Organic cytotoxic oligomers as payloads for antibody-drug conjugates and their use

Abstract

The present invention provides a cytotoxic oligomer for pharmaceutical use. The cytotoxic oligomer comprises a first organic cytotoxic entity, at least a second organic cytotoxic entity, and a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities. The monomers of the first and second organic cytotoxic entities exert cytotoxic effects on target cells by being actively transported into the target cells by the target cell's transporter protein and / or by inhibiting intracellular enzymes of the target cells. The covalent bond between the first and second organic cytotoxic entities is substantially resistant to cleavage under physiological conditions.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This patent application claims priority to European Patent Application No. 23187008.0, filed on 21 July 2023, the entirety of which is incorporated herein by reference.

[0002] (Sequence Listing) This application includes an electronic sequence listing in XML format compliant with the WIPO ST.26 standard, and has 17 sequences as part of the specification, the entire contents of which are incorporated herein by reference.

[0003] (Technical field) This invention relates to biologically active cytotoxic anti-cancer payloads or agents. In particular, this invention relates to organic cytotoxic substances, more specifically cyanobacteria and fungal toxins designed to be used as payloads for antibody-drug conjugates. This invention further relates to antibody-drug conjugates in which such biologically active cytotoxic anti-cancer payloads are conjugated to antibodies that target specific tumor antigens. [Background technology]

[0004] Currently, cancer is the second leading cause of death worldwide, accounting for approximately 10 million deaths annually. Due to global population growth and aging, the incidence of cancer has increased over the past few decades and is expected to continue rising.

[0005] Currently, most anticancer drugs used in conventional chemotherapy are generally cytotoxic because they cannot adequately distinguish between cancer cells and healthy tissue, and they have known serious side effects and significant limitations on patients' quality of life.

[0006] The need for anticancer drugs with a specific spectrum of action against malignant cells has driven the development of antibody-drug conjugates.

[0007] Antibody-drug conjugates (hereinafter also referred to 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 preserving healthy cells. An ADC is a biopharmaceutical in which a monoclonal antibody is conjugated to a biologically active and often cytotoxic drug portion, also referred to herein as the "payload," combining the target specificity of the monoclonal antibody with the cancer cell-killing ability of the cytotoxic drug.

[0008] Conceptually, ADCs are expected to target tumor cells through the target specificity of monoclonal antibodies, while preserving healthy tissue. 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 the release of the payload into surrounding tissue from lysed or dead cancer cells.

[0009] Current efforts to reduce or eliminate undesirable toxic side effects on healthy cells are primarily focused on improving the stability of the chemical bond between the cytotoxic payload and the monoclonal antibody, thereby avoiding premature release of the drug outside of tumor cells.

[0010] For example, International Patent Publication No. 2018 / 206715 and International Patent Publication No. 2018 / 219619 (their entire disclosures are incorporated herein by reference) disclose modified cyanobacteria-derived microcystin and nodularin as cytotoxic payloads, which each contain non-natural amino acids, etc., that provide chemical anchor groups for binding the payload to a linker or antibody, resulting in a uniform and stably loaded ADC with limited payload release in the bloodstream.

[0011] However, improved chemical bonding between antibodies and cytotoxic payloads only partially overcomes the aforementioned challenges of current ADCs. In particular, the problem of payload release after target cancer cell death remains unresolved. Furthermore, the high activity of cytotoxic substances creates unresolved health and safety risks in their manufacture and handling, requiring costly infrastructure and production.

[0012] An unpublished European Patent Application No. 22192517.5 (the entirety of which is incorporated herein by reference) teaches modifications of cyanobacteria or fungal toxins that reduce the uptake of the modified toxin into target cells by transporter proteins through structural modifications including organic inhibitor groups or effector molecules.

[0013] In this regard, an object of the present invention is to provide an improved organic cytotoxic substance for use as a payload for antibody-drug conjugates. Another object of the present invention is to provide an improved antibody-drug conjugate.

[0014] These objectives are addressed according to the subject matter of the independent claims. Preferred embodiments are the subject matter of the dependent claims and the description below. [Overview of the project]

[0015] In certain embodiments, the present invention provides pharmaceutically acceptable cytotoxic oligomers or pharmaceutically acceptable salts thereof. The pharmaceutically acceptable cytotoxic oligomer comprises a first organic cytotoxic entity, at least a second organic cytotoxic entity, and a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and at least a second organic cytotoxic entity, wherein each monomer of the first and second organic cytotoxic entities exerts a cytotoxic effect on target cells by (i) being actively transported into target cells by target cell transporter proteins and / or (ii) inhibiting intracellular enzymes of target cells, and the covalent bond between the first and at least a second organic cytotoxic entity is substantially resistant to cleavage under physiological conditions.

[0016] In certain embodiments, the present invention provides a method for producing the pharmaceutically active cytotoxic oligomers defined above. The method includes (A) providing a first organic cytotoxic entity and at least a second cytotoxic entity, each of which exerts a cytotoxic effect on a target cell by (i) being actively transported into the target cell by a target cell transporter protein and / or (ii) inhibiting an intracellular enzyme of the target cell, and (B) forming a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity that is substantially resistant to cleavage under physiological conditions, thereby forming a pharmaceutically active cytotoxic oligomer.

[0017] In certain embodiments, the present invention provides an application for producing antibody-drug conjugates, which involves covalently conjugating the pharmaceutically cytotoxic oligomer defined above to an antibody or its antigen-binding fragment.

[0018] In certain embodiments, the present invention provides an antibody-drug conjugate comprising an antibody or its antigen-binding fragment, and a pharmaceutically acceptable cytotoxic oligomer or a pharmaceutically acceptable salt thereof as defined above, wherein the pharmaceutically acceptable cytotoxic oligomer is covalently attached to the antibody or its antigen-binding fragment.

[0019] In certain embodiments, the present invention provides a method for producing an antibody-drug conjugate comprising: (a) providing a pharmaceutically acceptable cytotoxic oligomer or a pharmaceutically acceptable salt thereof as defined above; (b) providing an antibody or an antigen-binding fragment thereof; and (c) covalently attaching the pharmaceutically cytotoxic oligomer to the antibody or the antigen-binding fragment thereof.

[0020] In certain embodiments, the present invention provides applications for the use of the pharmaceutically acceptable cytotoxic oligomers or pharmaceutically acceptable salts thereof, or the antibody-drug conjugates defined above, in the treatment of malignant diseases, particularly cancer, and more specifically, malignant tumors.

[0021] In certain embodiments, the present invention provides a method for treating a malignant disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutically cytotoxic oligomer or a pharmaceutically acceptable salt thereof as defined above, or a therapeutically effective amount of the antibody-drug conjugate as defined above.

[0022] In any of the embodiments described above, or in some embodiments applied thereto, the first organic cytotoxic entity and the second organic cytotoxic entity are independently selected from the group consisting of cyanobacteria toxins, fungal toxins, or their variants or derivatives.

[0023] In any of the embodiments described above, or in some embodiments applied thereto, the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a cyclic oligopeptide having a plurality of amino acids, and the crosslinking entity covalently links one side chain of an amino acid of the first organic cytotoxic entity to one side chain of an amino acid of the second organic cytotoxic entity.

[0024] In some embodiments according to any of the above embodiments, or in some embodiments applied thereto, the intracellular enzyme in the target cell is a phosphatase or polymerase.

[0025] In some embodiments according to any of the above embodiments, or in some embodiments applied thereto, resistance to cleavage includes resistance to lysosomal degradation.

[0026] Preferred modifications of these embodiments can be derived from the description of the invention and the detailed description of the embodiments. [Description of the invention]

[0027] This invention is based on the inventors' finding that cytotoxins generally need to be taken up into cells via either active transport or passive diffusion by transporter proteins before they can exert their cytotoxic function. However, when a cytotoxin is bound to an antibody, thereby forming an ADC, the intracellular uptake of the cytotoxin changes to receptor-mediated endocytosis, such as cratrin-mediated endocytosis, caveolae-mediated endocytosis, or pinocytosis, which is a process in which, after the antibody has bound to the cell surface, the cell absorbs the ADC by inward budding of the cell membrane, thereby transporting the toxin into the target cell.

[0028] Based on this finding, the inventors describe in European Patent Application No. 22192517.5 the possibility of modifying organic cytotoxins, which in their isolated form require active transport to actively pass through the cell membrane and cannot enter cells by passive diffusion, by structural modifications that intentionally inhibit their reception by transporter proteins. In this way, the inventors confine organic cytotoxins with a structural "safety device" that prevents active uptake into cells and the exertion of cytotoxic functions, thereby protecting healthy cells from cytotoxic side effects.

[0029] In the course of further intensive research, the inventors newly discovered that a particularly advantageous structural modification for achieving this effect is the artificial oligomerization of organic cytotoxins via non-cleavable covalent bonds. First, the inventors found that artificial oligomerization strongly inhibits the active intracellular uptake of organic cytotoxins. Second, the inventors observed that the cytotoxicity of oligomerized cytotoxins shows a remarkable improvement in cytotoxicity through a synergistic mechanism that far exceeds the sum of monomeric cytotoxins.

[0030] Due to the cumulative pharmacological interaction of both effects, the cytotoxic oligomers of the present invention achieve a dose range optimized between efficacy and toxicity, i.e., a therapeutic range expanded by several orders of magnitude, compared to monomeric cytotoxins, and attain greater therapeutic efficacy without producing unacceptable side effects or toxicity. Please refer to the detailed description of the embodiments for further details.

[0031] Against this backdrop, one aspect of the present invention relates to a pharmaceutically acceptable cytotoxic oligomer or a pharmaceutically acceptable salt thereof. In particular, the present invention relates to a drug entity, also called a "payload," that contains or comprises a pharmaceutically acceptable cytotoxic oligomer or a pharmaceutically acceptable salt thereof.

[0032] The pharmaceutical cytotoxic oligomer of the present invention comprises a first organic cytotoxic entity, at least a second organic cytotoxic entity, and a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and at least the second organic cytotoxic entity.

[0033] In this specification, the terms "first," "second," and possibly "third," "fourth," etc., are used to distinguish one feature or element of the present invention from others, but do not have any other restrictive meaning unless the context specifically limits them. Therefore, the "first organic cytotoxic entity" described below may also be called the "second organic cytotoxic entity," and similarly, the "second organic cytotoxic entity" described below may also be called the "first organic cytotoxic entity," without departing from the spirit of the present invention.

[0034] In order to achieve the advantageous effects according to the present invention, each of the first organic cytotoxic entity and the second organic cytotoxic entity, in monomer form, (i) requires active transport into target cells by a transporter protein to exert a cytotoxic effect on target cells, or alternatively or additionally, (ii) exerts a cytotoxic effect by inhibiting an enzyme inhibitor, i.e., an intracellular enzyme of the target cell, and it is preferable that the intracellular enzyme is biologically essential for the target cell and that its inhibition exhibits cytotoxicity leading to the death of the target cell.

[0035] Furthermore, an essential feature of the present invention in this respect is that the covalent bond between the first organic cytotoxic entity and at least the second organic cytotoxic entity is substantially resistant to cleavage under physiological conditions.

[0036] In this specification, “physiological conditions” refer to the conditions of the internal environment that exist in nature, such as human blood flow, the intracellular environment, and / or the extracellular environment, and are in contrast to artificial laboratory conditions. In particular, physiological conditions include a temperature range of 20–40°C, atmospheric pressure of approximately 1 bar, pH 6–8, glucose concentration of 1–20 mM, and atmospheric oxygen concentration.

[0037] "Substantially resistant" means that spontaneous cleavage of the covalent bond, e.g., spontaneous hydrolysis, is extremely slow, and the half-life of the covalent bond at 25°C is typically several months or years, and in certain embodiments, several decades. In certain embodiments, "substantially resistant" means resistance to lysosomal degradation. In certain embodiments, "substantially resistant" means resistance to enzymatic and / or chemical cleavage, e.g., cleavage by hydrolase and / or oxidoreductase, and further, resistance to acid-inducible cleavage up to at least pH 4.5 or pH 4.0. In certain embodiments, "substantially resistant" means that the half-life of the covalent bond under physiological conditions is longer than the average or maximum residence time of cytotoxic oligomers in the human body, preferably at least twice, and more preferably several times longer.

[0038] In this way, unlike the corresponding cytotoxic monomers, the cytotoxic oligomers of the present invention are prevented from exhibiting nonspecific cytotoxic activity in the extracellular environment within the human body, such as during early release from ADCs or after release from dead cancer cells. In contrast, when the cytotoxic oligomers of the present invention are bound to an antibody, they are taken up by target cells via receptor-mediated endocytosis as an antibody payload, and after intracellular transport and lysosomal degradation of ADCs, the cytotoxic oligomers are released, exhibiting enhanced cytotoxicity and inducing tumor cell death. Thus, the present invention provides cytotoxic oligomers for use as ADC payloads with a significantly improved safety profile, thereby enabling an increase in ADC dosage to improve therapeutic efficacy without causing unacceptable side effects or toxicity. Furthermore, the safety of the manufacture and handling of the cytotoxic oligomers is also improved compared to monomer-type cytotoxins. Furthermore, this beneficial effect is not limited to ADCs; it can also be achieved when cytotoxic oligomers bind to other targeted entities, including but not limited to nanoparticles, liposomes, micelles, aptamers, peptides, and dendrimers, which bind to specific receptors present on the surface of tumor cells.

[0039] In the spirit of this invention, "oligomer" or "oligomeric" refers to a molecule obtained by covalently linking smaller molecules, i.e., "monomers." Therefore, "organic cytotoxic entity" refers to a cytotoxic entity derived from a cytotoxin monomer having an organic skeletal structure that is known to exhibit cytotoxicity to target cells, particularly cancer cells, after uptake by target cells, especially by transporter proteins. Each organic cytotoxic entity of a cytotoxic oligomer may also be called a "cytotoxic subunit." Below, a cytotoxic oligomer composed of two organic cytotoxic entities is called a "dimer," one composed of three is called a "trimer," and one composed of four is called a "tetramer." In preferred embodiments, the cytotoxic oligomer is a dimer, trimer, or tetramer, more preferably a dimer or trimer, and most preferably a dimer. When a cytotoxic oligomer is composed of the same organic cytotoxic entity, it is called a homooligomer; otherwise, it is called a heterooligomer.

[0040] Cytotoxin monomers having organic skeletal structures that require active intracellular uptake of the toxin by transporter proteins are known to those skilled in the art. For example, the transport of cytotoxin monomers or their organic skeletal structures can be mediated by organic anion transport polypeptides (OATPs) or their subtypes, such as OATP1B1, OATP1B3, and / or OATP1A2, respectively. These OATPs are mainly found in the human liver. Therefore, the inventors recognized that blocking the transport of payload toxins by OATPs is important in avoiding hepatotoxicity and improving the relative safety of ADCs.

[0041] In preferred embodiments of the present invention, the first organic cytotoxic entity and the second organic cytotoxic entity are independently selected from the group consisting of cyanobacteria toxins, fungal toxins, or their variants or derivatives. According to the common understanding in the prior art, “derivative” refers to an organic cytotoxic entity that has the same basic structure as a cyanobacteria toxin or fungal toxin, but has a functional group or other atom or group of atoms instead of a hydrogen atom or substituent, or in which one or more atoms or groups of atoms of the basic structure have been removed. Accordingly, “variant” refers to an organic cytotoxic entity in which the basic structure of a cyanobacteria toxin or fungal toxin is partially modified but is therapeutically equivalent.

[0042] In further preferred embodiments of the present invention, the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a cyclic oligopeptide having a plurality of amino acids, wherein the crosslinking entity covalently links one side chain of an amino acid of the first organic cytotoxic entity to one side chain of an amino acid of the second organic cytotoxic entity. In preferred embodiments, the cyclic oligopeptide comprises 4 to 10 amino acids, particularly 5 to 8 amino acids, for example, exactly 4, exactly 5, exactly 6, exactly 7, or exactly 8 amino acids. Preferably, the cyclic oligopeptide toxin comprises at least one non-proteinogenic amino acid, more preferably a plurality of non-proteinogenic amino acids. Even more preferably, the cyclic oligopeptide comprises a dipeptide sequence comprising a first amino acid selected from the group consisting of Adda, DM-Adda, dm-Adda, (6Z)Adda, and ADM-Adda, and a second amino acid selected from the group consisting of D-Glu and D-Glu(OCH3). More preferably, the first amino acid is selected from Adda, DM-Adda, dm-Adda, and ADM-Adda, and the second amino acid is D-Glu.

[0043] In certain embodiments, the first organic cytotoxic entity and / or the second organic cytotoxic entity is an enzyme inhibitor, particularly a protein phosphatase inhibitor or a polymerase inhibitor, such as an RNA polymerase inhibitor.

[0044] In a preferred embodiment of the present invention, the cyanobacterial toxin is microcystin. Microcystin is a cyclic heptapeptide derived from cyanobacteria that potently inhibits eukaryotic serine / threonine protein phosphatase types 1 and 2A, blocks many essential signal transduction pathways, and ultimately causes cytoskeleton collapse and cell death. Microcystin is mainly taken up by OATP1B1 and OATP1B3.

[0045] In another preferred embodiment of the present invention, the cyanobacterial toxin is nodularin. Nodularin is a cyclic pentapeptide derived from cyanobacteria that is evolutionarily related to microcystin and whose cytotoxic mechanism of action is the inhibition of protein phosphatase types 1 and 2A in the same manner. Nodularin is mainly taken up by OATP1B1.

[0046] In still another preferred embodiment, the fungal toxin is amanitin. Amanitin is a naturally occurring cyclic octapeptide derived from fungi belonging to the subgroup of amatoxins. The most representative α-amanitin is an inhibitor of RNA polymerase II, which binds to the bridging helix of RNA polymerase II and inhibits the translocation of RNA and DNA required to clear the site for the next synthesis cycle.

[0047] In a preferred embodiment of the present invention, the first organic cytotoxic entity and the second organic cytotoxic entity each comprise microcystin having the general structure below. cyclo(-Aa 1 -X 2 -Aa 3 -Z 4 -Aa 5 -Aa 6 -Aa 7 ).

[0048] As used herein, Aa 1 and Aa 3 each independently represent a D-amino acid that can be modified independently of each other, and Aa 5The group is selected from Adda, DM-Adda, dm-Adda, (6Z)Adda and ADM-Adda, and Aa 6 It is selected from the group consisting of D-Glu and D-Glu(OCH3), Aa 7 X 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. 2 and Z 4 These are L-amino acids that can be modified independently of each other. Mdha is N-methyldehydroalanine, and Mdhb is N-methyldehydrobutyrate. 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, MeSer is N-methyl-L-serine, Dhb is dehydrobutyrate, and MeLan is N-methyllanthionine. In this specification, "modified" or "modified amino acid" means an amino acid having a side chain having a binding site to a crosslinking entity, thereby adjacency with the crosslinking entity. In particular, the side chain of a modified amino acid may include chemical groups that constitute part of the covalent bond between the first and second cytotoxic entities, or are incorporated therein.

[0049] In some embodiments, Aa 1 is selected from the group consisting of D-Ala, D-Leu, D-Ser, Gly, or modified D-amino acids or modified Gly. In some embodiments, Aa 3 is selected from the group consisting of D-Asp and D-MeAsp or modified D-amino acids. D-MeAsp is D-erythro-β-methylaspartic acid. In some embodiments, Aa 5 This is selected from Adda, DM-Adda, dm-Adda and ADM-Adda. In some embodiments, Aa 6is D-Glu. Modified amino acids may be derived from, for example, amino acids having coupling functional groups that are not present in the corresponding natural cyanobacterial or fungal toxins, i.e., derivatives thereof. In this specification, “functional group” means the presence of one or more functional groups in a molecule. Monofunctional molecules have one functional group, bifunctional or difunctional molecules have two functional groups, trifunctional molecules have three functional groups, and so on. In organic chemistry, functional groups have a decisive influence on the reactivity of a molecule. Therefore, “coupling functional group” refers to a functional group that has a complementary reactivity with other coupling functional groups and / or is capable of or configured to form at least a portion of a covalent bond with them. For example, modified amino acids may be derived from amino acids selected from the group consisting of azidonorvaline, propargyltyrosine, azidodricin, azidophenylalanine, propargylcysteine, propargylserine, and azidoalanine, and the amino acid may be a D-amino acid or an L-amino acid.

[0050] Microcystin is a potent inhibitor of type 1 and type 2A protein phosphatases. Protein phosphatases 1 (PP1) and 2A (PP2A) are two of the major phosphatases that dephosphorylate serine and threonine residues in eukaryotic cells. For example, the IC50 of naturally occurring microcystin-LR (MC-LR, SEQ ID NO:16) against type 1 protein phosphatase is 0.03 nM, and the IC50 against type 2A protein phosphatase is 0.04 nM. In the prior art, A 5 and A 6 It has been established that the position of microcystin is essential and sufficient for the inhibitory effect on PP1 and PP2A.

[0051] Therefore, in a preferred embodiment, the crosslinking entity is Aa of the first organic cytotoxic entity. 1 , X 2 , Aa 3 , Z 4 or Aa 7 Either one of the side chains of the second organic cytotoxic entity Aa 1 , X2 , Aa 3 , Z 4 or Aa 7 It is covalently linked to one of the side chains.

[0052] In some embodiments, the crosslinking entity is the first organic cytotoxic entity Aa 1 , X 2 , Z 4 or Aa 7 Either one of the side chains of the second organic cytotoxic entity Aa 1 , X 2 , Z 4 or Aa 7 It is covalently linked to one of the side chains.

[0053] In some embodiments, the crosslinked entity is X of the first organic cytotoxic entity. 2 , Z 4 or Aa 7 Either one of the side chains of the second organic cytotoxic entity X 2 , Z 4 or Aa 7 It is covalently linked to one of the side chains.

[0054] In some embodiments, the crosslinked entity is X of the first organic cytotoxic entity. 2 or Z 4 Either one of the side chains of the second organic cytotoxic entity X 2 or Z 4 It is covalently linked to one of the side chains.

[0055] In some embodiments, the crosslinking entity is the first organic cytotoxic entity Aa 1 The side chain of the second organic cytotoxic entity Aa 1 , X 2 , Aa 3 , Z 4 or Aa 7 It covalently links to one of the side chains. In some embodiments, the crosslinking entity is X of the first organic cytotoxic entity. 2 The side chain of the second organic cytotoxic entity Aa 1 , X 2 , Aa3 , Z 4 or Aa 7 It is covalently linked to one of the side chains. In some embodiments, the crosslinking entity is Aa of the first organic cytotoxic entity. 3 The side chain of the second organic cytotoxic entity Aa 1 , X 2 , Aa 3 , Z 4 or Aa 7 It covalently links to one of the side chains. In some embodiments, the crosslinking entity is Z of the first organic cytotoxic entity. 4 The side chain of the second organic cytotoxic entity Aa 1 , X 2 , Aa 3 , Z 4 or Aa 7 It is covalently linked to one of the side chains. In some embodiments, the crosslinking entity is Aa of the first organic cytotoxic entity. 7 The side chain of the second organic cytotoxic entity Aa 1 , X 2 , Aa 3 , Z 4 or Aa 7 It is covalently linked to one of the side chains.

[0056] In other preferred embodiments of the present invention, the first organic cytotoxic entity and the second organic cytotoxic entity each comprise nodularin having the following general structure. cyclo[-Aa 1 -Aa 2 -Aa 3 -Aa 4 -Aa 5 ].

[0057] In this specification, Aa 1 These independently represent D-Asp, D-MeAsp, or modified D-amino acids, and Aa 2 represents Arg or Har or modified L-amino acid, Aa 3 The group is selected from Adda, DM-Adda, (6Z)Adda and MeAdda, and Aa 4is selected from the group consisting of D-Glu and D-Glu(OCH3), and Aa 5 is Dhb, Mdhb, or a modified L-amino acid. Har is homoarginine, (6Z)Adda is Adda that is a geometric isomer at C-6, and MeAdda is methylated Adda.

[0058] In the prior art, it has been established that the positions A3 and A4 are essential and sufficient for the inhibitory effect of nodularin on PP1 and PP2A. Therefore, in a preferred embodiment, the crosslinking moiety is the Aa of the first organic cytotoxic moiety 1 , Aa 2 or Aa 5 side chain is covalently linked to the side chain of any one of Aa 1 , Aa 2 or Aa 5 of the second organic cytotoxic moiety.

[0059] In some embodiments, the crosslinking moiety is the Aa of the first organic cytotoxic moiety 1 or Aa 5 side chain is covalently linked to the side chain of any one of Aa 1 or Aa 5 of the second organic cytotoxic moiety.

[0060] In some embodiments, the crosslinking moiety is the Aa of the first organic cytotoxic moiety 1 side chain is covalently linked to the side chain of any one of Aa 1 , Aa 2 or Aa 5 of the second organic cytotoxic moiety. In some embodiments, the crosslinking moiety is the Aa of the first organic cytotoxic moiety 2 side chain is covalently linked to the side chain of any one of Aa 1 , Aa 2 or Aa 5 of the second organic cytotoxic moiety. In some embodiments, the crosslinking moiety is the Aa of the first organic cytotoxic moiety 5 side chain is covalently linked to Aa 1 , Aa 2 or Aa5 It is covalently linked to one of the side chains.

[0061] In yet another preferred embodiment, the first organic cytotoxic unit and the second organic cytotoxic unit each comprise an amatoxin having the following general structure. cyclo{Aa 1 -cyclo[Aa 2 -Aa 3 -Aa 4 -Aa 5 -Aa 6 ]-Aa 7 -Aa 8 (SEQ ID NO:1).

[0062] In this specification, Aa 1 These are derivatives having side chains that independently contain isoleucine (Ile), hydroxyisoleucine (Hil), dihydroxyisoleucine (Dhil), or a binding site to a crosslinking entity, Aa 2 It is a derivative thereof having a side chain with a binding site to tryptophan (Trp), hydroxytryptophan (Htp), or a crosslinking entity, Aa 3 Glycine (Gly), Aa 4 isoleucine (Ile), Aa 5 Glycine (Gly), Aa 6 cysteine ​​(Cys), Aa 7 is asparagine (Asn) or aspartic acid (Asp), Aa 8 Aa is a derivative having a side chain with a binding site to proline (Pro), hydroxyproline (Hyp), or a crosslinking entity. 2 and Aa 6 They are connected by sulfoxide (S=O) bridges.

[0063] For example, the amatoxin may be any of the group consisting of α-amanitin, β-amanitin, γ-amanitin, δ-amanitin, ε-amanitin, amanulin, amanulinic acid, amaninamide, amanine, and proamanulin. Preferably, the amatoxin is amanitin.

[0064] In a preferred embodiment, the crosslinking entity is Aa of the first organic cytotoxic entity. 1 , Aa 2 or Aa 8 Either one of the side chains of the second organic cytotoxic entity Aa 1 , Aa 2 or Aa 8 It is covalently linked to one of the side chains.

[0065] In some embodiments, the crosslinking entity is the first organic cytotoxic entity Aa 2 or Aa 8 Either one of the side chains of the second organic cytotoxic entity Aa 2 or Aa 8 It is covalently linked to one of the side chains.

[0066] In some embodiments, the crosslinking entity is the first organic cytotoxic entity Aa 1 The side chain of the second organic cytotoxic entity Aa 1 , Aa 2 or Aa 8 It is covalently linked to one of the side chains. In some embodiments, the crosslinking entity is Aa of the first organic cytotoxic entity. 2 The side chain of the second organic cytotoxic entity Aa 1 , Aa 2 or Aa 8 It is covalently linked to one of the side chains. In some embodiments, the crosslinking entity is Aa of the first organic cytotoxic entity. 8 The side chain of the second organic cytotoxic entity Aa 1 , Aa 2 or Aa 8 It is covalently linked to one of the side chains.

[0067] In some embodiments following or applying any of the above embodiments, the cytotoxic oligomer comprises at least two crosslinking entities, each forming a covalent bond between a first organic cytotoxic entity and a second organic cytotoxic entity in the manner described above.

[0068] In some embodiments following or applying any of the above embodiments, the crosslinking entity covalently links a side chain at a certain amino acid position of a first organic cytotoxic entity to a side chain at the same amino acid position of a second organic cytotoxic entity. For example, Aa 1 and Aa 1 , X 2 and X 2 , Aa 2 and Aa 2 , Aa 3 and Aa 3 , Z 4 and Z 4 , Aa 7 and Aa 7 , Aa 8 and Aa 8 These are some examples.

[0069] In some embodiments following or applying any of the above embodiments, the crosslinking entity covalently links a side chain at a certain amino acid position of the first organic cytotoxic entity to a side chain at a different amino acid position of the second organic cytotoxic entity. For example, Aa of the first organic cytotoxic entity 2 or X 2 The side chain of the second organic cytotoxic entity Aa 1 , Aa 3 , Z 4 or Aa 7 Either one of the side chains, and the Z of the first organic cytotoxic entity. 4 The side chain of the second organic cytotoxic entity Aa 1 , Aa 2 or X 2 , Aa 3 or Aa 7 Examples include covalently linking to one of the side chains.

[0070] In some embodiments following or applying any of the above embodiments, the first organic cytotoxic entity and the second organic cytotoxic entity are substantially identical, i.e., the cytotoxic oligomer is a homooligomer, in particular a homodimer or homotrimer. In certain embodiments, “substantially identical” means that the organic cytotoxic entities contain or are composed of the same amino acid sequence. For example, the first cytotoxic entity and the second cytotoxic entity are derived from the same organic cytotoxic monomer.

[0071] In any embodiment following or applying any of the above embodiments, the first organic cytotoxic entity and the second organic cytotoxic entity are distinct from each other, i.e., the cytotoxic oligomer is a heterooligomer, particularly a heterodimer or heterotrimer. For example, the first organic cytotoxic entity and the second organic cytotoxic entity may contain or be composed of different amino acid sequences (e.g., different modified amino acids). In particular, the first cytotoxic entity and the second cytotoxic entity may be derived from different organic cytotoxic monomers.

[0072] In some embodiments following or applying any of the above embodiments, the crosslinking entity or covalent bond is, respectively, an amide bond, a thioamide bond, an ether bond, a thioether bond, a triazole, a dihydropyridazine, or a higher amine (e.g., secondary (2 0 ) Amine, tertiary (3 0 )amine or quaternary (4 0The crosslinked entity contains or comprises a covalent bond selected from the group consisting of a covalent bond containing an 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. The inventors have found that these types of covalent bonds confer resistance to cleavage under physiological conditions to the crosslinked entity and enhance the advantageous pharmacological effects of the cytotoxic oligomer according to the present invention. In certain embodiments, the crosslinked entity does not contain a disulfide bond, an ester bond, and / or an isourea bond. Furthermore, it is preferable that the crosslinked entity does not contain a substrate for intracellular enzymatic cleavage, such as a substrate or cleavage site for hydrolase and / or oxidoreductase, or a substrate for chemically induced cleavage, such as a substrate that responds to pH changes or reduction potential changes.

[0073] In some embodiments following or applying any of the above embodiments, the crosslinking entity or covalent bond each includes an organic crosslinking portion that covalently links a first organic cytotoxic entity and a second organic cytotoxic entity to each other. The organic crosslinking portion may also spatially separate the first organic cytotoxic entity and the second organic cytotoxic entity. The organic crosslinking portion is not particularly limited, as long as the covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity, which is a key requirement of the present invention, is substantially resistant to cleavage under physiological conditions. Suitable organic crosslinking portions are widely known to those skilled in the art or can be determined based on this disclosure.

[0074] The organic crosslinking portion may be linear (i.e., unbranched) or branched. A branched organic spacer unit may have three or more branches, with at least one branch terminally bound to a first organic cytotoxic entity and a second branch terminally bound to a second organic cytotoxic entity. A third or subsequent branch may terminally bind to a third or subsequent organic cytotoxic entity, or may have a coupling functional group at its terminal, i.e., a functional group for covalently bonding a cytotoxic oligomer to an antibody (via a linker, if necessary). The coupling functional group may be protected with a protecting group.

[0075] Preferably, the crosslinked portion is an organic molecule having a molecular size of about 50 Da to about 3,000 Da, more preferably about 50 Da to about 2,500 Da or about 50 Da to about 2,000 Da, and most preferably about 50 Da to about 1,500 Da or about 50 Da to about 1,000 Da. The crosslinked portion may contain a methylene group, a PEG group, or other groups for spatial separation of organic cytotoxic entities.

[0076] In some embodiments, at least one of the first organic cytotoxic entity, the second organic cytotoxic entity, and the crosslinking entity includes an additional coupling functional group, i.e., a functional group configured, i.e., capable of covalently conjugating the cytotoxic oligomer to an antibody (optionally via a linker). Suitable coupling functional groups for bioconjugating the cytotoxic oligomer to an antibody are widely known to those skilled in the art. For example, the additional coupling functional group may be a functional group selected from the group consisting of amino groups, carboxyl groups, hydroxyl groups, azide groups, alkyne groups, alkene groups, thiol groups, aldehyde groups, keto groups, tetrazine groups, hydrazine groups, or any combination thereof. The additional coupling functional group may be protected with a removable protecting group, such as a tert-butyloxycarbonyl (BOC) group, an acetyl (Ac) group, a tert-butyl ether (tBu), a methyl ester, a benzyl ester, or a tert-butyl ester. Further suitable protecting groups are known to those skilled in the art. In preferred embodiments, the additional coupling functional group is configured, or can be configured, for a bioorthogonal chemical coupling reaction such as a click chemistry reaction. Click chemistry reactions, as used herein, include, for example, copper(I) catalyzed azide-alkyne cycloaddition, strain-accelerated azide-alkyne cycloaddition, strain-accelerated alkyne-nitrone cycloaddition, [3+2] cycloaddition of alkenes and azides, retroelectrophilic Diels-Alder reaction of alkenes and tetrazines, and photoclick reaction of alkenes and tetrazoles. Therefore, the additional coupling functional group is preferably an azide, alkyne, alkene, or tetrazine.

[0077] As described above, cytotoxic oligomers may contain two or more organic cytotoxic entities and may form trimers, tetramers, or even higher-order oligomers. For example, a cytotoxic oligomer may contain at least a third organic cytotoxic entity, and a crosslinking entity may form covalent bonds between the first organic cytotoxic entity, the second organic cytotoxic entity, and at least the third organic cytotoxic entity (e.g., via branched crosslinking portions incorporated into the crosslinking entity as described above). Alternatively, a cytotoxic oligomer may contain a first crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity, and a second crosslinking entity that forms a covalent bond between the second organic cytotoxic entity and the third organic cytotoxic entity, wherein the covalent bonds between the first organic cytotoxic entity, the second organic cytotoxic entity, and at least the third organic cytotoxic entity are substantially resistant to cleavage under physiological conditions in either case.

[0078] In some embodiments of this specification, or in some embodiments applied thereto, resistance to cleavage includes resistance to lysosomal degradation.

[0079] In preferred embodiments according to any embodiment of this specification, or applicable thereto, resistance to cleavage includes resistance to enzymatic cleavage and resistance to acid-induced cleavage up to pH 4.5 or pH 4.

[0080] The above-mentioned pharmaceutical cytotoxic oligomers can be advantageously used as the cytotoxic drug portion ("payload") in antibody-drug conjugates.

[0081] In some embodiments, the organic cytotoxic oligomer is selected from the group consisting of organic cytotoxic oligomers described in the detailed description of the embodiments below.

[0082] Therefore, another aspect of the present invention relates to the use of the above-mentioned pharmacotoxic cytotoxic oligomers in the production of antibody-drug conjugates. In particular, this use involves covalently conjugating the pharmacotoxic cytotoxic oligomers to an antibody or its antigen-binding fragment.

[0083] From yet another perspective, the present invention provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and the above-mentioned pharmaceutically cytotoxic oligomer or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically cytotoxic oligomer is covalently attached to the antibody or its antigen-binding fragment.

[0084] In certain embodiments, the antibody-drug conjugate has the following general formula (I). Ab-[L-(T) m ] n (I)

[0085] In this specification, Ab is an antibody or its antigen-binding fragment, T is a pharmacokinetic oligomer, L is an organolinker, and m and n are integers from 1 to 10, independently of each other.

[0086] In some embodiments, m is an integer from 1 to 5, preferably from 1 to 3. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.

[0087] In some embodiments, n is an integer from 1 to 8, preferably from 2 to 8, and more preferably from 4 to 8.

[0088] The product m·n is also called the "drug-antibody ratio (DAR)". In preferred embodiments, the DAR is 1 to 16, i.e., at least one and up to 16 cytotoxic oligomers are bound to each antibody molecule. Preferably, the DAR is 2 to 12, more preferably 2 to 10 or 2 to 8, and most preferably 4 to 8.

[0089] For the purposes of this invention, the term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, bispecific or multispecific antibodies, and antibody fragments having an antigen-specific binding site, i.e., an antigen-binding site that immunospecifically binds to the antigen or a part thereof of a target cell. Preferably, the antibody is a monoclonal antibody (mAb) or its antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, scFv fragments, single-molecule antibodies (also called nanobodies), diabodies, linear antibodies, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity-determining regions), and any of the above epitope-binding fragments that immunospecifically bind to, for example, human or animal antigens, cancer cell antigens, senescent cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, etc. The antibody may be derived from mouse, human, humanized, chimeric, or other species. The antibody may be any type of immunoglobulin, e.g., IgG, IgE, IgM, IgD, or IgA; any class, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2; or a subclass of immunoglobulin.

[0090] In preferred embodiments, the antibody or its antigen-binding fragment is specific to 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 present only in cancer cells and not in healthy cells. Tumor-associated antigens are highly expressed in tumor cells and expressed at low levels in healthy cells. Such tumor-associated antigens and tumor-specific antigens are known to those skilled in the art. Examples of TAAs and TSAs include BMPR1B (osteogenesis imperfecta receptor type IB, Genbank accession number NM-001203), E16 (LAT1, SLC7A5, Genbank accession number NM-003486), STEAP1 (six-transmembrane antigen of the prostate, Genbank accession number NM-012449), 0772P (CA125, MUC16, Genbank accession number AF361486), MPF (MPF, MSLN, SMR, megakaryocyte growth factor, mesoserine, Genbank accession number NM-005823), Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM-006424), Sema 5b(F1110372, KIAa1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema entity, 7 thrombospongin repeats (type 1 and type 1-like), transmembrane entity (TM) and short cytoplasmic entity, (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, virtual protein F1120315, Genbank accession number NM-017763), STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, six-transmembrane epithelial antigen of the prostate 2, six-transmembrane prostate protein, Gen Genbank accession number AF455138), TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM-017636), CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP-003203 or NM-003212), CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Genbank accession number M26004), CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank accession number NM-000626 or 11038674), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 entity-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession number NM-030764, AY358130), HER2 (ErbB2, Genbank accession number M11730), NCA (CEACAM6, Genbank accession number M18728), MDP (DPEP1, Genbank accession number BC0170) 23) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971), Brevican (BCAN, BEHAB, Genbank accession number AF229053), EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession number NM-004442), ASLG659 (B7h, Genbank accession number AX092328), PSCA (Prostate stem cell antigen precursor, Genbank accession number AJ297436), GEDA (Genbank accession number AY260763), AAP14954 Lipoma HMGIC fusion partner-like protein / pid=AAP14954.1 Homo sapiens (human), BAFF-R (B cell activator 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-associated alpha), CXCR5 (Burkitt 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).1) Examples include LY64 (lymphocyte antigen 64 (RP105)), FcRH1 (Fc receptor-like protein 1), IRTA2 (FcRH5, immunoglobulin superfamily receptor translocation-related 2), TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR), and MUC1 (tumor-related MUC1 glycopeptide epitope).

[0091] In some embodiments, the antibody or its antigen-binding fragment is specific to senescent cell surface markers, i.e., molecules or proteins that are specifically present on the surface of senescent cells.

[0092] According to the common understanding in the field of ADCs, a “linker” is a bifunctional or polyfunctional organic molecule that enables the covalent conjugate ("linked") of a drug moiety or “payload,” such as the cytotoxic oligomer of the present invention, with an antibody or its antigen-binding fragment to form an antibody-drug complex. Therefore, a linker can be defined as the interface between the targeting moiety, i.e., the antibody or its antigen-binding fragment, and the drug moiety, i.e., the cytotoxic oligomer. The linker contains independent coupling functional groups for binding to the antibody or its antigen-binding fragment and for binding to the drug moiety, respectively. Thus, linkers can be used not only for efficient ADC construction but also to further adapt or modify ADCs in terms of therapeutic efficiency, mechanism of action, and therapeutic window. For example, the hydrophobicity of the entire drug-linker can be addressed, overcoming the limiting binding of drug loads. Innovative hydrophilic sources have been explored in various linker frameworks and different payloads, resulting in improved physicochemical properties, favorable pharmacokinetic profiles, major tolerability, avoidance of MDR-related resistance, and efficacy in heterogeneous tumors. One possibility for increasing the hydrophilicity of the drug-linker is the introduction of PEG (polyethylene glycol) and PEG units, respectively. Furthermore, the linker can also be used to control the timing of the payload's release from the antibody carrier.

[0093] Linkers belong to two genera: so-called "cleavage linkers" and "non-cleavage linkers."

[0094] Non-cleavable linkers are stably bound to the drug moiety at one end and to the antibody at the other. Therefore, the drug moiety is released from the antibody only during receptor-mediated endocytosis and lysosomal processing of the ADC. Non-cleavable linkers are independent of (bio)chemical signals, i.e., "triggers." When the antibody is degraded by lysosomal enzymes, the linker-payload complex is released into the intracellular environment and can exert cytotoxic activity. For example, when targeting hematological malignancies, ADCs based on non-cleavable linkers may be useful for conferring higher specificity and lower toxicity.

[0095] In the art, many non-cleavable linkers suitable for carrying out the present invention are known. Suitable examples include, but are not limited to, non-cleavable linkers based on maleimidocaproyl (mc) and 4-maleimidomethylcyclohexane-1-carboxylate (mcc), as well as PEG-containing non-cleavable linkers containing alkynes and piperazines.

[0096] Cleavage linkers are cleaved in response to specific biochemical or chemical signals ("triggers"). For example, cleavage linkers are cleaved by changes in redox potential or pH value, or in the presence of enzymes such as hydrolases or oxidoreductases. Cleavage linkers can be used to control drug release from ADCs.

[0097] Cleavage linkers can be classified into two subclasses: "chemically cleavage linkers" and "enzymatically cleavage linkers."

[0098] Chemically cleaved linkers can be further classified as acid-cleaved or reducing linkers. Both are clinically established by multiple FDA and EMA-approved ADCs based on acid-cleaved and reducing linkers.

[0099] Acid-cleaved linkers utilize the natural acidity of endosomes and lysosomes, which lies in the pH range of 4.5–6.2, in contrast to the neutral pH of plasma (7.4). Examples of approved ADCs with acid-cleaved linkers include Mylotarg and Besponsa. The most commonly used acid-cleaved linker is hydrazone, which is reliably stable at neutral pH and unstable at pH 4.5. Other acid-unstable groups include combinations of carbonate linkers with alcohol-containing payloads.

[0100] Reducing or disulfide linkers are the most prominent class of chemically cleavable linkers. This linker chemistry is stable at physiological pH but susceptible to nucleophilic attack from thiols. In plasma, only human serum albumin (HSA) contains thiols, but its reactivity is limited due to the low solvent exposure of these groups. In contrast, the cytoplasm is rich in glutathione (GSH), which has exposed, highly reactive thiol groups. Its presence allows for highly selective intracellular release of payloads from ADCs using disulfide linkers. Furthermore, elevated GSH levels are often characteristic of tumor-related oxidative stress, which can confer additional selectivity to this type of linker chemistry.

[0101] Enzyme-cleaved linkers utilize enzymes for payload release. An example of an enzyme used in applied technologies for payload release is cathepsin B, a cysteine ​​protease that cleaves peptide bonds in dipeptide substrates containing valine (Val) and citrulline (Cit) and in p-aminobenzyl alcohol (PAB). Valine-citrulline is the most commonly used dipeptide linker substrate in currently applied enzyme-dependent linker chemistry. Glycosidase-cleaved linkers are another example of enzyme-cleaved linkers commonly used in ADCs. Glucosidases are hydrolytic enzymes, usually confined to the lysosomal compartment, but like cathepsin B, they can be secreted by tumor cells in necrotic tissue. Less common examples include β-galactosidase-cleaved linkers and the more commonly used β-glucuronsidase-cleaved linkers, which are based on the hydrolysis of β-D-glucuronic acid residues at lysosomal pH.

[0102] Therefore, non-cleavable linkers are inherently extracellular, i.e., stable outside the cell. Cleavable linkers contain predefined cleavage sites that are cleaved by enzymatic activity, hydrolysis, or other metabolic conditions or triggers.

[0103] The linker may also include a spacer structure that spatially separates cytotoxic oligomers from the antibody. The use of linkers is well known to those skilled in the art, and they can easily select an appropriate linker based on their knowledge and this disclosure and teaching.

[0104] In certain embodiments, the linker includes a coupling functional group configured for covalent coupling to a cysteine ​​thiol, an amine (e.g., an N-terminal or amino acid side chain such as lysine), or other modifications of the antibody. The linker may be substituted with a sulfonic acid group or other substituent, which may improve the water solubility of the linker and facilitate the coupling reaction between the linker and the antibody. The coupling functional group may be configured for covalent coupling to a nucleophile on the antibody, i.e., an N-terminal amino group, a side-chain amino group (e.g., lysine), a side-chain thiol group (e.g., cysteine), and, in the case of glycosylated antibodies, a hydroxyl group or amino group of a glycan. Amino groups, thiol groups, and hydroxyl groups are nucleophilic and can react with electrophiles of the linker (e.g., N-hydroxysuccinimide (NHS) ester, hydroxybenzotriazole (HOBt) ester, haloformates, acid halides, alkyl and benzyl halides (e.g., haloacetamide), aldehydes, ketones, carboxyl and maleimide groups, etc.) to form a covalent bond. Certain antibodies possess reducible interchain disulfides, i.e., cysteine ​​crosslinks. Antibodies can be made receptive to conjugation with linkers by treatment with reducing agents such as DTT (dithiothreitol). Additional nucleophiles can be introduced into antibodies by the reaction of lysine with 2-iminothiolane (Trout's reagent), which converts 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-natural cysteine ​​residues, or by using commercially available chemical kits.

[0105] In certain embodiments, the linker is a cleavable linker. In preferred embodiments, the linker is less resistant to cleavage under physiological conditions than the crosslinking entity of the pharmacotoxic oligomer.

[0106] In certain embodiments, the linker is configured to release a pharmaceutically active cytotoxic oligomer from an antibody or its antigen-binding fragment during intracellular environmental changes, particularly receptor-mediated endocytosis and / or lysosomal processing of an antibody-drug conjugate. In some embodiments, the linker includes a cleavage site configured to cleave in response to a chemical or biochemical trigger selected from the group consisting of redox potential, enzymes, and pH values. Specifically, the linker may include a substrate for intracellular enzymatic cleavage, such as a hydrolase and / or oxidoreductase substrate or cleavage site, or a substrate for chemically triggered cleavage, such as a substrate that responds to a change in pH or reduction potential.

[0107] In some embodiments, the linker is a non-cleavable linker. This further ensures that the ADC payload is not released prematurely extracellularly, but only after intracellular uptake and lysosomal degradation of the antibody, thereby further improving the pharmaceutical properties of the ADC of the present invention, particularly its therapeutic window.

[0108] From another perspective, the present invention provides a method for producing the above-described cytotoxic oligomers for pharmaceutical use.

[0109] The method comprises the steps of (A) providing a first organic cytotoxic entity and at least a second organic cytotoxic entity, each of which is (i) an enzyme inhibitor and / or (ii) cytotoxic to target cells after uptake by a target cell transporter protein, and (B) forming a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity that is substantially resistant to cleavage under physiological conditions, thereby forming a cytotoxic oligomer.

[0110] In some embodiments, the first organic cytotoxic entity comprises a first coupling functional group, and the second organic cytotoxic monomer comprises a second coupling functional group, wherein the second coupling functional group has a chemical reactivity complementary to the first coupling functional group, and the first and second coupling functional groups can form a covalent bond with each other. Furthermore, step (B) may include combining the first organic cytotoxic entity and the second organic cytotoxic entity under conditions that the first and second coupling functional groups form a covalent bond with each other.

[0111] In some embodiments, step (A) further includes providing an organic crosslinking portion, and step (B) includes forming a covalent bond between a first organic cytotoxic entity and a second organic cytotoxic entity via the crosslinking portion. For example, the formation of the covalent bond may include covalently attaching the crosslinking portion to the first organic cytotoxic entity to form a first covalent bond, and covalently attaching the crosslinking portion to the second organic cytotoxic entity to form a second covalent bond, thereby crosslinking the first organic cytotoxic entity and the second organic cytotoxic entity with each other via the crosslinking portion.

[0112] Therefore, the crosslinked portion is at least a bifunctional molecule, i.e., a molecule containing at least two coupling functional groups (also called "functional groups") configured to form covalent bonds with a first organic cytotoxic entity and a second organic cytotoxic entity. Naturally, the crosslinked portion can be a polyfunctional molecule having multiple coupling functional groups configured to form covalent bonds with additional entities, such as an additional third or subsequent organic cytotoxic entity or linker. In some embodiments, the crosslinked portion is a bifunctional molecule having two coupling functional groups configured to form covalent bonds with a first organic cytotoxic entity and a second organic cytotoxic entity. In preferred embodiments, the crosslinked portion is a trifunctional molecule having three coupling functional groups configured to form covalent bonds with any of the first organic cytotoxic entity, the second organic cytotoxic entity, and a third organic cytotoxic entity or linker. In other preferred embodiments, the crosslinking portion is a tetrafunctional molecule having three coupling functional groups configured to form covalent bonds with either a first organic cytotoxic entity, a second organic cytotoxic entity, a third organic cytotoxic entity, and a fourth organic cytotoxic entity or linker.

[0113] In some embodiments, the first organic cytotoxic entity comprises a first coupling functional group, the second organic cytotoxic entity comprises a second coupling functional group, and the organic crosslinking portion comprises at least a first complementary coupling functional group and a second complementary coupling functional group, wherein the first complementary coupling functional group has a chemical reactivity complementary to the first coupling functional group, and the first coupling functional group and the first complementary coupling functional group can form a covalent bond with each other, and the second complementary coupling functional group has a chemical reactivity complementary to the second coupling functional group, and the second coupling functional group and the second complementary coupling functional group can form a covalent bond with each other. Furthermore, step (B) may include combining the first organic cytotoxic entity, the second organic cytotoxic entity and the organic crosslinking portion under conditions in which the first coupling functional group and the first complementary coupling functional group form a covalent bond with each other, preferably simultaneously forming a covalent bond with the second coupling functional group and the second complementary coupling functional group.

[0114] In some embodiments, the first coupling functional group is the same as the second coupling functional group, and therefore the first complementary coupling functional group is the same as the second complementary coupling functional group. In other embodiments, the first coupling functional group and the second coupling functional group are different from each other, and therefore the first complementary coupling functional group and the second complementary coupling functional group are also different from each other.

[0115] In embodiments in which step (B) involves forming a covalent bond between a first organic cytotoxic entity and a second organic cytotoxic entity via a crosslinking portion, it is preferable that the first coupling functional group and the second coupling functional group are identical or chemically complementary to each other, i.e., have orthogonal chemical reactivity, thereby preventing the first organic cytotoxic entity and the second organic cytotoxic entity from directly forming a covalent bond. Similarly, in such embodiments, the first complementary coupling functional group and the second complementary coupling functional group are also identical or chemically complementary to each other, thereby preventing the crosslinking portions from forming a covalent bond.

[0116] As already stated above, the crosslinked portion may further include a third or subsequent complementary coupling functional group that is identical or different from the first and second coupling functional groups, which has a chemical reactivity complementary to the third or subsequent organic cytotoxic entity and / or the third or subsequent coupling functional group of the linker. Other possible modifications will be readily apparent to those skilled in the art based on the above disclosure and teaching.

[0117] In some embodiments, the crosslinked portion includes at least one orthogonal coupling functional group, i.e., a functional group having a chemical reactivity that is non-complementary to the coupling functional groups of the first and second organic cytotoxic entities (e.g., the first coupling functional group and the second coupling functional group), thereby preventing the first and second organic cytotoxic entities from forming covalent bonds with the crosslinked portion via the orthogonal coupling functional group. In this way, the orthogonal coupling functional group remains on the crosslinked portion after the formation of the cytotoxic oligomer in step (B) and can be used to link the cytotoxic oligomer with other molecules, such as a linker and / or antibody. In this specification, the orthogonal coupling functional group may be a functional group protected by a reversible protecting group, as already described above.

[0118] Preferably, the first coupling functional group and the second coupling functional group, or optionally the first coupling functional group and the first complementary coupling functional group, and the second coupling functional group and the second complementary coupling functional group, and if present, the third or subsequent coupling functional group and the third or subsequent complementary coupling functional group are reciprocal reaction partners in any of the click chemistry reactions described above, for example, the first coupling functional group may be an azide group or tetrazine group, the second coupling functional group may be an alkyne group or alkene group, the first coupling functional group and the second coupling functional group may be an azide group or tetrazine group, the first complementary coupling functional group and the second complementary coupling functional group may be an alkyne group or alkene group, or vice versa. However, the first coupling functional group and the second coupling functional group, or optionally the first coupling functional group and the first complementary coupling functional group and the second coupling functional group and the second complementary coupling functional group, and if present, the third or subsequent coupling functional group and the third or subsequent complementary coupling functional group, can of course be selected from other mutually reactive functional groups described above, such as amino groups and carboxyl groups, aldehyde groups or keto groups. The orthogonal coupling functional group is preferably selected from the group consisting of amino groups, carboxyl groups, hydroxyl groups, hydroxylamine groups, isothiocyanate groups, aldehyde groups, keto groups and thiol groups, and may be protected with a detachable protecting group as needed.

[0119] The coupling functional groups of each organic cytotoxic entity, for example, the first coupling functional group and the second coupling functional group, may be amino acid functional groups naturally present in the corresponding first and / or second organic cytotoxic entities, such as an amino group, carboxyl group, hydroxyl group, aldehyde group, keto group, or thiol group. However, in preferred embodiments, the coupling functional group of the first organic cytotoxic entity is a functional group not present in the natural form of the first organic cytotoxic entity, and / or the coupling functional group of the second organic cytotoxic entity is a functional group not present in the natural form of the second organic cytotoxic entity. In embodiments where the first and / or second organic cytotoxic entities are microcystin or nodularin, the coupling functional group or functional group is preferably introduced into the cytotoxic entity biosynthetically or semisynthetically, as described, for example, in International Patent Publication 2018 / 219619, which is incorporated herein by reference to further illustrate the technical background to which the present invention belongs. Preferably, the coupling functional groups of the first and / or second organic cytotoxic entities are configured to be compatible with bioorthogonal conjugation, i.e., chemical conjugation reactions that can occur in vivo without interfering with intrinsic biochemical processes. In some embodiments, the coupling functional groups of the first and / or second organic cytotoxic entities are selected from the group consisting of azide groups, alkyne groups, alkene groups, phosphines, phosphonates, tetrazines, hydrazines, hydroxylamines, isothiocyanates, and any combination thereof.

[0120] In some embodiments, the first organic cytotoxic entity and / or the second organic cytotoxic entity have at least 90% or 95% sequence identity with the sequences listed in Table 1 below, i.e., the first organic cytotoxic entity and / or the second organic cytotoxic entity have sequence ID NO:2 to SEQ ID NO:15, NOD and [D-PrgMeAsp] 1 - It has at least 90% or 95% sequence identity with a sequence selected from the group consisting of NOD or SEQ ID NO:17.

[0121] From yet another perspective, the present invention provides a method for producing antibody-drug conjugates.

[0122] The method comprises (a) providing a pharmaceutically acceptable cytotoxic oligomer or a pharmaceutically acceptable salt thereof as defined above; (b) providing an antibody or an antigen-binding fragment thereof; and (c) covalently conjugating the pharmaceutically acceptable cytotoxic oligomer to the antibody or the antigen-binding fragment thereof.

[0123] In certain embodiments, step (a) includes carrying out the method for producing the pharmaceutically active cytotoxic oligomer as defined above.

[0124] In certain embodiments, step (c) includes a substep of (c1) providing a linker, a substep of (c2) covalently attaching a pharmacotoxic oligomer to the linker, and a substep of (c3) covalently attaching the linker to an antibody or its antigen-binding fragment. It should be understood that the order of the substeps is not necessarily predetermined by the numbering. For example, substep (c2) can be performed before or after substep (c3). However, it is preferable to perform substep (c2) before substep (c3), i.e., first covalently attaching the pharmacotoxic oligomer to the linker, and then covalently attaching the cytotoxic oligomer-linker complex to the antibody via the linker.

[0125] Appropriate experimental protocols for covalently conjugating pharmaceutical cytotoxic oligomers to antibodies or linkers, respectively, and for covalently conjugating linkers to antibodies, are well-established and readily available to those skilled in the art. See also the above description and the detailed description of the embodiments below for further information.

[0126] In some embodiments, covalently linking a linker and / or a pharmaceutically active cytotoxic oligomer to the linker is configured to release the pharmaceutically active cytotoxic oligomer from the antibody or its antigen-binding fragment during receptor-mediated endocytosis and / or lysosomal treatment of the antibody-drug conjugate. In particular, the linker may include a cleavage site configured to cleave in response to a chemical or biochemical trigger, as already described above.

[0127] In certain embodiments, at least one of the first organic cytotoxic entity, the second organic cytotoxic entity, and the crosslinking entity of the pharmacotoxic cytotoxic oligomer contains a first coupling functional group, and the linker contains a second coupling functional group having a chemical reactivity complementary to the first coupling functional group, and the two are configured to form a covalent bond with each other. Furthermore, substep c2) may include mixing the pharmacotoxic cytotoxic oligomer and the linker under conditions in which the first coupling functional group and the second coupling functional group form a covalent bond with each other.

[0128] In certain embodiments, the present invention provides the pharmaceutically acceptable cytotoxic oligomers or pharmaceutically acceptable salts thereof, or the antibody-drug conjugates defined above, for use in pharmaceuticals, more specifically, in the treatment of malignant diseases or age-related diseases.

[0129] In certain embodiments, the present invention provides a method for treating a malignant disease, comprising administering to a subject, particularly a human subject, a pharmaceutically effective amount or therapeutically effective administration regimen of the pharmaceutically cytotoxic oligomer or a pharmaceutically acceptable salt thereof as defined above, or a pharmaceutically effective amount of the antibody-drug conjugate as defined above.

[0130] In certain embodiments, the malignant disease is cancer. Examples of cancers to be treated herein include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, leukemias, or lymphoid malignancies. More specific examples include squamous cell carcinoma, e.g., epithelial squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric or gastrointestinal cancer (including gastrointestinal stromal tumors (GISTs)), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. Cancer may be characterized, for example, by overexpression of HER2 or ErbB receptors.

[0131] A pharmacokinetically effective dose may be, for example, 0.01 to 100 mg (mg / kg) per kg of body weight, more specifically 0.1 to 20 mg / kg or 1 to 10 mg / kg. A therapeutically effective administration regimen may 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.

[0132] It is understood that the various embodiments described in one aspect of the present invention are applicable to other aspects. Accordingly, the features and functions disclosed above and below in relation to cytotoxic oligomers may also be relevant to antibody-drug conjugates and their corresponding uses and manufacturing methods, and vice versa. [Brief explanation of the drawing]

[0133] [Figure 1] Structural formula of the microcystin homodimer according to the present invention. [Figure 2] Structural formula of another microcystin homodimer according to the present invention. [Figure 3] Structural formula of the microcystin heterodimer according to the present invention. [Figure 4] Structural formula of the microcystin homotrimer according to the present invention. [Figure 5]Schematic diagram of the semi-synthetic preparation of [D-EdaMeAsp]1-NOD(A and C) and [D-PrgMeAsp]1-NOD(B and D) monomers. [Figure 6] HPLC chromatogram of the product mixture obtained from the semi-synthetic preparation of [D-PrgMeAsp]1-NOD monomer. [Figure 7] Structural formula of the nodularin homodimer according to the present invention. [Figure 8] Structural formula of the nodularin homotrimer according to the present invention. [Figure 9] Structural formula of the amatoxin homodimer according to the present invention. [Figure 10] Structural formula of the amatoxin homotrimer according to the present invention. [Figure 11] Structural formula of the microcystin homodimer shown in Figure 2, conjugated with the cleavable linker maleimidocaproyl-Val-Ala-(p-aminobenzyl)carbamate, which is configured for antibody conjugation. [Figure 12] Column diagrams showing the results of general cytotoxicity assessments and protein phosphatase inhibition (B) in OATP1B1 and OATP1B3 expressing cell lines (A) for the microcystin homodimers shown in Figures 1 and 2, as well as the corresponding monomers and native microcystin MC-LR as reference examples. Detailed description of embodiments for carrying out the invention

[0134] The present invention will be described in further detail below based on embodiments with reference to the accompanying drawings. Note that the examples and drawings are not limiting.

[0135] Example 1: Synthesis of monomeric organic cytotoxic entities

[0136] The following examples include several microcystins, nodularins, and amatoxins as representative organic cytotoxic entities. These are actively transported into target cells by target cell transporter proteins and then exert cytotoxic activity by inhibiting intracellular enzymes in the target cells (see Table 1).

[0137] Unless otherwise specified in the following examples, microcystin (MC) monomers were obtained by precursor-directed biosynthesis as described in International Patent Publication No. 2018 / 219619. Briefly, naturally occurring microcystin-producing cyanobacteria strains, such as PCC 7820 (Pasteur Collection), were cultured under conditions suitable for strain growth. During culture, propargyltyrosine (PrgTyr), propargyllysine (PrgLys), azidophenylalanine (AzPhe), azidolicin (AzLys), azidonorvaline (AzNva), and N were added to an inorganic medium. ε - Either tert-butyloxycarbonyl-ornithine (Orn(Boc)) or D-azidolanine (D-AzAla) was added. Each of these amino acids contains a coupling functional group configured to covalently crosslink with other coupling functional groups having complementary chemical reactivity. The added amino acids are incorporated into microcystin during strain culture, thereby crosslinking the functional groups configured for covalent conjugation ("coupling functional groups") with Aa 1 , X 2 , Z 4 or Aa 7 Biosynthesis of a non-natural microcystin derivative having the ("coupling site") was achieved. The coupling functional group was Aa 3 It is also generally possible to introduce the microcystin derivatives at the site of cell formation. After culturing, the biomass was harvested and extracted, and the microcystin derivatives were purified by HPLC. The purified microcystin derivatives were used as the organic cytotoxic entities of the cytotoxic oligomers according to the present invention. In the case of Orn(Boc)-containing microcystin derivatives, the Boc group was removed by stirring in trifluoroacetic acid (TFA) / H2O / acetonitrile (can) (1:1:1, v / v / v) for 90 minutes to expose the free amino group, after which it was isolated by evaporation and RP-HPLC. Suitable microcystins can also be synthesized by total synthesis (e.g., Zemskov et al., J Org Chem 2017, 82, 3680-91).

[0138] Furthermore, semi-synthetic modified variants of microcystins, nodularins and amatoxins can be produced by chemically introducing a coupling functional group configured to covalently crosslink with other coupling functional groups having complementary chemical reactivity into naturally occurring cytotoxic monomers.

[0139] For example, a semi-synthetic modified variant of the natural microcystin monomer MC-LR has Aa 7 obtained by Michael addition of an amine, thiol or other Michael donor to the Mdha residue (Michael acceptor) at the position. This chemically introduces an additional coupling functional group (e.g., an amino group, an alkyne group or an azide group), enabling selective reaction with a complementary functional group as described in Example 11 below.

[0140] Furthermore, a semi-synthetic modified variant of a natural microcystin monomer can be obtained according to the modification described below for the carboxyl group at the Aa 1 position of a nodularin monomer, by chemical modification of the carboxyl group at the Aa 3 position.

[0141] The nodularin (NOD) monomer included in the examples is a natural nodularin monomer obtained by culturing a nodularin-producing cyanobacterial strain such as PCC 73104, then extracting and isolating nodularin from biomass by HPLC or the like. Suitable nodularin monomers can also be obtained by precursor-directed biosynthesis described in Patent International Publication No. WO 2018 / 219619. For example, during culture, N ω -nitro-L-arginine (Narg) or Orn(Boc) was added to the inorganic medium. Both NOD derivatives thus obtained yield a primary amino group by reduction of the nitro group of Narg or Boc deprotection of Orn(Boc), to be converted into [Orn] 2 -NOD (as described above and in Patent International Publication No. WO 2018 / 219619).

[0142] As in the case of the above microcystins, nodularins can also be semisynthetically modified via Michael addition with a Michael donor to the Mdhb / Dhb group (a Michael acceptor) at the Aa 5 position to introduce an additional coupling functional group. Thereby, as described in Example 37 below, an alkyne coupling group can be introduced at the Aa 5 position.

[0143] Furthermore, semisynthetically modified variants of nodularin monomers having an amino or alkyne coupling functional group at the Aa 1 position can be obtained by forming an amide bond between a carboxyl group and an amino group at the Aa 1 position, as described in Examples 34 and 35 below.

[0144] The amatoxin (AMA) monomers included in the examples are naturally occurring α-amanitin and amanidinamide (lacking the 6-OH of Trp2), a derivative thereof. Amatoxins are obtained, for example, by collecting wild fruit bodies from the natural environment of the genus Amanita (e.g., Amanita phalloides), or from cultured mycelium of the genus Amanita (e.g., Amanita exitialis) by the method described in Zhang et al. (FEMS Microbiol Lett 2005, 252(2), 223-08), followed by extraction and isolation of amatoxins from biomass by means such as HPLC. The α-amanitin monomer has the Aa 1 position (Dhil = (2S,3R,4R)-4,5-dihydroxyisoleucine), the Aa 2 position (Htp = 6-hydroxytryptophan), the Aa 8It has a hydroxyl group as a coupling functional group at position (Hyp = trans-4-hydroxyproline). However, the Hyp residue is not considered a suitable coupling site because it greatly affects the biological activity and corresponding cytotoxicity of the amatoxin (Matinkhoo et al., Chem. Eur. J. 2021, 27, 10282-92). Suitable amatoxins can also be synthesized by total synthesis (e.g., Siegert et al., Angew Chem 2020, 59, 5500-04; Lutz et al., Angew Chem 2020, 59, 11390-93).

[0145] As described in Examples 46 and 47 below, Aa 2 amino coupling group at position, Aa 1 A semi-synthetic modified mutant of the α-amanitin monomer having an alkyne coupling group at a specific position is obtained.

[0146] Table 1: Microcystin (MC), nodularin (NOD), and amatoxin (AMA) used as monomeric cytotoxic entities in the examples. PrgTyr = propargyltyrosine, PrgLys = propargyllysine, AzPhe = azidophenylalanine, AzLys = azidolicine, AzNva = azidonorvaline, Orn = ornithine, D-AzAla = D-azidoalanine, AzProMDap = 3-N-azidopropyl-2-N-methyl-2,3-diaminopropionic acid, D-EdaMeAsp = D-β-ethylene-1 ,2-diamine-β-methylaspartic acid, D-PrgMeAsp=D-erythro-β-propargylamine-β-methylaspartic acid, AeHtp=6-(2-aminoethoxy)tryptophan, PrgDhil=4,5-[(oxycarbonyl)-propargylamine]-dihydroxyisoleucine, PrgSMMeCys=S-propargyl-N-methyl-β-methylcysteine.

[0147] [Table 1]

[0148] Example 2: Synthesis of homomeric microcystin dimers using a bifunctional crosslinked moiety of MC-PrgTyr-Arg monomer

[0149] Homomeric dimers were prepared from the two MC-PrgTyr-Arg monomers (SEQ ID NO:2) described in Example 1 as both the first and second organic cytotoxic entities. The MC-PrgTyr-Arg monomer is X 2 It has an alkyne group as a coupling functional group at that position.

[0150] The MC-PrgTyr-Arg monomer is dimerized using a crosslinking molecule with structural formula BM-I, thereby creating a crosslinking entity that forms a covalent bond between a first organic cytotoxic entity and a second organic cytotoxic entity. For this purpose, the crosslinking molecule BM-I has two terminal azide groups as complementary coupling functional groups. [ka]

[0151] X of two MC-PrgTyr-Arg monomers 2 The bridging between the alkyne coupling functional group located at the 100-11 position and the two terminal azide groups (complementary coupling functional groups) of BM-I was achieved by a copper-catalyzed azide-alkyne addition reaction (CuAAC). Briefly, CuAAC was carried out using a reaction mixture of 2 equivalents (eq.) of monomer in dimethyl sulfoxide (DMSO), 1 equivalent of BM-I, 10 equivalents of 100 mM copper sulfate (CuSO4) aqueous solution, and 20 equivalents of 200 mM tris((1-hydroxypropyl-1H-1,2,3-triazole-4-yl)methyl)amine (THPTA) aqueous solution. The reaction was initiated by adding 10 equivalents of 100 mM sodium ascorbate aqueous solution. The reaction product was isolated by semi-preparative C-18 reverse-phase HPLC and lyophilized. The structural formula of the homo-microcystin dimer DM-I is shown in Figure 1.

[0152] Example 3: Synthesis of homo-microcystin dimers by crosslinking of MC-PrgLys-Arg monomers with a bifunctional crosslinking moiety

[0153] Homo-dimers derived from the two MC-PrgLys-Arg monomers (SEQ ID NO:3) described in Example 1 as both the first and second organic cytotoxic entities are prepared. The MC-PrgLys-Arg monomer is X 2 It has an alkyne group as a coupling functional group at the 11 position. The MC-PrgLys-Arg monomer is dimerized using a crosslinking molecule of structural formula BM-I in combination with the CuAAC protocol described in Example 2 above, thereby creating a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0154] Example 4: Synthesis of homo-type microcystin dimers by crosslinking of MC-AzPhe-Arg monomers with a bifunctional crosslinking moiety

[0155] Homodimers derived from the two MC-AzPhe-Arg monomers (SEQ ID NO:4) described in Example 1 as both the first and second organic cytotoxic entities are prepared. The MC-AzPhe-Arg monomer is X 2 It has an azide group as a coupling functional group at the 12th position. The MC-AzPhe-Arg monomer is dimerized using a crosslinking molecule of structural formula BM-II, thereby creating a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities. For this purpose, the crosslinking molecule BM-II has two terminal alkyne groups as complementary coupling functional groups. [ka]

[0156] X of two MC-AzPhe-Arg monomers 2Crosslinking between the azide coupling functional group present at the position and the two terminal alkyne groups (complementary coupling functional groups) of BM-II is achieved by CuAAC as described in Example 2 above. The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0157] Example 5: Synthesis of homodimeric microcystin dimers by crosslinking of MC-AzLys-Arg monomers via a bifunctional crosslinking moiety

[0158] Homodimeric dimers derived from two MC-AzLys-Arg monomers (SEQ ID NO: 5) described in Example 1 as both a first organic cytotoxic entity and a second organic cytotoxic entity are prepared. The MC-AzLys-Arg monomer has an azide group at the X 2 position as a coupling functional group. The MC-AzLys-Arg monomer is dimerized using the crosslinking molecule of structural formula BM-II in combination with the CuAAC protocol described in Example 2 above, whereby a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity is produced. The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0159] Example 6: Synthesis of heterodimeric microcystin dimers by crosslinking of MC-AzNva-Arg and MC-AzPhe-Arg monomers via a bifunctional crosslinking moiety

[0160] Heterodimeric dimers derived from the MC-AzNva-Arg monomer (SEQ ID NO: 6) described in Example 1 as the first organic cytotoxic entity and the MC-AzPhe-Arg monomer (SEQ ID NO: 4) described as the second organic cytotoxic entity are prepared. Both monomers have an azide group at the X 2It has an azide group as a coupling functional group at the 12th position. MC-AzNva-Arg monomer (1 equivalent) and MC-AzPhe-Arg (1 equivalent) are dimerized using a crosslinking molecule of structural formula BM-II in combination with the CuAAC protocol described in Example 2 above, thereby creating a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity. The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0161] Example 7: Synthesis of homo-type microcystin dimers by crosslinking of MC-Leu-PrgTyr monomers with a bifunctional crosslinking moiety

[0162] Homodimers derived from the two MC-Leu-PrgTyr monomers (SEQ ID NO:7), described in Example 1 as both the first and second organic cytotoxic entities, are prepared. The MC-Leu-PrgTyr monomer is Z 4 It has an alkyne group as a coupling functional group at the 11th position. The MC-Leu-PrgTyr monomer is dimerized using a crosslinking molecule of structural formula BM-I in combination with the CuAAC protocol described in Example 2 above, thereby creating a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0163] Example 8: Synthesis of homo-type microcystin dimers by crosslinking of MC-Leu-AzLys monomers with a bifunctional crosslinking moiety

[0164] Homodimers derived from the two MC-Leu-AzLys monomers (SEQ ID NO:8) described in Example 1 as both the first and second organic cytotoxic entities are prepared. The MC-Leu-AzLys monomer is Z 4It has an azide group as a coupling functional group at the 12th position. The MC-Leu-AzLys monomer is dimerized using a crosslinking molecule of structural formula BM-II in combination with the CuAAC protocol described in Example 2 above, thereby creating a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0165] Example 9: Synthesis of homo-type microcystin dimers by crosslinking of MC-Leu-Orn monomers with a bifunctional crosslinking moiety

[0166] Homodimers derived from the two MC-Leu-Orn monomers (SEQ ID NO:9) described in Example 1 as both the first and second organic cytotoxic entities are prepared. The MC-Leu-Orn monomer is Z 4 It has an amino group as a coupling functional group at the 12th position. The MC-Leu-Orn monomer is dimerized using a crosslinking molecule of structural formula BM-III, thereby creating a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities. For this purpose, the crosslinking molecule BM-III has two terminal carboxyl groups as complementary coupling functional groups. [ka]

[0167] Z of two MC-Leu-Orn monomers 4The crosslinking between the amino coupling functional group located at the 100° position and the two terminal carboxyl groups (complementary coupling functional groups) of BM-III is achieved by amide bond formation. Briefly, the crosslinking molecule BM-III (1 equivalent in DMF) is pre-reacted with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1.95 equivalents) and N,N-diisopropylethylamine (DIPEA; 4 equivalents) for 30 minutes. Then, an amino group-containing monomer (2 equivalents) is added to the solution and stirred overnight at room temperature. The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0168] Example 10: Synthesis of hetero-type microcystin dimers by crosslinking of MC-Leu-PrgLys and MC-Leu-PrgTyr monomers with a bifunctional crosslinking moiety

[0169] Heterodimers are prepared from the MC-Leu-PrgLys monomer (SEQ ID NO:10), described as the first organic cytotoxic entity in Example 1, and the MC-Leu-PrgTyr monomer (SEQ ID NO:7), described as the second organic cytotoxic entity. Both monomers are Z 4 It has an alkyne group as a coupling functional group at the 12th position. MC-Leu-PrgLys monomer (1 equivalent) and MC-Leu-PrgTyr monomer (1 equivalent) are dimerized using a crosslinking molecule of structural formula BM-I in combination with the CuAAC protocol described in Example 2 above, thereby creating a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity. The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0170] Example 11: [D-AzAla] by a bifunctional crosslinked moiety 1 MC-WR and [AzproMDap] 7 Synthesis of hetero-type microcystin dimers by crosslinking of MC-LR monomers

[0171] [D-AzAla] was described as the first organic cytotoxic entity in Example 1. 1 MC-WR monomer (SEQ ID NO:11) and described as a second organic cytotoxic entity [AzproMDap] 7 A heterodimer derived from the MC-LR monomer (SEQ ID NO:12) is produced.

[0172] [D-AzAla] 1 The MC-WR monomer is obtained by precursor-directed biosynthesis as described in Example 1. [AzproMDap] 7 The MC-LR monomer was semi-synthetically prepared from a natural microcystin MC-LR precursor (SEQ ID NO: 16) via an Aza-Michael addition reaction. Therefore, MC-LR (1 equivalent) was dissolved in DMSO and added to a 1% (w / v) K2CO3 aqueous solution containing 3-azido-1-propanamine (40 equivalents). The reaction was stirred overnight at 40°C. The resulting [AzproMDap] 7 MC-LR was isolated by semi-preparative C-18 reverse-phase HPLC and lyophilized.

[0173] [D-AzAla] 1 MC-WR monomer is Aa 1 It has an azide group as a coupling functional group at the position. [AzproMDap] 7 MC-LR monomer is Aa 7 It has an azide group as a coupling functional group at the 12th position. Both monomers (1 equivalent each) are dimerized using a crosslinking molecule of structural formula BM-II in combination with the CuAAC protocol described in Example 2 above, thereby creating a crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0174] Examples 12-21: Synthesis of homo- and hetero-microcystin dimers by crosslinking of monomers from Examples 2-11 using crosslinking molecules having trifunctional crosslinking moieties.

[0175] In Examples 12-21, the homo-dimers and hetero-dimers from Examples 2-11 were prepared again using different crosslinking molecules to create crosslinking entities that form a covalent bond between the first and second organic cytotoxic entities. In Examples 12-21, each crosslinking molecule has two coupling functional groups that have complementary chemical reactivity to the coupling functional groups of the microcystin monomer described in Examples 2-11, and a third coupling functional group configured to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0176] To this end, the crosslinking molecule BM-IV has two azide groups as complementary coupling functional groups for crosslinking microcystin monomers having alkyne coupling functional groups, and a terminal amino group as a third coupling functional group for linking the dimer to an antibody or its antigen-binding fragment. [ka]

[0177] The crosslinking molecule BM-V has two alkyne groups as complementary coupling functional groups for crosslinking microcystin monomers having azide coupling functional groups, and a terminal amino group as a third coupling functional group for linking the dimer to an antibody or its antigen-binding fragment. [ka]

[0178] The crosslinking molecule BM-VI has two carboxyl groups as complementary coupling functional groups for crosslinking microcystin monomers that have amino groups as coupling functional groups, and a terminal Boc-protected amino group as a third coupling functional group. After the dimerization reaction, the Boc group is removed in TFA / H2O / ACN (1:1:1, v / v / v), exposing a free amino group for linking the dimer to an antibody or its antigen-binding fragment. [ka]

[0179] Table 2 shows an overview of the dimers produced in Examples 12-21.

[0180] Table 2: Microcystin dimers produced in Examples 12-21. PrgTyr = propargyltyrosine, PrgLys = propargyllysine, AzPhe = azidophenylalanine, AzLys = azidridine, Aznva = azidonorvaline, Orn = ornithine, D-AzAla = D-azidoalanine, AzProMDap = 3-N-azidopropyl-2-N-methyl-2,3-diaminopropionic acid.

[0181] [Table 2]

[0182] Dimerization using the crosslinked molecule of structural formula BM-IV or the crosslinked molecule of structural formula BM-V is achieved by the CuAAC protocol described in Example 2, using 2 equivalents of microcystin monomer for homodimers and 1 equivalent of each microcystin monomer for heterodimers. Dimerization using the crosslinked molecule of structural formula BM-VI is achieved by the amide bond formation protocol described in Example 9.

[0183] The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0184] As an example, Figure 2 shows the structural formula DM-II of the homomicrocystin dimer of Example 12. CuAAC between the propargyl coupling functional group of the MC-PrgTyr-Arg monomer and the complementary azide coupling functional group of the crosslinking molecule BM-IV causes both microcystin entities to cross at position X via two opposing branches of the crosslinking moiety. 2Two triazole rings are covalently crosslinked. The third branch of the crosslinking portion has a terminal amino group that is orthogonal to the other coupling functional groups and remains available after dimer formation, and can be used to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0185] Example 22: Synthesis of heteromicrocystin dimers by direct crosslinking of monomers MC-PrgTyr-Arg and MC-Tyr-AzNva

[0186] Heterodimers are generated from the MC-PrgTyr-Arg monomer (SEQ ID NO:2) as the first organic cytotoxic entity and the MC-Tyr-AzNva monomer (SEQ ID NO:13) as the second organic cytotoxic entity described in Example 1.

[0187] The MC-PrgTyr-Arg monomer is at position X 2 It has an alkyne coupling functional group, and the MC-Tyr-AzNva monomer is at position Z 4 Both monomers possess an azide group as a coupling functional group, which allows for dimerization of both monomers by direct crosslinking using CuAAC. Therefore, CuAAC is performed using equimolar amounts of each monomer (1 equivalent in DMSO), 5 equivalents of 100 mM CuSO4 solution, and 10 equivalents of 200 mM THPTA solution. The reaction is initiated by adding 5 equivalents of 100 mM sodium ascorbate solution. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0188] Example 23: Synthesis of heteromicrocystin dimers by direct crosslinking of monomers MC-AzLys-Arg and MC-Leu-PrgLys

[0189] Heterodimers are generated from MC-AzLys-Arg (SEQ ID NO: 5) as the first organic cytotoxic entity and MC-Leu-PrgLys monomer (SEQ ID NO: 10) as the second organic cytotoxic entity described in Example 1.

[0190] The MC-AzLys-Arg monomer is at position X 2 It has an azide group as a coupling functional group, and the MC-Leu-PrgLys monomer is at position Z 4 The monomers have a propargyl group as a coupling functional group, which allows both monomers to be dimerized by direct crosslinking using the CuAAC protocol described in Example 22. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0191] As an example, Figure 3 shows the structural formula DM-III of the heteromicrocystin dimer of Example 23. Direct CuAAC between the azide coupling functional group of the MC-AzLys-Arg monomer and the complementary alkyne coupling functional group of the MC-Leu-PrgLys monomer leads to the formation of a cross-sectional area (X) in the amino acid side chain. 2 and Z 4 A crosslinking entity is formed, consisting of a triazole ring that covalently crosslinks both microcystin entities via this. Position Aa 3 The carboxyl group in the side chain of D-MeAsp can be used to link the dimer to an antibody or its antigen-binding fragment.

[0192] Example 24: Synthesis of heteromicrocystin dimers by direct crosslinking of monomers MC-AzLys-Arg and MC-PrgLys-Arg

[0193] Heterodimers are generated from the MC-AzLys-Arg monomer (SEQ ID NO:5) as the first organic cytotoxic entity and the MC-PrgLys-Arg monomer (SEQ ID NO:3) as the second organic cytotoxic entity described in Example 1.

[0194] The MC-AzLys-Arg monomer is at position X 2 It has an azide group as a coupling functional group, and the MC-PrgLys-Arg monomer is at position X 2The monomers have a propargyl group as a coupling functional group, which allows both monomers to be dimerized by direct crosslinking using the CuAAC protocol described in Example 22. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0195] Example 25: Synthesis of heteromicrocystin dimers by direct crosslinking of monomers MC-Leu-AzLys and MC-Leu-PrgLys

[0196] Heterodimers are generated from the MC-Leu-AzLys monomer (SEQ ID NO:8) as the first organic cytotoxic entity and the MC-Leu-PrgLys monomer (SEQ ID NO:10) as the second organic cytotoxic entity described in Example 1.

[0197] The MC-Leu-AzLys monomer is at position Z 4 It has an azide group as a coupling functional group, and the MC-Leu-PrgLys monomer is at position Z 4 The monomers have a propargyl group as a coupling functional group, which allows both monomers to be dimerized by direct crosslinking using the CuAAC protocol described in Example 21. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0198] Example 26: Synthesis of homomicrocystin trimer by crosslinking of MC-AzLys-Arg monomer with trifunctional crosslinking moiety

[0199] Homotrimers are derived from the three MC-AzLys-Arg monomers (SEQ ID NO: 5) described in Example 1 as the first, second, and third organic cytotoxic entities. The MC-AzLys-Arg monomer is located at position X 2 It has an azide group as a coupling functional group.

[0200] The MC-AzLys-Arg monomer is trimerized using a crosslinking molecule of structural formula BM-VII, thereby creating a crosslinking entity that forms covalent bonds between the first, second, and third organic cytotoxic entities. For this purpose, the crosslinking molecule BM-VII has three terminal alkyne groups as complementary coupling functional groups: [ka]

[0201] X of three MC-AzLys-Arg monomers 2 The crosslinking between the azide coupling functional group located at the 11th position and the three terminal alkyne groups (complementary coupling functional groups) of BM-VII is achieved by CuAAC. For trimer formation, CuAAC is performed in DMSO using 3 equivalents of monomer, 1 equivalent of crosslinking molecule, 15 equivalents of 100 mM CuSO4 solution, and 30 equivalents of 200 mM THPTA. The reaction is initiated by adding 15 equivalents of 100 mM sodium ascorbate solution. The reaction product is isolated by semi-preparative C-18RP-HPLC and lyophilized.

[0202] Example 27: Synthesis of homotrimer-type microcystin by crosslinking of MC-PrgTyr-Arg monomer using a trifunctional crosslinked moiety

[0203] The three homotrimers derived from the MC-PrgTyr-Arg monomers (SEQ ID NO:2) described in Example 1 are prepared as the first, second, and third organic cytotoxic entities. The MC-PrgTyr-Arg monomer is X 2 It has an alkyne group as a coupling functional group at the position.

[0204] The MC-PrgTyr-Arg monomer is trimerized using a crosslinking molecule with structural formula BM-VIII, thereby creating a crosslinking entity that forms covalent bonds between the first, second, and third organic cytotoxic entities. For this purpose, the crosslinking molecule BM-VIII has three terminal azide groups as complementary coupling functional groups: [ka]

[0205] X of three MC-PrgTyr-Arg monomers 2 The crosslinking between the alkyne coupling functional group located at the 11 position and the three terminal azide groups (complementary coupling functional groups) of BM-VIII is achieved by CuAAC according to the protocol described in Example 26 above. The reaction product is isolated by semi-preparative C-18RP-HPLC and lyophilized.

[0206] Example 28: Synthesis of homotrimer-type microcystin by crosslinking of MC-Leu-AzLys monomer using a trifunctional crosslinked moiety

[0207] The three homotrimers derived from the MC-Leu-AzLys monomers (SEQ ID NO:8) described in Example 1 are prepared as the first, second, and third organic cytotoxic entities. The MC-Leu-AzLys monomer is Z 4 It has an azide group as a coupling functional group at the position.

[0208] The MC-Leu-AzLys monomer is trimerized using a crosslinking molecule of structural formula BM-VII and the CuAAC protocol described in Example 26 above, thereby creating a crosslinking entity that forms covalent bonds between the first, second, and third organic cytotoxic entities. The reaction product is isolated by semipreparative C-18RP-HPLC and lyophilized.

[0209] Example 29: Synthesis of homotrimer-type microcystin by crosslinking of MC-Leu-PrgLys monomer using a trifunctional crosslinked moiety

[0210] The homotrimers derived from the three MC-Leu-PrgLys monomers (SEQ ID NO:10) described in Example 1 are prepared as the first, second, and third organic cytotoxic entities. The MC-Leu-PrgLys monomer is Z 4 It has an alkyne group as a coupling functional group at the position.

[0211] The MC-Leu-PrgLys monomer is trimerized using a crosslinking molecule of structural formula BM-VIII and the CuAAC protocol described in Example 26 above, thereby creating a crosslinking entity that forms covalent bonds between the first, second, and third organic cytotoxic entities. The reaction product is isolated by semipreparative C-18RP-HPLC and lyophilized.

[0212] Examples 30-33: Synthesis of homotrimer-type microcystins by crosslinking of microcystin monomers from Examples 26-29 with a tetrafunctional crosslinking moiety having additional antibody coupling functional groups.

[0213] In Examples 30-33, homotrimers from Examples 26-29 are prepared again using different crosslinking molecules to create crosslinking entities that form covalent bonds between the first, second, and third organic cytotoxic entities. In Examples 30-33, each crosslinking molecule has three coupling functional groups that have complementary chemical reactivity to the coupling functional groups of the microcystin monomers described in Examples 26-29, and an additional third coupling functional group configured to subsequently link the trimer to an antibody or its antigen-binding fragment.

[0214] For this purpose, the crosslinking molecule BM-IX has three branches, each having a terminal azide group as a complementary coupling functional group for crosslinking microcystin monomers having alkyne coupling functional groups, and a fourth branch having a terminal amino group as a fourth coupling functional group, and is used to link cytotoxic trimers to antibodies or their antigen-binding fragments: [ka]

[0215] The crosslinking molecule BM-X has three branches, each having a terminal azide group as a complementary coupling functional group for crosslinking microcystin monomers that have an alkyne group as a coupling functional group, and a fourth branch having a terminal amino group as a fourth coupling functional group, and is used to link cytotoxic trimers to antibodies or their antigen-binding fragments: [ka]

[0216] Table 3 shows an overview of the dimers produced in Examples 12-21.

[0217] Table 3: Microcystin trimers prepared in Examples 30-33. PrgTyr = propargyltyrosine, PrgLys = propargyllysine, AzLys = azidolicin.

[0218] [Table 3]

[0219] Trimerization using either the crosslinked molecule of structural formula BM-IX or the crosslinked molecule of structural formula BM-X is achieved by the CuAAC protocol described in Example 26 above. The reaction product is isolated by semipreparative C-18RP-HPLC and lyophilized.

[0220] As an example, Figure 4 shows the structural formula TM-I of the homotrimer type microcystin of Example 30. CuAAC between the azide coupling functional group of the MC-AzLys-Arg monomer and the complementary propargyl coupling functional group of the crosslinking molecule BM-IX allows X to be crosslinked through the branched crosslink portion. 2 At the 11th position, three triazole rings are formed, covalently crosslinking all three microcystin entities. The fourth branch of the crosslinking region has a terminal amino group that is orthogonal to the other coupling functional groups and remains available for subsequent linkage to the antibody or its antigen-binding fragment even after trimer formation.

[0221] Example 34: [D-EdaMeAsp] using a bifunctional crosslinked moiety 1 - Synthesis of homodimeric nodularins by crosslinking of NOD monomers

[0222] Two [D-EdaMeAsp] 1 -NOD monomer-derived homodimers are produced as the first and second organic cytotoxic entities. [D-EdaMeAsp] 1 -NOD monomer is a natural nodularin Aa 1 It is semi-synthetically prepared using the carboxyl group of D-MeAsp at the position as a coupling functional group.

[0223] For this purpose, natural nodularin (prepared biosynthetically as described in Example 1) is dissolved in DMF and stirred at 0°C. Hydroxybenztriazole (HOBt) (1.2 equivalents) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.2 equivalents) are added. After 30 minutes, N-Boc-ethylene-1,2-diamine (Boc-Eda) (1.2 equivalents) and DIPEA (2 equivalents) are added to the mixture. The reaction is maintained overnight. The reaction yields Aa 1 rank or Aa 4 [D-Boc-EdaMeAsp] has a protective terminal amine at or at the position of the protective terminal amine. 1 -NOD and [D-Boc-EdaGlu] 4-NOD, or [D-Boc-EdaMeAsp] as shown in Figure 5A 1 [D-Boc-EdaGlu] 4 -NOD product mixture is obtained. The target product is [D-Boc-EdaMeAsp] 1 -NOD is isolated by semi-preparative C-18RP-HPLC and lyophilized. The Boc protecting group is removed by the method described in Example 19, and [D-EdaMeAsp] 1 -NOD monomer is obtained. After isolation, [D-EdaMeAsp] 1 -NOD is freeze-dried.

[0224] Alternatively, [D-EdaMeAsp] 1 -NOD monomers are prepared semi-synthetically as shown in Figure 5C. For this purpose, natural nodularin is dissolved in 0.5 M hydrochloric acid (HCl) / methanol (MeOH) and stirred at room temperature for about 5 hours, so that the 4-position is more easily methylated than the 1-position [D-Glu(OMe)]. 4 -NOD is obtained. The product is isolated by semi-preparative C-18RP-HPLC and lyophilized. Next, [D-Glu(OMe)] 4 -NOD is dissolved in DMF and stirred at 0°C. HOBt (1.2 equivalents) and EDC (1.2 equivalents) are added. After 30 minutes, Boc-Eda (1.2 equivalents) and DIPEA (2 equivalents) are added to the mixture. The reaction is maintained overnight at 25°C. The reaction produces Aa 1 A protected amino group at position Aa 4 [D-Boc-EdaMeAsp] has a methyl ester at the position. 1 -D-Glu(OMe) 4]-NOD is obtained. After HPLC purification, the methyl ester is hydrolyzed using 10 equivalents of 50 mM NaOH methanol solution in CH2Cl2 at a final CH2Cl2 / MeOH ratio of approximately 9 / 1 (v / v) according to the method described by Theodorou et al. (Tetrahedron Lett. 2007, 48, 8230-8233). The solution is stirred at 4°C for several days, then neutralized with 10 equivalents of 50 mM HCl and concentrated. Subsequently, the Boc protecting group is removed by the method described in Example 19. The reaction product is [D-EdaMeAsp] 1 -NOD monomers are isolated by semi-preparative C-18RP-HPLC and lyophilized.

[0225] [D-EdaMeAsp] 1 -NOD monomer consists of two [D-EdaMeAsp] 1 - NOD monomer Aa 1 Dimerization is performed using the crosslinking molecule BM-III to facilitate crosslinking by amide bond formation between the amino coupling functional group at the position and the two terminal carboxyl groups (complementary coupling functional groups) of BM-III (see Example 9).

[0226] Alternatively, two [D-MeAsp-NHS] 1 Similar homodimers consisting of the NOD carboxylic acid-activated nodularin intermediate are prepared as the first and second organic cytotoxic entities.

[0227] [D-MeAsp-NHS] 1 -NOD-active esters are prepared semi-synthetically. For this purpose, natural nodularin (described in Example 1) is dissolved in DMF and stirred at 0°C. N-hydroxysuccinimide (NHS) (1.2 equivalents) and EDC (1.2 equivalents) are added. The reaction is maintained overnight at room temperature. The reaction produces Aa 1 rank or Aa 4 [D-MeAsp-NHS] has an NHS-activated carboxylic acid at or at both positions. 1 -NOD and [D-Glu-NHS] 4-NOD, or [D-MeAsp-NHS] 1 [D-Glu-NHS] 4 -NOD product mixture is obtained. The target product is [D-MeAsp-NHS] 1 -NOD is isolated by semi-preparative C-18RP-HPLC and lyophilized.

[0228] [D-MeAsp-NHS] 1 -NOD-active esters are dimerized using a crosslinking molecule with structural formula BM-XI, thereby creating a crosslinking entity that forms a covalent bond between a first and second organic cytotoxic entity. For this purpose, the crosslinking molecule BM-XI has two terminal amino groups as complementary coupling functional groups: [ka]

[0229] Aa of two NOD monomers 1 The crosslinking between the NHS-activated carboxyl coupling functional group at the position and the two terminal amino groups of BM-XI by complementary coupling functional groups is achieved by amide bond formation. Specifically, [D-MeAsp-NHS] 1 -NOD (2.2 equivalents) and BM-XI are dissolved in DMF / sodium phosphate buffer (0.1 mM, pH 8) (1:1, v / v) and stirred overnight at 25°C. The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0230] Example 35: [D-PrgMeAsp] by a bifunctional crosslinked moiety 1 - Synthesis of homomeric nodularin dimers by crosslinking of NOD monomers

[0231] Two [D-PrgMeAsp] are used as both the first and second organic cytotoxic entities. 1 - Homomeric dimers are produced from NOD monomers.

[0232] [D-PrgMeAsp] 1 -NOD monomers were prepared semi-synthetically as shown in Figure 5B. For this purpose, natural nodularin was dissolved in DMF and stirred at 0°C. HOBt (1.2 equivalents) and EDC (1.2 equivalents) were added. After 30 minutes, propargylamine (1.2 equivalents) and DIPEA (2 equivalents) were added to the mixture. The reaction was maintained overnight at 25°C. The reaction yielded Aa 1 [D-PrgMeAsp] has an alkyne (propargyl) group at the position. 1 -NOD and Aa 4 [D-PrgGlu] has an alkyne (propargyl) group at the position. 4 A mixture of NOD products was obtained.

[0233] The target product is [D-PrgMeAsp] 1 -NOD was isolated by semi-preparative C-18 reversed-phase HPLC. Figure 6 shows a typical reversed-phase HPLC chromatogram of the product mixture, where peak 1 corresponds to unmodified NOD and peak 2 corresponds to the target product [D-PrgMeAsp] with extracted ion chromatogram mass m / z = 862.5 (shown in the inset). 1 -NOD corresponds to an unwanted byproduct, peak 3 is [D-PrgGlu]. 4 -NOD-compatible. After isolation, [D-PrgAsp] 1 -NOD was freeze-dried.

[0234] Alternatively, [D-PrgMeAsp] 1 -NOD monomers were semi-synthetically prepared as shown in Figure 5D. Natural nodularin was methylated with 0.5 M HCl in MeOH as described in Example 34 above, and [D-Glu(OMe)] 4 -NOD was obtained. The isolated product was dissolved in DMF and stirred at 0°C. HOBt (1.2 equivalents) and EDC (1.2 equivalents) were added. After 30 minutes, propargylamine (1.2 equivalents) and DIPEA (2 equivalents) were added to the mixture. The reaction was maintained overnight at 25°C. The reaction yielded Aa 1 Alkyne (propargyl) group at position Aa 4[D-PrgMeAsp] has a methyl ester at the position. 1 -D-Glu(OMe) 4 ]-NOD was obtained. After HPLC purification, the methyl ester was hydrolyzed as described in Example 34 above, and the reaction product was isolated by semi-preparative C-18 reverse-phase HPLC and lyophilized.

[0235] [D-PrgMeAsp] 1 -NOD monomers were crosslinked using the structural formula BM-I and the CuAAC protocol described in Example 2, resulting in two [D-PrgMeAsp] 1 - NOD monomer Aa 1 A complementary coupling functional group is formed between the alkyne coupling functional group at the 11 position and the two terminal azide groups of BM-I, thereby creating a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities in the cytotoxic dimer. The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0236] Example 36: [Orn] by a bifunctional crosslinked portion 2 - Synthesis of homomeric nodularin dimers by crosslinking of NOD monomers

[0237] [Orn] described in Example 1 2 - Homomeric dimers derived from two NOD monomer molecules are prepared as both the primary and secondary organic cytotoxic entities. [Orn] 2 -NOD monomer is Aa 2 It has an amino group as a coupling functional group at the position. [Orn] 2 -NOD monomers are formed using BM-III crosslinking molecules as described in Example 9, with two [Orn] 2 - NOD monomer Aa 2 The molecule is crosslinked and dimerized by the formation of an amide bond through complementary coupling functional groups between the amino coupling functional group at the position and the two terminal carboxyl groups of BM-III.

[0238] Example 37: Synthesis of homomeric nodularin dimer by crosslinking of [PrgSMMeCys]5-NOD monomer with a bifunctional crosslinking moiety

[0239] The monomer [PrgSMMeCys]5-NOD (SEQ ID NO: 17) is semi-synthetically prepared from a natural NOD precursor via a Thia-Michael addition reaction. One equivalent of NOD is dissolved in DMSO and added to a 5% (w / v) K2CO3 aqueous solution containing 100 equivalents of propargylthiol. The reaction is stirred overnight at 40°C. The resulting [PrgSMMeCys]5-NOD is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0240] Homomeric dimers are produced from two [PrgSMMeCys]5-NOD monomers, which are both the first and second organic cytotoxic entities. The [PrgSMMeCys]5-NOD monomer is Aa 5 It has an alkyne group as a coupling functional group at the 11th position. The [PrgSMMeCys]5-NOD monomer is dimerized using a crosslinking molecule of structural formula BM-I along with the CuAAC protocol described in Example 2 above, thereby creating a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0241] Example 38: [D-PrgMeAsp] by a bifunctional crosslinked moiety 1 -NOD and [D-EdaMeAsp] 1 - Synthesis of heteromeric nodularin dimers by crosslinking of NOD monomers

[0242] [D-PrgMeAsp] is the primary organic cytotoxic entity. 1 -NOD monomer, as a second organic cytotoxic entity [D-EdaMeAsp] 1 - Heteromers are produced from NOD monomers.

[0243] [D-EdaMeAsp] 1 -NOD monomer and [D-PrgMeAsp] 1 -NOD monomers were semi-synthetically produced as described in Examples 34 and 35 above.

[0244] Monomer [D-EdaMeAsp] 1 -NOD and [D-PrgMeAsp] 1 -NOD is dimerized using a bifunctional crosslinking molecule with structural formula BM-XII, thereby creating a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities. For this purpose, the bifunctional crosslinking molecule BM-XII is [D-EdaMeAsp] 1 -NOD's amino group and terminal carboxyl group for conjugation by amide bond formation, and [D-PrgMeAsp] 1 -NOD has a terminal azide group for CuAAC with an alkyne (propargyl) group: [ka]

[0245] First, [D-EdaMeAsp] 1 -NOD monomer was prepared using the protocol described in Example 9, provided that BM-XII was 1 equivalent, HATU 0.95 equivalents, DIPEA 2 equivalents, and [D-EdaMeAsp] 1 - Except for the use of 1 equivalent of NOD, Aa 1 The amino group at the position and the terminal carboxyl group of the crosslinking molecule BM-XII conjugate via amide bond formation, thereby forming the first part of the covalent bond of the crosslinking entity. The intermediate product is isolated by C18 reverse-phase HPLC and lyophilized.

[0246] After that, [D-PrgMeAsp] 1 -NOD monomer was used with the protocol described in Example 2, except that equimolar amounts of monomer and intermediate product were used, [D-PrgMeAsp] 1 - NOD monomer Aa 1Dimerization is completed by achieving crosslinking through complementary coupling functional groups between the alkyne coupling functional group at the position and the terminal azide group of the crosslinking molecule BM-XII in the intermediate product.

[0247] In this way, the cross-linked entity is completed by forming a second covalent bond that cross-links the two nodularin entities with each other. The dimer is isolated by C18 reverse-phase HPLC and lyophilized.

[0248] Examples 39-43: Synthesis of homomeric and heteromeric nodularin dimers by crosslinking of monomers from Examples 34-38 with crosslinking molecules having a trifunctional crosslinking moiety.

[0249] In Examples 39-43, the homomeric and heteromeric nodularin dimers of Examples 34-38 are reproduced using different crosslinking molecules to create crosslinked entities that form a covalent bond between a first organic cytotoxic entity and a second organic cytotoxic entity. In Examples 39-43, each crosslinking molecule has two coupling functional groups that have complementary chemical reactivity to the coupling functional groups of the nodularin monomers described in Examples 34-38, and an additional third coupling functional group configured to subsequently bind the dimer to an antibody or its antigen-binding fragment.

[0250] For this purpose, the crosslinked molecules BM-VI described in Example 19 each contain an amine coupling functional group [D-EdaMeAsp] 1 -NOD monomer or [Orn] 2 - The NOD monomer has two carboxyl groups and a terminally protecting amino group as complementary coupling functional groups for crosslinking. After the dimerization reaction, the protecting group is removed as described in Example 19, exposing a free amino group for binding the dimer to an antibody or its antigen-binding fragment.

[0251] Another synthetic route uses the crosslinking molecule BM-XIII, which has two free amino groups and one Boc-protected amino group. The two amino groups are [D-MeAsp-NHS]1 -The activated carboxyl group of the NOD monomer is used as the first and second complementary coupling functional groups for crosslinking. After dimerization, the Boc group is removed as described in Example 19, and the free amino group is exposed as a third coupling functional group for binding the dimer to the antibody or its antigen-binding fragment: [ka]

[0252] The crosslinking molecules BM-IV described in Example 12 each contain an alkyne coupling functional group [D-PrgMeAsp] 1 -NOD monomer or [PrgSMMeCys] 5 -It has two azide groups, which are complementary coupling functional groups for crosslinking the NOD monomer, and a terminal amino group as a third coupling functional group for linking the dimer to an antibody or its antigen-binding fragment.

[0253] Furthermore, the crosslinking molecule BM-XIV contains an alkyne coupling functional group [D-PrgMeAsp] 1 - One azide group, which is a complementary coupling functional group for crosslinking NOD monomers, and [D-EdaMeAsp] 1 - It has a terminal carboxyl group for conjugation with the amino group of the NOD monomer. In addition, BM-XIV has a terminal Boc-protected amino group as a third coupling functional group. After dimerization, the Boc group is cleaved as described in Example 19, and a free amino group is exposed as a third coupling functional group, which allows the heterodimer to be linked to an antibody or its antigen-binding fragment. [ka]

[0254] Table 4 shows an overview of the dimers produced in Examples 39-43.

[0255] Table 4: Nodularin (NOD) dimers prepared in Examples 39-43. D-PrgMeAsp = D-erythro-β-propargylamine-β-methylaspartic acid.

[0256] [Table 4]

[0257] Dimerization using crosslinked molecules of structural formula BM-VI or BM-XIII is achieved by the protocol described for crosslinked molecules of structural formula BM-III or BM-XI in corresponding Example 34. Dimerization using crosslinked molecules of structural formula BM-IV is achieved by the CuAAC protocol described in Example 2 and corresponding Example 35. Dimerization using crosslinked molecules of structural formula BM-XIV is achieved by the protocol described for crosslinked molecules of structural formula BM-XII in corresponding Example 38.

[0258] The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0259] As an exemplary embodiment, Figure 7 shows the structural formula DM-IV of the homo-nodularin dimer of Example 39. As described above, the conjugation reaction is [D-EdaMeAsp] 1 -Between the amino group of NOD and the complementary carboxyl group of the crosslinking molecule BM-VI, or [D-MeAsp-NHS] 1 - This is achieved between the activated carboxyl coupling functional group of the NOD monomer and the complementary amino coupling functional group of the crosslinking molecule BM-XIII. In either case, two amide bonds are formed, and two nodularin entities are connected at position Aa via two opposing branches of the crosslinking portion. 1 The molecules are covalently crosslinked. Furthermore, the crosslinking molecules contain additional amino group substituents that remain available after dimer formation and can be used to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0260] Example 44: [D-PrgMeAsp] by a trifunctional crosslinked moiety 1 - Synthesis of homo-type nodularin trimers by crosslinking of NOD monomers

[0261] The three [D-PrgMeAsp] described in Example 34 1 - Homotype trimers derived from NOD monomers are prepared as the first, second, and third organic cytotoxic entities. [D-PrgMeAsp] 1 -NOD monomer is at position Aa 1 It has an alkyne group as a coupling functional group.

[0262] [D-PrgMeAsp] 1 -NOD monomers are trimmerized using a crosslinking molecule of structural formula BM-VIII in combination with the CuAAC protocol described in Example 26 above, thereby creating crosslinking entities that form covalent bonds between the first, second, and third organic cytotoxic entities. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0263] Example 45: [D-PrgMeAsp] by a tetrafunctional crosslinking moiety having an antibody coupling functional group 1 - Synthesis of homo-type nodularin trimers by crosslinking of NOD monomers

[0264] The three [D-PrgMeAsp] described in Example 35 1 Homo-nodularin trimers composed of NOD monomers are prepared as the first, second, and third organic cytotoxic entities. [D-PrgMeAsp] 1 -NOD monomer is at position Aa 1 It has an alkyne group as a coupling functional group.

[0265] [D-PrgMeAsp] 1-NOD monomers are trimmerized using a crosslinking molecule of structural formula BM-X in combination with the CuAAC protocol described in Example 26 above, thereby creating crosslinking entities that form covalent bonds between the first, second, and third organic cytotoxic entities via the three branches of the crosslinking molecule BM-X. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0266] As an exemplary embodiment, Figure 8 shows the structural formula TM-II of the homo-type nodularin trimer of Example 45. [D-PrgMeAsp] 1 CuAAC between the alkyne coupling functional group of the NOD monomer and the complementary azide coupling functional group of the crosslinking molecule BM-X forms three triazole rings, and all three microcystin entities are located at position Aa via the branched crosslinking portion. 1 The molecules are covalently crosslinked. The fourth branch of the crosslinked portion has a terminal amino group that is orthogonal to the other coupling functional groups and is available after trimer formation, and can be used to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0267] Example 46: [AeHtp] by a bifunctional crosslinked moiety 2 - Synthesis of homozygous amatoxin dimers by crosslinking of AMA monomers

[0268] Two [AeHtp] 2 - Homo-dimers derived from AMA monomer (SEQ ID NO:14) are prepared as the first and second organic cytotoxic entities.

[0269] [AeHtp] 2 -AMA monomer is the naturally derived AMA at position Aa as described in Example 1 2 It is semi-synthetically prepared using a hydroxyindole group as a coupling functional group.

[0270] [AeHtp] 2 -AMA monomers are produced in two steps. First, at position Aa 2The hydroxyindole group is etherified with alkyl halide 2-(Boc-amino)ethyl bromide (Boc-Ae-Br) or other protected aminoalkyl halides using the etherification protocol for amanitin derivatization (Faulstich et al., Biochemistry 1981, 20, 6498-504). Briefly, natural AMA is dissolved in anhydrous ethanol, a freshly prepared sodium ethoxide solution (1.1 equivalents) is added, and the solution is immediately evaporated. The residue solid and Boc-Ae-Br (4 equivalents) are dissolved in DMF and stirred overnight at 20°C. The reaction results in monoalkylation [Boc-AeHtp] 2 -AMA, dialkylation [(Boc-Ae)2Htp] 2 -A mixture of products of AMA and unmodified natural AMA is obtained. Target product [Boc-AeHtp] 2 -AMA is isolated by semi-preparative C-18 reverse-phase HPLC and lyophilized. In the second step, the Boc group is cleaved as described in Example 19, and [AeHtp] 2 - Obtain AMA monomer

[0271] Two [AeHtp] 2 - Position Aa of AMA-1 monomer 2 The crosslinking between the amino coupling functional group and the two terminal carboxyl groups of BM-III, which is a complementary coupling functional group, is achieved by amide bond formation as described in Example 9.

[0272] Example 47: [PrgDhil] by a bifunctional crosslinked moiety 1 - Synthesis of homozygous amatoxin dimers by crosslinking of AMA monomers

[0273] Two [PrgDhil] 1 - Homo-dimers derived from AMA monomer (SEQ ID NO:15) are prepared as the first and second organic cytotoxic entities.

[0274] [PrgDhil] 1 -AMA monomer is naturally derived AMA at position Aa 1It is semi-synthetically prepared using the primary hydroxyl group as a coupling functional group. First, natural AMA is dissolved in anhydrous DMF and stirred under nitrogen at 0°C. Next, bis(p-nitrophenyl (bis-PNP)) carbonate (1.2 equivalents) and DIPEA (1.2 equivalents) are added, and the reaction mixture is stirred at 0°C for 2 to 24 hours. After evaporating the DMF, the residue is washed with diethyl ether, and the precipitate is filtered to obtain the AMA-PNP intermediate. Next, propargylamine (2 equivalents) is dissolved in DMF, AMA-PNP (1 equivalent) and DIPEA (2 equivalents) are added, and the mixture is stirred at 20°C for 2 to 24 hours. Reaction product [PrgDhil] 1 -AMA is isolated by semi-preparative C-18 reverse-phase HPLC and lyophilized.

[0275] Alternatively, [PrgDhil] 1 -AMA is 3-isocyanatopropin-1-in and naturally derived AMA position Aa 1 It can also be prepared by reaction with the primary hydroxyl group. Simply put, dissolve dry AMA (1 equivalent) in dry DMF, then add 2 equivalents of 3-isocyanatopropyne-1-yne and 2 equivalents of dibutyltin dilaurate, and stir for several days. The reaction product is [PrgDhil] 1 -AMA is isolated by semi-preparative C-18 reverse-phase HPLC and lyophilized.

[0276] [PrgDhil] 1 -AMA monomers are dimerized using a crosslinking molecule of structural formula BM-I in combination with the CuAAC protocol described in Example 2, thereby creating a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0277] Example 48: [AeHtp] by a bifunctional crosslinked moiety 2 -AMA and [PrgDhil] 1 - Synthesis of heterogeneous amatoxin dimers by crosslinking of AMA monomers

[0278] [AeHtp] 2 -AMA (SEQ ID NO:15) is the first organic cytotoxic entity, [PrgDhil] 1 -A heterodimer is created in which AMA is the second organic cytotoxic entity.

[0279] [AeHtp] 2 -AMA monomers and [PrgDhil] 1 -AMA monomers are prepared semi-synthetically as described in Examples 46 and 47 above.

[0280] [AeHtp] 2 -AMA and [PrgDhil] 1 -AMA monomers are dimerized using a bifunctional crosslinking molecule with structural formula BM-XII, thereby creating a crosslinking entity that forms a covalent bond between the first and second organic cytotoxic entities. For this purpose, the bifunctional crosslinking molecule BM-XII forms an amide bond [AeHtp] 2 -The amino group of AMA and the terminal carboxyl group for conjugation, using the protocol described in Example 38 [PrgDhil] 1 - It has an alkyne (propargyl) group of AMA and a terminal azide group for CuAAC.

[0281] Examples 49-51: Synthesis of homo and hetero amatoxin dimers by crosslinking of amatoxin monomers from Examples 46-48 with trifunctional crosslinking moieties having antibody coupling functional groups.

[0282] In Examples 49-51, homozygous and heterozygous amatoxin dimers from Examples 46-48 are prepared again using different crosslinking molecules to create crosslinking entities that form a covalent bond between the first and second organic cytotoxic entities. In Examples 49-51, each crosslinking molecule has two coupling functional groups that have complementary chemical reactivity to the coupling functional groups of the amanitin monomer described in Examples 46-48, and an additional third coupling functional group for subsequently linking the dimer to an antibody or its antigen-binding fragment.

[0283] Table 5: Amatoxin (AMA) dimers prepared in Examples 49-51.

[0284] [Table 5]

[0285] Dimerization using the crosslinked molecule of structural formula BM-VI is achieved by the protocol described for the crosslinked molecule of structural formula BM-III in corresponding Example 46. Dimerization using the crosslinked molecule of structural formula BM-IV is achieved by the CuAAC protocol described in Example 2 and corresponding Example 47. Dimerization using the crosslinked molecule of structural formula BM-XIV is achieved by the protocol described for the crosslinked molecule of structural formula BM-XII in corresponding Example 48.

[0286] The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0287] As an exemplary embodiment, Figure 9 shows the structural formula DM-V of the homozygous amatoxin dimer of Example 49. 2 -A conjugation reaction between the amino coupling functional group of the AMA monomer and the complementary carboxyl coupling functional group of the crosslinking molecule BM-VI forms two amide bonds, and two amatoxin entities are located at position Aa via two opposing branches of the crosslinking portion. 2 The molecules are covalently crosslinked. Following dimerization and Boc deprotection, the crosslinked molecules have additional amino group substituents that can be used to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0288] Example 52: [AeHtp] by a trifunctional crosslinked moiety 2 - Synthesis of homozygous amatoxin trimers by crosslinking of AMA monomers

[0289] The three [AeHtp] described in Example 46 2- Homotype trimers derived from AMA monomer (SEQ ID NO:14) are prepared as the first, second, and third organic cytotoxic entities. [AeHtp] 2 -AMA monomer is at position Aa 2 It has an amino group as a coupling functional group.

[0290] [AeHtp] 2 -AMA monomers are trimerized using a crosslinking molecule with structural formula BM-XV. [ka]

[0291] Trimerization using a crosslinked molecule of structural formula BM-XV was performed using the protocol described in Example 47 above, [AeHtp] 2 - AMA monomer 3 equivalents, BM-XV 1 equivalent, HATU 2.95 equivalents, DIPEA 6 Adjust to the equivalent amount, [AeHtp] 2 - This is achieved by creating a crosslinked entity that forms a covalent bond between the first, second, and third organic cytotoxic entities by forming an amide bond between the amino group of the AMA monomer and the carboxyl group of the crosslinked molecule.

[0292] The reaction product is isolated by semi-preparative C-18 reversed-phase HPLC and lyophilized.

[0293] Example 53: By the trifunctional crosslinked portion [PrgDhil] 1 - Synthesis of homotrimer amatoxins obtained by crosslinking AMA monomers

[0294] Three [PrgDhil] are the first, second, and third organic cytotoxic entities. 1 - A homotrimer derived from AMA monomer (SEQ ID NO:15) is produced. [PrgDhil] 1 -AMA monomer is Aa 1 It has an alkyne group as a coupling functional group at the position.

[0295] [PrgDhil] 1 -AMA monomers are trimerized using a crosslinking molecule of structural formula BM-VIII, as described in Example 27 above. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0296] Examples 54-55: Synthesis of homotrimer amatoxins obtained by crosslinking the monomers of Examples 52-53 with a trifunctional crosslinked moiety having an additional antibody coupling functional group.

[0297] In Examples 54 and 55, the homotrimer amatoxins from Examples 52 and 53 are prepared again using different crosslinking molecules to create crosslinking entities that form covalent bonds between the first, second, and third organic cytotoxic entities. In Example 54, the crosslinking molecule with structural formula BM-XVI is used. [ka]

[0298] Therefore, the crosslinked molecule BM-XVI has three carboxyl groups as coupling functional groups that have complementary chemical reactivity to the amino coupling functional group of the amatoxin monomer described in Example 52, and further has a Bock-protected amino group as an additional fourth coupling functional group configured to subsequently link a cytotoxic trimer to an antibody or its antigen-binding fragment.

[0299] Table 6: Amatoxin (AMA) trimmers prepared in Examples 54 and 55

[0300] [Table 6]

[0301] Trimerization using a crosslinked molecule of structural formula BM-XVI was performed using the protocol described in Example 52, [AeHtp] 2- This is achieved by creating a crosslinked entity that forms a covalent bond between the first, second, and third organic cytotoxic entities by forming an amide bond between the amino group of the AMA monomer and the carboxyl group of the crosslinking molecule. After the dimerization reaction, the Bock group is removed as described in Example 19. The reaction product is isolated by semipreparative C-18 reversed-phase HPLC and lyophilized.

[0302] Trimerization using a crosslinked molecule of structural formula BM-X is achieved by CuAAC according to the protocol for the crosslinked molecule of structural formula BM-X described in Example 26.

[0303] As an example, Figure 10 shows the structural formula TM-III of the homotrimer amatoxin from Example 54. [AeHtp] 2 -A conjugation reaction between the terminal amino group of the AMA monomer and the complementary carboxyl coupling functional group of the crosslinked moiety BM-XVI leads to Aa through different branching of the crosslinked moiety. 2 Three amide bonds are formed at the 11th position, covalently crosslinking three amatoxin entities. Furthermore, the crosslinking molecule has additional Bock-protected amino group substituents that can be used to link the trimer to an antibody or its antigen-binding fragment after trimer formation and subsequent Bock deprotection.

[0304] Example 56: Synthesis of an ADC composed of a monoclonal anti-HER2 antibody and the organic cytotoxic dimer from Example 12

[0305] The organic cytotoxic dimer synthesized in Example 12 is used as the drug portion of the ADC. The organic cytotoxic dimer consists of two MC-PrgTyr-Arg cytotoxic entities, with X mediated by two branches of the cross-linking portion BM-IV. 2 This is a microcystin homodimer symmetrically crosslinked at the 11th position. The crosslinked portion further contains a free amino coupling functional group for binding the microcystin homodimer to the antibody. Figure 2 shows the corresponding structural formula DM-II.

[0306] The monoclonal antibody (mAb) trastuzumab (trade name "Herceptin") targets human epidermal growth factor receptor 2 (Her2, also known as ERBB2, erb-b2 receptor tyrosine kinase 2). The mAb is produced by synthesizing the genes encoding the heavy and light chain variable regions of trastuzumab and cloning them into a human heavy and light chain vector encoding human IgG1. The construct sequence is validated by DNA sequencing. The vector is transiently transfected and expressed in CHO cells. The mAb is purified from the cell culture supernatant (e.g., using protein A) to a purity of over 90%. The purified mAb is quantified by absorbance at 280 nm and characterized by SEC and SDS-PAGE. It is understood that the protocol can be applied to produce ADCs with other target specificities using genes encoding the heavy and light chain variable regions of other monoclonal antibodies without departing from the teachings of this invention.

[0307] The microcystin homodimer obtained according to Example 12 is conjugated to the cleavable linker maleimidocaproyl-Val-Ala-(p-aminobenzyl)-(para-nitrophenyl)-carbonate (mc-Val-Ala-PAB-PNP) by incubating 1 equivalent of microcystin dimer and 1 equivalent of linker in DMF and DIPEA (2 equivalents) at 25°C for 1 to 3 hours. In this way, the microcystin dimer is bound to the linker via the formation of a carbamate bond between the activated carbonate on the linker and the free amino group of the crosslinking portion of the microcystin dimer, yielding mc-Val-Ala-PABC-DII (PABC = p-aminobenzylcarbamate), hereafter referred to as "DM-II-L". The resulting linker-microcystin dimer conjugate is isolated by RP-HPLC and characterized for identity (MS) and purity (RP-LC) with a target purity of at least 95%. Figure 11 shows the structural formula of the linker-microcystin dimer conjugate DM-II-L. Other suitable linker and conjugation chemistry are known to those skilled in the art.

[0308] Next, the resulting linker-homodimer conjugate is conjugated to the monoclonal anti-HER2 antibody trastuzumab (trade name: Herceptin), a representative benchmark antibody of the applied technology, in order to form an ADC using the complementary coupling functional groups of the linker and monoclonal antibody of the linker-homodimer conjugate. For this purpose, the native (unmodified) mAb is dissolved in 5% tris(hydroxymethyl)aminomethane (TRIS) buffer (with 25 mM ethylenediaminetetraacetic acid (EDTA), pH 8.5) to a final concentration of 26.5 mg / mL. To reduce the mAb, 1 equivalent of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mM) is added if the final DAR is approximately 2, or 2 equivalents if it is approximately 4. The reaction is incubated at room temperature for 1.5 hours, followed by dilution to 5 mg / mL with PBS containing 2 mM EDTA (pH 7.4).

[0309] The buffer is desalted and replaced by gel filtration (Zeba® spin) with PBS containing 2 mM EDTA for DAR2, and with PBS further supplemented with 3% cyclodextrin (CD) for DAR4, to reduce antibody aggregation. For conjugation of the linker-microcystin dimer conjugate to the reduced mAb, for DAR2, 4 equivalents from 10 mM DM-II-L solution (in dimethylacetamide (DMA)) are added to the reduced mAb (5 mg / mL, PBS containing 2 mM EDTA) and incubated at room temperature for 2 hours. For DAR4, 8 equivalents of DM-II-L are added to the reduced mAb (5 mg / mL, PBS containing 2 mM EDTA with 3% CD) and incubated at room temperature for 3.5 hours. To improve solubility, a final concentration of 5% DMA is used for DAR2, and 10% (v / v) DMA is used for DAR4. The conjugation reaction is carried out by adding an equimolar amount of N-acetyl-L-cysteine ​​(NAC) to the toxin-linker conjugate used and stopping the reaction after 30 minutes. The reaction mixture is then desalted by gel filtration (G-25, PBS (DAR2) or PBS containing 3% CD (DAR4)). Subsequently, activated carbon (100 mg / mL, PBS or PBS containing 3% CD, pH 7.4) is added in an equal volume to the weight of the mAb. After 1 hour, the activated carbon is removed by centrifugation (4000 g, 15 minutes), and the supernatant is analyzed by SEC and HIC and aseptically filtered.

[0310] Example 57: Synthesis of an ADC composed of a monoclonal anti-HER2 antibody and the organic cytotoxic dimer from Example 18

[0311] The organic cytotoxic dimer synthesized in Example 18 is used as the drug portion of the ADC. The organic cytotoxic dimer consists of two MC-Leu-AzLys entities, with two branches of the cross-linking portion BM-V leading to Z 4 This is a microcystin homodimer that is symmetrically crosslinked at the 12-degree position. The crosslinked portion further contains a free amino coupling functional group for binding the microcystin homodimer to an antibody.

[0312] First, the microcystin homodimer obtained according to Example 18 is conjugated to a cleavable linker such as mc-Val-Ala-PAB-PNP via the free terminal amino group of the crosslinking portion BM-V, as described in Example 56 above. Other suitable linker and conjugation chemistry are known to those skilled in the art.

[0313] Next, the resulting linker-homodimer conjugate is conjugated to the monoclonal anti-HER2 antibody trastuzumab, as described in Example 56 above.

[0314] Example 58: Synthesis of an ADC composed of a monoclonal anti-HER2 antibody and the organic cytotoxic dimer from Example 40

[0315] The organic cytotoxic dimer synthesized in Example 40 is used as the drug portion of the ADC. The organic cytotoxic dimer consists of two [D-PrgMeAsp] 1 - Consists of NOD entities, with two branches of the bridging portion BM-IV passing through Aa 1 It is a nodularin homodimer that is symmetrically crosslinked at the 12-degree position. The crosslinked portion further contains a free amino coupling functional group for binding the nodularin homodimer to the antibody.

[0316] First, the nodularin homodimer obtained according to Example 40 is conjugated to a cleavable linker such as mc-Val-Ala-PAB-PNP via the free terminal amino group of the crosslinking portion BM-IV, as described in Example 56 above. Other suitable linker and conjugation chemistry are known to those skilled in the art.

[0317] Next, the resulting linker-homodimer conjugate is conjugated to the monoclonal anti-HER2 antibody trastuzumab, as described in Example 56 above.

[0318] Example 59: Synthesis of an ADC composed of a monoclonal anti-HER2 antibody and the organic cytotoxic dimer from Example 49

[0319] The organic cytotoxic dimer synthesized in Example 49 is used as a drug entity in the construction of ADCs. The organic cytotoxic dimer consists of two [AeHtp] 2 - Consists of AMA cytotoxic entities, which are connected via two branches of the cross-linking portion BM-VI to Aa 2 This is an amatoxin homodimer that is symmetrically crosslinked at the 12th position. The crosslinked portion further contains a free amino coupling functional group for binding the microcystin homodimer to the antibody (see Figure 9).

[0320] First, the amatoxin homodimer obtained according to Example 49 is conjugated to a cleavable linker such as mc-Val-Ala-PAB-PNP via the free terminal amino group of the crosslinking moiety BM-VI, as described in Example 57. Other suitable linker and conjugation chemistry are also known to those skilled in the art.

[0321] Next, the obtained linker-homodimer conjugate is conjugated to the anti-HER2 monoclonal antibody trastuzumab using the complementary coupling functional groups of the monoclonal antibody and the linker of the linker-homodimer conjugate, as described in Example 56 above, to form an ADC.

[0322] Example 60: Protocol for cell culture and subsequent cytotoxicity assay of OATP-expressing cancer cell lines

[0323] HEK293 cells stably transfected with the expression vectors pcDNA3.1(+)-OATPB1 and pcDNA3.1 / Hygro(-)-OATP1B3, as well as their respective empty vectors pcDNA3.1(+) and pcDNA3.1 / Hygro(-) (controls), were provided by Prof. Dr. Jo:rg Ko:nig (Friedrich Alexander University Erlangen-Nuremberg, Germany).

[0324] All cell lines were maintained at 37°C and 5% CO2 in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, a mixture of non-essential amino acids, and 2 mM glutamine.

[0325] HEK293 OATP1B1+ and the corresponding control cell lines were constitutively selected with 800 μg / mL of genetisine (G418), while HEK293 OATP1B3+ and its empty vector control were selected with 250 μg / mL of hygromycin B.

[0326] To measure the cytotoxicity of the organic cytotoxic oligomer and the corresponding cytotoxic monomer (for reference) of the present invention, 5 × 10⁶ units per well were used for each cell line. 4 Cells were seeded in triple duplication in 96-well plates without a selection marker. After 24 hours, sodium butyrate was added to a final concentration of 10 mM to induce transporter expression. The following day, the medium was removed and the cells were incubated for 48 hours in medium without a selection marker at eight different concentrations of each cytotoxic oligomer (0.01 nM to 3 μM) and the corresponding toxin monomer (control).

[0327] Subsequently, the cells were fixed, washed, and stained with sulforodamine B (SRB) (as described by Vichai et al., Nature protocols 2006, 1, 1112-1116). Briefly, 10 μL of trichloroacetic acid (10%) was added directly to each well and incubated at 4°C for 1 hour. The solution was then removed, and each well was washed three times with 200 μL of H2O. The wells were then dried, and 100 μL of 0.057% SRB solution (in 1% acetic acid) was added to each well and incubated at 4°C for 30 minutes. The solution was removed, and each well was washed three times with 200 μL of acetic acid (1%). After drying the wells, 200 μL of Tris buffer (10 mM, pH 10.5) was added, and the plate was shaken for 5 minutes. Next, the absorbance of SRB was measured at 510 nm using a TECAN Infinity M PLEX plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). The experiment was performed at least twice.

[0328] Cell viability and IC50 values ​​(IC50_Cytotox) were calculated using GraphPad PRISM 6 with nonlinear regression (sigmoid dose-response).

[0329] Example 61: Protocol for a protein phosphatase inhibition (PPI) assay

[0330] The PPI assay was performed according to a recently reported method (Heresztyn et al., Water Res 2001, 35, 3049-3056).

[0331] Prior to the measurements, various working solutions were prepared. The reaction buffer was newly prepared by mixing 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. The substrate solution was prepared by mixing the reaction buffer with stock solution of p-nitrophenyl phosphate (pNPP) (60 mM) and DTT solution (20 mM) (5:4:1 (v / v)), and the pNPP was newly 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) (65:20:5:10 (v / v)). The enzyme solution was prepared by diluting the protein phosphatase-1 catalytic subunit (rabbit-derived α-isoform, Sigma-Aldrich) to 10 units / mL with the enzyme dilution buffer and storing it on ice.

[0332] The assay was performed in a 384-well plate. Diluted organic cytotoxic oligomers and their corresponding cytotoxic monomers (control) (4 μL in DMSO) were pre-incubated with enzyme solution (4 μL) at 37°C for 5 minutes. Next, 40 μL of substrate solution was added to each well and incubated at 37°C for 2 hours. The cytotoxic substance concentrations at seven levels ranged from 0.001 nM to 1 μM, and the substrate concentration per well was kept constant at 20 mM. As a positive control, 4 μL of DMSO was used instead of MC solution, and substrate conversion was tracked without PPI. The negative control (blank) contained 4 μL of enzyme dilution buffer instead of diluting enzyme to prevent substrate hydrolysis. To measure PPI, absorbance at 405 nm was measured using a TECAN Infinity M PLEX plate reader. The experiment was performed at least twice, and the IC50 value (IC50_PPI) was calculated using nonlinear regression (sigmoid dose-response) with GraphPad PRISM 6.

[0333] Example 62: Protocol for RNA polymerase II (RNA Pol II) inhibition assay

[0334] The RNA Pol II inhibition assay was performed according to a recently reported method (Voss et al., BMC Molecular Biology 2014, 15:7). This paper describes a non-radioactive, robust, and reproducible eukaryotic in vitro transcription assay for highly sensitive detection and accurate quantification of newly transcribed unlabeled RNA.

[0335] After transcription, newly synthesized RNA is directly detected and quantified using the QuantiGene assay (Thermo Fisher Scientific Inc.). Alternatively, the RNA can be purified and then detected by PCR or qPCR after primer extension. This assay is particularly useful for quantifying potent transcription inhibitors such as α-amanitin. When applied to the activity evaluation of RNA polymerase II inhibitors, this novel method makes it possible to accurately estimate their relative potency.

[0336] Example 63: In vitro efficacy testing protocol for ADC

[0337] The ADCs constructed in Examples 56-59 were evaluated in vitro for efficacy using a cell viability assay in which each ADC was incubated at different concentrations ranging from picomolar to nanomolar or micromolar using tumor antigen-expressing cancer cell lines such as NCI-N87 that express the Her2 antigen.

[0338] In vitro efficacy studies of ADCs were conducted using, for example, the CellTiter-Glo luminescent cell viability assay (Promega GmbH, Walldorf, Germany), following the manufacturer's instructions.

[0339] Example 64: Bioassay Results

[0340] As representative examples, Figure 12 and Table 7 show the results of general cytotoxicity (IC50_OATP1B1 and IC50_OATP1B3 in Figure 12A and Table 7) and protein phosphatase inhibition assays (IC50_PPI in Figure 12B and Table 7) against OATP1B1 and OATP1B3 expressing cell lines obtained from the assays described in Examples 60 and 61 above. These are microcystin homodimers of Examples 2 and 12 having structural formulas DM-I and DM-II (see Figures 1 and 2), and were obtained by covalently crosslinking two MC-PrgTyr-Arg monomers as the first and second organic cytotoxic entities using a crosslinking molecule of structural formula BM-I (hereinafter referred to as "MC-PrgTyr-Arg-Dimer-1") and BM-IV (hereinafter referred to as "MC-PrgTyr-Arg-Dimer-2").

[0341] For reference, Figure 12 and Table 7 show the corresponding assay results for the non-conjugate reference monomer MC-PrgTyr-Arg and MC-LR, which is currently the most potent and most studied microcystin analog and has the highest protein phosphatase inhibitory activity described in the literature.

[0342] Table 7 shows the results of cytotoxicity assays and protein phosphatase inhibition assays (IC50_PPI = type 1 protein phosphatase) using OATP1B1-expressing cell lines (IC50_OATP1B1) and OATP1B3-expressing cell lines (IC50_OATP1B3) for MC-LR (reference), MC-PrgTyr-Arg monomer (reference), and the corresponding MC-PrgTyr-Arg dimer according to the present invention (Examples 2 and 12). "SE" indicates the standard error of at least two experiments.

[0343] [Table 7]

[0344] As another representative example, Table 8 shows the results for general cytotoxicity (IC50_OATP1B1 in Table 8) and protein phosphatase inhibition assays (IC50_PPI in Table 8) against OATP1B1-expressing cell lines obtained from the assays described in Examples 60 and 61 above. These were obtained using a cross-linking molecule of structural formula BM-IV, with two [D-PrgMeAsp] 1 - The nodularin homodimer of Example 40 (see below "[D-PrgMeAsp]") obtained by covalently crosslinking NOD monomers (first and second organic cytotoxic entities) 1 This concerns what is called "-NOD-dimer-1".

[0345] For reference, Table 8 lists the non-conjugate type reference monomer [D-PrgMeAsp] 1 -The corresponding assay results for NOD are also shown.

[0346] Table 8: [D-PrgMeAsp] in cytotoxicity assays (IC50_OATP1B1) and protein phosphatase inhibition assays (IC50_PPI = type 1 protein phosphatase) using OATP1B1-expressing cell lines. 1-NOD monomer (reference) and the corresponding [D-PrgMeAsp] according to the present invention 1 The results for NOD-dimer-1 (Example 40) are shown. "SE" indicates the standard error of at least two experiments.

[0347] [Table 8]

[0348] Tables 7 and 8 also show the ratios of IC50_OATP1B1 to IC50_PPI, and the ratio of IC50_OATP1B3 to IC50_PPI (the latter in Table 7 only), as indicators of PPI-normalized OATP uptake. These ratios were used to evaluate whether oligomerization exhibited the desired effect. In this context, if general cytotoxicity to OATP1B1 and / or OATP1B3-expressing cell lines is reduced (reflected as an increase in IC50_OATP1B1 or IC50_OATP1B3, respectively), and at the same time protein phosphatase inhibition is increased or essentially unchanged (reflected by a change or decrease in IC50_PPI), i.e., if the ratio is greater than the reference ratio, it indicates that the therapeutic window of the organic cytotoxic oligomer of the present invention is expanded for the OATP-expressing cell lines tested. Such an expansion of the therapeutic window occurs when the active cellular uptake of the organic cytotoxic oligomer of the present invention is relatively reduced relative to protein phosphatase inhibition, or when the protein phosphatase inhibition of the organic cytotoxic oligomer of the present invention is relatively increased relative to active cellular uptake. Furthermore, when the ratio of the IC50_PPI of the organic cytotoxic oligomer of the present invention to the IC50_PPI of the reference is less than 0.5 if the oligomer is a dimer, less than 0.33 if the oligomer is a trimer, etc. (generally the ratio is less than the reciprocal of the number of monomers in the oligomer), this indicates that the efficacy of the cytotoxic effect of the organic cytotoxic oligomer of the present invention based on the corresponding protein phosphatase inhibition is synergistically enhanced compared to the reference, beyond a mere increase in the number of toxins in the conjugate.

[0349] The results in Figure 12, Table 7, and Table 8 are for MC-PrgTyr-Arg-dimer-1, MC-PrgTyr-Arg-dimer-2, and [D-PrgMeAsp] according to the present invention. 1 The IC50_OATP1B1 and IC50_OATP1B3 values ​​of -NOD-dimer-1 are significantly increased compared to the monomer-type reference microcystin or nodularin, respectively. For IC50_OATP1B1, an increase of over 116 times was observed in MC-PrgTyr-Arg-dimer-1 compared to MC-LR, and an increase of over 175 times was observed in MC-PrgTyr-Arg-dimer-2. Compared to the corresponding MC-PrgTyr-Arg monomer, increases of over 24 times and 36 times were observed, respectively. Similar effects were observed for IC50_OATP1B3, with an increase of 134 times in MC-PrgTyr-Arg-dimer-1 compared to MC-LR, a 234-fold increase in MC-PrgTyr-Arg-dimer-2, and increases of 26 times and 45 times compared to MC-PrgTyr-Arg monomer, respectively. Furthermore, [D-PrgMeAsp] 1 -NOD-dimer-1 corresponds to [D-PrgMeAsp] 1 - The IC50_OATP1B1 value was more than nine times higher compared to the NOD monomer. These findings indicate that the microcystin and nodularin oligomers of the present invention significantly reduce general cytotoxicity to OATP-expressing cell lines compared to the monomer-type reference.

[0350] On the other hand, MC-PrgTyr-Arg-dimer and PrgMeAsp according to the present invention] 1 Protein phosphatase inhibition of NOD-dimer-1 is associated with the corresponding MC-PrgTyr-Arg monomer or [D-PrgMeAsp] 1 -Compared to the IC50_PPI of NOD monomer, MC-PrgTyr-Arg-dimer-1 is more than 20 times higher, MC-PrgTyr-Arg-dimer-2 is more than 50 times higher, [D-PrgMeAsp] 1The study showed an IC50_PPI that was more than 8 times lower with NOD-dimer-1, and also approximately 10 times lower with MC-PrgTyr-Arg-dimer-1 and approximately 16 times lower with MC-PrgTyr-Arg-dimer-2 compared to the IC50_PPI of the MC-LR reference cytotoxin, clearly demonstrating a significant enhancement of protein phosphatase inhibition.

[0351] MC-PrgTyr-Arg-dimer and [D-PrgMeAsp] according to the present invention 1 The general cytotoxicity of -NOD-dimer-1 against OATP1B1 and OATP1B3-expressing cell lines was greatly reduced, and the enzyme inhibitory effect was greatly enhanced. These results indicate that oligomerization yields the desired effect of significantly reducing active uptake into cells.

[0352] Furthermore, the IC50_PPI of the MC-PrgTyr-Arg-dimer is more than 20 to more than 50 times lower than that of the corresponding MC-PrgTyr-Arg monomer, [D-PrgMeAsp] 1 - The IC50_PPI of NOD-dimer-1 corresponds to [D-PrgMeAsp] 1 The fact that the IC50_PPI of the NOD monomer is more than eight times lower clearly indicates a synergistic effect on enzyme inhibition due to oligomerization, rather than simply the additive effect of the individual cytotoxicities of the two monomers.

[0353] Finally, the combination of a more than 20-fold to more than 50-fold lower IC50_PPI and the general cytotoxicity-to-protein phosphatase inhibition ratio (an indicator of PPI-normalized OATP uptake) against OATP1B1 and OATP1B3 expressing cell lines demonstrates that the MC-PrgTyr-Arg dimer according to the present invention expands the therapeutic window of OATP expressing cell lines by at least two orders of magnitude to up to four orders of magnitude compared to the MC-PrgTyr-Arg monomer (more than 700-fold to more than 2400-fold) and the MC-LR reference cytotoxin (more than 1000-fold to more than 3900-fold). Similarly, [D-PrgMeAsp] 1Regarding NOD-dimer-1, the IC50_PPI decreased by more than eight times, and when combined with the ratio of general cytotoxicity to protein phosphatase inhibition against OATP1B1-expressing cell lines, the therapeutic window was expanded by almost two orders of magnitude compared to the monomer, confirming that the effects of the present invention are generalizable.

[0354] Two types of MC-PrgTyr-Arg-dimers and [D-PrgMeAsp] 1 The results obtained with the NOD-dimer are understood to be representative of the beneficial effects of the organic cytotoxic oligomers of the present invention. Similar improvements and favorable effects with respect to general cytotoxicity, protein phosphatase inhibition and / or therapeutic window can be obtained with respect to other representative organic cytotoxic oligomers described in the other examples above.

[0355] These synergistic effects and improvements, as well as the resulting advantages, were not predictable to those skilled in the art.

[0356] Furthermore, it should be understood that the present invention is not limited to the embodiments described in detail. Rather, the present invention encompasses all novel features and all combinations of novel features, including, in particular, all combinations of features in the claims and specification, even if a feature or combination of features is not explicitly defined in the claims, specification, or embodiments.

[0357] For example, those skilled in the art will understand that the microcystin dimers described in Examples 2-21 can also be constructed using different microcystin analogs as monomers, and can be used as homomicrocystin dimers or heteromicrocystin dimers, or with different crosslinking moieties. In particular, these microcystin analogs may have different amino acid sequences than those included in Examples 2-21, for example, Aa 1 , X 2 , Aa 3 , Z 4 and / or Aa 7Microcystin analogs having different amino acids at the same position, or having different coupling functional groups such as alkenes, carboxyls, ketones, hydroxyls, hydroxylamines, isothiocyanates, tetrazines, thiols, and aldehydes. Furthermore, microcystin dimers may be formed by crosslinking monomers at different positions, for example, Aa of the first microcystin entity. 1 Position and X of the second microcystin entity 2 Location, or Aa 5 and Aa 6 It is also possible to bridge using any combination other than those mentioned above.

[0358] Similarly, the microcystin trimers described in Examples 26-33 can also be constructed as homomicrocystin trimers or heteromicrocystin trimers using different microcystin analogs as monomers. In particular, these microcystin analogs have different amino acid sequences than those included in Examples 2-33, for example, Aa 1 , X 2 , Aa 3 , Z 4 and / or Aa 7 Microcystin analogs having different amino acids at the same position may also be present, or they may have different coupling functional groups such as alkenes, alkyl halides, hydrazides, carboxyls, ketones, hydroxyls, hydroxylamines, isothiocyanates, tetrazines, thiols, or aldehydes. The crosslinking domain can also be covalently linked at different positions of the microcystin entity.

[0359] Furthermore, microcystin oligomers, such as microcystin tetramers, microcystin pentamers, and microcystin hexamers, can also be constructed as homomicrocystin oligomers or heteromicrocystin oligomers. This includes, but is not limited to, the microcystin entities used in Examples 2-33, as well as microcystin analogs having different amino acids and / or different coupling functional groups as described above.

[0360] The nodularin dimers described in Examples 34-43 can also be constructed as homonodularin dimers or heteronodularin dimers using different nodularin analogs as monomers. In particular, these nodularin analogs have different amino acid sequences than those included in Examples 34-43, for example, Aa 1 , Aa 2 and / or Aa 5 Nodularin analogs having different amino acids at the same position, or those having different coupling functional groups such as alkenes, carboxyls, ketones, hydroxyls, hydroxylamines, isothiocyanates, tetrazines, thiols, and aldehydes, may also be used.

[0361] The nodularin trimers described in Examples 44 and 45 can also be constructed as homonodularin trimers or heteronodularin trimers using different nodularin analogs as monomers, and in particular, nodularin analogs having different amino acid sequences from those included in Examples 34-45 can be used as cytotoxic entities.

[0362] Furthermore, nodularin oligomers, such as nodularin tetramers, nodularin pentamers, and nodularin hexamers, can also be constructed as homonodularin oligomers or heteronodularin oligomers, including, but not limited to, the nodularin entities used in Examples 34-45, as well as nodularin entities having different amino acids and / or different coupling functional groups as described above.

[0363] The amatoxin dimers described in Examples 46-51 can also be constructed as homoamatoxin dimers or heteroamatoxin dimers using different amatoxin entities as monomers. In particular, the amatoxin entity can have a different amino acid sequence than those included in Examples 46-51, for example, Aa 1 , Aa 3 , Aa 4 , Aa 5 , Aa 6 and / or Aa 7It may also have different amino acids at the position of, or it may have different coupling functional groups such as alkenes, carboxyls, ketones, hydroxyls, hydroxylamines, isothiocyanates, tetrazines, thiols, aldehydes, oximes, etc.

[0364] Furthermore, the amatoxin trimers described in Examples 52-55 can also be constructed as homoamatoxin trimers or heteroamatoxin trimers using different amatoxin entities as monomers, and amatoxin entities having different amino acid sequences and / or different coupling functional groups than those exemplified can be used. In addition, amatoxin oligomers, such as amatoxin tetramers, amatoxin pentamers, and amatoxin hexamers, can also be constructed as homoamatoxin oligomers or heteroamatoxin oligomers.

[0365] Furthermore, it is understood that the anti-HER2 antibody trastuzumab used in Examples 56-59 was intentionally selected because trastuzumab is an established benchmark antibody in the anti-TAA and anti-TSA antibody genera. The results obtained with trastuzumab-containing ADCs demonstrate that the present invention can also be carried out with other antibody species, particularly antibodies belonging to the anti-TAA and anti-TSA antibody genera.

[0366] Therefore, it is understood that the ADCs of Examples 56-59 can also be constructed using different monoclonal antibodies targeting different antigens expressed by different cancer cells, such as CD7 or BCMA. Naturally, ADCs can also be constructed using other homozygous and heterozygous microcystins, nodularins and / or amatoxin dimers and oligomers, as well as different linkers and other conjugation methods, as described above.

Claims

1. The first organic cytotoxic entity, At least a second organic cytotoxic entity, A crosslinking entity that forms a covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity, Equipped with, The monomers of the first organic cytotoxic entity and the second organic cytotoxic entity are (i) By being actively transported into the target cell by the target cell's transporter protein, and / or, (ii) By inhibiting the intracellular enzymes of the target cells, a cytotoxic effect is exerted on the target cells, The covalent bond between the first organic cytotoxic entity and the second organic cytotoxic entity is substantially resistant to cleavage under physiological conditions, thereby providing a pharmacokinetic cytotoxic oligomer.

2. The first organic cytotoxic entity and the second organic cytotoxic entity each comprise a cyclic oligopeptide having a plurality of amino acids. The pharmacokinetic cytotoxic oligomer according to claim 1, wherein the crosslinked entity covalently links one side chain of an amino acid of the first organic cytotoxic entity to one side chain of an amino acid of the second organic cytotoxic entity.

3. The first organic cytotoxic entity and the second organic cytotoxic entity each comprise a microcystin having the following general structure: cyclo(-Aa １ -X ２ -Aa ３ -Z ４ -Aa ５ -Aa ６ -Aa ７ )、 Aa １ and Aa ３ each independently represent a D-amino acid, and Aa ５ is selected from the group consisting of Adda, DM-Adda, dm-Adda, (6Z)Adda and ADM-Adda, and Aa ６ is selected from the group consisting of D-Glu and D-Glu(OCH ３ ), and Aa ７ is selected from the group consisting of Mdha, MdhB, Dha, L-Ser, L-MeSer, Dhb, (E)-Dhb, (Z)-Dhb, MeLan, Cys and Thr, or a modified L-amino acid, and X ２ and Z ４ are independently L-amino acids, The aforementioned crosslinking is Aa of the first organic cytotoxic entity. １ , X ２ Aa ３ Z ４ or Aa ７ The side chain of either of the Aa of the second organic cytotoxic entity １ , X ２ Aa ３ Z ４ or Aa ７ The pharmaceutical cytotoxic oligomer according to claim 1 or 2, which is covalently linked to one of the side chains.

4. The first organic cytotoxic entity and the second organic cytotoxic entity each comprise nodularin having the following general structure: cyclo[-Aa １ -Aa ２ -Aa ３ -Aa ４ -Aa ５ ]、 Aa １ These independently represent D-Asp, D-MeAsp, or modified D-amino acids, and Aa ２ Arg or Har or modified L-amino acid, Aa ３ The group is selected from Adda, DM-Adda, (6Z)Adda, and MeAdda, and Aa ４ D-Glu and D-Glu (OCH ３ Selected from the group consisting of ), Aa ５ This is Dhb or Mdhb or a modified L-amino acid. The crosslinked entity is Aa of the first organic cytotoxic entity. １ Aa ２ or Aa ５ The side chain of the second organic cytotoxic entity Aa １ Aa ２ or Aa ５ The pharmaceutical cytotoxic oligomer according to claim 1 or 2, which is covalently linked to one of the side chains.

5. The first organic cytotoxic entity and the second organic cytotoxic entity each comprise an amatoxin having the following general structure: cyclo{Aa １ -cyclo[Aa ２ -Aa ３ -Aa ４ -Aa ５ -Aa ６ ]-Aa ７ -Aa ８ }(SEQ ID NO:1)、 Independent of each other, Aa １ is Ile, Hil or Dhil, Aa ２ is Trp or Htp, Aa ３ Gly, Aa ４ Ile, Aa ５ Gly, Aa ６ is Cys, Aa ７ is Asn or Asp, Aa ８ is Pro or Hyp, Aa ２ and Aa ６ They are connected by sulfoxide (S=O) crosslinks. The crosslinked entity is Aa of the first organic cytotoxic entity. １ Aa ２ or Aa ８ The side chain of either of the Aa of the second organic cytotoxic entity １ Aa ２ or Aa ８ The pharmaceutical cytotoxic oligomer according to claim 1 or 2, which is covalently linked to one of the side chains.

6. The crosslinked entity comprises or consists of a covalent bond selected from the group consisting of an amide bond, a thioamide bond, an ether bond, a thioether bond, a covalent bond containing a triazole, dihydropyridazine or a higher 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, according to any one of claims 1 to 5.

7. The pharmacokinetic cytotoxic oligomer according to any one of claims 1 to 6, wherein the crosslinked entity comprises a crosslinking portion that connects the first organic cytotoxic entity and the second organic cytotoxic entity to each other, and the crosslinking portion is an organic molecule having a molecular size of about 50 Da to about 1,000 Da.

8. The crosslinked entity comprises a coupling functional group configured to covalently conjugate a pharmaceutical cytotoxic oligomer to an antibody, The cytotoxic oligomer for pharmaceutical use according to any one of claims 1 to 7, wherein the coupling functional 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.

9. The crosslinked entity comprises at least a third organic cytotoxic entity, wherein the crosslinked entity forms a covalent bond between the first organic cytotoxic entity, the second organic cytotoxic entity, and at least the third organic cytotoxic entity. The pharmaceutically active cytotoxic oligomer according to any one of claims 1 to 8, wherein the covalent bonds between the first organic cytotoxic entity, the second organic cytotoxic entity, and the at least further third organic cytotoxic entity are substantially resistant to cleavage under physiological conditions.

10. The cytotoxic oligomer for pharmaceutical use according to any one of claims 1 to 9, wherein the resistance to cleavage comprises resistance to enzymatic cleavage and resistance to acid-induced cleavage up to at least pH 4.

5.

11. The pharmaceutically cytotoxic oligomer according to any one of claims 1 to 10, wherein the intracellular enzyme of the target cell is a phosphatase or polymerase.

12. (A) Prepare a first organic cytotoxic entity and at least a second cytotoxic entity, and the first organic cytotoxic entity and at least the second organic cytotoxic entity are (i) By being actively transported into the target cell by the target cell's transporter protein, and / or (ii) By inhibiting the intracellular enzyme of the target cell, The steps involve exerting a cytotoxic effect on target cells, (B) The step of forming a covalent bond between the first organic cytotoxic entity and at least the second organic cytotoxic entity that is substantially resistant to cleavage under physiological conditions, thereby forming a cytotoxic oligomer, A method for producing a pharmaceutical cytotoxic oligomer according to any one of claims 1 to 11, comprising:

13. (A) further comprises the step of providing at least one organic crosslinked portion, wherein the crosslinked portion is an organic molecule having a molecular size of about 50 Da to about 1,000 Da. The method according to claim 12, further comprising the steps of (B) forming a first covalent portion by covalently bonding the crosslinking portion to the first organic cytotoxic entity, and forming at least a second covalent portion by covalently bonding the crosslinking portion to at least the second organic cytotoxic entity, thereby crosslinking the first organic cytotoxic entity and at least the second organic cytotoxic entity with each other via the crosslinking portion.

14. Antibodies or their antigen-binding fragments, A cytotoxic oligomer according to any one of claims 1 to 11, Equipped with, The aforementioned cytotoxic oligomer is an antibody-drug conjugate, in which the cytotoxic oligomer is covalently attached to an antibody or its antigen-binding fragment.

15. The following general formula (I) Ab-[L-(T) ｍ ] ｎ (I) It has, The antibody-drug conjugate according to claim 14, wherein Ab is the antibody or its antigen-binding fragment, T is the pharmaceutically active cytotoxic oligomer, L is an organolinker, m is an integer from 1 to 5, and n is an integer from 1 to 8.

16. The antibody-drug conjugate according to claim 14 or 15, wherein the linker is configured to release the pharmaceutically cytotoxic oligomer from the antibody or its antigen-binding fragment during receptor-mediated endocytosis and / or lysosomal treatment of the antibody-drug conjugate.

17. (a) the step of providing a pharmaceutically cytotoxic oligomer according to any one of claims 1 to 11, (b) Providing an antibody or an antigen-binding fragment thereof, (c) A method for producing an antibody-drug conjugate, comprising the step of covalently binding a pharmaceutical cytotoxic oligomer to the antibody or its antigen-binding fragment.

18. (c) is, (c1) To provide a linker, (c2) Covalently attaching the pharmacokinetic oligomer to the linker, (c3) Further comprising covalently binding the linker to the antibody or its antigen-binding fragment, The method according to claim 17.

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