -X 2 -A 3 -Z 4 -A 5 -A 6 -A 7 ) and a microcystin having the formula: Independently of each other, A 1 and A 3 each represents a D-amino acid, and A 5 is selected from the group consisting of Adda, DM-Adda, dm-Adda, (6Z)Adda, and ADM-Adda; 6 is selected from the group consisting of D-Glu and D-Glu(OCH3), and 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 modified L-amino acids; and X 2 and Z 4 are independently L-amino acids, and A 1 , X 2 , A 3 , Z 4 , and A 7 at least one of which is a modified amino acid having a side chain not present in the natural counterpart of microcystin, the side chain comprising a functional group configured or capable of covalently conjugating at least one structural modification.

[0030] In some embodiments according to or applied to any of the above embodiments, the chemical bond is a bioorthogonal chemical bond, preferably comprising a click chemistry reaction.

[0031] In some embodiments according to or as applied to any of the above embodiments, the organic inhibitory group is selected from the group consisting of an organic group that carries at least one charge at physiological pH values, a heterocycle such as biotin, an organic formamide, an alcohol, or any combination thereof.

[0032] In some embodiments according to or applied to any of the above embodiments, the transport protein comprises an organic anion transporting polypeptide (OATP), in particular OATP1B1 and / or OATP1B3.

[0033] In some embodiments according to or applied to any of the above embodiments, the target cell is a mammalian cell, particularly a cancer cell.

[0034] Preferred variations of these embodiments can be derived from the description of the invention and the detailed description of the preferred embodiments.

[0035] Description of the Invention A first aspect of the present invention relates to a method for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody drug conjugate.

[0036] The present invention provides a method comprising the steps of selecting a cyanobacterial or fungal toxin having a basic structure known to be cytotoxic to a target cell after uptake by a transport protein of the target cell, and modifying the basic structure with at least one structural modification, whereby the toxin is modified. Provided herein is a method comprising the steps of: selecting a cyanobacterial or fungal toxin having a basic structure known to be cytotoxic to a target cell after uptake by a transport protein of the target cell; and modifying the basic structure with at least one structural modification, where 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 transport protein, compared to the basic structure; and an organic coupling group configured for covalently conjugating the modified toxin to an antibody.

[0037] The present invention is based on the inventors' insight that, while cytotoxins generally must be taken up into cells either via active transport by transport proteins or by passive diffusion before they can exert their cytotoxic function, when conjugated to an antibody, internalization of the cytotoxin is altered to receptor-mediated endocytosis, such as clathrin-mediated endocytosis, caveolae-mediated endocytosis, or pinocytosis, and after antibody binding to the cell surface, the cell absorbs the ADC by inward budding of the plasma membrane, thereby transporting the toxin into the target cell. Based on this insight, the inventors recognized the possibility of modifying toxins that, in isolated form, require active transport across the cell membrane and cannot enter cells by passive diffusion with structural modifications that intentionally inhibit their acceptance by the corresponding transport proteins, thereby containing a structural "safety feature" that prevents the toxin from being actively taken up into cells and exerting its cytotoxic function. In this way, the modified toxins of the present invention are prevented from exerting nonspecific cytotoxic activity as isolated compounds in the extracellular environment of the human body, for example, upon premature release from ADCs into the bloodstream or after release from dead cancer cells. In contrast, when the modified toxins of the present invention are conjugated to antibodies via the provided organic coupling groups, they are taken up by target cells as the antibody payload via receptor-mediated endocytosis. After intracellular trafficking and processing of the ADC, the toxins are unloaded, releasing their cytotoxicity and inducing tumor cell death. In this way, the present invention provides modified toxins for use as ADC payloads with a significantly improved safety profile, thereby enabling increased ADC dosage and improved therapeutic efficacy without unacceptable side effects or toxicity. Furthermore, safety during the manufacture and processing of the modified toxins is also improved.

[0038] Those skilled in the art are familiar with toxins that have a basic structure that requires a transport protein for cellular uptake. For example, the transport of toxins or basic structures is mediated by organic anion transporting polypeptides (OATPs) or their subtypes, such as OATP1B1, OATP1B3, and / or OATP1A2. These OATPs are mainly found in the human liver. 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.

[0039] Generally, 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 naturally occurring toxin base structure modified with at least one anchor group configured for chemical conjugation to an antibody, for example, as described in WO 2018 / 206715.

[0040] Preferably, the toxin is an oligopeptide toxin, i.e., the basic structure comprises an oligopeptide comprising several amino acids, for example, 5 to 15 or 5 to 10 amino acids, particularly 5, 6, 7, or 8 amino acids. More preferably, the oligopeptide is cyclic. Preferably, the cyclic oligopeptide toxin comprises at least one non-proteinogenic amino acid.

[0041] Non-limiting examples of suitable cyanobacterial or fungal toxins within the meaning of the present invention include amatoxins such as microcystin, nodularin, and amanitin.

[0042] In a preferred embodiment, the cyanobacterial toxin is microcystin. Microcystin is a cyanobacterial cyclic heptapeptide that potently inhibits eukaryotic serine / threonine protein phosphatases type 1 and type 2A, causing disruption of many important signaling pathways, ultimately leading to cytoskeletal collapse and cell death. Microcystin is primarily taken up by OATP1B1 and OATP1B3.

[0043] In another preferred embodiment, the cyanobacterial toxin is nodularin, a cyanobacterial cyclic pentapeptide evolutionarily related to microcystins that also inhibits protein phosphatases type 1 and type 2A as its mode of cytotoxic action and is primarily taken up by OATP1B1.

[0044] In yet another preferred embodiment, the fungal toxin is amanitin, a fungal bicyclic octapeptide belonging to the amatoxin subgroup. Most notably, alpha-amanitin is an inhibitor of RNA polymerase II, binding to the cross-linked helix of RNA polymerase II and preventing the translocation of RNA and DNA necessary to free up the site for the next round of synthesis.

[0045] In a preferred embodiment, the method includes incorporating at least one structural modification into the side chain of at least one amino acid or attaching at least one structural modification to the side chain of at least one amino acid. As used herein, incorporating at least one structural modification into the side chain of an amino acid includes synthetically or biosynthetically introducing the structural modification or a portion thereof into the base structure, for example, via an amino acid building block or via providing an amino acid precursor containing the structural modification or a portion thereof, as described, for example, in WO 2018 / 219619. Attaching at least one structural modification to the side chain of an amino acid includes chemically binding the structural modification to a chemically bondable functional group of the side chain. The functional group may be a functional group of an amino acid naturally occurring in the base structure, such as an amino group, a carboxyl group, a hydroxyl group, an aldehyde group, a keto group, or a thiol group. The functional group may be a group not present in the natural counterpart of the base structure, for example, a functional group artificially introduced into the base structure, as described, for example, in WO 2018 / 219619. Preferably, the functional groups are configured for biorthogonal conjugation, i.e., chemical conjugation reactions that can occur within biological systems without interfering with biochemical processes. Preferably, the functional groups are selected from the group consisting of azide groups, alkyne groups, alkene groups, phosphines, phosphonates, tetrazines, hydrazines, hydrazones, oximes, and any combination thereof.

[0046] In preferred embodiments, at least one structural modification comprises a single organic moiety that includes both an organic inhibitor or an effector molecule and an organic coupling group, respectively. In some embodiments described below, the organic inhibitor and the organic coupling group can 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 can be part of the organic inhibitor.

[0047] In other preferred embodiments, the at least one structural modification comprises a first structural modification comprising an organic inhibitory group or effector molecule and a second structural modification comprising an organic coupling group. Thus, modifying the base structure can comprise incorporating or attaching a first structural modification to the side chain of a first amino acid in the base structure, and incorporating or attaching a second structural modification to the side chain of a second amino acid in the base structure that is different from the first amino acid. In the preferred embodiments described below, the first structural modification comprises attaching an organic molecule containing or consisting of an organic inhibitory group or effector molecule to the side chain of the first amino acid in the base structure, and incorporating a second structural modification containing or consisting of an organic coupling group configured for covalent conjugation of a toxin to an antibody, particularly a functional group configured for biorthogonal binding, to the side chain of the second amino acid.

[0048] As used herein, the selection of a cyanobacterial or fungal toxin can, in principle, be a mental step or a step performed in silico. Alternatively, modifying a base structure with at least one structural modification can include substeps for producing a modified toxin, such as (i) providing a base structure and (ii) modifying the base structure with at least one structural modification. Advantageously, the step of modifying the base structure with at least one structural modification can be performed by semisynthesis, also known as partial chemical synthesis, in which the base structure is isolated from a natural source, such as a cyanobacterial or fungal cell culture, as described in WO 2018 / 219619, and then at least one structural modification is chemically introduced into the base structure to produce the modified toxin. In this way, the present invention provides unprecedented access to structurally diverse and improved toxins for use in ADCs.

[0049] In preferred embodiments, the organic inhibitor group comprises an organic group that carries at least one charge at physiological pH values, i.e., pH 6-8 in aqueous media at 20-40°C. In some embodiments, the at least one charge is positive. In some embodiments, the at least one charge is negative.

[0050] Preferably, the organic inhibitory group comprises an amphoteric or zwitterionic group having at least one cationic charge and at least one anionic charge at physiological pH values. The cationic charge may be based, for example, on primary, secondary, and / or tertiary amines, as well as quaternary ammonium cations and guanidine. The anionic moiety may be based, for example, on sulfonates, sulfinates, phenolic hydroxyl groups, thiols, carboxylates, and / or thiocarboxylates. Preferably, the organic amphoteric group is an amino acid group, with at least one positive charge based on an amino group and at least one negative charge based on a carboxylic acid group. In some embodiments, the organic inhibitory group comprises multiple amino acids.

[0051] The inventors have discovered that the above-described organic inhibitory groups are potent inhibitors of transport proteins and also surprisingly enhance the cytotoxic effect of the modified toxin compared to the base structure without structural modifications. This synergistic dual effect allows the modified toxins of the present invention to have an improved therapeutic window compared to conventional toxins, achieving greater therapeutic efficacy without unacceptable side effects or toxicity. In this regard, please also refer to the detailed description of the invention below.

[0052] In a more preferred embodiment, the organic inhibitor comprises a heterocyclic ring, particularly a saturated or unsaturated, optionally substituted, five- or six-membered ring containing at least one or at least two heteroatoms, particularly one to three heteroatoms selected from the group N, O, and S. Preferably, the heterocyclic ring contains at least one sulfur atom and / or at least one nitrogen atom in the ring. The organic inhibitor may comprise a ring system containing or consisting of two fused heterocyclic rings, saturated or unsaturated, and optionally substituted. For example, the organic inhibitor may be biotin.

[0053] In a further preferred embodiment, the organic inhibiting group comprises an organic formamide and / or alcohol. Suitable formamides and alcohols are exemplified in the detailed description of the preferred embodiment below.

[0054] In some embodiments, at least one structural modification is provided to comprise an effector molecule that is a known inhibitor of active transporter, or a functional part or derivative of such an inhibitor.In some embodiments, the effector molecule comprises a competitive or allosteric inhibitory ligand of transport protein, or a part or derivative thereof that at least partially has the competitive or allosteric inhibitory function of the ligand.In particular, the effector molecule is a known inhibitor of OATP, and has either a specific inhibitory activity against an OATP subtype, or an inhibitory activity against two or more OATP subtypes.Suitable inhibitors are known to those skilled in the art, and include, but are not limited to, pravastatin, erlotinib, cyclosporin A, rifampicin, nystatin, doxorubicin, ritonavir, and flavonoids and nostocyclopeptides, or functional parts or derivatives thereof.

[0055] As described above, the organic coupling group is a functional group, i.e., a substituent or moiety with characteristic chemical reactivity. The same functional group undergoes the same or similar chemical reaction regardless of the remaining parts of the modified toxin molecule. This allows for systematic prediction of the chemical reaction and behavior of the modified toxin upon chemical conjugation to an antibody. Preferably, the organic coupling group is capable of or configured for bioorthogonal chemical coupling reactions, such as click chemistry reactions. Preferably, the organic coupling group is selected from the group consisting of amino groups, carboxyl groups, hydroxyl groups, azide groups, alkyne groups, alkene groups, tetrazine groups, thiol groups, aldehyde groups, keto groups, oximes, hydrazines, hydrazones, phosphines, phosphonates, and any combination thereof.

[0056] A second aspect of the present invention relates to a modified cyanobacterial or fungal toxin obtainable by the above method.

[0057] The modified cyanobacterial or fungal toxins comprise a base structure that is cytotoxic to a target cell after uptake by a transport protein of the target cell, and at least one structural modification of the base structure, where 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 transport protein, compared to the base structure, and an organic coupling group configured to covalently conjugate the modified toxin to an antibody.

[0058] In a preferred embodiment, the toxin or base structure, respectively, has the general formula cyclo(-A 1 -X 2 -A 3 -Z 4 -A 5 -A 6 -A 7 ), and a microcystin having the formula: Independently of each other, A 1 and A 3 represents the D-amino acid obtained by modification, and A 5is selected from the group consisting of Adda, DM-Adda, dm-Adda, (6Z)Adda, and ADM-Adda; 6 is selected from the group consisting of D-Glu and D-Glu(OCH3), and 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 L-amino acids that may be modified independently of each other. In some embodiments, A 1 is selected from the group consisting of D-Ala, D-Leu, and D-Ser, or modified D-amino acids. 3 is selected from the group consisting of D-Asp, D-MeAsp, or modified D-amino acids. D-MeAsp is D-erythro-β-methylaspartic acid, Mdha is N-methyldehydroalanine, Mdhb is N-methyldehydrobutyric acid, Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid, ADM-Adda is O-acetyl-9-O-desmethyl-Adda, DM-Adda is 9-O-desmethyl-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 not present in the naturally occurring counterpart of microcystin or the basic structure, and comprising a chemical group covalently conjugated to at least one structural modification, or a functional group configured to be covalently conjugated 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, at least one of X is a modified amino acid.2 , Z 4 , and A 7 At least one of is a modified amino acid. The modified amino acid may be selected from the group consisting of, for example, azidonorvaline, propargyltyrosine, azidolysine, azidophenylalanine, propargylcysteine, propargylserine, azidoalanine, or other amino acids not present in the corresponding naturally occurring cyanobacterial or fungal toxin or basic structure and having a functional group configured or capable of covalent conjugation.

[0059] Microcystin is a potent inhibitor of type 1 and type 2A protein phosphatases. Protein phosphatase type 1 (PP1) and type 2A (PP2A) are the two major phosphatases that dephosphorylate serine and threonine residues in eukaryotic cells. For example, the IC50 of naturally occurring microcystin-LR is 0.03 nM for type 1 protein phosphatase and 0.04 nM for type 2A protein phosphatase. In the art, the IC50 of position A 5 and A 6 It has been established that at least one structural modification of microcystin at position A is essential for the inhibitory effect of microcystins on PP1 and PP2A. 1 , X 2 , A 3 , Z 4 and / or A 7 Preferably, at least one structural modification is provided at position A 1 , X 2 , Z 4 and / or A 7 and most preferably at position X 2 or Z 4 For example, at least one structural modification is incorporated into or covalently attached to the side chain of the amino acid at that position. 1 , X 2 , A 3 , Z 4 and / or A 7Preferably, at least one structural modification is at position A 1 , X 2 , Z 4 and / or A 7 and most preferably at position X 2 and / or Z 4 Preferably, at least one structural modification is provided at a maximum of two positions, for example at exactly one position.

[0060] In another preferred embodiment, the toxin or base structure, respectively, has the general structure cyclo[-A 1 -*A 2 -A 3 -A 4 -*A 5 ], is a nodularin having the formula Independently, A 1 is D-Asp or D-MeAsp or a modified D-amino acid, A2 is Arg or Har or a modified L-amino acid, and A 3 is selected from the group consisting of Adda, DM-Adda, (6Z)Adda and MeAdda, and A 4 is selected from the group consisting of D-Glu and D-Glu(OCH3), and A 5 is Dhb or Mdhb or modified L-amino acid, and "*" represents an isopeptide bond.

[0061] In the art, position A 3 and A 4 It has been established that the cleavage site at position A is essential for the inhibitory effect of nodularin on PP1 and PP2A. 1 , A2, and / or A 5 For example, at least one structural modification is incorporated into or covalently attached to the side chain of the amino acid at that position. 1 , A2 and / or A 5Preferably, at least one structural modification is provided at a maximum of two positions, for example at exactly one position. Preferably, at least one structural modification is provided at position A2.

[0062] In yet another preferred embodiment, the toxin or base structure, respectively, has the general structure cyclo{A 1 -cyclo[A 2 -A 3 -A 4 -A 5 -A 6 ]-A 7 -A 8}(SEQ ID NO:1), is an amatoxin having the formula Independently, A 1 isoleucine (Ile), hydroxyisoleucine (Hyile) or dihydroxyisoleucine (Dihyile), A 2 is tryptophan (Trp) or hydroxytryptophan (Hytrp), A 3 is glycine (Gly), A4 is isoleucine (Ile), A 5 is glycine (Gly), A 6 is cysteine ​​(Cys), A 7 is asparagine (Asn) or aspartic acid (Asp), A8 is proline (Pro) or hydroxyproline (Hypro), and A 2 and A 6 and are cross-linked by a sulfoxide (S=O) bridge.

[0063] For example, the amatoxin can be one of the group consisting of α-amanitin, β-amanitin, γ-amanitin, δ-amanitin, ε-amanitin, amanulin, amanuric acid, amaninamide, amanine, and proamanitin. Preferably, the amatoxin is amanitin.

[0064] In a preferred embodiment, at least one structural modification of amatoxin or amanitin is at position A 2For example, at least one structural modification is incorporated into or covalently attached to the side chain of the amino acid at that position, e.g., via the hydroxy or amino group of the side chain. Preferably, at least one structural modification is provided at position A 2 It is provided by hydroxytryptophan (Hytrp).

[0065] Toxin potency is generally determined by the correlation between cellular uptake and inhibitory effect (e.g., protein phosphatase inhibition). This correlation can be expressed, for example, in the form of an IC50 value, corresponding to the toxin concentration required to kill 50% of cells exposed to the toxin, hereinafter referred to as IC50_cytotox. However, because the IC50_cytotox value depends not only on cellular uptake but also on the level of inhibitory effect, a high IC50_cytotox value may be due to either insufficient cellular uptake or an insufficient inhibitory effect of the toxin, or both, while a low IC50_cytotox value may be due to insufficient cellular uptake or a strong inhibitory effect, or both. Thus, the inventors have recognized that a more appropriate characterization of the cytotoxic properties of a modified toxin is expressed as the relationship between the IC50_cytotox value and the inhibitory concentration of toxin required to achieve half of the maximal inhibitory effect, e.g., 50% protein phosphatase inhibition, hereinafter referred to as IC50_PPI. Therefore, if the ratio of IC50_cytotox / IC50_PPI of the modified toxin is greater than that of the base structure, cellular uptake is impaired relative to inhibitory function, which is a preferred characteristic of the modified toxin of the present invention. Therefore, a preferred embodiment of the method described herein includes determining the ratio IC50_cytotox / IC50_PPI of the modified toxin and / or the base structure, and, if necessary, comparing or correlating the IC50_cytotox / IC50_PPI of the modified toxin with the IC50_cytotox / IC50_PPI of the base structure. Preferably, the IC50_cytotox / IC50_PPI ratio of the modified toxin is at least 2-fold, more preferably at least 3-fold or at least 4-fold, and particularly preferably at least 5-fold, that of the base structure. Methods for determining the IC50 value of an inhibitor are well known in the art. In this regard, please also refer to the detailed description of the preferred embodiments of the invention below.

[0066] In one embodiment, at least one structural modification comprises a group B-In, where the group In represents an inhibitory group or an effector molecule that reduces the uptake of the toxin into the target cell by the transport protein, and the group B represents a bridging group that covalently links the group In to the toxin or the base structure, respectively. Preferably, the group B is a triazole, a peptide bond, a disulfide, a C1-C 10 Alkyl groups, C1-C 10 In a preferred embodiment, group B comprises a group selected from the group consisting of an alkylaryl group, an (oxyethylene)1-10 group, an amide bond, a thioamide bond, an ether bond, a thioether bond, a triazole, a dihydropyridazine, or a covalent bond containing a higher amine such as a secondary (20) amine, a tertiary (30) amine, a quaternary (40) amine, a carbamate bond, a thiocarbamate bond, a urea bond, a thiourea bond, a phosphate ester bond, a phosphamide bond, a sulfonamide bond, an oxime bond, or any combination thereof. In a preferred embodiment, group B comprises a triazole.

[0067] In some embodiments, the group In is C-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-C6 alkyl-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-C4 alkyl-CH(NH3 + )-CO2 - , [ka] is selected from the group consisting of The "*" indicates the point of covalent attachment to the bridging group B, and the "+" and "-" indicate the charge at physiological pH values.

[0068] In certain embodiments, the at least one structural modification may also include a label for in vivo visualization, such as a fluorescent label or a near-infrared dye, which allows for imaging of deep tissues in vivo using, for example, emission in the near-infrared region.

[0069] As already explained above, the 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 into a target cell by a transport protein compared to the base structure, i.e., the toxin lacking the first structural modification, and a second structural modification comprising an organic coupling group configured to covalently bind the modified toxin to an antibody. In particular, the first and second structural modifications may each be provided at different positions of microcystin, nodularin, or amatoxin. For example, the base structure may be microcystin and the first structural modification may be provided at position X. 2 and the second structural modification can be provided at position A 1 , A 3 , Z 4 , or A 7 Alternatively, the first structural modification may be provided at position Z 4 and the second structural modification can be located at position A 1 , X 2 , A 3 or A 7Preferably, one of the first and second structural modifications can be located at position X 2 and the other structural modification is at position Z 4 As described above, the first and second structural modifications can be attached to or incorporated into the side chain of the amino acid at that position, independently of one another. In some embodiments, the first structural modification is covalently attached to the side chain of the amino acid, while the second structural modification is incorporated into the side chain of the amino acid by incorporating a modified amino acid having a side chain containing the second structural modification, for example, as described in WO 2018 / 206715. In particular, the second structural modification can comprise an alkyne group or an azide group, or any group configured to covalently conjugate the modified toxin to the antibody.

[0070] In some embodiments, the second structural modification may comprise a linker incorporating an organic coupling group configured for covalent conjugation of the modified toxin to an antibody, or a linker disposed between the side chain of an amino acid and the organic coupling group.

[0071] According to common understanding in the field of ADCs, a "linker" is a bifunctional or multifunctional moiety that can be used to covalently attach a modified toxin to an antibody to form an antibody-drug conjugate. There are two genera of linkers: so-called "cleavable linkers" and "non-cleavable linkers." Non-cleavable linkers are essentially stable extracellularly, i.e., outside the cell. Cleavable linkers contain a predefined cleavage site that can be cleaved by enzymatic activity, hydrolysis, or other metabolic conditions. The linker may also contain a spacer structure that spatially separates the modified toxin or base structure from the antibody. The use of linkers is well known in the art, and skilled artisans can easily select an appropriate linker based on their own knowledge and this disclosure.

[0072] For example, the linker may comprise an organic coupling group configured to covalently bond with an amino acid side chain such as a cysteine ​​thiol, an amine (e.g., at the N-terminus), or a lysine, or other modifications of an antibody, as described below. In some preferred embodiments, the linker is a non-cleavable linker. The linker may be substituted with sulfonic acid or other substituents that can increase the water solubility of the reagent and facilitate the coupling reaction between the linker reagent and the antibody. The organic coupling group may be configured to covalently bond with a nucleophilic group on the antibody, including, but not limited to, the N-terminal amino group, a side chain amino group such as a lysine, a side chain thiol group such as a cysteine, and a carbohydrate hydroxyl or amino group in the case of a glycosylated antibody. Amino, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linkers to form covalent bonds, including, but not limited to, active esters such as NHS esters, HOBt esters, haloformates, and acid halides; alkyl and benzyl halides such as haloacetamides; aldehydes; ketones; carboxyl groups; and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for linker conjugation by treatment with a reducing agent such as DTT (dithiothreitol). Nucleophilic groups can be added to antibodies by reacting lysines with 2-iminothiolane (Traut's reagent), converting amines to thiols. Reactive thiol groups can be introduced into antibodies by introducing one, two, three, four, or more cysteine ​​residues, for example, by preparing mutant antibodies containing one or more non-native cysteine ​​residues or by using commercially available chemical kits.

[0073] In some embodiments, the second structural modification, particularly the organic coupling group, is configured to be attached to the antibody via a click chemistry reaction. Click chemistry reactions within the meaning of the present invention include, for example, copper(I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted alkyne-nitrone cycloaddition, alkene and azide [3+2] cycloaddition, alkene and tetrazine reverse demand Diels-Alder reaction, and alkene and tetrazole photoclick reaction. Thus, the organic coupling group is preferably an azide, alkyne, alkene, or tetrazine.

[0074] In certain embodiments, the at least one structural modification is a single structural modification, i.e., the organic inhibitor group comprises or consists of an organic coupling group or a portion thereof, or vice versa. In some preferred embodiments, the organic coupling group forms at least a portion of the organic inhibitor group, such that at least a portion of the organic inhibitor group is configured to covalently conjugate the modified toxin to the antibody. In this way, when the modified toxin is covalently bound to the antibody in the ADC via the organic coupling group, the function of the organic inhibitor group can be at least partially intentionally impaired. However, upon cleavage of the conjugation bond either intracellularly or extracellularly, the organic inhibitor group is restored, preventing the released modified toxin from being internalized by healthy cells. In this way, both promotion of internalization of the modified toxin when bound to the antibody and inhibition of internalization of the modified toxin after release from the antibody are achieved. In some embodiments described below, the single structural modification comprises a functional group as an organic coupling group configured for covalent attachment to the antibody, the functional group being selected from the group consisting of -NH, -COO, or -OH, where "-" indicates the point of attachment to the remainder of the single structural modification and "+" and "-" indicate the charge at physiological pH values.

[0075] As described above, the base structure may be a naturally occurring toxin or a non-naturally occurring toxin. The latter may be, for example, a naturally occurring toxin base structure modified with at least one functional group configured to be linked to a modifying molecule by chemical bonding, where the at least one structural modification is generated by a chemical bonding reaction between the functional group and the modifying molecule. In a preferred embodiment, the at least one functional group is an azide group or an alkyne group. However, the functional group may be any of the functional groups disclosed above.

[0076] The above-described modified toxins can be advantageously used as cytotoxic drugs in antibody-drug conjugates to reduce the uptake of the toxin into cells by transport proteins compared to toxins having the basic structure without at least one structural modification.

[0077] Thus, 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, which use comprises covalently attaching the modified toxin to an antibody or fragment thereof comprising an antigen-specific binding site, thereby forming the antibody drug conjugate.

[0078] Thus, a fourth aspect of the present invention provides an antibody-drug conjugate comprising a modified toxin as described above as a drug and an antibody or fragment thereof comprising an antigen-specific binding site, wherein the antibody or fragment thereof is attached, i.e. covalently conjugated, to the modified toxin via an organic coupling group.

[0079] In some embodiments described below, the at least one structural modification is one single structural modification, and the antibody is attached to the single structural modification via, for example, an amide, ester, ether, triazole, or carbamate bond.

[0080] In the present invention, the term "antibody" is used in its broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, bispecific antibodies, or multispecific antibodies, as well as antibody fragments having an antigen-specific binding site, i.e., an antigen-binding site that immunospecifically binds to an antigen on a target cell or a portion thereof. Examples of antibody fragments include Fab, Fab', F(ab'), scFv fragments, single-domain antibodies also known as 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 cancer cell antigens, viral antigens, microbial antigens, etc., single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Antibodies can be murine, human, humanized, chimeric, or derived from other species. The antibody can be any type of immunoglobulin, such as IgG, IgE, IgM, IgD, or IgA, or any class or subclass of immunoglobulin, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0081] In a preferred embodiment, 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 are expressed at high levels on tumor cells but at low levels on healthy cells. Such tumor-associated antigens and tumor-specific antigens are known in the art. Examples of TAAs and TSAs include BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM-001203), E16 (LAT1, SLC7A 5, 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, SEMA 5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 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 type 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 protein, Genbank accession number AY275463). k 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, teratocarcinoma-derived growth factor, Genbank accession numbers 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 beta), B29, Genbank accession numbers nM-000626 or 11038674), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession numbers nM-030764, AY358130), HER2 (ErbB2, Genbank accession number M11730), NCA (CEACAM6, Genbank accession number M18728), MDP (DPEP1, Genbank accession number BC017023), IL20Ra (IL20Ra, ZCYTOR7, Genbank accession number AF184 971), 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), AAP14954.1 Homo lipoma HMGIC fusion partner-like protein / pid=AAP14954. sapiens (human), BAFF-R (B cell-activating factor receptor, BLyS 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 alpha), CXCR5 (Burkitt's lymphoma receptor 1), HLA-DOB (beta subunit of MHC 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).These include, but are not limited to, LY64 (lymphocyte antigen 64 (RP105)), FcRH1 (Fc receptor-like protein 1), IRTA2 (FcRH5, immunoglobulin superfamily receptor translocation-associated 2), TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR), and MUC1 (tumor-associated MUC1 glycopeptide epitope).

[0082] In certain embodiments, the antibody drug conjugate has the general formula Ab-(LT) n and Ab is an antibody or antigen-binding fragment thereof, T is a modified cyanobacterial or fungal toxin, L is an organic linker that provides a covalent bond between the antibody or antigen-binding fragment thereof and the modified cyanobacterial or fungal toxin defined above, and n is an integer from 1 to 16.

[0083] 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.

[0084] The number "n" is also called the "drug-antibody ratio" (DAR).

[0085] In a preferred embodiment, the antibody-drug conjugate has a drug-to-antibody ratio (DAR) of 2 to 12, i.e., at least 2 and up to 12 modified toxins are conjugated per antibody. Preferably, the DAR is 2 to 10, more preferably 2 to 8, and most preferably 4 to 8.

[0086] A fifth aspect of the invention provides a modified cyanobacterial or fungal toxin, or an antibody drug conjugate as described above, for use in the treatment of malignant disease.

[0087] A sixth aspect of the present invention provides a method of treating a human subject suffering from a malignant disease, the method comprising administering to the subject a therapeutically effective amount or dosage regimen of an antibody drug conjugate as defined above.

[0088] In some embodiments, the malignant disease is cancer. Examples of cancers that can be 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, such as lung cancer including epithelial squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), gastrointestinal stromal tumor (GIST), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. Cancer can be characterized by, for example, the overexpression of HER2 or ErbB receptor.

[0089] A pharmaceutically effective amount can be, for example, 0.01 to 100 mg per kg of body weight (mg / kg), more specifically 0.1 to 20 mg / kg or 1 to 10 mg / kg. A therapeutically effective dosing regimen can include, for example, a single or multiple dose of 0.01 to 100 mg / kg, more specifically 0.1 to 20 mg / kg or 1 to 10 mg / kg.

[0090] In a seventh aspect, the present invention provides a method for producing a cyanobacterial or fungal toxin modified as described above.

[0091] The method includes the steps of: (A) providing a toxin having a base structure known to be cytotoxic to a target cell after uptake by a transport protein of the target cell; and (B) introducing at least one structural modification into the toxin or base structure, respectively, thereby forming a modified toxin.

[0092] In some embodiments, the toxin or base structure, respectively, provided in step (A) comprises at least one first functional group configured for and / or capable of covalent conjugation, and in method step (B), at least one structural modification is introduced into the toxin or base structure, respectively, via covalent conjugation to the at least one first functional group.

[0093] In some embodiments, method step (B) comprises introducing at least a first and a second structural modification to the toxin or base structure, respectively, wherein the first structural modification comprises an organic inhibitory group and the second structural modification comprises an organic coupling group configured to covalently conjugate the modified toxin to the antibody.

[0094] Additionally, method step (B) may include providing a modifying molecule, the modifying molecule comprising an organic inhibitor or effector group, and / or an organic coupling group, respectively, and covalently attaching the modifying molecule to at least one first functional group, thereby creating at least one structural modification.

[0095] The modifying molecule may include a second functional group that interacts 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 interacting partners in any of the above-described click chemistry reactions, such as an azide or tetrazine and an alkyne or alkene. However, the first and second functional groups may also be selected from any of the other interacting functional groups described above, such as an amino group and a carboxyl group, an aldehyde group, or a keto group.

[0096] Furthermore, it will be understood that various embodiments described with respect to one aspect of the invention are also applicable to other aspects of the invention. Thus, the features and functions disclosed above and below in relation to methods for modifying cyanobacterial or fungal toxins also relate to the modified cyanobacterial or fungal toxins, the use of the modified toxins in the manufacture of antibody drug conjugates, methods for making the modified cyanobacterial or fungal toxins, antibody drug conjugates and medical uses thereof, and vice versa. [Brief explanation of the drawings]

[0097] [Figure 1] Structural formulas of MC-(azidonorvaline)R (A), MC-propargyltyrosine)R (B), and MC-L(azidolysine) (C), which are used as reference toxins with cytotoxic basic structures. [Figure 2] 1 shows the structural formula of the modified toxin MC-(azidonorvaline)R produced by modifying MC-(azidonorvaline)R with propargylamine according to the present invention. [Figure 3] 1 is a scheme for the structural modification of MC-(propargyltyrosine)R according to the present invention. [Figure 4] 1 is a scheme of another structural modification of MC-(propargyltyrosine)R according to the present invention. [Figure 5] 1 is a structural formula of [AzProMDap]7MC-LR produced from MC-LR by Michael addition with 3-azidopropan-1-amine in accordance with the present invention. [Figure 6] The structural formula of the modified toxin according to the invention is embodied as a dimer obtained by cross-linking two MC-PrgTyr-Arg monomers with a cross-linking molecule. [Figure 7] 7 is a structural formula of an MC-dimer-linker conjugate consisting of the MC-dimer of FIG. 6 conjugated with a cleavable mc-Val-Ala-PAB-PNP linker. DETAILED DESCRIPTION OF THE INVENTION

[0098] The invention will be explained in more detail below on the basis of exemplary embodiments with reference to the accompanying drawings, in which: These examples and drawings are not intended to limit the invention. [Example] [Example]

[0099] Example 1: Preparation of MC-(azidonorvaline)R, MC-propargyltyrosine)R, MC-L (azidolysine), and MC-LR as reference toxins with cytotoxic base structures

[0100] Three reference toxins with basic structures known to be cytotoxic to OATP-expressing cancer cell lines were prepared: microcystin MC-(azidonorvaline)R or "MC-(Aznva)R" shown in Figure 1A, MC-(propargyltyrosine)R or "MC-(Prtyr)R" shown in Figure 1B, and MC-L (azidolysine) or "MC-L(Azlys)" shown in Figure 1C. Each basic structure contains a cytosine at position X. 2 Functional groups configured for covalent attachment of modifying moieties via click chemistry, i.e., the azide in MC-AznvaR and the alkyne in MC-PrtyrR, or at position Z 4 The functional groups configured for covalent attachment of a modifying moiety via click chemistry, i.e., the azide of MC-LAzlys, are included. Thus, the basic structures can be described as cyclo(D-Ala-Aznva-D-MeAsp-R-Adda-D-Glu-Mdha) for MC-(Aznva)R (SEQ ID NO:2), cyclo(D-Ala-Prtyr-D-MeAsp-R-Adda-D-Glu-Mdha) for MC-(Prtyr)R (SEQ ID NO:3), and cyclo(D-Ala-Leu-D-MeAsp-Azlys-Adda-D-Glu-Mdha) for MC-L(Azlys) (SEQ ID NO:4).

[0101] MC-(Aznva)R, MC-(Prtyr)R, and MC-L(AzLys) were produced essentially as described in WO 2018 / 219619. Briefly, a cyanobacterial strain producing natural microcystins was cultured under conditions that allowed for the growth of the strain. A non-limiting example of a suitable strain is PCC7820, available from the Pasteur Culture Collection (PCC). During cultivation, the mineral culture medium was supplemented with either the modified amino acids azidonorvaline (Aznva), propargyltyrosine (Prtyr), or azidolysine (Azlys). These modified amino acids were inserted at position X of the microcystins produced by the strains. 2 and Z 4 represent modified components of the amino acid sequence, which are incorporated into microcystins instead of the natural amino acid substrate during the cultivation of the strain, resulting in the amino acid sequence at position X, respectively. 2 and Z 4 This leads to the biosynthesis of non-naturally occurring microcystins bearing the respective modified amino acids. After cultivation, the biomass was harvested and extracted, and the non-natural microcystins that serve as the basic structures were purified by HPLC.

[0102] X 2 or Z 4 In addition to the modified microcystins MC-(Aznva)R, MC-(Prtyr)R, and MC-L(AzLys), the native microcystin MC-LR was obtained by standard cultivation of the strain and isolated by HPLC after biomass extraction.

[0103] Below, four basic structures were modified according to the present invention by structural modifications with organic blocking and coupling groups. [Example]

[0104] Example 2: Structural modification of MC-(Aznva)R, MC-(Prtyr)R, and MC-L(Azlys) using copper-catalyzed azide-alkyne cycloaddition (CuAAC)

[0105] According to the present invention, a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) can be used to prepare a compound at position X. 2 MC-(Aznva)R and MC-(Prtyr)R, and position Z 4 Several different structural modifications were introduced into MC-L(Azlys) to provide a variety of modified toxins.

[0106] The following modifying molecules were used for the structural modification of MC-(Aznva)R. Propargyl lysine (Prglys) as an example of structural modification with amino and carboxyl groups as amphoteric organic inhibitors and organic coupling groups. Biotin alkyne (Albio) as an example of structural modification with biotin as a heterocyclic blocking group and organic coupling group. Propargylamine (Prgam) is an example of structural modification with an inhibitory group that is positively charged at physiological pH and an amino group as an organic coupling group.

[0107] The following modifying molecules were used for the structural modification of MC-(Prtyr)R. Azidoethanol (Azeth) as an example of structural modification with an alcohol as a blocking group and a hydroxy group as an organic coupling group. Biotin-PEG-3-Azide (Azbio) as an example of structural modification with biotin as a heterocyclic blocking group and an organic coupling group. Azidohomoalanine (AzHala) as an example of structural modification with amino and carboxyl groups as amphoteric organic blocking and coupling groups. Azidophenylalanine (AzPhe) is another example of structural modification with amino and carboxyl groups as amphoteric organic blocking and coupling groups. N1-azidospermine (AzSpe) is another example of structural modification with an inhibitory group that is positively charged at physiological pH and an amino group as an organic coupling group. Azidopropylformamide (AzProam) as an example of structural modification using an organic formamide as both a blocking group and an organic coupling group. Azidoacetic acid (Azacac) is another example of a structural modification that has an inhibitory group that is negatively charged at physiological pH and a carboxyl group as the organic coupling group. 2-Amino-3-azidobutanoic acid (AzAbu) is another example of a structural modification having an amphoteric organic inhibitor group and an amino group and a carboxyl group as organic coupling groups. 3-Amino-4-azidobutyric acid (AzDbu) is another example of a structural modification having an amphoteric organic inhibitor group and an amino group and a carboxyl group as organic coupling groups. 3-Azido-D-alanine (AzDala) is another example of a structural modification that has an amphoteric organic inhibitor group and an amino group and a carboxyl group as organic coupling groups. 3-Azido-L-alanine (AzLala) is another example of a structural modification that has an amphoteric organic inhibitor group and an amino group and a carboxyl group as organic coupling groups. 6-Azido-L-lysine (Azlys) is another example of a structural modification that has an amphoteric organic inhibitor group and an amino group and a carboxyl group as organic coupling groups. 3-Azido-propan-1-amine (Azpro) is an example of a structural modification that has an inhibitory group that is positively charged at physiological pH and an amino group as the organic coupling group. MC-(Prtyr)R is another example of structural modification using an amphoteric organic inhibitor (guanidinium group) that is positively charged at physiological pH and a carboxyl group as an organic coupling group to artificially create a microcystin dimer. Structural modification (i.e., dimerization) was achieved using the bridging molecules BM-I (1,4-diazidoacetylpiperazine) or BM-II (1-azidoacetyl-4-(2-azido-L-lysine)-piperazine), with BM-II carrying an additional amino group as an organic coupling group.

[0108] The following modifying molecules were used for the structural modification of MC-L (Azlys). Prgam is an example of structural modification with an inhibitory group that is positively charged at physiological pH and an amino group as an organic coupling group.

[0109] Propargyl lysine (Prglys) is an example of a structural modification that has an amphoteric organic inhibitor group and an amino group and a carboxyl group as organic coupling groups.

[0110] In both cases, CuAAC was performed using MC-(Prtyr)R (0.32 mg, 0.3 μmol, 1 eq.) or MC-(Aznval)R (0.31 mg, 0.3 μmol, 1 eq.) in 300 μL DMSO with 150 mL of either the above "clickable" modified molecule (10 mM, 1.5 μmol, 5 eq.) in a mixture of 150 μL DMSO, 930 μL HO, 1.5 μL 100 mM CuSO solution (0.5 eq.), 3.75 μL 200 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) solution (2.5 eq.), and 75 μL 100 mM aminoguanidine (25 eq.).

[0111] The reaction was initiated by adding 37.5 μL of 100 mM sodium ascorbate solution (12.5 eq.). The modified toxin was isolated by either C-18 solid-phase extraction (SPE) or semi-preparative C-18 reverse-phase (RP)-HPLC and lyophilized.

[0112] FIG. 2 exemplarily shows the structural formula of the modified toxin MC-(AznvaPrgam)R obtained by modifying MC-(azidonorvaline)R with propargylamine according to the present invention. [Example]

[0113] Example 3: Synthesis of modified MC-(Prtyr)R compound 1

[0114] According to the present invention, MC-(Prtyr)R was modified with azidoacetyl-L-Lys(7-DCCA)-OH, an example of a structural modification in which the organic coupling group is a heterocyclic carboxyl group and negatively charged at physiological pH. The structural modification also contains a fluorescent structure. The synthetic route is shown in Figure 3 and described in more detail below.

[0115] 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) and mixed, followed by the addition of 400 μL of 0.1 M sodium phosphate buffer (pH 8). 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. 25 H 35 It has the molecular structure N3O7 and a monoisotopic mass of 489,2475 g / mol.

[0116] Synthesis of HL-Lys(7-DCCA)-OH (1b): Compound 1a (3.5 mg, 7.15 μmol, 1 eq.) 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 h. The solvent was evaporated, and the remaining solid was triturated with diethyl ether. The precipitate was isolated by centrifugation to give C. 20 H 27 Compound 1b was obtained with the molecular structure of N3O5 and a monoisotopic mass of 389,1951 g / mol.

[0117] Synthesis of azidoacetic acid N-succinimidyl ester (1c): N-hydroxysuccinimide (NHS; 62.6 mg, 0.54 mMol, 1.1 eq.) was dissolved in 750 μL of tetrahydrofuran (THF) and azidoacetic acid (37 μL, 0.49 mMol, 1 eq.) was added. N,N'-dicyclohexylcarbodiimide (DCC; 112.3 mg, 0.54 mMol, 1.1 eq.) was dissolved in 750 μL of THF and added dropwise to the reaction mixture. After 17 h, the reaction mixture was separated by suction filtration and the filtrate was evaporated. To further remove impurities, the product was dissolved in 20 mL of LDCME and extracted twice with 10 mL of saturated NaHCO3 and 50 mL of brine. The organic layer was dried over Na2SO4. C was obtained by evaporating the filtrate. 22 H 28 Compound 1b was obtained, which has the molecular structure of N6O6 and a monoisotopic mass of 472,2070 g / mol.

[0118] Synthesis of azidoacetyl-L-Lys(7-DCCA)-OH (1d): Compound 1b (2.8 mg, 7.2 μmol, 1 eq.) was dissolved in 500 μL of DMF along with 1c (4.28 mg, 21.6 μmol, 3 eq.). 500 μL of 0.1 M sodium phosphate buffer, pH 8, was then added, and the reaction was stirred at room temperature for 16 h. 22 H 28 Compound 1d, which has the molecular structure of N6O6 and a monoisotopic mass of 472,2070 g / mol, was isolated by semi-preparative C-18 RP-HPLC and lyophilized.

[0119] Synthesis of compound 1 via CuAAC: Copper-catalyzed azide-alkyne cycloaddition (CuAAC) was carried out 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 of DMSO, 75 μL of HO, 25 μL of 100 mM CuSO solution (5 eq.), and 25 μL of 200 mM THPTA solution (10 eq.). The reaction was initiated by adding 25 μL of 100 mM sodium ascorbate solution (5 eq.). 77 H 102 N 16O 19 The reaction product, Compound 1, with a molecular structure of and a monoisotopic mass of 1554,7507 g / mol, was isolated by semi-preparative C-18 RP-HPLC and lyophilized. [Example]

[0120] Example 4: Synthesis of modified MC-(Prtyr)R compound 2

[0121] According to the present invention, MC-(Prtyr)R was modified with HL-Lys(7-DCCA)-azidopropylamide, an example of a heterocyclic modification in which the organic coupling group is an amino group and is positively charged at physiological pH. The modification also contains a fluorescent moiety. The synthetic route is shown in Figure 3 and described in more detail below.

[0122] 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 LMF and stirred in the dark for 15 minutes. Next, 3-azidopropanamine (0.7 μL, 6.75 μmol, 1.1 eq.) was added and stirred in the dark for 16 hours. 28 H 41 Compound 2a, with the molecular structure of N7O6 and a monoisotopic mass of 571,3118 g / mol, was isolated by C-18 SPE.

[0123] Synthesis of HL-Lys(7-DCCA)-azidopropylamide (2b): Compound 2A (3.5 mg, 7.15 μmol, 1 eq.) was dissolved in 700 μL of dichloromethane (DCM), followed by the addition of 300 μL of TFA. The reaction was stirred at room temperature for 2 h. The solvent was evaporated, and the remaining solid was triturated with diethyl ether. The precipitate was isolated by centrifugation to give C. 23 H33 Compound 2b was obtained, which has the molecular structure of N7O4 and a monoisotopic mass of 471,2594 g / mol.

[0124] Synthesis of compound 2 via CuAAC: Copper-catalyzed azide-alkyne cycloaddition (CuAAC) was carried out 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 of DMSO, 75 μL of HO, 25 μL of 100 mM CuSO solution (5 eq.), and 25 μL of 200 mM THPTA solution (10 eq.). The reaction was initiated by adding 25 μL of 100 mM sodium ascorbate solution (5 eq.). 78 H 107 N 17 O 17 The reaction product, compound 2, with a molecular structure of 1554,7507 g / mol monoisotopic mass, was isolated by semi-preparative C-18 RP-HPLC and lyophilized. [Example]

[0125] Example 5: MdhA 7 Structural modification of MC-LR by Michael addition to the α,β-unsaturated carbonyl group of

[0126] Several different structural modifications were performed to the MC-LR A via Michael addition reactions. 7 The reaction is carried out by introducing the ATP-binding domain into the MdhA site, thereby providing a variety of modified toxins according to the present invention. 7 It occurs between an α,β-unsaturated carbonyl group (Michael acceptor) and a nucleophile (Michael donor).

[0127] The following modifying molecules were used for the structural modification of MC-LR.

[0128] Cysteine ​​(Cys) is an example of structural modification with amino and carboxyl groups as amphoteric organic inhibitors and organic coupling groups, resulting in [Mlan 7 ]MC-LR (Mlan = N-methyllanthionine).

[0129] Ammonia (NH3) is another example of structural modification with an amino group as an organic coupling group and an inhibitory group that is positively charged at physiological pH, resulting in [Mdap 7 ]MC-LR (Mdap = N-methyl-2,3-diaminopropionic acid).

[0130] N-type amines as another example of structural modification with amphiphilic organic inhibitors and amino and carboxyl groups as organic coupling groups α -tert-butoxycarbonyl-L-lysine (Boc-Lys), resulting in [LysMalA 7 ]MC-LR(LysMala=N ε -lysino-N-methylalanine).

[0131] Tris(2-carboxyethyl)phosphine (TCEP) is another example of structural modification with a negatively charged inhibitor at physiological pH and a carboxyl group as an organic coupling group, resulting in [TCEPMalA 7 ]MC-LR (TCEPMala = tris(2-carboxyethyl)phosphino-N-methylalanine).

[0132] In each case, a Michael addition was performed with MC-LR (1 eq.) in 50 μL of DMSO and added to 150 μL of 1% (w / v) aqueous K2CO3 solution containing Michael donor (40-100 eq.). The reaction was stirred overnight at 40 °C. The resulting MC-Michael adduct was acidified with 10% HCl, isolated by semi-preparative C-18 RP-HPLC, and lyophilized.

[0133] For the Michael donor Boc-Lys, the isolated product was subjected to a Michael reaction using 1 mL of TFA / HO / ACN (1:1:1, v / v / v) at room temperature for 1-2 h, followed by cleavage of the Boc protecting group to give the final product [LysMalA]. 7 ]MC-LR was isolated by semi-preparative C-18 RP-HPLC and lyophilized. [Example]

[0134] Example 6: Copper-catalyzed azide-alkyne cycloaddition (CuAAC) to [AzProMDap 7 Structural modification of ]MC-LR

[0135] 3-Azido-propan-1-amine (Azpro) was added to the A of MC-LR by Michael addition reaction as described in Example 5 above. 7 Introduced in the place, [AzProMDap 7 ]MC-LR (AzProMdap = 3-N-azidopropyl-2-N-methyl-2,3-diaminopropionic acid) was obtained.

[0136] Then click AzProMDap 7 ]MC-LR was further modified with Prglys as an example of structural modification using CuAAC as an amphoteric organic inhibitor and amino and carboxyl groups as organic coupling groups.

[0137] [AzProMDap 7 CuAAC between ]MC-LR and Prglys was performed as described in Example 2 above, and [AzPro(Prglys)Mlap 7 ]MC-LR was obtained. [Example]

[0138] Example 7: Protocol for cell culture of OATP-expressing cancer cell lines and subsequent cytotoxicity testing

[0139] 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 kindly provided by Prof. Jo:rg Ko:nig (Friedrich-Alexander-University Erlangen-Nuremberg, Germany).

[0140] All cell lines were maintained in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, a non-essential amino acid mixture, and 2 mM glutamine at 37°C and 5% CO .

[0141] HEK293 OATP1B1+ and corresponding control cell lines were continuously selected with 800 μg / mL geneticin (G418), while 250 μg / mL hygromycin B was used for selection of HEK293 OATP1B3+ and its empty vector control.

[0142] To test the cytotoxicity of the modified MC-(Aznva)R and MC-(Prtyr)R toxins described in Examples 2-4, each cell line was plated at 5x10 cells per well. 4 Each cell was seeded in triplicate in a 96-well plate without a selection marker. After 24 hours, 10 mM sodium butyrate was added to induce transporter expression. The next day, the medium was removed, and the cells were incubated for 48 hours with eight different concentrations of each of the modified toxins (0.01 nM to 3 μM), as well as unmodified MC-(Aznva)R and MC-(Prtyr)R as controls, in medium without a selection marker.

[0143] Next, the cells were fixed, washed, and stained with sulforhodamine B (SRB) as previously described (Vichai et al., Nature Protocols 2006, 1, 1112-1116). Briefly, 10 μL of 10% trichloroacetic acid was added directly to each well and incubated at 4°C for 1 h. The solution was then removed, and each well was washed three times with 200 μL of HO. The wells were then dried, and 100 μL of 0.057% SRB solution (in 1% acetic acid) was added per well and incubated at 4°C for 30 min. The solution was then removed, and each well was washed three times with 200 μL of 1% acetic acid. After drying the wells, 200 μL of Tris buffer (10 mM, pH 10.5) was added, and the plate was shaken for 5 min. 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 twice.

[0144] Cell viability and IC50 values ​​(IC50_cytotox) were calculated in GRAPHPAD PRISM6 using nonlinear regression (sigmoidal dose-response). [Example]

[0145] Example 8: Protein Phosphatase Inhibition (PPI) Assay Protocol

[0146] PPI assays were performed as recently described (Heresztyn et al., Water Res 2001, 35, 3049-3056).

[0147] 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 paranitrophenyl phosphate (pNPP) stock solution (60 mM) and DTT solution (20 mM) (5:4:1, (v / v)), and pNPP was freshly prepared. The enzyme dilution buffer was prepared by mixing 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) in a 65:20:5:10 (v / v) ratio. Protein phosphatase-1 catalytic subunit (α isoform from rabbit, Sigma-Aldrich) was diluted to 10 units / mL in the enzyme dilution buffer and stored on ice.

[0148] The assay was performed in a 384-well plate. Diluted modified toxins, as well as unmodified MC-(Aznva)R, MC-(Prtyr)R, MC-L(Azlys), and MC-LR (4 μL in dimethyl sulfoxide (DMSO)), were preincubated with enzyme solution (4 μL) for 5 minutes at 37°C. Next, 40 μL of substrate solution was added to each well and incubated for 2 hours at 37°C. Seven toxin concentrations ranged from 0.001 nM to 1 μM, with a constant substrate concentration of 20 mM 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 diluted enzyme to prevent substrate hydrolysis. To determine PPI, absorbance was measured at 405 nm using a TECAN Infinity M PLEX plate reader. Experiments were performed at least twice, and IC50 values ​​(IC50_PPI) were calculated using nonlinear regression (sigmoidal dose-response) in GraphPad PRISM 6. [Example]

[0149] Example 9: Bioassay Results

[0150] Tables 1-5 below separately summarize the results of the assays described in Examples 7 and 8 above for general cytotoxicity (IC50_Cytotox) and protein phosphatase inhibition (IC50_PPI) against OATP1B1- and OATP1B3-expressing cell lines for the base structures MC-(Aznva)R (Table 1), MC-(Prtyr)R (Tables 2-3), MC-L(Azlys) (Table 4), and MC-LR (Table 5) and the corresponding modified toxins derived therefrom according to the present invention. The tables also show the ratios of IC50_Cytotox values ​​to IC50_PPI values, which were used to evaluate whether the structural modifications exhibited the desired effect, as well as the ratios of these IC50 values ​​for the modified toxins and the corresponding base structures. In this context, a greater IC50_Cytotox / IC50_PPI ratio for the modified toxin than that for the base structure indicates an enhanced therapeutic window for the modified toxin against the cell line. Such an expansion of the therapeutic window occurs when the cellular uptake of the modified toxin is decreased relative to protein phosphatase inhibition and / or when the protein phosphatase inhibition of the modified toxin is increased relative to cellular uptake. Furthermore, a ratio of IC50_PPI of the modified toxin to IC50_PPI of the base structure less than 1 indicates an enhanced potency of the cytotoxic mode of action of the modified toxin based on the inhibition of the corresponding protein phosphatase compared to the base structure.

[0151] The results of the structural modifications of the base structure microcystin MC-(Aznva)R (Table 1) show that the therapeutic window for both OATP-expressing cell lines can be improved for all microcystins modified according to the present invention compared to the base structure. Furthermore, the PPI of two of the three modified microcystins was improved as indicated by a lower IC50_PPI compared to the IC50_PPI of the base structure, while the IC50_PPI of the third modified microcystin was unaffected by the structural modifications.

[0152] The results of the structural modifications of the base structure MC-(Prtyr)R (Tables 2-3) show that all microcystins modified according to the present invention can extend the therapeutic window of at least one OATP-expressing cell line compared to the base structure. Four structural modifications extended the therapeutic window of one of the two OATP-expressing cell lines, while 14 structural modifications extended the therapeutic window of both OATP-expressing cell lines.

[0153] Furthermore, the results show that the PPI of eight of the microcystins modified according to the present invention can be improved as indicated by the lower IC50_PPI compared to the IC50_PPI of the base structure.

[0154] The results of the structural modification of the base structure MC-L(Azlys) (Table 4) show that for both microcystins modified according to the present invention, the therapeutic window for both OATP-expressing cell lines can be improved compared to the base structure.

[0155] Furthermore, the results show that the PPI of both microcystins modified according to the present invention can be improved as indicated by the lower IC50_PPI compared to the IC50_PPI of the base structure.

[0156] The results of the structural modifications of the base structure MC-LR (Table 5) showed that for all microcystins modified according to the present invention, the therapeutic window of both OATP-expressing cell lines could be improved compared to the base structure.

[0157] Furthermore, [AzPro(Prlys)Mlap 7 ] The PPI of MC-LR was improved compared to the base structure, as indicated by the lower IC50_PPI compared to that of MC-LR.

[0158] Thus, an increase in therapeutic window of up to 2400-fold, or more than three orders of magnitude, has been achieved with the modified toxin according to the present invention. These improvements and the associated advantages would not have been anticipated by one of ordinary skill in the art. [Example]

[0159] Example 10: ADC synthesis

[0160] According to a further embodiment of the present invention, an ADC was synthesized using the monoclonal antibody (mAb) trastuzumab (trade name: Herceptin), which targets Her2 (human epidermal growth factor receptor 2 = ERBB2, erb-b2 receptor tyrosine kinase 2) as a representative benchmark mAb in ADC development, and a homomeric dimer of MC-PrgTyr-Arg ("MC dimer") as a representative example of a modified microcystin according to the present invention as a payload. The MC dimer was conjugated to the mAb using a cleavable linker.

[0161] The MC dimer is located at position X. 2 It was produced by crosslinking two MC-PrgTyr-Arg monomers using the trifunctional crosslinking moiety BM-II, which uses an alkyne group as the coupling functional group and has two terminal azide groups as complementary coupling functional groups, to form a covalent bond between the MC-PrgTyr-Arg monomers.

[0162] [ka]

[0163] Position X of two MC-PrgTyr-Arg monomers 2The cross-linking between the alkyne coupling functional group on MC-1 and the two terminal azide groups of the bridge moiety BM-II as complementary coupling functional groups was achieved by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Briefly, CuAAC was carried out using a reaction mixture of 2 eq. (eq.) of the monomer in DMSO, 1 eq. of the bridge moiety BM-II, 10 eq. of a 100 mM CuSO4 aqueous solution, and 20 eq. of a 200 mM tris((1-hydroxy-propyl-1H-1,2,3-triazol-4-yl)methyl)amine (THPTA) aqueous solution. The reaction was initiated by adding 10 eq. of a 100 mM sodium ascorbate aqueous solution. The reaction product was isolated by semi-preparative C-18 RP-HPLC and lyophilized. The structural formula of the MC dimer is shown in Figure 6.

[0164] For monoclonal antibody production, genes encoding the heavy and light chain variable regions of trastuzumab were synthesized and cloned into human heavy and light chain vectors encoding human IgG1. Both constructs were verified by DNA sequencing. Vector DNA was prepared for transient transfection into CHO cells. mAbs were purified from cell culture supernatants (e.g., with Protein A) aiming for a purity of 90% or greater. Purified mAbs were quantified by absorbance at 280 nm and characterized by SEC and SDS-PAGE.

[0165] The MC-dimer-linker conjugate was obtained by coupling the MC-dimer to a cleavable maleimidocaproyl-Val-Ala-(p-aminobenzyl)-(para-nitrophenyl)-carbonate (mc-Val-Ala-PAB-PNP) linker by incubating 1 eq. of MC-dimer and 1 eq. of linker in DMF and DIPEA (2 eq.) for 1–3 h at 25 °C. The amino group incorporated into the MC-dimer via the bridging moiety BM-I was then attached to the mc-Val-Ala-PAB-PNP linker via carbamate bond formation between the activated carbonate ester on the linker and the amino group of the MC-dimer, yielding the mc-Val-Ala-PABC-MC dimer (PABC = p-aminobenzylcarbamate). The resulting MC-dimer-linker conjugate was isolated by RP-HPLC, and its identity was confirmed by mass spectrometry. The linker-MC-dimer conjugate was then purified by RP-LC aiming for a purity of 95% or higher. Figure 7 shows the structural formula of the MC-dimer-linker conjugate.

[0166] For ADC synthesis, the natural (non-artificial) mAb trastuzumab was dissolved in 5% tris(hydroxymethyl)aminomethane (TRIS) buffer (0.5 M, supplemented with 25 mM ethylenediaminetetraacetic acid (EDTA), pH 8.5) to a final concentration of 26.5 mg / mL. For mAb reduction, 1 eq. of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mM) was added to a final DAR of approximately 2, or 2 eq. of TCEP to a final DAR of approximately 4. The reaction was incubated at room temperature for 1.5 h and then diluted with 2 mM EDTA in PBS (pH 7.4) to a final concentration of 5 mg / mL.

[0167] 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 a DAR of approximately 1000, or with additional 3% cyclodextrin (CD) for a DAR of approximately 4. To conjugate MC-dimer linker to reduced mAb with a DAR of approximately 2, 4 eq. of MC-dimer linker taken from a 10 mM MC-dimer linker solution in dimethylacetamide (DMA) was added to the reduced mAb (5 mg / mL in PBS with 2 mM EDTA) and the reaction was incubated at room temperature for 2 h. To achieve a DAR of approximately 4, 8 eq. of linker-MC dimer was used and added to the reduced mAb (5 mg / mL in PBS with 2 mM EDTA, 3% CD) and incubated at room temperature for 3.5 h. To improve solubility, a final concentration of 5% DMA was used for DAR 2 and 10% DMA for DAR 4. The conjugation reaction was stopped for 30 min by adding an equimolar amount of N-acetyl-L-cysteine ​​(NAC) to the MC dimer-linker. The reaction mixture was then desalted by gel filtration on a NAP column and eluted with PBS (DAR2) or PBS containing 3% CD (DAR4). An amount of activated charcoal (100 mg / mL in PBS or PBS containing 3% CD, pH 7.4) equal to the amount of mAb was then added. After 1 h, the charcoal was removed by centrifugation (4000 g, 15 min). The supernatant containing the ADC was analyzed by SEC and HIC and sterile filtered.

[0168] The efficacy of the ADCs was tested in vitro using cell viability assays (e.g., using the CellTiter-Glo Luminescent Cell Viability Assay (Promega GmbH, Walldorf, Germany)) with tumor antigen-expressing cancer cell lines such as NCI-N87 expressing the Her2 antigen, and it was found that, upon conjugation to antibodies, the modified toxins of the invention were selectively internalized by target cells and efficiently caused tumor cell death, achieving EC50 values ​​in the picomolar range.

[0169] The results demonstrate that the present invention provides modified toxins for use as payloads in ADCs with a significantly improved safety profile, thereby improving the therapeutic efficacy of ADCs without resulting in unacceptable side effects or toxicity.

[0170] The present invention is not limited by the description based on the embodiments, but rather includes every novel feature and every novel combination of features, and in particular includes every combination of features in the claims and the description, even if that feature or combination of features is not explicitly defined in the claims, the description or the embodiments.

[0171] Table 1: Results of cytotoxicity assay (IC50_Cytotox) and protein phosphatase inhibition assay (IC50_PPI) for the base structure MC-(Aznva)R (reference) and its modifications. "SE" indicates standard error of at least two experiments.

[0172] [Table 1]

[0173] Table 2: Results of cytotoxicity assay (IC50_Cytotox) and protein phosphatase inhibition assay (IC50_PPI) for the base structure MC-(Prtyr)R (reference) and its modifications. "SE" indicates standard error of at least two experiments. Azidoethanol (Azeth), biotin-PEG-3-azide (Azbio), azidohomoalanine (AzHala), azidophenylalanine (AzPhe), N1-azidospermine (AzSpe), azidopropylformamide (AzProam), azidoacetic acid (Azacac), 2-amino-3-azidobutanoic acid (AzAbu), 3-amino-4- Azidobutyric acid (AzDbu), 3-azido-D-alanine (AzDala), 3-azido-L-alanine (AzLala), 6-azido-L-lysine (Azlys), 3-azido-propan-1-amine (Azpro), BM-I (1,4-diazidoacetylpiperazine), BM-II (1-azidoacetyl-4-(2-azido-L-lysinyl)-piperazine).

[0174] [Table 2-1] [Table 2-2]

[0175] Table 3: Results of cytotoxicity assay (IC50_Cytotox) and protein phosphatase inhibition assay (IC50_PPI) for the base structure MC-(Prtyr)R (reference) and its modifications. "SE" indicates standard error of at least two experiments.

[0176] [Table 3]

[0177] Table 4: Results of cytotoxicity tests (IC50_Cytotox) and protein phosphatase inhibition tests (IC50_PPI) for the base structure MC-L (Azlys) (reference) and its modifications. "SE" indicates the standard error of at least two experiments. Modifications: propargylamine (Prgam), propargyllysine (Prglys).

[0178] [Table 4]

[0179] Table 5: Results of cytotoxicity assay (IC50_Cytotox) and protein phosphatase inhibition assay (IC50_PPI) for the base structure MC-LR (reference) and its modifications. "SE" indicates the standard error of experiments repeated at least twice. Modifications: N-methyllanthionine (Mlan), N-methyl-2,3-diaminopropionic acid (Mdap), N ε -lysine-N-methylalanine) (LysMala), tris(2-carboxyethyl)phosphino-N-methylalanine (TCEPMala), propargyl lysine (PrgLys).

[0180] [Table 5]

Claims

1. 1. A method for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody drug conjugate, comprising: selecting a cyanobacterial or fungal toxin having a basic structure known to be cytotoxic to a target cell after being taken up by a transport protein of the target cell; modifying the base structure with at least one structural modification; The at least one structural modification is an organic inhibitory group or effector molecule that reduces uptake of the modified toxin into the target cell by the transport protein compared to the base structure; an organic coupling group configured to covalently conjugate the modified toxin to an antibody; 1. A method for modifying a cyanobacterial or fungal toxin for use as a payload in an antibody drug conjugate, comprising:

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

3. 3. The method of claim 1 or 2, wherein the base structure is a derivative of a naturally occurring toxin, the derivative comprising modification of the naturally occurring toxin with at least one functional group configured for or capable of chemical conjugation to further modify the base structure with at least one structural modification.

4. The basic structure has the general formula cyclo(-A １ -X ２ -A ３ -Z ４ -A ５ -A ６ -A ７ ) and a microcystin having the formula: Independently of each other, A １ and A ３ each represents a D-amino acid, and A ５ is selected from the group consisting of Adda, DM-Adda, dm-Adda, (6Z)Adda, and ADM-Adda; ６ is D-Glu and D-Glu(OCH ３ ) selected from the group consisting of 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; ２ and Z ４ are each independently an L-amino acid, A １ , X ２ , A ３ , Z ４ , and A ７ 4. The method of claim 1, wherein at least one of the structural modifications is a modified amino acid having a side chain not present in the naturally occurring counterpart of the microcystin, the side chain comprising a functional group configured or capable of covalently conjugating the at least one structural modification.

5. 5. The method of claim 1, wherein the at least one structural modification comprises an organic moiety that includes both an organic inhibiting group and an organic coupling group.

6. the at least one structural modification comprises a first structural modification comprising the organic inhibitor or effector molecule and a second structural modification comprising the organic coupling group; 3. The method of claim 2, wherein modifying the base structure comprises incorporating the first structural modification into or attaching the first structural modification to a side chain of a first amino acid, and incorporating the second structural modification into or attaching the second structural modification to a side chain of a second amino acid different from the first amino acid.

7. 7. The method of claim 1, wherein the organic inhibitor group carries at least one charge at physiological pH values.

8. 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. 10. The method of claim 1, wherein the organic inhibitor group comprises an amphoteric group having at least one cationic charge and at least one anionic charge at physiological pH values.

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

12. 12. The method of claim 1, wherein the organic inhibitor comprises a plurality of amino acids.

13. 13. The method of any one of claims 1 to 12, wherein the effector molecule comprises a competitive or allosteric inhibitory ligand of the transport protein or a functional portion thereof.

14. 14. The method of any one of claims 1 to 13, wherein the organic coupling group is selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, an azide group, an alkyne group, an alkene group, a thiol group, an aldehyde group, a keto group, a tetrazine group, and any combination thereof.

15. a basic structure that is cytotoxic to target cells after being incorporated into a transport protein of the target cells; at least one structural modification of said base structure, said at least one structural modification comprising: at least one structural modification of said base structure comprising an organic inhibitory group or effector molecule that reduces uptake of the modified toxin into a target cell by said transport protein compared to said base structure; an organic coupling group configured for covalently conjugating the toxin to the antibody; Modified cyanobacterial or fungal toxins.

16. The at least one structural modification is comprising a B—In group, "In" represents an inhibitory group or effector molecule that reduces the uptake of toxin into the target cell by the transport protein; "B" represents a bridging group that covalently bridges the inhibitory group or effector molecule In to the base structure; The modified toxin of claim 15.

17. The bridging group B is a triazole, a peptide bond, a disulfide, C １ ~C １０ Alkyl group, C １ ~C １０ Alkylaryl group, (oxyethylene) １～１０ groups, amide bonds, thioamide bonds, ether bonds, thioether bonds, triazoles, dihydropyridazines or higher amines (e.g., secondary (2 ０ ) amine, tertiary (3 ０ ) amine or quaternary (4 ０ 17. The modified toxin of claim 16, comprising a group selected from the group consisting of: a hydroxyl group, a thiocarbamate group, a urea group, a thiourea group, a phosphate ester group, a phosphamide group, a sulfonamide group, an oxime group, or any combination thereof.

18. 18. The modified toxin of claim 16 or 17, wherein the bridging group B comprises a triazole.

19. 19. The modified toxin of any one of claims 15 to 18, wherein the basic structure is selected from the group consisting of microcystin, nodularin, or amanitin.

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

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

22. (A) providing a toxin having a base structure and at least one functional group capable of covalent conjugation known to be cytotoxic to a target cell after incorporation into a transport protein of the target cell; (B) introducing at least one structural modification into the toxin via covalent conjugation to a functional group of the toxin; 21. A method for producing a modified cyanobacterial or fungal toxin according to any one of claims 15 to 20, comprising:

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

24. (a) providing a monoclonal antibody or 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 said organic coupling group.

25. (a) providing a toxin having (i) a basic structure known to be cytotoxic to a target cell after incorporation into a transport 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 a first functional group, the at least one structural modification comprising an organic inhibitory group or an effector molecule that reduces uptake of the modified toxin into a target cell by a transport protein compared to the base structure; (c) covalently attaching a toxin to the antibody via a second functional group, thereby producing an antibody drug conjugate; A method for producing an antibody-drug conjugate comprising:

26. 24. A modified cyanobacterial or fungal toxin according to any one of claims 15 to 20, or an antibody drug conjugate according to claim 23, for use in the treatment of malignant disease.

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