Organic cytotoxin oligomers and uses thereof as a payload in antibody-drug conjugates

Abstract

Pharmaceutical cytotoxin oligomers are provided. The pharmaceutically acceptable cytotoxin oligomer comprises a first organic cytotoxin entity, at least a second organic cytotoxin entity, and a cross-linking entity forming a covalent linkage between the first organic cytotoxin entity and the second organic cytotoxin entity, wherein the monomer of the first organic cytotoxin entity and the monomer of the second organic cytotoxin entity each exert a cytotoxic effect on a target cell by actively transporting into the target cell via a transporter of the target cell and / or by inhibiting an intracellular enzyme of the target cell. The covalent linkage between the first organic cytotoxin entity and the second organic cytotoxin entity is substantially resistant to cleavage under physiological conditions.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480048129.1 (22) Application Date 2024.07.21 (30) Priority Data 23187008.0 2023.07.21 EP (85) PCT International Application Entering National Phase Date 2026.01.20 (86) PCT International Application Application Data PCT / EP2024 / 070651 2024.07.21 (87) PCT International Application Publication Data WO2025 / 021727 EN 2025.01.30 (71) Applicant: Sinlis Biopharmaceuticals GmbH Address: Berlin, Germany (72) Inventors: T. Niedermeier, S. Schuster, D. Enke, H. Enke (74) Patent Agency: China Patent Agency (Hong Kong) Limited 72001 Patent Attorney Chu Mingming Lin Yibin (51) Int.Cl. A61K 47 / 54 (2006.01) A61K 47 / 55 (2006.01) A61K 47 / 68 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title Organic Cytotoxin Oligomers and Their Use as a Loading Material in Antibody-Drug Conjugates (57) Abstract A pharmaceutical cytotoxin oligomer is provided. The pharmaceutical cytotoxin oligomer comprises a first organic cytotoxin entity, at least a second organic cytotoxin entity, and a cross-linked entity forming a covalent link between the first organic cytotoxin entity and the second organic cytotoxin entity, wherein the monomers of the first organic cytotoxin entity and the second organic cytotoxin entity each exert cytotoxic effects on the target cell by active transport to the target cell via a target cell transporter and / or by inhibiting intracellular enzymes of the target cell. The covalent link between the first organic cytotoxin entity and the second organic cytotoxin entity significantly resists lysis under physiological conditions. Claims (3 pages), Description (43 pages), Sequence List (electronic publication), Drawings (8 pages) CN 121620394 A 2026.03.06 CN 1 21 62 03 94 A 1. A pharmaceutical cytotoxic oligomer comprising a first organic cytotoxic entity, at least a second organic cytotoxic entity, and a cross-linked entity forming a covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity, wherein the monomers of the first organic cytotoxic entity and the second organic cytotoxic entity i) are actively transported to the target cell via a transport protein of the target cell, and / or ii) exert cytotoxic effects on the target cell by inhibiting intracellular enzymes of the target cell.The covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity significantly resists cleavage under physiological conditions. 2. The pharmaceutical cytotoxic oligomer according to claim 1, wherein the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a cyclic oligopeptide having a plurality of amino acids, and wherein the crosslinking entity covalently links a side chain of one of the amino acids of the first organic cytotoxic entity to a side chain of one of the amino acids of the second organic cytotoxic entity. 3. The pharmaceutical cytotoxin oligomer according to any one of claims 1 or 2, wherein the first organic cytotoxin entity and the second organic cytotoxin entity each comprise a microcystin ring having the following universal structure (-Aa1-X2-Aa3-Z4-Aa5-Aa6-Aa7), wherein, independently of each other, Aa1 and Aa3 each represent a D-amino acid, Aa5 is selected from Adda, DM-Adda, dm-Adda, (6Z) Adda and ADM-Adda, Aa6 is selected from D-Glu and D-Glu(OCH3), Aa7 is selected from Mdha, MdhB, Dha, L-Ser, L-MeSer, Dhb, (E)-Dhb, (Z)-Dhb, MeLan, Cys and Thr or modified L-amino acids, and X2 and Z4 are independently L-amino acids. The crosslinking portion covalently links one of the side chains of the first organic cytotoxic entity (Aa1, X2, Aa3, Z4, or Aa7) to one of the side chains of the second organic cytotoxic entity (Aa1, X2, Aa3, Z4, or Aa7). 4. The pharmaceutical cytotoxin oligomer according to any one of claims 1 or 2, wherein the first organic cytotoxin entity and the second organic cytotoxin entity each comprise a phycocyanin ring having the following general structure [-Aa1-Aa2-Aa3-Aa4-Aa5], wherein, independently of each other, Aa1 is D-Asp or D-MeAsp or a modified D-amino acid, Aa2 is Arg or Har or a modified L-amino acid, Aa3 is selected from Adda, DM-Adda, (6Z)Adda and MeAdda, Aa4 is selected from D-Glu and D-Glu(OCH3), and Aa5 is Dhb or Mdhb or a modified L-amino acid, wherein the crosslinking entity covalently links the side chain of Aa1, Aa2 or Aa5 of the first organic cytotoxin entity to the side chain of one of Aa1, Aa2 or Aa5 of the second organic cytotoxin entity. 5. The pharmaceutical cytotoxin oligomer according to any one of claims 1 or 2.The first and second organic cytotoxic entities each comprise an amatoxin ring {Aa1-ring[Aa2-Aa3-Aa4-Aa5-Aa6]-Aa7-Aa8} (SEQ ID NO:1) having the following general structure, wherein, independently of each other, Aa1 is Ile, Hil, or Dhil, Aa2 is Trp or Htp, Aa3 is Gly, Aa4 is Ile, Aa5 is Gly, Aa6 is Cys, Aa7 is Asn or Asp, and Aa8 is Pro or Hyp, crosslinked between Aa2 and Aa6 via a sulfoxide (S=O) bridge, wherein the crosslinked entity covalently links a side chain of one of Aa1, Aa2, or Aa8 of the first organic cytotoxic entity to a side chain of one of Aa1, Aa2, or Aa8 of the second organic cytotoxic entity. Claims 1 / 3 Page 2 CN 121620394 A 6. The pharmaceutical cytotoxin oligomer according to any one of claims 1-5, wherein the crosslinked entity comprises or is composed of a covalent link selected from or consisting of: amide bond, thioamide bond, ether bond, thioether bond, triazole-containing covalent bond, dihydropyridazine or higher amine, carbamate bond, thiocarbamate bond, urea bond, thiourea bond, phosphate ester bond, phosphoramide bond, sulfonamide bond, oxime bond, or any combination thereof. 7. The pharmaceutical cytotoxin oligomer according to any one of claims 1-6, wherein the crosslinked entity comprises a bridging portion connecting the first organic cytotoxin entity and the second organic cytotoxin entity to each other, wherein the bridging portion is an organic molecule with a molecular size of about 50 Da to about 1,000 Da. 8. The pharmaceutical cytotoxin oligomer according to any one of claims 1-7, wherein the crosslinking entity comprises a coupling function configured to covalently conjugate the pharmaceutical cytotoxin oligomer to an antibody, wherein the coupling function is selected from amino, carboxyl, hydroxyl, azide, alkynyl, alkenyl, thiol, aldehyde, ketone, tetrazinyl, and any combination thereof. 9. The pharmaceutical cytotoxin oligomer according to any one of claims 1-8, comprising at least a third organic cytotoxin entity, wherein the crosslinking entity forms a covalent link between the first organic cytotoxin entity, the second organic cytotoxin entity, and at least the third organic cytotoxin entity, wherein the covalent link between the first organic cytotoxin entity, the second organic cytotoxin entity, and at least the third organic cytotoxin entity significantly resists cleavage under physiological conditions. 10. The pharmaceutical cytotoxin oligomer according to any one of claims 1-9, wherein resistance to cleavage comprises resistance to enzymatic cleavage and resistance to acid-induced cleavage at a pH value as low as pH 4.5. 11. The pharmaceutical cytotoxin oligomer according to any one of claims 1-10,The intracellular enzyme of the target cell is a phosphatase or a polymerase. 12. A method for producing a pharmaceutical cytotoxic oligomer according to any one of claims 1-11, comprising A) providing a first organic cytotoxic entity and at least a second cytotoxic entity, wherein the first organic cytotoxic entity and the at least second organic cytotoxic entity each i) are actively transported to the target cell via a transport protein of the target cell, and / or ii) exert cytotoxic effects on the target cell by inhibiting the intracellular enzyme of the target cell, B) forming a covalent link between the first organic cytotoxic entity and the at least second organic cytotoxic entity that is significantly resistant to cleavage under physiological conditions, thereby forming the cytotoxic oligomer. 13. The method of claim 12, wherein A) further comprises providing at least one organic bridging portion, wherein the bridging portion is an organic molecule with a molecular size of about 50 Da to about 1,000 Da, and B) further comprises crosslinking the first organic cytotoxic entity and at least the second organic cytotoxic entity to each other by covalently conjugating the bridging portion to the first organic cytotoxic entity to form a first portion covalently linked, and by covalently conjugating the bridging portion to the at least second organic cytotoxic entity to form at least a second portion covalently linked. 14. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a pharmaceutical cytotoxic oligomer according to any one of claims 1-11, wherein the pharmaceutical cytotoxic oligomer is covalently linked to the antibody or the antigen-binding fragment thereof. Claims 2 / 3 Page 3 CN 121620394 A 15. The antibody-drug conjugate according to claim 14, having the general formula (I) Ab-[L-(T)m]n (I) where Ab is the antibody or its antigen-binding fragment; T is the pharmaceutical cytotoxin oligomer; L is an organic linker; and m is an integer from 1 to 5, and n is an integer from 1 to 8. 16. The antibody-drug conjugate according to any one of claims 14 or 15, wherein the linker is configured to release the pharmaceutical cytotoxin oligomer from the antibody or its antigen-binding fragment during receptor-mediated endocytosis and / or lysosomal processing of the antibody-drug conjugate. 17. A method for producing an antibody-drug conjugate, comprising a) providing a pharmaceutical cytotoxin oligomer according to any one of claims 1-11, b) providing an antibody or its antigen-binding fragment, and c) covalently linking the pharmaceutical cytotoxin oligomer to the antibody or its antigen-binding fragment. 18. The method of claim 17, wherein c) further comprises c1) providing a connector, and c2) covalently attaching the pharmaceutical cytotoxin oligomer to the connector.c3) The adapter is covalently linked to the antibody or its antigen-binding fragment. Claims 3 / 3 Page 4 CN 121620394 A Organic cytotoxic oligomers and their use as payloads in antibody-drug conjugates

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

[0002] Sequence listing This application contains an electronic sequence listing of 17 sequences in xml format according to the WIPO ST.26 standard, which is incorporated herein by reference in its entirety as part of the specification. Technical Field

[0003] The present invention relates to bioactive cytotoxic anticancer payloads or drugs. In particular, the present invention relates to organic cytotoxic toxins, more particularly modified cyanobacterial and fungal toxins, used as payloads in antibody-drug conjugates. The present invention further relates to antibody-drug conjugates comprising the bioactive cytotoxic anticancer payload conjugated to an antibody specifically targeting a tumor antigen. Background Art

[0004] Today, cancer is the second leading cause of death worldwide, causing approximately 10 million deaths annually. Cancer incidence has increased over the past few decades due to global population growth and aging, and is expected to continue to rise.

[0005] A drawback of most anticancer drugs currently used in conventional chemotherapy is that they are often cytotoxic, as they are insufficient to distinguish between cancer cells and healthy tissue, have known serious side effects, and significantly limit 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, also referred to below as “ADCs,” are a class of biological drugs designed as targeted therapies for the treatment of cancer. Unlike chemotherapy, ADCs are designed to target and kill tumor cells without harming healthy cells. ADCs are essentially biological drugs composed of a monoclonal antibody linked to a biologically active and typically cytotoxic drug portion (also referred to herein as a “payload”), thus combining the target specificity of the monoclonal antibody with the cancer-killing ability of the cytotoxic drug.

[0008] Conceptually, ADCs, mediated by the target specificity of monoclonal antibodies, are expected to target tumor cells without harming healthy tissue. However, off-target effects are frequently observed during clinical application, for example, by spontaneous or enzymatic hydrolysis of the chemical bond between the drug and antibody during transport and metabolism after systemic administration, or by the release of the payload from lysed or killed cancer cells into surrounding tissues.

[0009] Current efforts to reduce or eliminate undesirable toxic side effects on healthy cells mainly involve improving the stability of the chemical bond between the cytotoxic payload and the monoclonal antibody.To avoid premature release of the drug outside tumor cells.

[0010] For example, WO 2018 / 206715A2 and WO 2018 / 219619A1, the disclosures of which are incorporated herein by reference in their entirety, disclose modified cyanobacterial microcystin and arthrophyllin as cytotoxic payloads, which, in particular, incorporate non-naturally occurring amino acids, provide chemical anchoring groups for linking the payload to a linker or antibody, respectively, resulting in a homogeneous and stable ADC with limited payload release in the bloodstream.

[0011] However, the improved chemical linking between the antibody and the cytotoxic payload only partially overcomes the aforementioned drawbacks of current ADCs. In particular, it does not address the problem of payload release after the death of targeted cancer cells. Furthermore, the high potency of cytotoxins creates unresolved health and safety risks in their manufacture and handling, resulting in costly infrastructure and production.

[0012] European Patent Application No. 22192517.5, which has not been published to date, the disclosure of which is incorporated herein by reference in its entirety, teaches the modification of cyanobacterial or fungal toxins with structural modifications comprising organic inhibitory groups or effector molecules, said organic inhibitory groups or effector molecules reducing the uptake of the modified toxin into target cells via transport proteins.

[0013] In this respect, an object of the present invention is to further provide improved organic cytotoxins for use as payloads in antibody-drug conjugates. Another object of the present invention is to provide an improved antibody-drug conjugate.

[0014] These objects are achieved according to the subject matter of the independent claims. Preferred embodiments are the subject matter of the dependent claims and are described below.

[0015] In some embodiments, the present invention provides a pharmaceutical cytotoxin oligomer or a pharmaceutically acceptable salt thereof, said pharmaceutical cytotoxin oligomer comprising a first organic cytotoxin entity, at least a second organic cytotoxin entity, and a cross-linked entity forming a covalent link between the first organic cytotoxin entity and at least the second organic cytotoxin entity, wherein each monomer of the first organic cytotoxin entity and the second organic cytotoxin entity i) is actively transported to the target cell via a transporter protein of the target cell and / or ii) exerts cytotoxic effects on the target cell by inhibiting intracellular enzymes of the target cell, wherein the covalent link between the first organic cytotoxin entity and at least the second organic cytotoxin entity significantly resists lysis under physiological conditions.

[0016] In some embodiments, the present invention provides a method for producing a pharmaceutical cytotoxin oligomer as defined above, comprising A) providing a first organic cytotoxin entity and at least a second cytotoxin entity,The first organic cytotoxic entity and the second organic cytotoxic entity each i) are actively transported to the target cell via a transporter protein of the target cell and / or ii) exert cytotoxic effects on the target cell by inhibiting intracellular enzymes of the target cell, and B) form a covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity that is significantly resistant to cleavage under physiological conditions, thereby forming the pharmaceutical cytotoxic oligomer.

[0017] In some embodiments, the present invention provides the use of pharmaceutical cytotoxic oligomers as defined above for the production of antibody-drug conjugates, including covalently conjugating pharmaceutical cytotoxic oligomers to an antibody or an antigen-binding fragment thereof.

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

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

[0020] In some embodiments, the present invention provides a pharmaceutically acceptable salt of a pharmaceutically acceptable cytotoxic oligomer as defined above or an antibody-drug conjugate as defined above for treating malignant diseases, particularly cancer, and more particularly malignant tumors.

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

[0022] In some embodiments according to or applicable to any of the above embodiments, the first organocytotoxic entity and the second organocytotoxic entity are independently selected from cyanobacterial toxins, fungal toxins, or variants or derivatives thereof.

[0023] In some embodiments according to or applicable to any of the above embodiments, the first organocytotoxic entity and the second organocytotoxic entity each comprise a cyclic oligopeptide having a plurality of amino acids, wherein the crosslinking entity covalently links a side chain of one of the amino acids of the first organocytotoxic entity to a side chain of one of the amino acids of the second organocytotoxic entity.

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

[0025] In some embodiments according to or applicable to any of the above embodiments, resistance to lysis includes resistance to lysosomal degradation.

[0026] Preferred variations of these embodiments may be obtained from the description of the invention and specific implementation.

[0027] Description of the Invention The present invention is based on the inventors’ insight that cytotoxins typically must be taken up by cells via active transport mediated by transport proteins or by passive diffusion before they can exert their cytotoxic function. However, when cytotoxins are conjugated with antibodies to form ADCs, the internalization of cytotoxins becomes receptor-mediated endocytosis, such as clathrin-mediated endocytosis, cell membrane celluloid-mediated endocytosis, or pinocytosis (the process by which cells absorb ADCs by budding inward from the plasma membrane after the antibody binds to the cell surface, thereby transporting the toxin to the target cell).

[0028] Based on this insight, the inventors described the potential of modifying organic cytotoxins in European Patent Application No. 22192517.5, wherein the cytotoxins, in a segregated form, require active transport across the cell membrane and cannot enter the cell by passive diffusion, having structural modifications that intentionally weaken their acceptance by transport proteins. In this way, the inventors equipped organic cytotoxins with a structural “safety catch” that prevents them from being actively taken up into the cell and exerting their cytotoxic function, thereby protecting healthy cells from cytotoxic side effects.

[0029] In further in-depth research, the inventors have now discovered that a particularly advantageous structural modification for this effect is the artificial oligomerization of organic cytotoxins through non-cleavable covalent linkages. First, the inventors found that artificial oligomerization strongly inhibits active cellular uptake of organic cytotoxins. Second, the inventors observed a surprising improvement in the cytotoxicity of oligomerized cytotoxins in a synergistic manner, wherein the cytotoxicity of oligomerized cytotoxins significantly exceeds the total cytotoxicity of monomeric cytotoxin equivalents.

[0030] Due to the cumulative pharmacological interaction of the two effects, the cytotoxic oligomers of the present invention extend the therapeutic window (i.e., the dose range optimized between efficacy and toxicity) by several orders of magnitude compared to monomeric cytotoxic counterparts, achieving greater therapeutic benefits without causing unacceptable side effects or toxicity. In this regard, reference is also made to specific embodiments.

[0031] In this context, one aspect of the present invention relates to a pharmaceutically acceptable cytotoxic oligomer or a pharmaceutically acceptable salt thereof. Specifically, the present invention relates to a pharmaceutical portion comprising a pharmaceutically acceptable cytotoxic oligomer or a pharmaceutically acceptable salt thereof or composed thereof.Also referred to as "payload".

[0032] The pharmaceutical cytotoxic oligomer of the present invention comprises a first organic cytotoxic entity, at least a second organic cytotoxic entity, and a cross-linked entity forming a covalent link between the first organic cytotoxic entity and at least the second organic cytotoxic entity. Specification 3 / 43 pages 7 CN 121620394 A

[0033] The terms "first" and "second", and as appropriate, "third", "fourth", etc., are used herein to distinguish one feature or element of the invention from another feature or element, but should not be considered as limiting unless the context otherwise indicates. Thus, the "first organic cytotoxic entity" described below may be referred to as the "second organic cytotoxic entity", and correspondingly, the "second organic cytotoxic entity" described below may be referred to as the "first organic cytotoxic entity" without departing from the teachings of the invention.

[0034] To achieve the advantageous effects according to the invention, it is necessary that the first organic cytotoxic entity and the second organic cytotoxic entity, each in monomeric form, i) be actively transported to target cells via transport proteins to exert their cytotoxic effect on the target cells, and, alternatively or additionally, ii) be enzyme inhibitors, i.e., exert their cytotoxic effect by inhibiting intracellular enzymes of the target cells, said intracellular enzymes preferably being intracellular enzymes whose biological activity is essential to the target cells, such that their inhibition is cytotoxic and leads to the death of the target cells.

[0035] In addition, an essential feature of the invention in this respect is that the covalent link between the first organic cytotoxic entity and at least the second organic cytotoxic entity is significantly resistant to lysis under physiological conditions.

[0036] As used herein, the term “physiological conditions” refers to in vivo environmental conditions that are naturally present, for example, in human bloodstream, intracellular environment, and / or extracellular environment, as opposed to artificial laboratory conditions. Specifically, physiological conditions include a temperature range of 20–40 degrees Celsius, an atmospheric pressure of about 1 bar, a pH of 6–8, a glucose concentration of 1–20 mM, and an atmospheric oxygen concentration.

[0037] The term “significant resistance” means spontaneous cleavage of the covalently linked linker, such as extremely slow spontaneous hydrolysis, with a half-life typically lasting months or years, and in some embodiments decades, at 25°C. In some embodiments, the term “significant resistance” means resistance to lysosomal degradation. In some embodiments, “significant resistance” means resistance to enzymatic and / or chemical cleavage, such as resistance to cleavage by hydrolases and / or oxidoreductases, and / or resistance to acid-induced cleavage at pH values ​​as low as pH 4.5 or pH 4.0. In some embodiments, “significant resistance” means that the half-life of the covalently linked linker under physiological conditions is longer than the average or maximum residence time of the cytotoxic oligomer in the human body, preferably at least twice as long, more preferably several times.

[0038] In this way,In contrast to corresponding cytotoxic monomers, the cytotoxic oligomers of the present invention are prevented from exhibiting nonspecific cytotoxic activity in the human intracellular extracellular environment, such as when prematurely released from an ADC into the bloodstream or after release from dead cancer cells. In contrast, when the cytotoxic oligomers of the present invention bind to antibodies, they are internalized as antibody payloads by target cells via receptor-mediated endocytosis, wherein the cytotoxic oligomers are released following intracellular transport and lysosomal degradation of the ADC, and the enhanced cytotoxicity of the cytotoxic oligomers is released to trigger tumor cell death. In this way, the present invention provides cytotoxic oligomers as ADC payloads with significantly improved safety characteristics, allowing for increased ADC dosage to improve therapeutic benefit without resulting in unacceptable side effects or toxicity. Furthermore, safety is improved in the production and processing of the cytotoxic oligomers compared to monomeric cytotoxic counterparts. It should be understood that the beneficial effects are not limited to ADCs, but can also be achieved when the cytotoxic oligomer is coupled with other targeting moieties (i.e., molecules or macromolecules that bind to specific receptors located on the surface of tumor cells), including but not limited to nanoparticles, liposomes, micelles, aptamers, peptides, and dendritic polymers.

[0039] In the sense of this invention, the term "oligomer" or "oligomeric" refers to a molecule obtained by covalently linking smaller molecular "monomers". Thus, the term "organic cytotoxic entity" refers to a cytotoxic moiety of a cytotoxic monomer with an organic basic structure that is known to be cytotoxic to target cells (especially cancer cells) after uptake by transport proteins of target cells. Each organic cytotoxic entity of a cytotoxic oligomer may also be referred to as a "cytotoxic subunit". In the following, a cytotoxic oligomer consisting of two organic cytotoxic entities is referred to as a "dimer", a cytotoxic oligomer consisting of three organic cytotoxic entities is referred to as a "trimer", a cytotoxic oligomer consisting of four organic cytotoxic entities is referred to as a "tetramer", and so on. In a preferred embodiment, the cytotoxin oligomer is a dimer, trimer, or tetramer, more preferably a dimer or trimer, and most preferably a dimer. If the cytotoxin oligomer is composed of the same organic cytotoxin entity, the cytotoxin oligomer is called a homooligomer; otherwise, the cytotoxin oligomer is called a heterooligomer.

[0040] Cytotoxin monomers with an organic basic structure that require transport proteins for active cell uptake of toxins are known to those skilled in the art. For example, the transport of cytotoxin monomers or organic basic structures can be mediated by organic anion transport polypeptides (OATP) or their subtypes, such as OATP1B1, OATP1B3, and / or OATP1A2. These OATPs are primarily found in the human liver. Therefore,The inventors have achieved the importance of blocking OATP transport of the payload toxin to avoid hepatotoxicity and improve the relative safety of ADCs.

[0041] In a preferred embodiment of the invention, the first organic cytotoxic entity and the second organic cytotoxic entity are independently selected from cyanobacterial toxins, fungal toxins, or variants or derivatives thereof. As is generally understood in the art, the term "derivative" refers to an organic cytotoxic entity having the same basic structure as a cyanobacterial toxin or fungal toxin but having a functional group or another atom or atomic group instead of a hydrogen atom or substituent, or wherein one or more atoms or atomic groups of the basic structure are removed. Thus, the term "variant" refers to an organic cytotoxic entity having a partially modified but therapeutically equivalent basic structure of a cyanobacterial toxin or fungal toxin.

[0042] In a further preferred embodiment of the 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 a side chain of one of the amino acids of the first organic cytotoxic entity to a side chain of one of the amino acids of the second organic cytotoxic entity. In a preferred embodiment, the cyclic oligopeptide comprises 4 to 10 amino acids, particularly 5 to 8 amino acids, for example, precisely 4, precisely 5, precisely 6, precisely 7, or precisely 8 amino acids. Preferably, the cyclic oligopeptide toxin comprises at least one non-protein amino acid, preferably several non-protein amino acids. More preferably, the cyclic oligopeptide comprises a dipeptide sequence wherein the first amino acid is selected from Adda, DM-Adda, dm-Adda, (6Z)Adda, and ADM-Adda, and the second amino acid is selected from 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 some embodiments, the first organic cytotoxic entity and / or the second organic cytotoxic entity are enzyme inhibitors, particularly protein phosphatase inhibitors or polymerase inhibitors, such as RNA polymerase inhibitors.

[0044] In a preferred embodiment of the invention, the cyanobacterial toxin is a microcystin toxin. Microcystin is a cyclic heptapeptide from cyanobacteria that effectively inhibits eukaryotic serine / threonine protein phosphatases type 1 and 2A, leading to the disruption of many essential signal transduction pathways and ultimately resulting in cytoskeleton collapse and cell death. Microcystin is primarily taken up via OATP1B1 and OATP1B3.

[0045] In other preferred embodiments of the invention, the cyanobacterial toxin is an arthrococcus toxin. Arthrococcus toxin is a cyclic pentapeptide from cyanobacteria that is evolutionarily related to microcystin.It also inhibits protein phosphatases type 1 and 2A as its cytotoxic mode of action. Arthrocarpus toxins are primarily taken up by OATP1B1.

[0046] In another preferred embodiment of the invention, the fungal toxin is amatoxin. Amatoxin is a fungal bicyclic octapeptide belonging to the amatoxin subgroup. The most prominent α-amatoxin is an inhibitor of RNA polymerase II, which binds to the bridging helix of RNA polymerase II and inhibits the translocation of RNA and DNA required to empty the site for the next synthetic run.

[0047] In a preferred embodiment of the invention, the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a microcystin ring having the following universal structure (-Aa1-X2-Aa3-Z4-Aa5-Aa6-Aa7). Specification 5 / 43 pages 9 CN 121620394 A

[0048] In this document, Aa1 and Aa3 each represent D-amino acids independently of each other, which may be modified; Aa5 is selected from Adda, DM-Adda, dm-Adda, (6Z)Adda and ADM-Adda; Aa6 is selected from D-Glu and D-Glu(OCH3); Aa7 is selected from Mdha, MdhB, Dha, L-Ser, L-MeSer, Dhb, (E)-Dhb, (Z)-Dhb, MeLan, Cys and Thr or modified L-amino acids; and X2 and Z4 are L-amino acids independently of each other, which may be modified. 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-demethyl-Adda; DM-Adda is 9-O-demethyl-Adda; dm-Adda is Adda demethylated at C-2, C-6, or C-8; Dha is dehydroalanine; MeSer is N-methyl-L-serine; Dhb is dehydrobutyrate; and MeLan is N-methyl-lanothionine. As used herein, “modified” or “modified amino acid” means an amino acid whose side chain has a binding site with the crosslinked entity and is adjacent to the crosslinked entity. Specifically, the side chain of the modified amino acid may be a chemical group incorporated into the covalent link between the first and second cytotoxic entities, or a chemical group forming part of the covalent link between the first and second cytotoxic entities.

[0049] In some embodiments, Aa1 is selected from D-Ala, D-Leu, D-Ser, Gly, or modified D-amino acids or modified Gly. In some embodiments, Aa3 is selected from D-Asp and D-MeAsp or modified D-amino acids. D-MeAsp is D-erythrose-β-methylaspartic acid. In some embodiments,Aa5 is selected from Adda, DM-Adda, dm-Adda, and ADM-Adda. In some embodiments, Aa6 is D-Glu. The modified amino acid may be derived, for example, from an amino acid with a coupled functionality in its side chain that is not present in the corresponding natural cyanobacterial or fungal toxin, i.e., a derivative thereof. As used herein, “functionality” refers to the presence of one or more functional groups in a molecule. A monofunctional molecule has one functional group, a bifunctional (or difunctional) molecule has two functional groups, a trifunctional molecule has three functional groups, and so on. In organic chemistry, functionality has a decisive influence on the reactivity of a molecule. Therefore, “coupled functionality” is a functional group that can and / or is configured to form a covalent link with at least a portion of another coupled functionality having complementary reactivity. For example, the modified amino acids may be derived from the group consisting of azidovaline, propargyltyrosine, azidolysine, azidophenylalanine, propargylcysteine, propargylserine, and azidoalanine (as D-amino acids or L-amino acids, if applicable).

[0050] Microcystins are potent inhibitors of type 1 and type 2A protein phosphatases. Protein phosphatases 1 (PP1) and 2A (PP2A) are two of the major phosphatases in eukaryotic cells that dephosphorylate serine and threonine residues. For example, the IC50 of naturally occurring microcystin-LR (MC-LR, SEQ ID NO:16) is 0.03 nM for type 1 protein phosphatases and 0.04 nM for type 2A protein phosphatases. Positions A5 and A6 are determined in the art to be necessary and sufficient for the inhibitory effect of microcystins against PP1 and PP2A.

[0051] Therefore, in a preferred embodiment, the crosslinking entity covalently links a side chain of one of Aa1, X2, Aa3, Z4, or Aa7 of the first organic cytotoxic entity to a side chain of one of Aa1, X2, Aa3, Z4, or Aa7 of the second organic cytotoxic entity.

[0052] In some embodiments, the crosslinking entity covalently links a side chain of one of Aa1, X2, Z4, or Aa7 of the first organic cytotoxic entity to a side chain of one of Aa1, X2, Z4, or Aa7 of the second organic cytotoxic entity.

[0053] In some embodiments, the crosslinking entity covalently links a side chain of one of X2, Z4, or Aa7 of the first organic cytotoxic entity to a side chain of one of X2, Z4, or Aa7 of the second organic cytotoxic entity.

[0054] In some embodiments, the crosslinking entity covalently links a side chain of one of X2 or Z4 of the first organic cytotoxic entity to a side chain of one of X2 or Z4 of the second organic cytotoxic entity.

[0055] In some embodiments,The crosslinking entity covalently links the Aa1 side chain of the first organic cytotoxic entity to one of the side chains of the second organic cytotoxic entity, namely Aa1, X2, Aa3, Z4, or Aa7. In some embodiments, the crosslinking entity covalently links the X2 side chain of the first organic cytotoxic entity to one of the side chains of the second organic cytotoxic entity, namely Aa1, X2, Aa3, Z4, or Aa7. In some embodiments, the crosslinking entity covalently links the Aa3 side chain of the first organic cytotoxic entity to one of the side chains of the second organic cytotoxic entity, namely Aa1, X2, Aa3, Z4, or Aa7. In some embodiments, the crosslinking entity covalently links the Z4 side chain of the first organic cytotoxic entity to one of the side chains of the second organic cytotoxic entity, namely Aa1, X2, Aa3, Z4, or Aa7. In some embodiments, the crosslinking entity covalently links the side chain of Aa7 of the first organic cytotoxic entity to the side chain of one of Aa1, X2, Aa3, Z4, or Aa7 of the second organic cytotoxic entity.

[0056] In other preferred embodiments of the invention, the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a phycocyanin ring having the following general structure [-Aa1-Aa2-Aa3-Aa4-Aa5].

[0057] Hereinafter, independently of each other, Aa1 is D-Asp or D-MeAsp or a modified D-amino acid, Aa2 is Arg or Har or a modified L-amino acid, Aa3 is selected from Adda, DM-Adda, (6Z)Adda, and MeAdda, Aa4 is selected from D-Glu and D-Glu (OCH3), and Aa5 is Dhb or Mdhb or a modified L-amino acid. Har is high-arginine, (6Z)Adda is the geometric isomer of Adda at C-6, and MeAdda is methylated Adda.

[0058] It is determined in the art that positions A3 and A4 are necessary and sufficient for the inhibitory effect of arthrotoxicogens on PP1 and PP2A. Therefore, in a preferred embodiment, the crosslinking entity covalently links a side chain of Aa1, Aa2, or Aa5 of the first organic cytotoxic entity to a side chain of one of Aa1, Aa2, or Aa5 of the second organic cytotoxic entity.

[0059] In some embodiments, the crosslinking entity covalently links a side chain of one of Aa1 or Aa5 of the first organic cytotoxic entity to a side chain of one of Aa1 or Aa5 of the second organic cytotoxic entity.

[0060] In some embodiments,The crosslinking entity covalently links the side chain of Aa1 of the first organic cytotoxic entity to the side chain of one of Aa1, Aa2, or Aa5 of the second organic cytotoxic entity. In some embodiments, the crosslinking entity covalently links the side chain of Aa2 of the first organic cytotoxic entity to the side chain of one of Aa1, Aa2, or Aa5 of the second organic cytotoxic entity. In some embodiments, the crosslinking entity covalently links the side chain of Aa5 of the first organic cytotoxic entity to the side chain of one of Aa1, Aa2, or Aa5 of the second organic cytotoxic entity.

[0061] In still other preferred embodiments, the first organic cytotoxic unit and the second organic cytotoxic unit each comprise an amatoxin ring having the following general structure: {Aa1-ring[Aa2-Aa3-Aa4-Aa5-Aa6]-Aa7-Aa8} (SEQ ID NO:1).

[0062] In this document, independently of each other, Aa1 is Ile, hydroxyisoleucine (Hil) or dihydroxyisoleucine (Dhil) or a derivative thereof with a side chain having a binding site with said crosslinking entity, Aa2 is Trp or hydroxytryptophan (Htp) or a derivative thereof with a side chain having a binding site with said crosslinking entity, Aa3 is Gly, Aa4 is Ile, Aa5 is Gly, Aa6 is Cys, Aa7 is Asn or Asp, and Aa8 is Pro or hydroxyproline (Hyp) or a derivative thereof with a side chain having a binding site with said crosslinking entity, crosslinked between Aa2 and Aa6 by a sulfoxide (S=O) bridge.

[0063] For example, amatoxins can be one of the groups consisting of α-amatoxin, β-amatoxin, γ-amatoxin, δ-amatoxin, ε-amatoxin, amatoxin, amatoxinic acid, amatoxin, amatoxin, and protoamatoxin. Preferably, the amatoxin is amatoxin.

[0064] In a preferred embodiment, the crosslinking entity covalently links one of the side chains of the first organic cytotoxic entity (Aa1, Aa2, or Aa8) to one of the side chains of the second organic cytotoxic entity (Aa1, Aa2, or Aa8).

[0065] In some embodiments, the crosslinking entity covalently links one of the side chains of the first organic cytotoxic entity (Aa2 or Aa8) to one of the side chains of the second organic cytotoxic entity (Aa2 or Aa8).

[0066] In some embodiments, the crosslinking entity covalently links the Aa1 side chain of the first organic cytotoxic entity to the side chain of one of the Aa1, Aa2, or Aa8 of the second organic cytotoxic entity. In some embodiments,The crosslinking entity covalently links the side chain of Aa2 of the first organic cytotoxic entity to the side chain of one of Aa1, Aa2, or Aa8 of the second organic cytotoxic entity. In some embodiments, the crosslinking entity covalently links the side chain of Aa8 of the first organic cytotoxic entity to the side chain of one of Aa1, Aa2, or Aa8 of the second organic cytotoxic entity.

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

[0068] In some embodiments according to or applicable to any of the above embodiments, the crosslinking entity covalently links the side chain at one amino acid position of the first organic cytotoxic entity to the side chain at the same amino acid position of the second organic cytotoxic entity, for example, Aa1 with Aa1, X2 with X2, Aa2 with Aa2, Aa3 with Aa3, Z4 with Z4, Aa7 with Aa7, Aa8 with Aa8, etc. (if applicable).

[0069] In some embodiments according to or applicable to any of the above embodiments, the crosslinking entity covalently links a side chain at one amino acid position of the first organic cytotoxic moiety to a side chain at a different amino acid position of the second organic cytotoxic moiety, for example, the side chain of Aa2 or X2 of the first organic cytotoxic entity to a side chain of any one of Aa1, Aa3, Z4, or Aa7 of the second organic cytotoxic entity, the side chain of Z4 of the first organic cytotoxic entity to a side chain of any one of Aa1, Aa2, or X2, Aa3, or Aa7 of the second organic cytotoxic entity, etc.

[0070] In some embodiments according to or applicable to any of the above embodiments, the first organic cytotoxic entity and the second organic cytotoxic entity are substantially identical, i.e., the cytotoxic oligomers are homooligomers, particularly homodimers or homotrimers. In some 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 other embodiments according to or applicable to any of the above embodiments, the first organic cytotoxic entity and the second organic cytotoxic entity are different 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 different amino acid sequences (e.g., including different modified amino acids) or be composed of them. In particular,The first cytotoxic entity and the second cytotoxic entity are derived from different organic cytotoxic monomers.

[0072] In some embodiments according to or applicable to any of the above embodiments, the crosslinked entity or covalent link comprises, or is composed of, a covalent link selected from amide bonds, thioamide bonds, ether bonds, thioether bonds, triazole-containing covalent links, dihydropyridazine or higher amines such as secondary (20) amines, tertiary (30) amines or quaternary (40) amines, carbamate bonds, thiocarbamate bonds, urea bonds, thiourea bonds, phosphate ester bonds, phosphoramide bonds, sulfonamide bonds, oxime bonds or any combination thereof. The inventors have found that these types of covalent links result in the crosslinked entity being resistant to cleavage under physiological conditions, which enhances the beneficial pharmacological effects of the cytotoxic oligomers according to the invention. In some embodiments, the crosslinked entity does not contain disulfide bonds, ester bonds and / or isourea bonds. Furthermore, the crosslinked entity preferably does not contain substrates for intracellular enzymatic cleavage, such as hydrolases and / or oxidoreductase substrates or cleavage sites, or substrates for chemically triggered cleavage (e.g., in response to pH changes or reduction potential changes).

[0073] In some embodiments according to or applicable to any of the above embodiments, the crosslinked entity or covalent link respectively comprises an organic bridging portion that covalently links the first organic cytotoxic entity and the second organic cytotoxic entity to each other. The organic bridging portion may also spatially separate the first and second organic cytotoxic entities from each other. The organic bridging portion is not particularly limited, provided that the main requirement of the invention is met, namely, that the covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity is significantly resistant to cleavage under physiological conditions. Suitable organic bridging portions are well known to those skilled in the art or can be determined based on this disclosure.

[0074] The organic bridging portion may be linear (i.e., unbranched) or branched. The organic spacer unit of the branched chain may have three or more branches, wherein at least one branch is terminally connected to the first organic cytotoxic entity, and a second branch is terminally connected to the second organic cytotoxic entity. A third or more branches may be terminally connected to a third or more organic cytotoxic entities, and / or may have ends containing coupling functionality, i.e., functional groups configured to covalently couple (optionally via a linker) the cytotoxic oligomer to the antibody. The coupling functionality may optionally be protected with a protecting group.

[0075] Preferably,The bridging portion is an organic molecule with 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, most preferably about 50 Da to about 1,500 Da, or about 50 Da to about 1,000 Da. The bridging portion may contain a methylene group, a PEG group, or other groups for spatially separating the organic cytotoxic entity.

[0076] In some embodiments, at least one of the first organic cytotoxic entity, the second organic cytotoxic entity, and the crosslinked entity contains an additional coupling functionality, i.e., a functional group configured (i.e., capable of) covalently conjugating the cytotoxic oligomer to an antibody (optionally via a linker). Suitable coupling functionalities for bioconjugating the cytotoxic oligomer to an antibody are well known to those skilled in the art. For example, the additional coupling functionality may be a functional group selected from amino, carboxyl, hydroxyl, azide, alkynyl, alkenyl, thiol, aldehyde, ketone, tetraazinyl, hydrazyl, or any combination thereof. Additional coupling functionality can be protected with removable protecting groups, including but not limited to tert-butyloxycarbonyl (BOC), acetyl (Ac), tert-butyl ether (tBu), methyl ester, benzyl ester, or tert-butyl ester. Further suitable protecting groups are known to those skilled in the art. In a preferred embodiment, the additional coupling functionality is configured to, or is capable of, undergoing a bioorthogonal chemical coupling reaction, such as a click chemistry reaction. In the sense of the invention, a click chemistry reaction is, for example, copper (I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted alkyne-nitroketone cycloaddition, alkene and azide [3+2] cycloaddition, alkene and tetrazine reverse electron-demanding Diels-Alder reaction, alkene and tetrazolium photoclick reaction. Therefore, the additional coupling functionality is preferably an azide, alkyne, alkene, or tetrazine.

[0077] As already mentioned above, the cytotoxic oligomer may comprise more than two organic cytotoxic entities, forming a trimer, tetramer, or even higher oligomer. For example, the cytotoxin oligomer may comprise at least a third additional organic cytotoxin entity, wherein the crosslinking entity forms a covalent link between the first organic cytotoxin entity, the second organic cytotoxin entity, and at least the third additional organic cytotoxin entity, for example, through a branched bridging portion included in the crosslinking entity as described above. Alternatively, the cytotoxin oligomer may comprise a first crosslinking entity forming a covalent link between the first organic cytotoxin entity and the second cytotoxin entity, and a second crosslinking entity forming a covalent link between the second cytotoxin entity and the third cytotoxin entity, wherein in each case,The covalent linkage between the first organic cytotoxic entity, the second organic cytotoxic entity, and at least the additional third organic cytotoxic entity significantly resists lysis under physiological conditions.

[0078] In some embodiments according to or applicable to any of the embodiments herein, resistance to lysis includes resistance to lysosomal degradation.

[0079] In a preferred embodiment according to or applicable to any of the embodiments herein, resistance to lysis includes resistance to enzymatic lysis and resistance to acid-induced lysis at pH values ​​as low as pH 4.5 or pH 4.

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

[0081] In some embodiments, the organic cytotoxic oligomers are selected from those described in the detailed description on page 9 / 43 of CN 121620394 A below.

[0082] Therefore, another aspect of the invention relates to the use of pharmaceutical cytotoxic oligomers as described above for the production of antibody-drug conjugates. Specifically, the use includes covalently conjugating a pharmaceutical cytotoxin oligomer to an antibody or an antigen-binding fragment thereof.

[0083] In another aspect, the present invention provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a pharmaceutical cytotoxin oligomer or a pharmaceutically acceptable salt thereof as described above, wherein the pharmaceutical cytotoxin oligomer is covalently linked to the antibody or the antigen-binding fragment thereof.

[0084] In some embodiments, the antibody-drug conjugate has the general formula (I) Ab-[L-(T)m]n (I), wherein Ab is an antibody or an antigen-binding fragment thereof; T is a pharmaceutical cytotoxin oligomer; L is an organic linker; and m and n are independently integers from 1 to 10.

[0085] 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.

[0086] In some embodiments, n is an integer from 1 to 8, preferably from 2 to 8, more preferably from 4 to 8.

[0087] The product m∙n is also referred to as the “drug-to-antibody ratio” (DAR). In a preferred embodiment, the DAR is 1-16, meaning that each antibody binds at least one and at most sixteen cytotoxic oligomers. Preferably, the DAR is 2-12, more preferably 2-10 or 2-8, and most preferably 4-8.

[0088] For the purposes of this invention, the term “antibody” is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, bispecific antibodies, or multispecific antibodies, as well as antibody fragments having an antigen-specific binding site (i.e., an antigen-binding site for an antigen or a portion thereof that specifically binds to an immune target cell). Preferably,An antibody is a monoclonal antibody (mAb) or an antigen-binding fragment thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; single solid antibodies (also known as nanobodies); biantibodies; linear antibodies; fragments produced from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity-determining regions); and epitope-binding fragments of any of the above, which are immune-specifically bound to, for example, any human or animal antigen, cancer cell antigen, senescent cell antigen, viral antigen, or microbial antigen; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or antibodies derived from other species. Antibodies can be any type of immunoglobulin, such as IgG, IgE, IgM, IgD, or IgA; any class, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2; or a subclass of immunoglobulin.

[0089] In a preferred embodiment, the antibody or its antigen-binding fragment is specific to a tumor-associated antigen (TAA) (i.e., anti-TAA antibody) or a tumor-specific antigen (TSA) (i.e., anti-TSA antibody). Tumor-specific antigens are found only on cancer cells and not on healthy cells. Tumor-associated antigens are expressed at elevated levels on tumor cells but at lower levels on healthy cells. Such tumor-associated antigens and tumor-specific antigens are known in the art. Examples of TAA and TSA include, but are not limited to, BMPR1B (bone morphogenetic protein receptor-IB, Genbank accession number NM-001203), E16 (LAT1, SLC7A5, Genbank accession number NM-003486), STEAP1 (prostate six-transmembrane epithelial antigen, Genbank accession number NM-012449), 0772P (CA125, MUC16, Genbank accession number AF361486), MPF (MPF, MSLN, SMR, megakaryocyte enhancer factor, mesothelin, Genbank accession number NM-005823), Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM-006424), and Sema 5b. (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema entity, seven platelet-reactive protein repeats (type 1 and type 1-like), transmembrane entity (TM) and short cytoplasmic entity, (Semaphorin) 5B,Genbank accession number AB040878), PSCA hlg (2700050C12Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628), ETBR (endothelin B receptor, Genbank accession number AY275463), MSG783 (RNF124, hypothesized protein F1120315, Genbank accession number NM-017763), STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related gene 1, prostate six-transmembrane epithelial antigen 2, six-transmembrane prostate protein, 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, teratogenic 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 β), B29, GenBank accession number NM-000626 or 11038674), FcRH2 (IFGP4, IRTA4, SPAP1A) (phosphatase-anchored protein 1a containing SH2 entity), 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 BC017023), IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971), short proteoglycans (BCAN, BEHAB, Genbank accession number AF229053), EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession number NM-004442), ASLG659 (B7h,Genbank accession number AX092328), PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436), GEDA (Genbank accession number AY260763), AAP14954 lipoma HMGIC fusion-partner-like protein / pid=AAP14954.1 Homo sapiens (human), BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456), BAFF receptor / pid=NP—443177.1—Homo sapiens, CD22 (B cell receptor CD22-B isotype, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number AK026467), CD79a (CD79A, CD79α, immunoglobulin-associated α), CXCR5 (Burkitt lymphoma receptor 1), HLA-DOB (MHC Class II molecules, β subunit), P2X5 (purinergic receptor P2X ligand-gated ion channel 5), CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP-001773.1), LY64 (lymphocyte antigen 64 (RP105)), FcRH1 (Fc receptor-like protein 1), IRTA2 (FcRH5, immunoglobulin superfamily receptor translocation-associated 2), TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR), MUC1 (tumor-associated MUC1 glycopeptide epitope).

[0090] In some embodiments, the antibody or its antigen-binding fragment is specific for aging surface markers, i.e. molecules or proteins specifically present on the surface of senescent cells.

[0091] According to the common understanding in the field of ADCs, a "connector" is a bifunctional or multifunctional organic molecule that allows the drug portion or "payload," such as the cytotoxic oligomer of the present invention, to be covalently conjugated ("linked") with an antibody or its antigen-binding fragment to form an antibody-drug conjugate. Thus, a connector can be defined as the interface between the target portion, i.e., the antibody or its antigen-binding fragment, and the drug portion, i.e., the cytotoxic oligomer. Connectors contain separate coupling functionalities, each for binding the antibody or its antigen-binding fragment and binding the drug portion, respectively. Therefore, connectors can be used to further modify and / or customize ADCs from the perspectives of efficient ADC construction as well as therapeutic efficacy, mechanism of action, and therapeutic window, for example, to address the overall hydrophobicity of the drug-connector and overcome the limiting constraints of restricted drug loading. Novel hydrophilic sources have been investigated in various connector frameworks and with different payloads, resulting in improved physicochemical properties, favorable pharmacokinetic characteristics, higher tolerability, avoidance of MDR-related resistance, and efficacy against heterogeneous tumors. For example,One possibility for increasing the hydrophilicity of the drug-connector is to introduce PEG (polyethylene glycol) and PEG units, respectively. Furthermore, the connector can be used to control the timing of payload release from the antibody carrier.

[0092] There are two types of connectors, namely the so-called "cleavable connectors" and "non-cleavable connectors".

[0093] A non-cleavable connector is stably connected to the drug moiety at one end and to the antibody at the other end. Therefore, the drug moiety is released from the antibody only during receptor-mediated endocytosis and lysosomal processing of the ADC. The non-cleavable connector specification 11 / 43 pages 15 CN 121620394 A is independent of (bio)chemical signals (also known as "triggers"). Once the antibody is degraded by lysosomal enzymes, the connector-payload conjugate is released into the intracellular environment and freely exerts its cytotoxic activity. When targeting hematologic malignancies, for example, ADCs based on non-cleavable connectors may be more useful in conferring greater specificity and lower toxicity.

[0094] The applied techniques provide those skilled in the art with a large number of non-cleavable connectors suitable for implementing the present invention. Suitable examples include, but are not limited to, non-cleavable linkers based on maleimide hexanoyl (MC) and 4-maleimide methylcyclohexane-1-carboxylate (MCC) and non-cleavable linkers containing PEG as well as acetylene and piperazine.

[0095] Cleavable linkers are cleaved in response to specific biochemical or chemical signals (“triggers”). For example, cleavable linkers are cleaved in response to changes in redox potential or pH or in the presence of enzymes such as hydrolases or oxidoreductases. Cleavable linkers can be used to control the release of drugs from ADCs.

[0096] Cleavable linkers can be classified into two subclasses: “chemically cleavable linkers” and “enzymatically cleavable linkers”.

[0097] Chemically cleavable linkers can be further classified into acid-cleavable or reducible linkers. Both have been clinically established through several FDA and EMA-approved ADCs based on acid-cleavable and reducible linkers.

[0098] Acid-cleavable linkers utilize the natural acidity of endosomes and lysosomes in the pH range of 4.5–6.2, in contrast to the neutral pH of plasma at 7.4. Approved examples of ADCs with acid-cleavable linkers are Mylotarg and Besponsa. The most commonly used acid-cleavable linker is hydrazone, which is reliably stable at neutral pH and unstable at pH 4.5. Other acid-labile groups include combinations of carbonate linkers with alcohol-containing payloads.

[0099] Reducible or disulfide linkers are the most prevalent class of chemically cleavable linkers. These linkers are chemically stable at physiological pH but are sensitive to nucleophilic attacks from thiols. In plasma, only human serum albumin (HSA) contains thiols, but their reactivity is limited due to reduced solvent exposure to these groups. In contrast, cytosols are rich in glutathione (GSH),It has exposed and highly reactive thiol groups. Its presence provides an opportunity to use disulfide linkers to release the payload intracellularly with high selectivity from ADCs. Furthermore, elevated GSH levels are often characteristic of tumor-associated oxidative stress, which can provide additional selectivity for this type of linker chemistry.

[0100] Enzyme-cleavable linkers use enzymes to release the payload. Enzymes utilized in the applied techniques for payload release are, for example, cathepsin B, a cysteine ​​protease that cleaves peptide bonds in dipeptide substrates containing valine (Val) and citrulline (Cit) and p-aminobenzyl alcohol (PAB). Valine-citrulline is the most commonly used dipeptide linker substrate in currently applied enzyme-dependent linker chemistry. Glycosidase-cleavable linkers are another example of enzyme-cleavable linkers commonly used in ADCs. Glucosidases are hydrolases that are usually confined to lysosomal compartments, but like cathepsin B, they can be secreted by tumor cells in necrotic tissue. Non-limiting examples are β-galactosidase cleavable linkers, and most commonly, β-glucuronidase cleavable linkers, which are based on the hydrolysis of β-D-glucuronic acid residues at lysosomal pH.

[0101] Thus, non-cleavable linkers are substantially stable outside the cell (i.e., extracellular). Cleavable linkers include predefined cleavage sites that can be cleaved by enzymatic activity, hydrolysis, or other metabolic conditions or triggers.

[0102] Linkers may also include spacer structures that spatially separate the cytotoxic oligomer from the antibody. The use of linkers is well known in the art, and those skilled in the art can readily select suitable linkers based on their knowledge and the present disclosure and teachings.

[0103] In some embodiments, the linker comprises a coupling functionality configured to covalently couple with any other modification of the cysteine ​​thiol, amine (e.g., N-terminus or amino acid side chain such as lysine), or antibody. The linker may be substituted with sulfonate substituents or other substituents that may increase the water solubility of the linker and promote the coupling reaction between the linker and the antibody. The coupling functionality can be configured to covalently couple with nucleophilic groups on the antibody, including but not limited to, N-terminal amino groups, side-chain amino groups (e.g., the side-chain amino group of lysine), side-chain thiols (e.g., the side-chain thiols of cysteine), and hydroxyl or amino groups of carbohydrates (in the case of the antibody described in the glycosylation specification, page 12 / 43, 16 CN 121620394 A). The amino, thiols, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups, such as linkers, to form covalent bonds. These electrophilic groups include, but are not limited to, active esters, such as N-hydroxysuccinimide (NHS) esters, hydroxybenzotriazole (HOBt) esters, haloformates and acid halides, alkyl and benzyl halides such as haloacetamides, aldehydes, ketones, carboxyl groups, and maleimide groups. Some antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. These can be addressed by treatment with a reducing agent such as DTT (dithiothreitol).The antibody is made reactive to conjugate with the linker. Additional nucleophilic groups can be introduced into the antibody via a reaction of lysine with 2-iminothiacyclopentane (Trout's reagent), resulting in the conversion of the amine to a thiol. Reactive thiol groups can be introduced into the antibody by introducing one, two, three, four, or more cysteine ​​residues, for example, by preparing a mutant antibody containing one or more non-natural cysteine ​​residues or by using a commercially available chemical kit.

[0104] In some embodiments, the linker is a cleavable linker. In a preferred embodiment, the linker is less resistant to cleavage under physiological conditions than the cross-linked entity of the pharmaceutical cytotoxin oligomer.

[0105] In some embodiments, the linker is configured to release the pharmaceutical cytotoxin oligomer from the antibody or its antigen-binding fragment in an intracellular environment, particularly during receptor-mediated endocytosis and / or lysosomal processing of the 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 redox potential, enzymes, and pH. Specifically, the linker may include a substrate for intracellular enzymatic cleavage, such as a hydrolase and / or an oxidoreductase substrate or cleavage site, or a substrate for chemically triggered cleavage, such as in response to a change in pH or a change in reduction potential.

[0106] In some embodiments, the linker is a non-cleavable linker. In this way, it can be further ensured that the payload of the ADC is not released prematurely from the extracellular environment, but only after intracellular uptake and lysosomal degradation of the antibody, thereby further improving the drug properties, particularly the therapeutic window of the ADC of the present invention.

[0107] In another aspect, the present invention provides a method for producing pharmaceutical cytotoxic oligomers as described above.

[0108] The method comprises the steps of: A) providing a first organic cytotoxic entity and at least a second cytotoxic entity, wherein the first organic cytotoxic entity and the second organic cytotoxic entity are each i) enzyme inhibitors and / or ii) cytotoxic to target cells after uptake via transport proteins of target cells, B) forming a covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity that is significantly resistant to cleavage under physiological conditions, thereby forming a cytotoxic oligomer.

[0109] In some embodiments, the first organic cytotoxic entity comprises a first coupling function, and the second organic cytotoxic monomer comprises a second coupling function, wherein the second coupling function is chemically complementary to the first coupling function, allowing the first and second coupling functions to form covalent bonds with each other. Additionally, step B) may include combining the first organic cytotoxic entity and the second organic cytotoxic entity under conditions that cause the first and second coupling functions to form covalent bonds with each other.

[0110] In some embodiments, step A) further includes providing an organic bridging portion,Step B) includes forming a covalent connection between the first organic cytotoxic entity and the second organic cytotoxic entity via a bridging portion. For example, forming a covalent connection may include forming a first portion by covalently attaching the bridging portion to the first organic cytotoxic entity and forming a second portion by covalently attaching the bridging portion to the second organic cytotoxic entity, thereby crosslinking the first organic cytotoxic entity and the second organic cytotoxic entity with each other via the bridging portion.

[0111] Therefore, the bridging portion is at least a bifunctional molecule, i.e., containing at least two coupling functionalities, also referred to as "functional groups", configured (i.e., capable of) forming covalent bonds with the first organic cytotoxic entity and the second organic cytotoxic entity. Naturally, the bridging portion may also be a multifunctional molecule having multiple coupling functionalities, configured, for example, to form covalent bonds with another entity, such as another third or further organic cytotoxic entity or connector. In some embodiments, the bridging portion is a bifunctional molecule with two coupling functionalities, configured to form covalent bonds with the first and second organocytotoxic entities. In a preferred embodiment, the bridging portion is a trifunctional molecule with three coupling functionalities, configured to form covalent bonds, for example, with the first, second, and third organocytotoxic entities or a connector. In other preferred embodiments, the bridging portion is a tetrafunctional molecule with three coupling functionalities, configured to form covalent bonds, for example, with the first, second, third, and fourth organocytotoxic entities or a connector.

[0112] In some embodiments, the first organic cytotoxic entity comprises a first coupling function, the second organic cytotoxic entity comprises a second coupling function, and the organic bridging portion comprises at least a first complementary coupling function and a second complementary coupling function, wherein the first complementary coupling function is chemically reactive with the first coupling function, allowing the first coupling function and the first complementary coupling function to form a covalent bond with each other, and the second complementary coupling function is chemically reactive with the second coupling function, allowing the second coupling function and the second complementary coupling function to form a covalent bond with each other. Additionally, step B) may include combining the first organic cytotoxic entity, the second organic cytotoxic entity, and the organic bridging portion under conditions that cause the first coupling function and the first complementary coupling function to form a covalent bond with each other, and preferably simultaneously cause the second coupling function and the second complementary coupling function to form a covalent bond with each other.

[0113] In some embodiments, the first coupling function is the same functional group as the second coupling function.Therefore, the first complementary coupling function is the same functional group as the second complementary coupling function. In other embodiments, the first coupling function and the second coupling function are different from each other, and therefore the first complementary coupling function and the second complementary coupling function are different from each other.

[0114] In an embodiment where B) includes forming a covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity through a bridging portion, it is preferred that the first coupling function and the second coupling function are the same or chemically non-complementary to each other, also known as having orthogonal chemical reactivity, thereby preventing the first organic cytotoxic entity and the second organic cytotoxic entity from directly forming covalent bonds with each other. Similarly, in such an embodiment, the first complementary coupling function and the second complementary coupling function are the same or chemically non-complementary to each other, thereby preventing the bridging portion from forming covalent bonds with each other.

[0115] As noted above, the bridging portion may also include a third or further complementary coupling functionality, which may be the same as or different from the first and second coupling functionalities, and has complementary chemical reactivity with the third or further coupling functionality of the third or further organic cytotoxic entity and / or connector. Further possible variations will be apparent to those skilled in the art based on the disclosure and teachings above.

[0116] In some embodiments, the bridging portion includes at least one orthogonal coupling functionality that has non-complementary chemical reactivity with the coupling functionality of the first and second organic cytotoxic entities, such as the first and second coupling functionalities, thereby preventing the first and second organic cytotoxic entities from forming covalent bonds with the bridging portion through the orthogonal coupling functionality. In this way, the orthogonal coupling functionality remains available on the bridging portion after the formation of the cytotoxic oligomer in step B), and can therefore be used, for example, to couple the cytotoxic oligomer to another molecule, such as a connector and / or antibody. As used herein, orthogonal coupling functionality can also be a functional group protected as described above in the release-protected specification (page 14 / 43, CN 121620394 A).

[0117] Preferably, the first coupling functionality and the second coupling functionality, or as the case may be, the first coupling functionality and the first complementary coupling functionality, as well as the second coupling functionality and the second complementary coupling functionality, and if present, the third or further coupling functionality and the third or further complementary coupling functionality, are mutually reactive pairs in any of the click chemistry reactions described above, for example, an azide or tetrazine as the first coupling functionality and an alkyne or alkene as the second coupling functionality; an azide or tetrazine as the first coupling functionality and the second coupling functionality and an alkyne or alkene as the first complementary coupling functionality and the second complementary coupling functionality, or, for example, the reverse. However, the first coupling functionality and the second coupling functionality, or as the case may be,The first coupling functionality and the first complementary coupling functionality, as well as the second coupling functionality and the second complementary coupling functionality, and, if present, the third or further coupling functionality and the third or further complementary coupling functionality, may of course be selected, for example, from any other reactive functional groups described above, such as amino and carboxyl, aldehyde or ketone groups. Orthogonal coupling functionality is preferably selected from amino, carboxyl, hydroxyl, hydroxyamino, isothiocyanate, aldehyde, ketone and thiol groups, optionally protected with a releasable protecting group.

[0118] The coupling functionality of each organic cytotoxic entity, such as the first coupling functionality and the second coupling functionality, may be the functional group of an amino acid naturally present in the corresponding first and / or second organic cytotoxic entity, such as amino, carboxyl, hydroxyl, aldehyde, ketone or thiol group. However, in a preferred embodiment, the coupling functionality of the first organic cytotoxic entity is a functional group not present in the natural counterpart of the first organic cytotoxic entity, and / or the coupling functionality of the second organic cytotoxic entity is a functional group not present in the natural counterpart of the second organic cytotoxic entity. In embodiments where the first and / or second organic cytotoxic entities are microcystins or arthrophyllins, the coupling functionality or functional group is preferably introduced into the cytotoxic entity biosynthetically or semi-synthetically, for example, as described in WO 2018 / 219619 A1, the disclosure of which is incorporated herein by reference in its entirety to more fully describe the technical background to which the invention relates. Preferably, the coupling functionality of the first organic cytotoxic entity and / or the second organic cytotoxic entity is configured as a bioorthogonal conjugation, i.e., a chemical conjugation reaction that occurs within the living system without interfering with natural biochemical processes. In some embodiments, the coupling functionality of the first organic cytotoxic entity and / or the second organic cytotoxic entity is selected from azide, alkynyl, alkenyl, phosphine, phosphonate, tetrazine, hydrazine, hydroxylamine, isothiocyanate, and any combination thereof.

[0119] In some embodiments, the first organic cytotoxic entity and / or the second organic cytotoxic entity has 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 has at least 90% or 95% sequence identity with the sequences selected from SEQ ID NO:2 to SEQ ID NO:15, NOD and [D-PrgMeAsp]1-NOD or SEQ ID NO:17.

[0120] In another aspect, the present invention provides a method for producing antibody-drug conjugates.

[0121] The method comprises the steps of: a) providing a pharmaceutical cytotoxic oligomer as defined above or a pharmaceutically acceptable salt thereof, b) providing an antibody or an antigen-binding fragment thereof,c) Covalently linking the pharmaceutical cytotoxin oligomer to an antibody or its antigen-binding fragment.

[0122] In some embodiments, method step a) includes performing a method for producing a pharmaceutical cytotoxin oligomer as defined above.

[0123] In some embodiments, method step c) includes the following sub-steps: c1) providing a connector, c2) covalently linking the pharmaceutical cytotoxin oligomer to the connector, and c3) covalently linking the connector to an antibody or its antigen-binding fragment. It should be understood that the order of the sub-steps is not necessarily predetermined by numbering. For example, it is possible to perform sub-step c2) before or after sub-step c3). However, it is preferred that sub-step c2) is performed before sub-step c3), i.e., the pharmaceutical cytotoxin oligomer is first covalently linked to the connector, and then the cytotoxin oligomer-connector conjugate is covalently linked to the antibody via the connector.

[0124] Suitable laboratory protocols for covalently conjugating pharmaceutical cytotoxin oligomers to antibodies or adapters, respectively, and for covalently conjugating adapters to antibodies, are established in the art and readily available to those skilled in the art. In this regard, reference is also made to the description above and the specific embodiments described below.

[0125] In some embodiments, the covalent linking of the adapter and / or pharmaceutical cytotoxin oligomer to the adapter is configured to release the pharmaceutical cytotoxin oligomer from the antibody or its antigen-binding fragment during receptor-mediated endocytosis and / or lysosomal processing of the antibody-drug conjugate. Specifically, the adapter may include a cleavage site configured to cleave in response to a chemical or biochemical trigger, as described above.

[0126] In some embodiments, at least one of the first organic cytotoxin entity, the second organic cytotoxin entity, and the crosslinked entity of the pharmaceutical cytotoxin oligomer comprises a first coupling functionality, and the adapter comprises a second coupling functionality having a complementary chemical reactivity to the first coupling functionality for forming covalent bonds with each other. Additionally, substep c2) may include combining the pharmaceutical cytotoxic oligomer and the linker under conditions that cause the first and second coupling functionalities to form covalent bonds with each other.

[0127] In some embodiments, the present invention provides a pharmaceutical cytotoxic oligomer as defined above or a pharmaceutically acceptable salt thereof or an antibody-drug conjugate as defined above, for use as a medicine, more particularly for treating malignant diseases or age-related pathogenesis.

[0128] In some embodiments, the present invention provides a method for treating a subject, particularly a human subject, a malignant disease, comprising administering to the subject a pharmaceutically effective amount or a therapeutically effective dosing regimen of a pharmaceutical cytotoxic oligomer as defined above or a pharmaceutically acceptable salt thereof or an antibody-drug conjugate as defined above.

[0129] In some embodiments,The malignant disease referred to is cancer. Examples of cancers treated in this article include, but are not limited to, carcinoma, lymphoma, germ cell tumor, sarcoma, and leukemia or lymphoma. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer, gastrointestinal stromal tumor (GIST)), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic cancer, anal cancer, penile cancer, and head and neck cancer. For example, cancer may be characterized by overexpression of the HER2 or ErbB receptor.

[0130] The pharmaceutically effective amount may be, for example, 0.01-100 mg / kg body weight (mg / kg), more particularly 0.1-20 mg / kg or 1-10 mg / kg. A therapeutically effective dosing regimen may, for example, comprise a single dose or multiple doses of 0.01-100 mg / kg, more particularly 0.1-20 mg / kg or 1-10 mg / kg.

[0131] It should be understood that the various embodiments described for one aspect of the invention are also applicable to other aspects of the invention. Therefore, the features and functions disclosed above and below in conjunction with cytotoxic oligomers can also relate to antibody-drug conjugates and their corresponding uses and methods of manufacture.And vice versa.

[0132] 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; Specification 16 / 43 pages 20 CN 121620394 A Figure 4: Structural formula of the microcystin homotrimer according to the present invention; Figure 5: Scheme for semi-synthetic production 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 production of [D-PrgMeAsp]1-NOD monomers; Figure 7: Structural formula of the arthrophyllin homodimer according to the present invention; Figure 8: Structural formula of the arthrophyllin 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 of Figure 2 conjugated with a cleavable linker maleimide hexanoyl-Val-Ala-(p-amino-benzyl)carbamate configured as an antibody; Figure 12: Bar graph showing the results of evaluating general cytotoxicity (A) and protein phosphatase inhibition (B) of cell lines expressing OATP1B1 and OATP1B3, with reference to the microcystin homodimers of Figures 1 and 2, their corresponding monomers, and the natural microcystin MC-LR. Detailed Description

[0133] The invention will now be explained in more detail based on exemplary embodiments with reference to the accompanying drawings. The examples and drawings should not be considered limiting.

[0134] Example 1: Synthesis of Monomeric Organic Cytotoxic Entities The following examples include several microcystins, arthrophyllins, and amatoxins as representative classes of organic cytotoxic entities that exert their cytotoxic activity by actively transporting to target cells via target cell transport proteins and then inhibiting intracellular enzymes of the target cells (Table 1).

[0135] Unless otherwise described in the following examples, the microcystin (MC) monomers are obtained via precursor-guided biosynthesis as described in WO 2018 / 219619 A1. In short, cyanobacterial strains that produce natural microcystins, such as PCC 7820 (Pasteur Culture Collection), are cultured under conditions that allow for strain growth. During culture,The inorganic culture medium is supplemented with, for example, one of the amino acids propargyltyrosine (PrgTyr), propargyllysine (PrgLys), azidophenylalanine (AzPhe), azidolysine (AzLys), azidovaline (AzNva), Nε-tert-butoxycarbonyl-ornithine (Orn (Boc)), or D-azidoalanine (D-AzAla). Each of these amino acids contains a coupling function configured to covalently cross-link with another coupling function having complementary chemical reactivity. The supplemented amino acids are incorporated into the microcystin during strain culture, resulting in the biosynthesis of non-naturally occurring microcystin derivatives having functional groups ("coupling functions") configured to covalently bind at positions Aa1, X2, Z4, or Aa7 ("coupling sites"). Incorporation of a coupling function at position Aa3 is also generally possible. After culture, biomass is harvested and extracted, and the microcystin derivatives are purified by HPLC. Purified microcystin derivatives are used as organic cytotoxin entities to produce cytotoxin oligomers according to the invention. In the case of microcystin derivatives containing Orn(Boc), the Boc group is cleaved in trifluoroacetic acid (TFA) / H2O / acetonitrile (can) (1:1:1, v / v / v) and stirred for 90 minutes to expose the free amino functionality, followed by evaporation and separation by RP-HPLC. Suitable microcystins can also be synthesized by total chemical synthesis (e.g., Zemskov et al., J Org Chem 2017, 82, 3680–91).

[0136] Furthermore, semi-synthetic modified variants of microcystins, arthrophyllins, and amatoxins can be generated from naturally occurring cytotoxin monomers by chemical incorporation configured to covalently crosslink with another coupling functionality having complementary chemical reactivity. Specification 17 / 43 pages 21 CN 121620394 A

[0137] For example, a semi-synthetic modified variant of the naturally occurring microcystin monomer MC-LR is obtained by Michael addition of an amine, thiol, or other Michael donor to a Mdha residue (Michael acceptor) at position Aa7. Thus, additional coupling functionalities (e.g., amines, alkynes, or azides) are chemically incorporated, allowing selective reactions with complementary functionalities, as described in Example 11 below.

[0138] In addition, a semi-synthetic modified variant of the natural microcystin monomer can be obtained by chemically modifying the carboxyl group at position Aa3, according to the modification described below for the carboxyl group at position Aa1 of the nodosum toxin monomer.

[0139] The nodosum toxin (NOD) monomer included in the examples is a naturally occurring nodosum toxin monomer, which is obtained by culturing a cyanobacterial strain that produces nodosum toxin, such as PCC 73104 (as described elsewhere).The phycotoxins were subsequently obtained from the biomass by, for example, extraction and separation by HPLC. Suitable phycotoxin monomers can also be obtained through precursor-guided biosynthesis, as described in WO 2018 / 219619 A1. For example, during cultivation, the inorganic medium is supplemented with, for example, Nω-nitro-L-arginine (Narg) or Orn(Boc). In this way, the two obtained NOD-derived compounds can be converted to [Orn]2-NOD by reducing the nitro group of Narg or by deprotecting the Boc of Orn(Boc) to obtain a primary amino group (as described above and in WO 2018 / 219619 A1).

[0140] As described above for microcystins, phycotoxins can also be semi-synthetically modified by Michael addition at position Aa5 with a Michael donor and an Mdhb / Dhb group (Michael acceptor) to incorporate additional coupling functionality. Thus, an alkyne coupling group is obtained at position Aa5, as described in Example 37 below.

[0141] Additionally, semi-synthetic modified variants of the amatoxin monomer having amino or alkyne coupling functionality at position Aa1 are obtained by forming an amide bond between the carboxyl and amino groups at position Aa1, as described in Examples 34 and 35 below.

[0142] The amatoxin (AMA) monomers included in the examples are naturally occurring α-amatoxin and its derivative amatoxinamide lacking 6-OH at Trp2. Amatoxins are obtained from wild fruiting bodies of Amanita species (e.g., death amanita) collected from natural habitats or from cultured mycelia of Amanita species (e.g., death amanita) as described by Zhang et al. (FEMS Microbiol Lett 2005, 252(2), 223-08), and subsequently from biomass by extraction and separation of amatoxins, for example, by HPLC. The α-amanitin monomer possesses hydroxyl groups at positions Aa1 (Dhil = (2S,3R,4R)-4,5-dihydroxy-isoleucine), Aa2 (Htp = 6-hydroxytryptophan), and Aa8 (Hyp = trans-4-hydroxyproline) as coupling functional groups. However, the Hyp residues have a major influence on the biological activity and corresponding cytotoxicity of amanitin.Therefore, it is not considered a suitable linker site (Matinkhoo et al., Chem. Eur. J. 2021, 27, 10282-92). Suitable amatoxins can also be synthesized by total chemical synthesis (e.g., Siegert et al., Angew Chem 2020, 59, 5500-04; Lutz et al., Angew Chem 2020, 59, 11390-93).

[0143] Semi-synthetic modified variants of α-amatoxin monomers having an amino coupling group at position Aa2 and an alkyne coupling group at position Aa1 were obtained as described in Examples 46 and 47 below.

[0144] Table 1: Microcystin (MC), Nodoxin (NOD), and Amatoxin (AMA) used as monomeric cytotoxic entities in the examples. PrgTyr = propargyltyrosine, PrgLys = propargyllysine, AzPhe = azidophenylalanine, AzLys = azidolysine, AzNva = azidovaline, Orn = ornithine, D-AzAla = D-azidoalanine, AzProMDap = 3-N-azidopropyl-2-N-methyl-2,3-diaminopropionic acid, D-EdaMeAsp = D-β-ethylidene-1,2-diamine-β-methylaspartic acid, D-PrgMeAsp = D-erythrose-β-propargylamine-β-methylaspartic acid, AeHtp = 6-(2-aminoethoxy)tryptophan, PrgDhil = 4,5-[(oxycarbonyl)-propargylamine]-dihydroxyisoleucine, PrgSMMeCys =S-propargyl-N-methyl-β-methylcysteine. Specification 18 / 43 pages 22 CN 121620394 A Specification 19 / 43 pages 23 CN 121620394 A

[0145] Example 2: Synthesis of homologous microcystin dimers by crosslinking MC-PrgTyr-Arg monomers with bifunctional bridging to produce homologous dimers derived from the MC-PrgTyr-Arg monomers (SEQ ID NO:2) described in two Examples 1 as the first and second organic cytotoxic entities. The MC-PrgTyr-Arg monomers have an alkyne group at position X2 as coupling functionality.

[0146] The MC-PrgTyr-Arg monomer is dimerized using a bridging molecule of structural formula BM-I to produce a cross-linked entity, which forms a covalent link between a first organic cytotoxic entity and a second organic cytotoxic entity as described on page 20 / 43 of the specification, CN 121620394 A. For this purpose,The bridging molecule BM-I carries two terminal azide groups as complementary coupling functions: (BM-I).

[0147] The crosslinking between the alkyne coupling function at position X2 of the two MC-PrgTyr-Arg monomers and the two terminal azide groups of BM-I as complementary coupling functions is achieved by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In short, CuAAC is carried out using a reaction mixture of 2 eq. of monomer / dimethyl sulfoxide (DMSO), 1 eq. of BM-I, 10 eq. of 100 mM CuSO4 aqueous solution and 20 eq. of 200 mM tris((1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl)amine (THPTA) aqueous solution. The reaction is initiated by adding 10 eq. of 100 mM sodium ascorbate aqueous solution. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC. The structural formula DM-I of the homomicrocystin dimer is shown in Figure 1.

[0148] Example 3: Synthesis of homomicrocystin dimers by crosslinking MC-PrgLys-Arg monomers with bifunctional bridging links to produce homodimers derived from the MC-PrgLys-Arg monomers (SEQ ID NO:3) described in two Examples 1 as the first and second organic cytotoxic entities. The MC-PrgLys-Arg monomers have an alkyne group at position X2 as coupling functionality. The MC-PrgLys-Arg monomers were dimerized using a bridging molecule of structural formula BM-I in conjunction with the CuAAC scheme described in Example 2 above to produce crosslinked entities that form a covalent link between the first and second organic cytotoxic entities. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0149] Example 4: Synthesis of homologous microcystin dimers by crosslinking MC-AzPhe-Arg monomers with bifunctional bridging portions to produce homologous dimers derived from the MC-AzPhe-Arg monomers (SEQ ID NO:4) described in two Examples 1 as the first and second organic cytotoxic entities. The MC-AzPhe-Arg monomers have an azide group at position X2 as coupling functionality. The MC-AzPhe-Arg monomers are dimerized using a bridging molecule of structural formula BM-II to produce crosslinked entities that form a covalent link between the first and second organic cytotoxic entities. For this purpose,The bridging molecule BM-II carries two terminal alkyne groups as complementary coupling functionality: (BM-II).

[0150] The crosslinking between the azide coupling functionality at position X2 of the two MC-AzPhe-Arg monomers and the two terminal alkyne groups of BM-II as complementary coupling functionality was accomplished by CuAAC as described in Example 2 above. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0151] Example 5: Synthesis of homologous microcystin dimers by crosslinking MC-AzLys-Arg monomers with bifunctional bridging portions to produce homologous dimers derived from the two MC-AzLys-Arg monomers (SEQ ID NO:5) described in Example 1 as the first organic cytotoxic entity and the second organic cytotoxic entity. The MC-AzLys-Arg monomers have an azide group at position X2 as coupling functionality. The MC-AzLys-Arg monomer was dimerized using a bridging molecule of structural formula BM-II in accordance with the CuAAC scheme described in Example 2 of the specification (page 21 / 43, CN 121620394 A) to produce a cross-linked entity, which forms a covalent link between the first and second organic cytotoxic entities. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0152] Example 6: Heterocystic microcystin dimers were synthesized by using monomers with bifunctional bridging portions to cross-link MC-AzNva-Arg and MC-AzPhe-Arg to produce heterodimers 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 in Example 1 as the second organic cytotoxic entity. Both monomers have an azide group at position X2 as coupling functionality. MC-AzNva-Arg monomers (1 eq.) and MC-AzPhe-Arg (1 eq.) were dimerized using a bridging molecule of structural formula BM-II in conjunction with the CuAAC scheme described in Example 2 above to produce cross-linked entities.It forms a covalent link between the first and second organic cytotoxic entities. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0153] Example 7: Synthesis of homologous microcystin dimers by crosslinking MC-Leu-PrgTyr monomers with bifunctional bridging portions to produce homologous dimers derived from the first and second organic cytotoxic entities derived from the MC-Leu-PrgTyr monomers described in two Examples 1 (SEQ ID NO:7). The MC-Leu-PrgTyr monomer has an alkyne group at position Z4 as a coupling function. The MC-Leu-PrgTyr monomer is dimerized using a bridging molecule of structural formula BM-I in conjunction with the CuAAC scheme described in Example 2 above to produce a crosslinked entity that forms a covalent link between the first and second organic cytotoxic entities. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0154] Example 8: Synthesis of homologous microcystin dimers by crosslinking MC-Leu-AzLys monomers with bifunctional bridging links to produce homologous dimers derived from the MC-Leu-AzLys monomers (SEQ ID NO: 8) described in two Examples 1 as the first and second organic cytotoxic entities. The MC-Leu-AzLys monomers have an azide group at position Z4 as coupling functionality. The MC-Leu-AzLys monomers are dimerized using a bridging molecule of formula BM-II in conjunction with the CuAAC scheme described in Example 2 above to produce crosslinked entities that form a covalent link between the first and second organic cytotoxic entities. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0155] Example 9: Synthesis of homologous microcystin dimers by crosslinking MC-Leu-Orn monomers with bifunctional bridging portions to produce homologous dimers derived from the MC-Leu-Orn monomers (SEQ ID NO: 9) described in two Examples 1 as the first and second organic cytotoxic entities. The MC-Leu-Orn monomers have an amino group at position Z4 as coupling functionality. The MC-Leu-Orn monomers are dimerized using a bridging molecule of the structural formula BM-III to produce crosslinked entities that form a covalent link between the first and second organic cytotoxic entities. For this purpose, the bridging molecule BM-III carries two terminal carboxyl groups as complementary coupling functionality: (BM-III).

[0156] The crosslinking between the amino coupling functionality at position Z4 of the two MC-Leu-Orn monomers and the two terminal carboxyl groups of BM-III as complementary coupling functionality is accomplished by forming an amide bond. In short, the bridging molecule BM-III (1 eq.,In DMF, the mixture was pre-incubated for 30 minutes with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (HATU) (1.95 eq.) and N,N-diisopropylethylamine (DIPEA; 4 eq.). Afterward, an amino-containing monomer (2 eq., see specification 22 / 43 pages 26 CN 121620394 A) was added to the solution, and the mixture was stirred overnight at room temperature. The reaction product was separated by semi-preparative C-18 RP-HPLC and lyophilized.

[0157] Example 10: A heterodimer derived from the MC-Leu-PrgLys monomer (SEQ ID NO: 10) described in Example 1 as the first organic cytotoxic entity and the MC-Leu-PrgTyr monomer (SEQ ID NO: 7) described in Example 1 as the second organic cytotoxic entity was synthesized by crosslinking MC-Leu-PrgLys and MC-Leu-PrgTyr monomer (SEQ ID NO: 7) with bifunctional bridging portions. Both monomers have an alkyne group at position Z4 as coupling functionality. The MC-Leu-PrgLys monomer (1 eq.) and the MC-Leu-PrgTyr monomer (1 eq.) were dimerized using a bridging molecule of formula BM-I in conjunction with the CuAAC scheme described in Example 2 above to produce a crosslinked entity that forms a covalent link between the first and second organic cytotoxic entities. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0158] Example 11: Heteromeric microcystin dimers were synthesized by crosslinking monomers of [D-AzAla]1MC-WR and [AzproMDap]7MC-LR with bifunctional bridging. Heteromeric dimers derived from the monomers [D-AzAla]1MC-WR (SEQ ID NO: 11) as the first organic cytotoxic entity and [AzproMDap]7MC-LR (SEQ ID NO: 12) as the second organic cytotoxic entity, as described in Example 1, were produced.

[0159] The monomer [D-AzAla]1MC-WR was obtained by precursor-guided biosynthesis as described in Example 1. The monomer [AzproMDap]7MC-LR was semi-synthetically prepared from the natural microcystin MC-LR precursor (SEQ ID NO: 16) by aza-Michael addition. For this purpose,MC-LR (1 eq.) was dissolved in DMSO and added to a 1% (w / v) aqueous solution of K2CO3 containing 3-azido-1-propylamine (40 eq.). The reaction was stirred overnight at 40°C. The obtained [AzproMDap]7MC-LR was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0160] The [D-AzAla]1MC-WR monomer has an azide as a coupling function at position Aa1. The [AzproMDap]7MC-LR monomer has an azide as a coupling function at position Aa7. The monomers (1 eq. each) were dimerized using a bridging molecule of the formula BM-II with the CuAAC scheme described in Example 2 above to produce a cross-linked entity that forms a covalent link between the first and second organic cytotoxic entities. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0161] Examples 12-21: Synthesis of Homo- and Hetero-microcystin Dimers of Examples 2-11 by Crosslinking Monomers of Examples 2-11 with Trifunctional Bridging Molecules Having Additional Antibody-Conjugation Functionality In Examples 12-21, the homo- and hetero-microcystins of Examples 2-11 were regenerated using different bridging molecules to produce crosslinked entities that form a covalent link between a first organic cytotoxin entity and a second organic cytotoxin entity. In Examples 12-21, each bridging molecule carries two coupling functionalities that are chemically reactive to the coupling functionalities of the microcystin monomers described in Examples 2-11, and an additional third coupling functionality, configured to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0162] For this purpose, bridging molecule BM-IV carries two azides as complementary coupling functions for crosslinking microcystin monomers containing alkyne coupling functionality, and a terminal amino group as a third coupling function for linking the dimer to an antibody or its antigen-binding fragment: (BM-IV).

[0163] Bridging molecule BM-V carries two alkynes as complementary coupling functions for crosslinking microcystin monomers containing azide coupling functionality (see page 23 / 43 of the specification, CN 121620394 A), and a terminal amino group as a third coupling function for linking the dimer to an antibody or its antigen-binding fragment: (BM-V).

[0164] Bridging molecule BM-VI carries two carboxyl groups as complementary coupling functions for crosslinking microcystin monomers containing an amino group as a coupling function, and a terminal Boc-protected amino group as a third coupling function. Following the dimerization reaction, the Boc group is cleaved in TFA / H2O / ACN (1:1:1, v / v / v).Exposing free amino groups is used to link the dimer to an antibody or its antigen-binding fragment: (BM-VI).

[0165] Table 2 provides an overview of the dimers produced in Examples 12-21.

[0166] Table 2: Microcystin dimers produced in Examples 12-21. PrgTyr = propargyltyrosine, PrgLys = propargyllysine, AzPhe = azidophenylalanine, AzLys = azidolysine, Aznva = azidovaline, Orn = ornithine, D-AzAla = D-azidoalanine, AzProMDap = 3-N-azidopropyl-2-N-methyl-2,3-diaminopropionic acid.

[0167] Dimerization using bridging molecules of structural formula BM-IV or structural formula BM-V was achieved via the CuAAC scheme described in Example 2, wherein 2 eq. of microcystin monomers were used for homodimers, and 1 eq. of each microcystin monomer was used for heterodimers. Dimerization using bridging molecules of structural formula BM-VI was achieved via the amide bond formation scheme described in Example 9.

[0168] The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC. Specification 24 / 43 pages 28 CN 121620394 A

[0169] As an illustrative embodiment, Figure 2 shows the structural formula DM-II of the homocystin dimer of Example 12. Two triazole rings are formed between the propargyl coupling functionality of the MC-PrgTyr-Arg monomer and the complementary azide coupling functionality of the bridging molecule BM-IV, which covalently crosslink two microcystin entities at position X2 through two opposing branches of the bridging portion. The third branch of the bridging portion carries a terminal amino group that is orthogonally reactive with the other coupling functions, thus remaining usable after dimer formation for subsequent linking of the dimer to an antibody or its antigen-binding fragment.

[0170] Example 22: Synthesis of heteromicrocystin dimers by direct crosslinking of monomers MC-PrgTyr-Arg and MC-Tyr-AzNva to produce heterodimers derived from the MC-PrgTyr-Arg monomer (SEQ ID NO:2) described in Example 1 as the first organic cytotoxic entity and the MC-Tyr-AzNva monomer (SEQ ID NO:13) described in Example 1 as the second organic cytotoxic entity.

[0171] The monomer MC-PrgTyr-Arg has alkyne coupling functionality at position X2, while the monomer MC-Tyr-AzNva has an azide group as coupling functionality at position Z4. This allows the two monomers to dimerize by direct crosslinking using CuAAC. For this purpose, equimolar amounts of each monomer (1 eq.,CuAAC was performed in DMSO, 100 mM CuSO4 solution (5 eq.), and 200 mM THPTA solution (10 eq.). The reaction was initiated by adding 100 mM sodium ascorbate solution (5 eq.). The reaction product was separated by semi-preparative C-18 RP-HPLC and lyophilized.

[0172] Example 23: Synthesis of heterodimers of microcystin by direct crosslinking of monomers MC-AzLys-Arg and MC-Leu-PrgLys to produce heterodimers derived 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.

[0173] The monomer MC-AzLys-Arg has an azide group at position X2 as a coupling function, while the monomer MC-Leu-PrgLys has a propyne group at position Z4 as a coupling function. This allows the two monomers to dimerize by direct crosslinking using the CuAAC scheme described in Example 22 above. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0174] As an illustrative embodiment, Figure 3 shows the structural formula DM-III of the heteromicrocystin dimer of Example 23. Direct CuAAC between the azide coupling function of the MC-AzLys-Arg monomer and the complementary alkyne coupling function of the MC-Leu-PrgLys monomer forms a crosslinked entity composed of a triazole ring, which covalently crosslinks the two microcystin entities through the amino acid side chains at positions X2 and Z4. The carboxyl group in the side chain at position Aa3 of D-MeAsp can be used to link the dimer to an antibody or its antigen-binding fragment.

[0175] Example 24: Synthesis of heterodimers of microcystin by direct crosslinking of monomers MC-AzLys-Arg and MC-PrgLys-Arg. Heterodimers derived from the MC-AzLys-Arg monomer (SEQ ID NO:5) described in Example 1 as the first organic cytotoxic entity and the MC-PrgLys-Arg monomer (SEQ ID NO:3) described in Example 1 as the second organic cytotoxic entity were produced.

[0176] The monomer MC-AzLys-Arg has an azide group at position X2 as a coupling function, while the monomer MC-PrgLys-Arg has a propargyl group at position X2 as a coupling function.This allows the two monomers to dimerize directly by crosslinking using the CuAAC scheme described in Example 22 above. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0177] Example 25: Synthesis of heterodimers of microcystin by directly crosslinking monomers MC-Leu-AzLys and MC-Leu-PrgLys to produce heterodimers derived from the MC-Leu-AzLys monomer (SEQ ID NO:8) described in Example 1 as the first organic cytotoxic entity and the MC-Leu-PrgLys monomer (SEQ ID NO:10) described in Example 1 as the second organic cytotoxic entity.

[0178] The monomer MC-Leu-AzLys has an azide group at position Z4 as a coupling function, while the monomer MC-Leu-PrgLys has a propargyl group at position Z4 as a coupling function. This allows the two monomers to dimerize directly by crosslinking using the CuAAC scheme described in Example 21 above. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0179] Example 26: Synthesis of homologous microcystin trimers by using monomers of MC-AzLys-Arg partially crosslinked with trifunctional bridging monomers to generate homologous trimers 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 has an azide group at position X2 as a coupling function.

[0180] The MC-AzLys-Arg monomers are trimerized using a bridging molecule of the structural formula BM-VII to produce cross-linked entities that form covalent links between the first, second, and third organocytotoxin entities. For this purpose, the bridging molecule BM-VII carries three terminal alkyne groups as complementary coupling functions: (BM-VII).

[0181] Cross-linking between the azide coupling function at position X2 of the three MC-AzLys-Arg monomers and the three terminal alkyne groups of BM-VII as complementary coupling functions is achieved via CuAAC. For trimer formation,CuAAC was performed using 3 eq. of monomer / DMSO, 1 eq. of bridging molecule, 100 mM CuSO4 solution (15 eq.), and 200 mM THPTA (30 eq.). The reaction was initiated by adding 100 mM sodium ascorbate solution (15 eq.). The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0182] Example 27: Synthesis of homologous microcystin trimers by crosslinking MC-PrgTyr-Arg monomers with trifunctional bridging portions to produce homologous trimers derived from the MC-PrgTyr-Arg monomers (SEQ ID NO:2) described in three Examples 1 as the first, second, and third organic cytotoxic entities. The MC-PrgTyr-Arg monomers have an alkyne group at position X2 as coupling functionality.

[0183] The MC-PrgTyr-Arg monomers were trimerized using a bridging molecule of the formula BM-VIII to produce cross-linked entities that form covalent links between the first, second, and third organocytotoxin entities. For this purpose, the bridging molecule BM-VIII carries three terminal azide groups as complementary coupling functions: (BM-VIII).

[0184] The cross-linking between the alkyne coupling function at position X2 of the three MC-PrgTyr-Arg monomers and the three terminal azide groups of BM-VIII as complementary coupling functions was achieved by CuAAC according to the scheme described in Example 26 above. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0185] Example 28: Synthesis of homologous microcystin by crosslinking MC-Leu-AzLys monomers with trifunctional bridging portions. (Instructions for Use, Pages 26 / 43, 30 CN 121620394 A) Trimers were generated from the MC-Leu-AzLys monomers (SEQ ID NO: 8) described in three Examples 1 as the first, second, and third organic cytotoxin entities. The MC-Leu-AzLys monomers have an azide group at position Z4 as a coupling function.

[0186] The MC-Leu-AzLys monomers were trimerized using a bridging molecule of structural formula BM-VII in conjunction with the CuAAC scheme described in Example 26 above to generate crosslinked entities.It forms a covalent link between the first, second, and third organocytotoxin entities. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0187] Example 29: Synthesis of homologous microcystin trimers by crosslinking MC-Leu-PrgLys monomers with trifunctional bridging portions to produce homologous trimers derived from the first, second, and third organocytotoxin entities from the three MC-Leu-PrgLys monomers (SEQ ID NO: 10) described in Example 1. The MC-Leu-PrgLys monomers have an alkyne group at position Z4 as a coupling functionality.

[0188] The MC-Leu-PrgLys monomers were trimerized using a bridging molecule of structural formula BM-VIII in conjunction with the CuAAC scheme described in Example 26 above to produce crosslinked entities that form a covalent link between the first, second, and third organocytotoxin entities. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0189] Examples 30-33: Synthesis of Homologous Microcystin Trimers by Crosslinking Monomers of Examples 26-29 with Tetrafunctional Bridging Portions Having Additional Antibody-Conjugation Functionality In Examples 30-33, the homologous trimers of Examples 26-29 were regenerated using different bridging molecules to produce crosslinked entities that form covalent links between the first, second, and third organic cytotoxic entities. In Examples 30-33, each bridging molecule carries three coupling functionalities that are chemically reactive to the coupling functionalities of the microcystin monomers described in Examples 26-29, and an additional third coupling functionality, configured to subsequently link the trimer to an antibody or its antigen-binding fragment.

[0190] For this purpose, the bridging molecule BM-IX has three branches, each carrying a terminal azido group as a complementary coupling function for crosslinking microcystin monomers containing alkyne coupling functionality, and a fourth branch carrying a terminal amino group as a fourth coupling function for linking the cytotoxin trimer to an antibody or its antigen-binding fragment: (BM-IX).

[0191] The bridging molecule BM-X has three branches, each carrying a terminal azido group as a complementary coupling function for crosslinking microcystin monomers containing alkyne as coupling functionality, and a fourth branch carrying a terminal amino group as a fourth coupling function for linking the cytotoxin trimer to an antibody or its antigen-binding fragment: (BM-X). Specification 27 / 43 pages 31 CN 121620394 A

[0192] Table 3 provides an overview of the dimers produced in Examples 12-21.

[0193] Table 3: Microcystin trimers produced in Examples 30-33. PrgTyr = propargyltyrosine, PrgLys = propargyllysine.AzLys = azidolysine.

[0194] Trimerization using a bridging molecule of formula BM-IX or formula BM-X was achieved via the CuAAC scheme described in Example 26 above. The reaction product was separated and lyophilized by a semi-preparative C-18 RP-HPLC.

[0195] As an illustrative embodiment, Figure 4 shows the structural formula TM-I of the homologous microcystin trimer of Example 30. CuAAC between the azido coupling functionality of the MC-AzLys-Arg monomer and the complementary propargyl coupling functionality of the bridging molecule BM-IX results in three triazole rings, which covalently crosslink all three microcystin entities at position X2 via a branched bridging portion. The fourth branch of the bridging portion carries a terminal amino group that is orthogonally reactive with the other coupling functionalities, thereby remaining usable after trimer formation for subsequent linking of the dimer to an antibody or its antigen-binding fragment.

[0196] Example 34: Synthesis of homologous arthrotoxicin dimers by crosslinking [D-EdaMeAsp]1-NOD monomers with bifunctional bridging. Homologous dimers derived from two [D-EdaMeAsp]1-NOD monomers as the first and second organic cytotoxic entities were produced. The [D-EdaMeAsp]1-NOD monomer was semi-synthetically produced at position Aa1 of the naturally occurring arthrotoxicin using the carboxyl group of D-MeAsp as the coupling functionality.

[0197] For this purpose, natural arthrotoxicin (biosynthetically produced as described in Example 1) was dissolved in DMF and stirred at 0°C. Hydroxybenzotriazole (HOBt) (1.2 eq.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.2 eq.) were added. After 30 minutes, N-Boc-ethylene-1,2-diamine (Boc-Eda) (1.2 eq.) and DIPEA (2 eq.) were added to the mixture. The reaction was allowed to proceed overnight. The reaction resulted in a mixture of products containing [D-Boc-EdaMeAsp]1-NOD and [D-Boc-EdaGlu]4-NOD with protected terminal amines at positions Aa1 or Aa4, or [D-Boc-EdaMeAsp]1,[D-Boc-EdaGlu]4-NOD with protected terminal amines at both of the stated positions, as shown in Figure 5A. The desired product [D-Boc-EdaMeAsp]1-NOD was separated and lyophilized using a semi-preparative C-18 RP-HPLC. As described in Example 19, the Boc-protecting group was cleaved to yield the [D-EdaMeAsp]1-NOD monomer. After separation, [D-EdaMeAsp]1-NOD was lyophilized.

[0198] Alternatively, the [D-EdaMeAsp]1-NOD monomer is produced semi-synthetically, as shown in FIG5C. For this purpose,Naturally occurring phycocyanin was dissolved in 0.5 M hydrochloric acid (HCl) / methanol (MeOH) and stirred at room temperature for approximately 5 hours to give [D-Glu(OMe)]4-NOD, since position 4 is more readily methylated than position 1. The product was separated by semi-preparative C-18 RP-HPLC and lyophilized. [D-Glu(OMe)]4-NOD was then dissolved in DMF and stirred at 0°C. HOBt (1.2 eq.) and EDC (1.2 eq.) were added. After 30 minutes, Boc-Eda (1.2 eq.) and DIPEA (2 eq.) were added to the mixture. The reaction was maintained overnight at 25°C. The reaction yielded the product [D-Boc-EdaMeAsp1-D-Glu(OMe)4]-NOD, which has an amino group protected at position Aa1 and a methyl ester at position Aa4. Following HPLC purification, the methyl ester was hydrolyzed using 10 eq. of a 50 mM NaOH methanol solution in CH2Cl2 (the final CH2Cl2 / MeOH ratio was approximately 9 / 1 (v / v)), as described by Theodorou et al. (Tetrahedron Lett. 2007, 48, 8230–8233). The solution was stirred at 4°C for several days, then neutralized by adding 10 eq. of 50 mM HCl and evaporated. Subsequently, the Boc- protecting group was cleaved, as described in Example 19. The reaction product [D-EdaMeAsp]1-NOD monomer was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0199] The [D-EdaMeAsp]1-NOD monomer is dimerized using the bridging molecule BM-III to crosslink by forming an amide bond between the amino coupling functionality at position Aa1 of the two [D-EdaMeAsp]1-NOD monomers and the two terminal carboxyl groups of BM-III as described in Example 9, which serve as complementary coupling functionality.

[0200] Alternatively, a similar homodimer is generated as a first organic cytotoxic entity and a second organic cytotoxic entity derived from an activated [D-MeAsp-NHS]1-NOD carboxylic acid intermediate.

[0201] The [D-MeAsp-NHS]1-NOD active ester is generated semi-synthetically. For this purpose,Naturally occurring arthrophyllin (described in Example 1) was dissolved in DMF and stirred at 0°C. N-hydroxysuccinimide (NHS) (1.2 eq.) and EDC (1.2 eq.) were added. The reaction was kept at room temperature overnight. The reaction resulted in a mixture of products containing [D-MeAsp-NHS]1-NOD and [D-Glu-NHS]4-NOD with NHS-activated carboxylic acids at position Aa1 or position Aa4, or [D-MeAsp-NHS]1, [D-Glu-NHS]4-NOD with NHS-activated carboxylic acids at both said positions. The desired product [D-MeAsp-NHS]1-NOD was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0202] The active ester of [D-MeAsp-NHS]1-NOD was dimerized using a bridging molecule of structural formula BM-XI to produce a cross-linked entity that forms a covalent link between the first and second organic cytotoxic entities. For this purpose, the bridging molecule BM-XI carries two terminal amino groups as complementary coupling functions: (BM-XI).

[0203] The crosslinking between the NHS-activated carboxyl coupling function at position Aa1 of the two NOD monomers and the two terminal amino groups of BM-XI as complementary coupling functions is accomplished by amide bond formation. In short, [D-MeAsp-NHS]1-NOD (2.2 eq.) and BM-XI were dissolved in DMF / sodium phosphate buffer (0.1 mM, pH 8) (1:1, v / v) and stirred overnight at 25°C. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0204] Example 35: Synthesis of homologous arthrotoxicin dimers by using monomers of [D-PrgMeAsp]1-NOD with bifunctional bridging to generate homologous dimers derived from two [D-PrgMeAsp]1-NOD monomers as the first and second organic cytotoxic entities.

[0205] The [D-PrgMeAsp]1-NOD monomers were semi-synthetically generated, as shown in FIG5B. For this purpose, naturally occurring arthrotoxicin was dissolved in DMF and stirred at 0°C. HOBt (1.2 eq.) and EDC (1.2 eq.) were added. After 30 minutes,Proprynilamine (1.2 eq.) and DIPEA (2 eq.) were added to the mixture. The reaction was maintained overnight at 25°C. The reaction yielded a mixture of products containing [D-PrgMeAsp]1-NOD with an alkynyl group (propynyl) at position Aa1 and [D-PrgGlu]4-NOD with an alkynyl group (propynyl) at position Aa4.

[0206] The desired product [D-PrgMeAsp]1-NOD was then separated by a semi-preparative C-18 RP-HPLC. Figure 6 shows a representative RP-HPLC chromatogram of the product mixture, where peak 1 corresponds to unmodified NOD, peak 2 corresponds to the desired product [D-PrgMeAsp]1-NOD, the extracted ion chromatogram mass is m / z = 862.5 (as shown in the inset), and peak 3 corresponds to the unwanted byproduct [D-PrgGlu]4-NOD. After separation, [D-PrgAsp]1-NOD was lyophilized.

[0207] Alternatively, the [D-PrgMeAsp]1-NOD monomer was semi-synthetically produced, as shown in Figure 5D. The naturally occurring arthrophyllin was methylated with 0.5 M HCl / MeOH as described in Example 34 above to give [D-Glu(OMe)]4-NOD. The separated product was dissolved in DMF and stirred at 0°C. HOBt (1.2 eq.) and EDC (1.2 eq.) were added. After 30 minutes, propylamine (1.2 eq.) and DIPEA (2 eq.) were added to the mixture. The reaction was maintained overnight at 25°C. The reaction yielded a product [D-PrgMeAsp1-D-Glu(OMe)4]-NOD, having an alkyne group (propynyl) at position Aa1 and a methyl ester at position Aa4. After HPLC purification, the methyl ester was hydrolyzed as described in Example 34 above, and the reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0208] The [D-PrgMeAsp]1-NOD monomer was dimerized using a bridging molecule of structural formula BM-I in conjunction with the CuAAC scheme described in Example 2 to achieve crosslinking between the alkyne coupling functionality at position Aa1 of the two [D-PrgMeAsp]1-NOD monomers and the two terminal azide groups of BM-I as complementary coupling functionality, thereby producing a crosslinked entity.It forms a covalent link between the first and second organic cytotoxic entities of the cytotoxic dimer. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0209] Example 36: Synthesis of homologous phycotoxicity dimers by crosslinking [Orn]2-NOD monomers with bifunctional bridging links to produce homologous dimers derived from the two [Orn]2-NOD monomers described in Example 1 as the first and second organic cytotoxic entities. The [Orn]2-NOD monomers have an amino group at position A2 as a coupling function. The [Orn]2-NOD monomers are dimerized using the bridging molecule BM-III to crosslink by forming an amide bond between the amino coupling function at position Aa2 of the two [Orn]2-NOD monomers and the two terminal carboxyl groups of BM-III as the complementary coupling function.

[0210] Example 37: Synthesis of Homologous Cytotoxin Dimers from Natural NOD Precursor by Bifunctional Bridging of [PrgSMMeCys]5-NOD Monomers The monomer [PrgSMMeCys]5-NOD (SEQ ID NO:17) was semi-synthetically prepared from a natural NOD precursor via thia-Michael addition. NOD (1 eq.) was dissolved in DMSO and added to a 5% (w / v) aqueous solution of K2CO3 containing propargyl thiol (100 eq.). The reaction was stirred overnight at 40°C. The obtained [PrgSMMeCys]5-NOD was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0211] Homodimers derived from two [PrgSMMeCys]5-NOD monomers as a first and a second organic cytotoxin entity were produced. The [PrgSMMeCys]5-NOD monomer has an alkyne group at position Aa5 as a coupling function. The [PrgSMMeCys]5-NOD monomer is dimerized using a bridging molecule of structural formula BM-I in conjunction with the CuAAC scheme described in Example 2 above to produce a cross-linked entity, which forms a covalent link between the first and second organic cytotoxic entities. The reaction product is separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0212] Example 38: Synthesis of heterodimers of [D-PrgMeAsp]1-NOD and [D-EdaMeAsp]1-NOD by using monomers with bifunctional bridging portions to produce heterodimers derived from [D-PrgMeAsp]1-NOD monomer as the first organic cytotoxic entity and [D-EdaMeAsp]1-NOD monomer as the second organic cytotoxic entity.

[0213] The [D-EdaMeAsp]1-NOD monomer and the [D-PrgMeAsp]1-NOD monomer are produced semi-synthetically.As described in Examples 34 and 35 above.

[0214] The monomers [D-EdaMeAsp]1-NOD and [D-PrgMeAsp]1-NOD are dimerized using a bifunctional bridging molecule of the structural formula BM-XII to produce a cross-linked entity that forms a covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity. For this purpose, the bifunctional bridging molecule BM-XII carries a terminal carboxyl group for forming an amino conjugation with [D-EdaMeAsp]1-NOD via an amide bond and a terminal azide group for CuAAC with the alkynyl group (propynyl) of [D-PrgMeAsp]1-NOD: Specification 30 / 43 pages 34 CN 121620394 A (BM-XII).

[0215] First, in addition to using 1 eq. BM-XII, 0.95 eq. HATU, 2 eq. DIPEA, and 1 eq. [D-EdaMeAsp]1-NOD, using the scheme described in Example 9, the D-EdaMeAsp]1-NOD monomer is conjugated by an amide bond formed between the amino group at position Aa1 and the terminal carboxyl group of the bridging molecule BM-XII, thereby forming the first covalently linked part of the crosslinked entity. The intermediate product was separated and lyophilized by C18 RP-HPLC.

[0216] Subsequently, except for using equimolar amounts of monomer and intermediate product, dimerization was completed by conjugating the [D-PrgMeAsp]1-NOD monomer with the intermediate product via CuAAC, to achieve crosslinking between the alkyne coupling functionality at position Aa1 of the [D-PrgMeAsp]1-NOD monomer and the terminal azide group of the bridging molecule BM-XII of the intermediate product as the complementary coupling functionality.

[0217] In this manner, the crosslinked entity was completed by forming a second covalently linked portion, which crosslinks the two phycocyanin entities to each other through the crosslinked entity. The dimer was separated and lyophilized by C18 RP-HPLC.

[0218] Examples 39-43: Synthesis of Homologous and Heterologous Arthropodium Dimers of Examples 34-38 by Crosslinking with Trifunctional Bridging Molecules Having Additional Antibody-Conjugation Functionality In Examples 39-43, the homologous and heterologous arthropodium dimers of Examples 34-38 were regenerated using different bridging molecules to produce crosslinked entities that form a covalent link between a first organic cytotoxin entity and a second organic cytotoxin entity. In Examples 39-43, each bridging molecule carries two coupling functions that are chemically reactive to the coupling functions of the arthropodium monomers described in Examples 34-38, and an additional third coupling function, configured to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0219] For this purpose,The bridging molecule BM-VI described in Example 19 carries two carboxyl groups as complementary coupling functions for crosslinking [D-EdaMeAsp]1-NOD monomers or [Orn]2-NOD monomers, each containing amine coupling functions, and a terminal protected amino group. After dimerization, the protecting group is cleaved, as described in Example 19, exposing the free amino group for linking the dimer to an antibody or its antigen-binding fragment.

[0220] In an alternative synthetic route, the bridging molecule BM-XIII is used, which carries two free amino groups and a Boc-protected amino group. The two amino groups serve as first and second complementary coupling functions for crosslinking [D-MeAsp-NHS]1-NOD monomers by activating the carboxyl group as a coupling function. After dimerization, the Boc group is cleaved, as described in Example 19, exposing the free amino group as a third coupling function for linking the dimer to an antibody or its antigen-binding fragment: (BM-XIII).

[0221] The bridging molecule BM-IV described in Example 12 carries two azido groups as complementary coupling functions for crosslinking [D-PrgMeAsp]1-NOD monomers or [PrgSMMeCys]5-NOD monomers, each containing alkyne coupling functionality, and a terminal amino group as a third coupling function for linking the dimer to an antibody or its antigen-binding fragment.

[0222] In addition, the bridging molecule BM-XIV carries one azido group as a complementary coupling function for crosslinking [D-PrgMeAsp]1-NOD monomers containing alkyne coupling functionality, and a terminal carboxyl group for coupling with the amino group of the [D-EdaMeAsp]1-NOD monomer. Furthermore, BM-XIV carries a terminal Boc-protected amino group as a third coupling function. After dimerization, the Boc group is cleaved, as described in Example 19.Exposing free amino groups as a third coupling function is used to link the heterodimer to the antibody or its antigen-binding fragment: (BM-XIV).

[0223] Table 4 provides an overview of the dimers produced in Examples 39-43.

[0224] Table 4: Nodular toxin (NOD) dimers produced in Examples 39-43. D-PrgMeAsp = D-erythrose-β-propargylamine-β-methylaspartic acid.

[0225] Dimerization using bridging molecules of structural formula BM-VI or BM-XIII was achieved by the scheme described in the corresponding Example 34 for bridging molecules of structural formula BM-III or BM-XI. Dimerization using bridging molecules of structural formula BM-IV was achieved by the CuAAC scheme described in Example 2 and the corresponding Example 35. Dimerization using bridging molecules of structural formula BM-XIV was achieved by the scheme described in the corresponding Example 38 for bridging molecules of structural formula BM-XII.

[0226] The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0227] As an illustrative embodiment, Figure 7 shows the structural formula DM-IV of the homologous arthrotoxic toxin dimer of Example 39. As described above, a conjugation reaction is achieved between the amino group of [D-EdaMeAsp]1-NOD and the complementary carboxyl group of the bridging molecule BM-VI, or between the activated carboxyl group coupling functionality of the [D-MeAsp-NHS]1-NOD monomer and the complementary amino group coupling functionality of the bridging molecule BM-XIII. In both cases, two amide bonds are formed, which covalently crosslink the two arthrotoxic toxin entities at position Aa1 through two opposing branches of the bridging portion. Furthermore, the bridging molecule contains additional amino substituents that remain after dimer formation and can be used to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0228] Example 44: Synthesis of homologous phycotoxin trimers by crosslinking [D-PrgMeAsp]1-NOD monomers with trifunctional bridging portions to produce homologous trimers derived from the [D-PrgMeAsp]1-NOD monomers described in the three Examples 34 as the first, second, and third organocytotoxin entities. The [D-PrgMeAsp]1-NOD monomers have an alkyne group at position Aa1 as coupling functionality.

[0229] The [D-PrgMeAsp]1-NOD monomers were trimerized using a bridging molecule of structural formula BM-VIII in conjunction with the CuAAC scheme described in Example 26 above to produce crosslinked entities.It forms a covalent link between the first, second, and third organic cytotoxic entities. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0230] Example 45: Synthesis of homologous phycotoxin trimers by crosslinking [D-PrgMeAsp]1-NOD monomers with additional antibody-coupling functionality. Homologous phycotoxin trimers derived from the first, second, and third organic cytotoxic entities are produced by crosslinking the monomers of [D-PrgMeAsp]1-NOD described in the three examples 35. The [D-PrgMeAsp]1-NOD monomers have an alkyne group at position Aa1 as a coupling function.

[0231] The [D-PrgMeAsp]1-NOD monomer was trimerized using a bridging molecule of the formula BM-X in conjunction with the CuAAC scheme described in Example 26 above to produce cross-linked entities, which are covalently linked between the first, second, and third organocytoxin entities through three branches of the bridging molecule BM-X. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0232] As an illustrative embodiment, Figure 8 shows the structural formula TM-II of the homologous cystotoxin trimer of Example 45. The CuAAC between the alkyne coupling functionality of the [D-PrgMeAsp]1-NOD monomer and the complementary azide coupling functionality of the bridging molecule BM-X results in three triazole rings, which covalently crosslink all three microcystin entities at position Aa1 through the bridging portion of the branched chain. The fourth branch of the bridging portion carries a terminal amino group that is orthogonally reactive with other coupling functionalities, thereby remaining usable after trimer formation for subsequent linking of the dimer to an antibody or its antigen-binding fragment.

[0233] Example 46: Synthesis of homoamanita toxin dimers by crosslinking [AeHtp]2-AMA monomers with bifunctional bridging portions to produce homodimers derived from two [AeHtp]2-AMA monomers (SEQ ID NO: 14) as the first and second organic cytotoxic entities.

[0234] The [AeHtp]2-AMA monomer was semi-synthetically produced at position Aa2 of the naturally occurring AMA described in Example 1 using a hydroxyindole group as the coupling functionality.

[0235] The [AeHtp]2-AMA monomer was produced in two steps. First, using the etherification scheme for amanitain derivatization described by Faulstich et al. (Biochemistry 1981, 20, 6498-504), the hydroxyindole group at position Aa2 is etherified with the alkyl halide 2-(Boc-amino)ethyl bromide (Boc-Ae-Br) or any other protected aminoalkyl halide. In short,Natural AMA was dissolved in anhydrous ethanol. A freshly prepared sodium acetate solution (1.1 eq.) was added, followed by direct evaporation of the solution. The remaining solid and Boc-Ae-Br (4 eq.) were dissolved in DMF and stirred overnight at 20°C. The reaction resulted in a mixture of monoalkylated [Boc-AeHtp]2-AMA, dialkylated [(Boc-Ae)2Htp]2-AMA, and unmodified natural AMA. The desired product [Boc-AeHtp]2-AMA was separated by semi-preparative C-18 RP-HPLC and lyophilized. In a second step, the Boc group was cleaved as described in Example 19, yielding the [AeHtp]2-AMA monomer.

[0236] The crosslinking between the amino coupling functionality at position Aa2 of the two [AeHtp]2-AMA-1 monomers and the two terminal carboxyl groups of BM-III as described in Example 9, which serve as complementary coupling functionality, is accomplished by amide bond formation.

[0237] Example 47: Synthesis of homoamanita toxin dimers by using monomers with bifunctional bridging crosslinking of [PrgDhil]1-AMA. Homodimers derived from two [PrgDhil]1-AMA monomers (SEQ ID NO: 15) as the first and second organic cytotoxic entities.

[0238] The [PrgDhil]1-AMA monomer was semi-synthetically produced at position Aa1 of naturally occurring AMA using a primary hydroxyl group as the coupling functionality. First, natural AMA was dissolved in anhydrous DMF and stirred at 0°C under nitrogen. Then, bis(p-nitrophenyl(bis-PNP)) carbonate (1.2 eq.) and DIPEA (1.2 eq.) were added, and the reaction mixture was stirred at 0°C for 2–24 h. After evaporation of DMF, the residue was ground in diethyl ether, and the precipitate was separated by filtration to obtain the AMA-PNP intermediate. Then, propargylamine (2 eq.) was dissolved in DMF, and AMA-PNP (1 eq.) and DIPEA (2 eq.) were added to the solution and stirred at 20°C for 2–24 h. The reaction product [PrgDhil]1-AMA was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0239] Alternatively, [PrgDhil]1-AMA can be produced by reacting 3-propan-1-isocyanate with the primary hydroxyl group at position Aa1 of naturally occurring AMA. (See page 33 / 43 of the specification, CN 121620394 A group) In short, dissolve anhydrous AMA (1 eq.) in anhydrous DMF. Then,2 eq. of propyne-1-isocyanate and 2 eq. of dibutyltin dilaurate were added and stirred for several days. The reaction product [PrgDhil]1-AMA was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0240] The [PrgDhil]1-AMA monomer was dimerized using a bridging molecule of structural formula BM-I in conjunction with the CuAAC scheme described in Example 2 to produce a cross-linked entity, which forms a covalent link between the first and second organic cytotoxic entities. The reaction product was separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0241] Example 48: A heterodimer derived from [AeHtp]2-AMA (SEQ ID NO:15) as the first organic cytotoxic entity and [PrgDhil]1-AMA as the second organic cytotoxic entity was synthesized by crosslinking monomers of [AeHtp]2-AMA and [PrgDhil]1-AMA with bifunctional bridging portions.

[0242] The [AeHtp]2-AMA monomer and the [PrgDhil]1-AMA monomer were produced semi-synthetically as described in Examples 46 and 47 above.

[0243] The monomers of [AeHtp]2-AMA and [PrgDhil]1-AMA were dimerized using a bifunctional bridging molecule of structure BM-XII to produce a crosslinked entity that forms a covalent link between the first organic cytotoxic entity and the second organic cytotoxic entity. For this purpose, the bifunctional bridging molecule BM-XII carries a terminal carboxyl group for forming an amino conjugation with [AeHtp]2-AMA via an amide bond, and a terminal azide group for CuAAC with the alkyne group (propyne) of [PrgDhil]1-AMA using the scheme described in Example 38.

[0244] Examples 49-51: Synthesis of homologous and heterologous amatoxin dimers by crosslinking the monomers of Examples 46-48 with a trifunctional bridging portion having additional antibody-coupling functionality In Examples 49-51, the homologous and heterologous amatoxin dimers of Examples 46-48 were regenerated using different bridging molecules to produce crosslinked entities that form a covalent link between a first organic cytotoxic entity and a second organic cytotoxic entity. In Examples 49-51, each bridging molecule carries two coupling functionalities that are chemically reactive with the coupling functionalities of the amatoxin monomers described in Examples 46-48, and an additional third coupling functionality.The configuration is such that the dimer is subsequently linked to an antibody or its antigen-binding fragment.

[0245] Table 5: The dimerization of the amatoxin (AMA) dimer produced in Examples 49-51 using a bridging molecule of structural formula BM-VI was achieved by the scheme described for the bridging molecule of structural formula BM-III in corresponding Example 46. The dimerization using a bridging molecule of structural formula BM-IV was achieved by the CuAAC scheme described in Example 2 and corresponding Example 47. The dimerization using a bridging molecule of structural formula BM-XIV was achieved by the scheme described for the bridging molecule of structural formula BM-XII in corresponding Example 48.

[0246] The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0247] As an illustrative embodiment, Figure 9 shows the structural formula DM-V of the homologous amatoxin dimer of Example 49. The conjugation reaction between the amino coupling functionality of the [AeHtp]2-AMA monomer and the complementary carboxyl coupling functionality of the bridging molecule BM-VI forms two amide bonds, which covalently crosslink two Amanita toxin entities at position Aa2 via two opposing branches of the bridging portion. After deprotection of the dimerized Boc, the bridging molecule contains additional amino substituents that can be used to subsequently link the dimer to an antibody or its antigen-binding fragment.

[0248] Example 52: Synthesis of homoamanita toxin trimers by crosslinking [AeHtp]2-AMA monomers with trifunctional bridging portions to produce homoamanita toxin trimers derived from the [AeHtp]2-AMA monomers (SEQ ID NO:14) described in three Examples 46 as the first, second, and third organic cytotoxic entities. The [AeHtp]2-AMA monomer has an amino group at position Aa2 as coupling functionality.

[0249] The [AeHtp]2-AMA monomer was trimerized using a bridging molecule with the structural formula BM-XV: (BM-XV).

[0250] The trimerization using the bridging molecule with the structural formula BM-XV was carried out using the scheme described in Example 47 above, with 3 eq. of the [AeHtp]2-AMA monomer, 1 eq. of BM-XV, 2.95 eq. of HATU, and 6 eq. of DIPEA, achieved by forming an amide bond between the amino group of the [AeHtp]2-AMA monomer and the carboxyl group of the bridging molecule, to produce a cross-linked entity.It forms a covalent link between the first, second, and third organic cytotoxic entities.

[0251] The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0252] Example 53: Synthesis of homoamanita toxin trimers by crosslinking [PrgDhil]1-AMA monomers with trifunctional bridging portions to produce homotrimers derived from three [PrgDhil]1-AMA monomers (SEQ ID NO:15) as the first, second, and third organic cytotoxic entities. The [PrgDhil]1-AMA monomers have an alkyne group at position Aa1 as coupling functionality.

[0253] The [PrgDhil]1-AMA monomers were trimerized using a bridging molecule of formula BM-VIII as described in Example 27 above. The reaction products were separated and lyophilized by semi-preparative C-18 RP-HPLC.

[0254] Examples 54-55: Synthesis of homologous amatoxin trimers by crosslinking monomers of Examples 52-53 with a trifunctional bridging portion having additional antibody-coupled functionality. In Examples 54 and 55, the homologous amatoxin trimers of Examples 52 and 53 were regenerated using different bridging molecules to produce crosslinked entities that form covalent links between the first, second, and third organic cytotoxic entities. In Example 54, the bridging molecule of the structural formula BM-XVI was used: Specification 35 / 43 pages 39 CN 121620394 A (BM-XVI).

[0255] Thus, the bridging molecule BM-XVI carries three carboxyl groups as coupling functions that are chemically reactive with the amino coupling functions of the amatoxin monomers described in Example 52, and a Boc-protected amino group as an additional fourth coupling function, configured to subsequently link the cytotoxic trimer to an antibody or its antigen-binding fragment.

[0256] Table 6: The trimerization of the amatoxin (AMA) trimer produced in Examples 54 and 55 using the bridging molecule of structural formula BM-XVI was achieved by forming an amide bond between the amino group of the [AeHtp]2-AMA monomer and the carboxyl group of the bridging molecule using the scheme described in Example 52, to produce a cross-linked entity that forms a covalent link between the first, second, and third organocytotoxic entities. After the dimerization reaction, the Boc group was cleaved, as described in Example 19. The reaction product was separated and lyophilized by a semi-preparative C-18 RP-HPLC.

[0257] The trimerization using the bridging molecule of structural formula BM-X was achieved by CuAAC using the scheme described for the bridging molecule of structural formula BM-X in the corresponding Example 26.

[0258] As an illustrative embodiment,Figure 10 shows the structural formula TM-III of the homoamanita toxin trimer of Example 54. Three amide bonds are formed by a conjugation reaction between the terminal amino group of the [AeHtp]2-AMA monomer and the complementary carboxyl group of the bridging portion BM-XVI, which covalently crosslink the three amanita toxin entities at position Aa2 through different branches of the bridging portion. Furthermore, the bridging molecule contains additional Boc-protected amino substituents, which remain after trimer formation and subsequent Boc deprotection and can be used to link the trimer to an antibody or its antigen-binding fragment.

[0259] Example 56: Synthesis of an ADC consisting of a monoclonal anti-HER2 antibody and the organic cytotoxin dimer of Example 12 as the drug component. In constructing the ADC, the organic cytotoxin dimer synthesized in Example 12 was used as the drug component. The organic cytotoxin dimer is a microcystin homodimer composed of two MC-PrgTyr-Arg cytotoxin entities symmetrically crosslinked at position X2 through two branches of the bridging portion BM-IV. The bridging portion further includes free amino-coupled functionality for linking the microcystin homodimer to the antibody. Figure 2 shows the corresponding structural formula DM-II.

[0260] The monoclonal antibody (mAb) trastuzumab (trademark “Herceptin”) targets human epidermal growth factor receptor 2 (Her2, also known as ERBB2, erb-b2 receptor tyrosine kinase 2). For mAb production, genes encoding the heavy and light chain variable regions of trastuzumab were synthesized and cloned into human heavy and light chain vectors encoding human IgG1. The sequences of the constructs were verified by DNA sequencing. The vectors were transiently transfected and expressed in CHO cells. The mAb was purified from cell culture supernatant (e.g., using protein A) to a purity exceeding 90%. The purified mAb was quantified by absorbance at 280 nm and characterized by SEC and SDS-PAGE. It should be understood that the present invention, page 36 / 43, CN 121620394 A, may be modified using genes encoding the heavy and light chain variable regions of other monoclonal antibodies to produce ADCs with other target specificities without departing from the teachings of the present invention.

[0261] The microcystin homodimer obtained according to Example 12 was conjugated to the cleavable linker maleimide hexanoyl-Val-Ala-(p-amino-benzyl)-(p-nitro-phenyl)-carbonate (mc-Val-Ala-PAB-PNP) by incubating 1 eq. of microcystin dimer and 1 eq. of linker in DMF and DIPEA (2 eq.) at 25°C for 1-3 hours. In this manner, the microcystin dimer is coupled to the linker by forming a carbamate bond between the activated carbonate on the linker and the free amino group of the bridging portion of the microcystin dimer.mc-Val-Ala-PABC-DII (PABC = p-amino-benzylcarbamate), hereinafter referred to as "DM-II-L", was obtained. The obtained linker-microcystin dimer conjugate was separated 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.

[0262] Subsequently, the obtained linker-homodimeric conjugate was conjugated with the monoclonal anti-HER2 antibody trastuzumab (trademark: Herceptin, a representative benchmark antibody of the applied technology) using the complementary coupling functionality of the monoclonal antibody and the linker of the linker-homodimeric conjugate to form an ADC. For this purpose, native (unmodified) mAb was dissolved in 5% tris(hydroxymethyl)aminomethane (TRIS) buffer supplemented with 25 mM EDTA to pH 8.5 to a final concentration of 26.5 mg / mL. For mAb reduction, 1 eq. of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP, 10 mM) was added for a final DAR of approximately 2, or 2 eq. for a final DAR of approximately 4. The reaction was incubated at room temperature for 1.5 hours, followed by dilution to 5 mg / mL with 2 mM EDTA / PBS (pH 7.4).

[0263] Buffer exchange and desalting were performed by gel filtration (Zeba™ spin) with PBS containing 2 mM EDTA (for DAR 2) and additionally supplemented with 3% cyclodextrin (CD) (for DAR 4) to reduce antibody aggregation. For the conjugation of the linker-microcystin dimer conjugate with the reduced mAb, for DAR approx. 2, 4 eq. of DM-II-L obtained from a 10 mM DM-II-L solution was added to the reduced mAb (5 mg / mL, PBS containing 2 mM EDTA), and the reaction mixture was incubated at room temperature for 2 h. For DAR approx. 4, 8 eq. of DM-II-L was used and added to the reduced mAb (5 mg / mL, 3% CD, PBS containing 2 mM EDTA), and the mixture was incubated at room temperature for 3.5 h. For better solubility, a final concentration of 5% DMA was used for DAR 2, and 10% DMA (v / v) was used for DAR 4. The conjugation reaction was quenched for 30 min by adding an equimolar amount of N-acetyl-L-cysteine ​​(NAC) relative to the toxin-linker conjugate used. The reaction mixture was then desalted by gel filtration (G-25, PBS (DAR 2) or in PBS with 3% CD (DAR 4)). Afterwards,Add an equal amount of activated carbon (100 mg / mL in PBS or 3% CD PBS, pH 7.4) to the weight of the mAb. After 1 hour, remove the activated carbon by centrifugation (4000 g, 15 min), analyze the supernatant by SEC and HIC, and filter aseptically.

[0264] Example 57: Synthesis of an ADC consisting of a monoclonal anti-HER2 antibody and the organic cytotoxin dimer of Example 18 as the drug moiety. In constructing the ADC, the organic cytotoxin dimer synthesized in Example 18 was used as the drug moiety. The organic cytotoxin dimer is a microcystin homodimer composed of two MC-Leu-AzLys entities symmetrically cross-linked at position Z4 by two branches of the bridging portion BM-V. The bridging portion further includes free amino coupling functionality for linking the microcystin homodimer to the antibody.

[0265] First, the microcystin homodimer obtained according to Example 18 was conjugated with a cleavable linker, such as mc-Val-Ala-PAB-PNP, via the free terminal amino group of the bridging portion BM-V as described in Example 56 above. Other suitable linkers and conjugation chemistry are known to those skilled in the art.

[0266] Subsequently, the resulting linker-homodimer conjugate was coupled with the monoclonal anti-HER2 antibody trastuzumab as described in Example 56 above.

[0267] Example 58: Synthesis of an ADC consisting of a monoclonal anti-HER2 antibody and the organic cytotoxic dimer of Example 40 as the drug part. In constructing the ADC, the organic cytotoxic dimer synthesized in Example 40 was used as the drug part. The organic cytotoxin dimer is a homodimer of Arachnium argentis toxin composed of two [D-PrgMeAsp]1-NOD entities symmetrically cross-linked at position Aa1 via two branches of the bridging portion BM-IV. The bridging portion further includes free amino-coupling functionality for linking the Arachnium argentis toxin homodimer to an antibody.

[0268] First, the Arachnium argentis toxin homodimer obtained according to Example 40 is conjugated to a cleavable linker, such as mc-Val-Ala-PAB-PNP, via the free terminal amino of the bridging portion BM-IV as described in Example 56 above. Other suitable linkers and conjugation chemistry are known to those skilled in the art.

[0269] Subsequently, the resulting linker-homodimer conjugate is conjugated to the monoclonal anti-HER2 antibody trastuzumab as described in Example 56 above.

[0270] Example 59: Synthesis of an ADC consisting of a monoclonal anti-HER2 antibody and the organic cytotoxin dimer of Example 49 as the drug component. In constructing the ADC,The organic cytotoxin dimer synthesized in Example 49 was used as the pharmaceutical part. The organic cytotoxin dimer is an amatoxin homodimer composed of two [AeHtp]2-AMA cytotoxin entities symmetrically cross-linked at position Aa2 by two branches of the bridging portion BM-VI. The bridging portion further includes free amino-coupled functionality for linking the microcystin homodimer to an antibody (see Figure 9).

[0271] First, the amatoxin homodimer obtained according to Example 49 was conjugated with a cleavable linker such as mc-Val-Ala-PAB-PNP by the free terminal amino group of the bridging portion BM-VI as described in Example 57. Other suitable linkers and conjugation chemistry are known to those skilled in the art.

[0272] Subsequently, using the complementary coupling functionality of the monoclonal antibody and the adapter of the adapter-homodimeric conjugate as described in Example 56 above, the resulting adapter-homodimeric conjugate was conjugated with the monoclonal anti-HER2 antibody trastuzumab to form an ADC.

[0273] Example 60: Protocol for cell culture and subsequent cytotoxicity assay of cancer cell lines expressing OATP HEK293 cells stably transfected with expression vectors pcDNA3.1(+)-OATPB1 and pcDNA3.1 / Hygro(-)-OATP1B3, and empty vectors pcDNA3.1(+) and pcDNA3.1 / Hygro(-) as controls, was provided by Prof. Dr. Jörg König (Friedrich-Alexander-University Erlangen-Nürnberg, Germany).

[0274] All cell lines were maintained in minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, a mixture of non-essential amino acids, and 2 mM glutamine at 37°C and 5% CO2.

[0275] HEK293 OATP1B1+ and the corresponding control cell lines were selected consistently with 800 µg / mL hygromycin (G418), while 250 µg / mL hygromycin B was used to select HEK293 OATP1B3+ and its empty vector control.

[0276] To determine the cytotoxicity of the organic cytotoxic oligomers of the present invention and the corresponding cytotoxic monomers as reference, each cell line was seeded in triplicate at 5 × 10⁴ cells per well in 96-well plates without selection markers. After 24 hours, transporter expression was induced by adding sodium butyrate to a final concentration of 10 mM. The next day, the culture medium was removed, and the cells were incubated for 48 hours in a marker-free medium with eight different concentrations of each cytotoxin oligomer (0.01 nM to 3 μM) and the corresponding toxin monomer as a control.

[0277] Then, the cells were fixed as described above.Wash and stain with sulfonylrhodamine B (SRB) (Vichai et al., Nature protocols 2006, 1, 1112-1116). Briefly, 10 µL of trichloroacetic acid (10%) was added directly to each well (page 38 / 43, CN 121620394 A, instructions) and incubated at 4°C for 1 hour. Then, the solution was 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, 200 µL of Tris buffer (10 mM, pH 10.5) was added, and the plate was shaken for five minutes. Then, the absorbance of SRB was measured at 510 nm using a TECAN Infinity M PLEX plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). At least two copies were used in the experiment.

[0278] Cell viability and IC50 values ​​(IC50_cytotox) were calculated using a GraphPad PRISM 6 with nonlinear regression (S-shaped dose response).

[0279] Example 61: Protein phosphatase inhibition (PPI) assay was performed according to the recently described protocol (Heresztyn et al., Water Res 2001, 35, 3049-3056).

[0280] Different working solutions were prepared prior to the measurement. The reaction buffer was freshly prepared by combining Tris buffer (250 mM, pH 8.1), magnesium chloride solution (200 mM), manganese chloride solution (10 mM), and bovine serum albumin (BSA) solution (5 mg / ml) (50:26:4:20 (v / v)) and stored on ice. For the substrate solution, the reaction buffer was mixed with p-nitrophenyl phosphate (pNPP) stock solution (60 mM) and DTT solution (20 mM) (5:4:1 (v / v)), wherein the pNPP was freshly prepared. Enzyme dilution buffer was prepared by combining solutions of ethylene glycol-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA; 0.19 mM, in Tris buffer (80 mM, pH 7.0)), manganese chloride (10 mM), dithiothreitol (DTT; 20 mM), and BSA (5 mg / ml) (65:20:5:10 (v / v)). For the enzyme solution, the protein phosphatase-1 catalytic subunit (derived from the rabbit α-isotype) was diluted with the enzyme dilution buffer.Dilute to 10 units / mL (Sigma-Aldrich) and store on ice.

[0281] Assays were performed in 384-well plates. The diluted organic cytotoxic oligomer and the corresponding cytotoxic monomer (4 μL, in DMSO) as a control were pre-incubated with enzyme solution (4 μL) at 37°C for 5 min. Then, 40 μL of substrate solution was added to each well and incubated at 37°C for 2 h. The concentrations of the seven cytotoxic agents were in the range of 0.001 nM to 1 μM, while a constant substrate concentration of 20 mM was used per well. As a positive control, 4 μL of DMSO was used instead of MC solution to track substrate conversion without PPI. The negative control (blank) contained 4 μL of enzyme dilution buffer instead of diluted enzyme to prevent any hydrolysis of the substrate. To determine the PPI, absorbance was measured at 405 nm using a TECAN Infinity M PLEX plate reader. At least two copies were used in the experiment, and the IC50 value (IC50_PPI) was calculated using GraphPad PRISM 6 with nonlinear regression (S-shaped dose response).

[0282] Example 62: RNA polymerase II (RNA Pol II) inhibition assay protocol as recently described (Voss et al. BMC Molecular Biology 2014, 15:7) was used for the RNA Pol II inhibition assay. This publication describes a non-radioactive, robust, and reproducible eukaryotic in vitro transcription assay for sensitive detection and accurate quantification of newly transcribed unlabeled RNA.

[0283] After transcription, the newly synthesized RNA was directly detected and quantified using the QuantiGene assay (Thermo Fisher Scientific Inc.). Alternatively, the RNA was purified and primers extended, followed by PCR detection or qPCR quantification. This assay can be particularly used to quantify strong transcription inhibitors, such as α-amanitin. When applied to evaluate the activity of RNA polymerase II inhibitors, this novel method allows for accurate estimation of their relative potency.

[0284] Example 63: Protocol for in vitro testing of ADC efficacy. The efficacy of the ADCs constructed in Examples 56-59 was tested in vitro using a cell viability assay, wherein cancer cell lines expressing tumor antigens, such as NCI-N87 expressing Her2 antigen, were incubated with different concentrations of each ADC in the range of picomolar to nanomolar or micromolar. Specification 39 / 43 pages 43 CN 121620394 A

[0285] According to the manufacturer's instructions,In vitro efficacy testing of the ADC was performed using, for example, the CellTiter-Glo Luminescent Cell Viability Assay (Promega GmbH, Walldorf, Germany).

[0286] Example 64: Bioassay Results As a representative example, Figure 12 and Table 7 show the microcystin homodimers (see Figures 1 and 2) of Examples 2 and 12 having structural formulas DM-I and DM-II, which were bridged from two MC-PrgTyr-Arg monomers (SEQ ID NO: 1) using bridging molecules of structural formulas BM-I (hereinafter referred to as "MC-PrgTyr-Arg-dimer-1") and BM-IV (hereinafter referred to as "MC-PrgTyr-Arg-dimer-2") NO:2) is a covalently cross-linked derivative of the first and second organic cytotoxic entities, and the results were obtained from the general cytotoxicity assays (IC50_OATP1B1 and IC50_OATP1B3, in Figures 12A and Table 7) and protein phosphatase inhibition assays (IC50_PPI, in Figures 12B and Table 7) for OATP1B1 and OATP1B3 expression cell lines described in Examples 60 and 61 above.

[0287] For reference, Figures 12 and Table 7 also show the corresponding assay results for the unconjugated reference monomer MC-PrgTyr-Arg and for MC-LR, which is one of the most potent and well-studied microcystin analogues known in the art with the highest protein phosphatase inhibition described in the literature.

[0288] Table 7: Results of cytotoxicity assays and protein phosphatase inhibition assays (IC50_OATP1B1 = type 1 protein phosphatase) from OATP1B1 expression cell lines (IC50_OATP1B1) and OATP1B3 expression cell lines (IC50_OATP1B3) for MC-LR (reference), MC-PrgTyr-Arg monomers (reference) and the corresponding MC-PrgTyr-Arg dimers according to the present invention (Examples 2 and 12). “SE” indicates the standard error of at least two replicate experiments.

[0289] As another representative example, Table 8 shows that for the arthrotoxic homodimer of Example 40, which is derived from two [D-PrgMeAsp]1-NOD monomers as first and second organic cytotoxic entities using a bridging molecule of formula BM-IV (hereinafter referred to as "[D-PrgMeAsp]1-NOD-dimer-1"), the general cytotoxicity assay (IC50_OATP1B1, ) for OATP1B1-expressing cell lines described in Examples 60 and 61 above is used.In Table 8) and the results obtained by the protein phosphatase inhibition assay (IC50_PPI, in Table 8) on pages 40 / 43 of the specification.

[0290] For reference, Table 8 shows the corresponding assay results for the unconjugated reference monomer [D-PrgMeAsp]1-NOD.

[0291] Table 8: Results of cytotoxicity assay (IC50_OATP1B1) and protein phosphatase inhibition assay (IC50_PPI = type 1 protein phosphatase) from the OATP1B1 expression cell line for the [D-PrgMeAsp]1-NOD monomer (reference) and the corresponding [D-PrgMeAsp]1-NOD-dimer-1 according to the present invention (Example 40). “SE” indicates the standard error of at least two replicate experiments.

[0292] Tables 7 and 8 also show the ratios of IC50_OATP1B1 and IC50_PPI, and the ratio of IC50_OATP1B3 and IC50_PPI (the latter ratio is only shown in Table 7) as measures of PPI-normalized OATP uptake. These ratios are used to evaluate whether oligomerization exhibits the desired effect. In this context, if the general cytotoxicity to OATP1B1 and / or OATP1B3 expressing cell lines decreases (reflected by an increase in IC50_OATP1B1 or IC50_OATP1B3, respectively) while protein phosphatase inhibition increases or remains substantially unchanged (reflected by no change or decrease in IC50_PPI), i.e., the ratios are greater than a reference ratio, then the therapeutic window of the organic cytotoxic oligomers of the present invention is improved with respect to the tested OATP-expressing cell lines. Such an improvement in the therapeutic window occurs because the active cellular uptake of the organic cytotoxic oligomers of the present invention decreases relative to protein phosphatase inhibition and / or because the protein phosphatase inhibition of the organic cytotoxic oligomers of the present invention increases relative to active cellular uptake. Furthermore, if the ratio of the IC50_PPI of the organic cytotoxic oligomer of the present invention to the reference IC50_PPI is <0.5 when the oligomer is a dimer, or <0.33 when the oligomer is a trimer, etc. (generally, if the ratio is <1 / number of monomers in the oligomer), it indicates that the cytotoxic efficacy of the organic cytotoxic oligomer of the present invention, based on the inhibition of the corresponding protein phosphatase, is enhanced in a synergistic manner compared to the reference by exceeding the increase in the number of toxins in the conjugate.

[0293] The results in Figure 12, Table 7, and Table 8 show thatThe IC50_OATP1B1 and IC50_OATP1B3 values ​​of MC-PrgTyr-Arg-dimer-1, MC-PrgTyr-Arg-dimer-2, and [D-PrgMeAsp]1-NOD-dimer-1 according to the present invention are significantly increased compared with monomeric reference microcystins or arthrococci. For IC50_OATP1B1, the increase compared with MC-LR is more than 116 times for MC-PrgTyr-Arg-dimer-1, more than 175 times for MC-PrgTyr-Arg-dimer-2, and 24 times and more than 36 times compared with the corresponding MC-PrgTyr-Arg monomers, respectively. Similar effects were observed in the IC50_OATP1B3 case, with a 134-fold increase for MC-PrgTyr-Arg-dimer-1 and a 234-fold increase for MC-PrgTyr-Arg-dimer-2 compared to MC-LR, and increases of 26-fold and 45-fold respectively compared to the MC-PrgTyr-Arg monomer. Furthermore, in the case of [D-PrgMeAsp]1-NOD-dimer-1, the IC50_OATP1B1 value was more than 9-fold increased compared to the corresponding [D-PrgMeAsp]1-NOD monomer. This indicates that the microcystins and arthrophyllin oligomers of the present invention tested exhibit significantly reduced general cytotoxicity to OATP-expressing cell lines compared to the monomer reference.

[0294] However, on the other hand, the results clearly show that the protein phosphatase inhibition of the MC-PrgTyr-Arg-dimer and PrgMeAsp]1-NOD-dimer-1 according to the present invention is significantly increased, as demonstrated by comparing the IC50_PPI of the corresponding MC-PrgTyr-Arg monomer or the [D-PrgMeAsp]1-NOD monomer, respectively, with an IC50_PPI of less than 1 / 20 for MC-PrgTyr-Arg-dimer-1, less than 1 / 50 for MC-PrgTyr-Arg-dimer-2, and less than 1 / 8 for [D-PrgMeAsp]1-NOD-dimer-1, and by comparing the IC50_PPI of the MC-LR reference cytotoxin.The IC50_PPI is approximately 1 / 10 for MC-PrgTyr-Arg-dimer-1 and approximately 1 / 16 for MC-PrgTyr-Arg-dimer-2.

[0295] Given the strong reduction in general cytotoxicity and strong increase in enzyme inhibition of the MC-PrgTyr-Arg-dimer and [D-PrgMeAsp]1-NOD-dimer-1 to OATP1B1 and OATP1B3 expressing cell lines according to the invention, these results indicate that oligomerization leads to a significant reduction in the desired effect of active uptake into cells.

[0296] In addition, the IC50_PPI of the MC-PrgTyr-Arg-dimer being less than 1 / 20 to less than 1 / 50 compared to the IC50_PPI of the corresponding MC-PrgTyr-Arg monomer, and the IC50_PPI of the [D-PrgMeAsp]1-NOD-dimer being less than 1 / 8 compared to the IC50_PPI of the corresponding [D-PrgMeAsp]1-NOD monomer, clearly demonstrate the synergistic effect of oligomerization on enzyme inhibition, rather than simply the additive effect of the individual cytotoxicity accumulation from the two monomers.

[0297] Finally, the ratio of general cytotoxicity and protein phosphatase inhibition of IC50_PPI binding to OATP1B1 and OATP1B3 expressing cell lines at less than 1 / 20 to less than 1 / 50 (as a measure of PPI-normalized OATP uptake) indicates that the therapeutic window for both OATP-expressing cell lines is amplified by at least two orders of magnitude and at most four orders of magnitude compared to MC-PrgTyr-Arg monomer (more than 700-fold to more than 2400-fold) and MC-LR reference cytotoxicity (more than 1000-fold to more than 3900-fold) by the MC-PrgTyr-Arg dimer according to the invention. Similarly, the results obtained for [D-PrgMeAsp]1-NOD-dimer-1 (showing a ratio of general cytotoxicity and protein phosphatase inhibition of less than 1 / 8 of IC50_PPI binding to OATP1B1 expressing cell lines) reflect an almost two-order-of-magnitude amplification of the therapeutic window compared to the monomer, thus confirming the effects observed according to the invention in a generalizable manner.

[0298] It should be understood that the results obtained with the two MC-PrgTyr-Arg-dimers and [D-PrgMeAsp]1-NOD-dimer generally represent the advantageous effects of the organic cytotoxic oligomers of the present invention. Similar improvements and advantageous effects regarding general cytotoxicity, protein phosphatase inhibition, and / or therapeutic window are obtained with other representative organic cytotoxic oligomers described in other embodiments above.

[0299] Those skilled in the art will not anticipate these synergistic effects and improvements, and the associated advantages.

[0300] Furthermore, it should be understood that the invention is not limited to the embodiments described in the detailed description. Rather,This invention includes each new feature and each new combination of features, particularly each combination of features in the claims and specification, even if the feature or combination of features is not explicitly defined in the claims, specification, or embodiments.

[0301] 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 homomicrocystin dimers or heteromicrocystin dimers) as monomers and / or with different bridging portions (if any). In particular, such microcystin analogs may have different amino acid sequences than those included in Examples 2-21, for example, having different amino acids at positions Aa1, X2, Aa3, Z4, and Aa7 and / or microcystin analogs with different coupling functionalities such as alkenes, carboxyl groups, ketones, hydroxyl groups, hydroxylamines, isothiocyanates, tetrazines, thiols, and aldehydes. Furthermore, microcystin dimers can also be constructed by crosslinking monomers at different positions, for example, position Aa1 of the first microcystin entity and position X2 of the second microcystin entity, or any other combination excluding positions Aa5 and Aa6.

[0302] Similarly, the microcystin trimers described in Examples 26-33 can also be constructed as homo- or hetero-microcystin trimers using different microcystin analogs as monomers. In particular, such microcystin analogs may have different amino acid sequences than those included in Examples 2-33, for example, having different amino acids at positions Aa1, X2, Aa3, Z4 and / or Aa7 and / or having different coupling functionalities such as alkenes, alkyl halides, hydrazides, carboxyl groups, ketones, hydroxyl groups, hydroxylamines, isothiocyanates, tetrazines, thiols, and aldehydes. Cross-linking domains can also covalently connect different positions of the microcystin entity.

[0303] Furthermore, microcystin oligomers, such as microcystin tetramers, microcystin pentamers, and microcystin hexamers, can also be constructed as homo- or hetero-microcystin oligomers, including but not limited to the microcystin entities used in Examples 2-33 or microcystin analogs with different amino acids and / or different coupling functionalities, as noted above.

[0304] The arthrophyllin dimers described in Examples 34-43 can also be constructed as homo- or hetero-arthrophyllin dimers using different arthrophyllin analogs as monomers. In particular, such arthrophyllin analogs may have different amino acid sequences than those included in Examples 34-43.For example, arthrotoxic analogs with different amino acids at positions Aa1, Aa2, and / or Aa5 and / or with different coupling functionalities such as alkenes, carboxyl groups, ketones, hydroxyl groups, hydroxylamines, isothiocyanates, tetrazines, thiols, and aldehydes.

[0305] The arthrotoxic trimers described in Examples 44 and 45 can also be constructed as homologous or heterologous arthrotoxic trimers using different arthrotoxic analogs as monomers, particularly using arthrotoxic analogs having amino acid sequences different from those included in Examples 34-45 as cytotoxic entities.

[0306] In addition, oligomers of Arachnium toxin, such as tetramers, pentamers, and hexamers, can also be constructed as homologous or heterologous Arachnium toxin oligomers, including but not limited to the Arachnium toxin entities used in Examples 34-45 or Arachnium toxin entities with different amino acids and / or different coupling functionalities, as noted above.

[0307] The amatoxin dimers described in Examples 46-51 can also be constructed as homologous or heterologous amatoxin dimers using different amatoxin entities as monomers. Specifically, the amatoxin entities may have amino acid sequences different from those included in Examples 46-51, for example, having different amino acids at positions Aa1, Aa3, Aa4, Aa5, Aa6 and / or Aa7 and / or having different coupling functionalities such as alkenes, carboxyl groups, ketones, hydroxyl groups, hydroxylamines, isothiocyanates, tetrazines, thiols, aldehydes, and oximes.

[0308] 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, particularly using amatoxin entities having different amino acid sequences and / or different coupling functionalities than those representatively shown in the examples. In addition, amatoxin oligomers, such as amatoxin tetramers, amatoxin pentamers, amatoxin hexamers, etc., can also be constructed as homoamatoxin oligomers or heteroamatoxin oligomers.

[0309] It should also be understood that the anti-HER2 antibody trastuzumab used in Examples 56-59 was intentionally chosen because trastuzumab is a benchmark antibody for the anti-TAA and anti-TSA antibody classes. Therefore, the results obtained with the ADC including trastuzumab confirm that the invention can also be implemented with other antibody classes, particularly anti-TAA and anti-TSA antibody classes.

[0310] Therefore, it should also be understood that the ADCs of Examples 56-59 can also be constructed using different monoclonal antibodies targeting different antigens (e.g., CD7 and BCMA) expressed by different cancer cells. Naturally,ADCs can also be constructed using other homologous and heterologous microcystins, arthrotoxins and / or amatoxin dimers and oligomers (as described above) and with different linkers and, for example, other conjugation methods. Instruction manual, page 43 / 43, page 47, CN 121620394 A, Figure 1, Figure 2; Instruction manual, Figure 1 / 8, page 48, CN 121620394 A, Figure 3, Figure 4; Instruction manual, Figure 2 / 8, page 49, CN 121620394 A, Figure 5; Instruction manual, Figure 3 / 8, page 50, CN 121620394 A, Figure 5 (continued); Instruction manual, Figure 4 / 8, page 51, CN 121620394 A, Figure 6, Figure 7; Instruction manual, Figure 5 / 8, page 52, CN 121620394 A, Figure 8, Figure 9; Instruction manual, Figure 6 / 8, page 53, CN 121620394 A, Figure 10; Instruction manual, Figure 7 / 8, page 54, CN 121620394 A, Figure 11, Figure 12; Instruction manual, Figure 8 / 8, page 55, CN 121620394 A.

Claims

1. A pharmaceutical cytotoxic oligomer comprising a first organic cytotoxic entity, at least a second organic cytotoxic entity, and a cross-linking entity forming a covalent linkage between the first organic cytotoxic entity and the second organic cytotoxic entity, wherein monomers of the first organic cytotoxic entity and the second organic cytotoxic entity i) exert a cytotoxic effect on a target cell by active transport into a target cell via a transporter protein of the target cell, and / or ii) exert a cytotoxic effect on a target cell by inhibiting an intracellular enzyme of the target cell, wherein the covalent linkage between the first organic cytotoxic entity and the second organic cytotoxic entity is significantly resistant to cleavage under physiological conditions.

2. The pharmaceutical cytotoxic oligomer of claim 1, wherein the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a cyclic oligopeptide having a plurality of amino acids, and wherein the cross-linking entity covalently links a side chain of one of the amino acids of the first organic cytotoxic entity with a side chain of one of the amino acids of the second organic cytotoxic entity.

3. The pharmaceutical cytotoxic oligomer of any one of claims 1 or 2, wherein the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a microcystin having the general structure 4. The pharmaceutical cytotoxic oligomer of any one of claims 1 or 2, wherein the first organic cytotoxic entity and the second organic cytotoxic entity each comprise a nodularin having the general structure ring (-Aa 1 -X 2 -Aa 3 -Z 4 -Aa 5 -Aa 6 -Aa 7 ), wherein independently of each other, Aa 1 and Aa 3 each represents a D-amino acid, Aa 5 is selected from Adda, DM-Adda, dm-Adda, (6Z)Adda and ADM-Adda, Aa 6 is selected from D-Glu and D-Glu(OCH3), Aa 7 is selected from 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 of each other L-amino acids, The cross-linking portion transfers the Aa of the first organic cytotoxic entity 1 X 2 Aa 3 Z 4 or Aa 7 One of the side chains and the Aa of the second organic cytotoxic entity 1 X 2 Aa 3 Z 4 or Aa 7 One of the side chains is covalently connected.

5. The pharmaceutical cytotoxic oligomer of any one of claims 1 or 2, wherein the first organic cytotoxic entity and the second organic cytotoxic entity each comprise an amatoxin having the general structure ring [-Aa 1 -Aa 2 -Aa 3 -Aa 4 -Aa 5 ], wherein, independently of each other, Aa 1 is D-Asp or D-MeAsp or a modified D-amino acid, Aa 2 is Arg or Har or a modified L-amino acid, Aa 3 is selected from Adda, DM-Adda, (6Z)Adda and MeAdda, Aa 4 is selected from D-Glu and D-Glu(OCH3), Aa 5 is Dhb or Mdhb or a modified L-amino acid, The cross-linked entity will transfer the Aa of the first organic cytotoxic entity 1 Aa 2 or Aa 5 The side chain of the second organic cytotoxic entity and Aa 1 Aa 2 or Aa 5 One of the side chains is covalently connected. an amide bond, a thioamide bond, an ether bond, a thioether bond, a covalent bond containing a triazole, a dihydropyridazine or a higher amine, a carbamate bond, a thiocarbamate bond, a urea bond, a thiourea bond, a phosphate bond, a phosphoramide bond, a sulfonamide bond, an oxime bond, or any combination thereof. Ring {Aa 1 - Ring [Aa 2 - Aa 3 - Aa 4 - Aa 5 - Aa 6 - Aa 7 - Aa 8} (SEQ ID NO: 1), wherein, independently of each other, Aa 1 is lie, Hil or Dhil, Aa 2 is Trp or Htp, Aa 3 is Gly, Aa 4 is lie, Aa 5 is Gly, Aa 6 is Cys, Aa 7 is Asn or Asp, and Aa 8 is Pro or Hyp, in Aa 2 and Aa 6 are cross-linked via a sulfoxide (S=0) bridge between Aa wherein said cross-linking entity covalently links a side chain of one of Aa 1 , Aa 2 , or Aa 8 of said first organic cell toxin entity to a side chain of one of Aa 1 , Aa 2 , or Aa 8 of said second organic cell toxin entity.

6. The cytotoxic oligomer for use in medicine according to any one of claims 1 to 5, wherein the cross-linking entity comprises or consists of covalent linkages selected from the group consisting of:

7. The pharmaceutical cytotoxic oligomer of any one of claims 1-6, wherein the cross-linking entity comprises a bridging moiety interconnecting the first organic cytotoxic entity and the second organic cytotoxic entity to each other, wherein the bridging moiety is an organic molecule having a molecular size of about 50 Da to about 1,000 Da.

8. The pharmaceutical cytotoxic oligomer of any one of claims 1-7, wherein the cross-linking entity comprises a coupling functionality configured to covalently conjugate the pharmaceutical cytotoxic oligomer to an antibody, wherein the coupling functionality is selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, an azido group, an alkyne group, an alkene group, a thiol group, an aldehyde group, a ketone group, a tetrazine group, and any combination thereof.

9. The pharmaceutical cytotoxic oligomer of any one of claims 1-8, comprising at least a further third organic cytotoxic entity, wherein the cross-linking entity forms a covalent linkage between the first organic cytotoxic entity, the second organic cytotoxic entity, and at least the further third organic cytotoxic entity, ​ wherein the covalent linkage between the first organic cytotoxic entity, the second organic cytotoxic entity, and at least the additional third organic cytotoxic entity is significantly resistant to cleavage under physiological conditions.

10. The pharmaceutically cytotoxic oligomer of any one of claims 1-9, wherein the resistance to cleavage comprises resistance to enzymatic cleavage and resistance to acid-induced cleavage at a pH as low as at least pH 4.

5.

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

12. A method for producing the pharmaceutically cytotoxic oligomer of any one of claims 1-11, comprising A) providing a first organic cytotoxic entity and at least a second cytotoxic entity, wherein the first organic cytotoxic entity and the at least second organic cytotoxic entity each i) exert a cytotoxic effect on a target cell by being actively transported into a target cell via a transporter protein of the target cell, and / or ii) exert a cytotoxic effect on a target cell by inhibiting an intracellular enzyme of the target cell, B) forming a covalent linkage between the first organic cytotoxic entity and the at least second organic cytotoxic entity that is significantly resistant to cleavage under physiological conditions, thereby forming the cytotoxic oligomer.

13. The method of claim 12, wherein A) further comprises providing at least one organic bridging moiety, wherein the bridging moiety is an organic molecule having a molecular size of about 50 Da to about 1,000 Da, and B) further comprises forming a covalently linked first moiety by covalently conjugating the bridging moiety to the first organic cytotoxic entity and forming a covalently linked at least second moiety by covalently conjugating the bridging moiety to the at least second organic cytotoxic entity, thereby cross-linking the first organic cytotoxic entity and at least the second organic cytotoxic entity to each other via the bridging moiety.

14. An antibody-drug conjugate, comprising an antibody or antigen binding fragment thereof, a pharmaceutically cytotoxic oligomer according to any one of claims 1-11, wherein the pharmaceutically cytotoxic oligomer is covalently linked to the antibody or antigen binding fragment thereof.

15. The antibody-drug conjugate of claim 14, having the general formula (I) wherein Ab is the antibody or antigen binding fragment thereof; T is the pharmaceutically cytotoxic oligomer; L is an organic linker; and m is an integer from 1-5, and n is an integer from 1-8. Ab-[L-(T) m ] n (I) 16. The antibody-drug conjugate of any one of claims 14 or 15, wherein the linker is configured to release the pharmaceutically cytotoxic oligomer from the antibody or antigen binding fragment thereof upon receptor-mediated endocytosis and / or lysosomal processing of the antibody-drug conjugate.

17. A method for producing an antibody-drug conjugate, comprising a) providing a pharmaceutically cytotoxic oligomer according to any one of claims 1-11, b) providing an antibody or antigen binding fragment thereof, c) covalently linking the pharmaceutically cytotoxic oligomer to the antibody or antigen binding fragment thereof. ​ 18. The method of claim 17, wherein c) further comprises cl) providing a linker, c2) covalently linking said cytotoxic oligomer to said linker, c3) covalently linking said linker to said antibody or antigen binding fragment thereof.

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