Methods of increasing therapeutic index of amatoxin-antibody conjugates

Abstract

The present invention relates to therapeutic regimens that increase the therapeutic index of antibody-drug conjugates, in particular antibody-drug conjugates comprising amatoxin. The invention also relates to an antibody-drug conjugate based on amatoxin for use in said therapeutic regimen.

Description

Field of the Invention

[0001] The present application relates to the field of cancer immunotherapy. In particular, the present application relates to a dosing regimen for treating cancer in patients to increase the therapeutic index of an antibody-drug conjugate based on amatoxin. The present application also relates to an antibody-drug conjugate based on amatoxin for such a treatment regimen.

[0002] background

[0003] Currently approved antibody-drug conjugates (ADCs) such as Enhertu (HER2), Trodelvy (Trop-2) and Blenrep (BCMA) are based on only a few cytotoxic mechanisms, mainly with microtubule or DNA-targeted toxins as payloads. Amanita toxins are a group of well-known toxins consisting of nine structurally related toxins naturally present in basidiomycetes mushrooms of the genus Amanita. The most prominent members are α-amanitin and β-amanitin, identified more than four decades ago in the green death cap mushroom Amanita Phalloides. Amanita toxins are well known to the public and have been intensively studied in health and medical sciences due to their toxic effects after mushroom ingestion. They are the cause of 95% of all fatal mushroom poisonings worldwide. Amanita toxins are highly heat-resistant, resistant to enzyme and acid degradation, and show excellent solubility in water. In particular, α-amanitin has been shown to be the main cause of the severe liver and kidney damage observed after poisoning by A. phalloides. In the liver, it is taken up by a specific transporter (organic anion transporting polypeptide (OATP1B3)) located on hepatocytes, leading to their cytolysis.

[0004] Due to its hydrophilic nature, amanitin does not passively penetrate cells and has shown no or only poor uptake for most other human cell types except hepatocytes. However, it has been reported that in addition to the liver, other organs, mainly the kidneys, are also affected. Since amanitin toxins are not further metabolized, they are mainly excreted through the kidneys and urine within the first few days after ingestion, resulting in severe nephrotoxicity.

[0005] Amanita aniline toxins are bicyclic octapeptides containing three unique amino acids: dihydroxy-isoleucine, hydroxy-tryptophan and hydroxy-proline. They bind with the highest affinity to eukaryotic RNA polymerase II, resulting in a significant reduction of transcription and protein synthesis by more than 1000-fold. The OH groups of the hydroxylated side chains are responsible for the high hydrophilicity of the molecule and, to a certain extent, for its binding to RNA polymerase II. Due to this highly specific inhibition, amanita aniline has been widely used as a tool to study gene expression and transcriptional regulation.

[0006] Importantly, the effects induced by amanitin are not restricted to dividing cells and also act in quiescent (non-dividing) cells, unlike the DNA- or tubulin-targeting toxins currently used in most approved ADCs.

[0007] With this new mode of action, amanitin-based ADCs therefore have several advantages over commercially available ADCs: First, amanitin-based ADCs also affect non-proliferating and quiescent cells. Second, amanitin is not a known substrate for efflux transporters. Therefore, amanitin-based ADCs are also effective in tumors that are resistant to other lines of therapy. Third, there are no known resistance mechanisms for amanitin as an ADC payload, which may be due to the strict structure-activity requirements of RNA polymerase II in eukaryotic cells, which provide little room for escape mutations in RNA polymerase II.

[0008] Amanita toxin is well suited for use as a payload for ADC based on its physicochemical properties: first, amanita toxin allows attachment of linkers without reducing its cytotoxic potential, second, amanita toxin has a high cytotoxic potential to achieve cell killing at low concentrations, and third, when conjugated to antibodies, amanita toxin exhibits its cytotoxic potential only inside cells.

[0009] In addition to its promising efficacy profile, amanitin also shows some favorable physicochemical properties. Compared with all other toxins commonly used as ADC payloads (warheads), the molecule is very hydrophilic. This is conducive to conjugation in aqueous buffers and prevents ADC aggregation, which makes it easy to handle and reduces its immunogenic potential. It is also shown that amanitin is a very poor substrate for initiating multidrug resistance (MDR) processes, and amanitin conjugates are highly effective in tumor cells expressing MDR.

[0010] Amanitin is highly stable in plasma, and due to its low molecular weight and hydrophilicity, it is cleared very quickly via the kidneys, which makes it unlikely to accumulate in other tissues. Another safety feature is that it cannot enter normal cells via passive uptake. Compared with the antibody conjugated with amanitin, the free toxin shows a 20,000-fold reduced cytotoxicity. When conjugated with an antibody, amanitin or amanitin toxin is no longer a substrate for cellular uptake mediated by OATP1B3, which especially reduces hepatotoxicity, however, some hepatotoxicity can be observed in animal models, as demonstrated by the transient increase of liver enzymes such as ALT, AST or LDH after the administration of an ADC based on amanitin, which narrows the therapeutic window of such ADCs based on amanitin. Therefore, there is still a continuous medical demand for further improving tolerance and thus improving the therapeutic index or therapeutic window of the ADC based on amanitin toxin. SUMMARY OF THE INVENTION

[0012] As shown in this application, the inventors unexpectedly discovered that by administering a first subtherapeutic dose of a first amanita toxin-based ADC prior to administering a therapeutic dose of a second amanita toxin-based ADC, the hepatotoxicity of the second amanita toxin-based ADC can be further reduced, resulting in an increase in the therapeutic index of the second amanita toxin-based ADC. The inventors additionally surprisingly discovered that this effect can also be achieved if the first amanita toxin-based ADC and the second amanita toxin-based ADC do bind to different target proteins, as demonstrated by using a non-binding antibody to the first ADC of the method of the present invention.

[0013] Therefore, one object of the present invention is to provide a method for improving the therapeutic index of an antibody-drug conjugate, wherein the method of the present invention comprises the step of administering to a patient a first dose of a first antibody-drug conjugate, wherein the first dose of the first antibody-drug conjugate is administered between about 30 days to about 15 days prior to administering a second dose of the antibody-drug conjugate, and wherein the antibody-drug conjugate has the following structure:

[0014] Ab-(ZL-Ama) n ,

[0015] wherein the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently linked) to a linker (L), conjugated to an Amanita phalloides toxin moiety (Ama) via a chemical moiety (Z), and n is from about 1, 2, 3, 4 to about 5, 6, 7, 8.

[0016] The present invention also provides a first and / or second Amanita toxin-based ADC as disclosed herein for use in the method of the present invention for improving the therapeutic index of the second Amanita toxin-based ADC, wherein the first Amanita toxin-based ADC and the second Amanita toxin-based ADC may be the same or may be different from each other.

[0017] Description of the drawings

[0018] Figure 1 Structural formulas of various Amanita toxins. The bold numbers (1 to 8) indicate the standard numbering of the eight amino acids that form the Amanita toxins. The standard names of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters α to γ, Greek letters α to δ, and numbers 1' to 7', respectively).

[0019] Figure 2(A) Experimental study design for studies in CB17-SCID mice with and without pretreatment of amanita phalloides-based ADCs. (B) Two independent experiments with 3 (upper panel) or 5 (lower panel) mice / group were performed using different doses of anti-DIG amanita phalloides-ADCs. The number of mice with macroscopic changes in the liver and the survival rate are depicted; liver abnormalities in all mice could not be examined due to the autolytic state. p values ​​were calculated using the Gehan-Breslow-Wiloxon test.

[0020] Figure 3 Repeated dosing reduces organ damage induced by amatoxin-ADC in cynomolgus monkeys. (A) Experimental setup comparing single-dose toxicity at the maximum tolerated dose (MTD) of amatoxin-ADC (left) with toxicity occurring after the same dose was given as the final step of a dose-escalation regimen. (B) Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels in serum of cynomolgus monkeys after iv treatment with amatoxin-ADC, which was given as a single dose (circles) or as the final step of a dose-escalation regimen (triangles). Dashed line: baseline level in untreated animals. Means and SEMs of three biological replicates are shown. p value: Welch t test; *: p≤0.05. (C) Experimental setup comparing the MTD toxicity of amatoxin-ADC given after different rounds of treatment. (D) AST, ALT and LDH levels in serum of cynomolgus monkeys after MTD iv treatment with amanita phalloides toxin-ADC, which was given in the first (triangle), second (open circle), third (open triangle) or fourth (polygon) round of treatment. Means and SEMs of three biological replicates are depicted.

[0021] Figure 4Repeated dosing with an amanita tinctoria toxin-based anti-PSMA ADC improves anti-tumor efficacy in the scLNCaP CDX model. (A) Experimental setup for in vivo efficacy studies comparing single versus multiple doses (once a week for 4 weeks, q7dx4) of an amanita tinctoria toxin-based anti-PSMA ADC in the sc human prostate adenocarcinoma LNCaP CDX model; (B) Tumor growth of scLNCaP tumors measured by caliper for 84 days (n=18 animals / group), triangles: single dose treatment, squares: multiple dose treatment, solid circles: PBS control. Means and SD are shown. p-value: Welch t-test corrected for multiple testing by the Holm-Sídák method; statistically significant from day 21 to day 56. **p<0.01; (C) Survival probability of mice bearing LNCaP sc tumors treated with single (triangle) or multiple doses (square) of an amanita tinctoria toxin-based anti-PSMA ADC; circles: PBS control. p-value: Gehan-Breslow-Wiloxon test; ****: p < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0023] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0024] Below, the key element of the present invention will be described.These key elements are listed together with specific embodiments, however, it should be understood that they can be combined in any way and in any quantity to create other embodiments.The examples and preferred embodiments of various descriptions should not be interpreted as limiting the present invention to only the embodiments clearly described.This specification should be understood as supporting and covering the embodiments that will clearly describe the embodiments and any number of disclosed and / or preferred key element combinations.In addition, unless the context indicates otherwise, any arrangement and combination of all described key elements in this application should be considered to be disclosed by the specification of the application.

[0025] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated members, integers, or steps, but not the exclusion of any other unstated members, integers, or steps. The term "consisting of is a specific embodiment of the term "comprising", in which any other unstated members, integers, or steps are excluded. In the context of the present invention, the term "comprising" encompasses the term "consisting of". Thus, the term "comprising" encompasses "including" as well as "consisting of", for example, a composition "comprising" X may consist of X alone, or may include additional things, such as X+Y.

[0026] Unless otherwise indicated herein or clearly contradictory to the context, the terms "a" and "an" and "the" and similar indicators used in the context of describing the present invention (especially in the context of the claims) should be interpreted as covering both the singular and the plural. The enumeration of the range of values ​​herein is intended to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were individually enumerated herein. The language in this specification should not be interpreted as indicating that any non-claimed element is essential to the practice of the present invention. The word "substantially" does not exclude "completely", for example, a composition that is "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" can be omitted from the definition of the present invention.

[0027] As used herein, the term "about" with respect to a numerical value x means x ± 10%.

[0028] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar to or equivalent to those described herein and materials can be used in the practice or testing of the present invention, suitable methods and materials are described as follows. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification, including definitions, will replace any other definitions. In addition, materials, methods and examples are illustrative only and are not intended to be restrictive.

[0029] In addition, the content of the prior art documents cited herein is incorporated by reference. This refers in particular to the prior art documents of open standards or conventional methods. In this case, the main purpose of incorporating by reference is to provide sufficient disclosure that can be realized and to avoid lengthy repetition. The chemical terms used throughout this application are according to the "Compendium of Chemical Terminology" published by the International Union of Pure and Applied Chemistry, ISBN: 0-9678550-9-8.

[0030] According to a first aspect, the present invention relates to a method of increasing the therapeutic index of an antibody-drug conjugate, wherein the method comprises the step of administering a first dose of a first antibody-drug conjugate to a patient suffering from cancer, wherein the first dose of the first antibody-drug conjugate is administered between about 30 days to about 15 days prior to administering a second dose of the antibody-drug conjugate.

[0031] Thus, in a first aspect, the present invention relates to a method of increasing the therapeutic index of an antibody-drug conjugate, wherein the method comprises the step of administering a first dose of a first antibody-drug conjugate to a patient suffering from cancer, wherein said first dose of said first antibody-drug conjugate is administered between about 30 days to about 15 days prior to administration of at least a second dose of the antibody-drug conjugate.

[0032] According to one embodiment, the first dose and the second dose of the antibody-drug conjugate (ADC) in the method according to the present invention include the same antibody-drug conjugate. As used herein, the term "therapeutic index" refers to the ratio of the toxic dose at which 50% of individuals show a toxic effect of the drug to the minimum concentration or amount of the drug at which 50% of individuals show a therapeutic effect. TI can also be expressed, for example, as TI=TD 50 :ED 50 , and is a quantitative measure of the relative safety of a drug, where TD 50 The median toxic dose of a drug or toxin is the dose at which toxicity occurs in 50% of cases and the ED 50Refers to the median effective dose, which is the dose that produces a qualitative effect (all or nothing) in 50% of individuals receiving a given drug (such as an ADC based on amanita phalloides toxin used according to the present invention). It is a comparison of the amount of therapeutic agent that causes a therapeutic effect to the amount that causes toxicity. Therefore, a larger TI corresponds to an increased relative safety of a given drug. In some aspects, the therapeutic index may include a comparison of the amount of a therapeutic agent (such as a first ADC and / or a second ADC as disclosed herein) that causes a therapeutic effect (e.g., killing cancer cells) to the amount of a therapeutic agent that causes toxicity (e.g., liver toxicity). Therefore, increasing the therapeutic index of the first ADC and / or the second ADC according to the present invention is advantageous because it increases the safety of the corresponding ADC, and preferably, the method of the present invention is used to increase the therapeutic index of the second ADC.

[0033] In one aspect, the present invention relates to said first and / or second ADC for use in the method of the present invention for increasing the therapeutic index of a first ADC and / or a second ADC, preferably a first ADC and / or a second ADC according to the present invention for increasing the therapeutic index of said second ADC as disclosed herein.

[0034] In some embodiments, the invention relates to a first ADC and / or a second ADC for use in a method of increasing the therapeutic index of an antibody-drug conjugate, wherein the method comprises administering a first dose of a first antibody-drug conjugate to a patient suffering from cancer, wherein the first dose of the first antibody-drug conjugate is administered between about 30 days to about 15 days prior to administering at least a second dose of the antibody-drug conjugate.

[0035] The term "same ADC" used according to the method of the present invention refers to the fact that the first ADC and the second ADC used in the method of the present invention are the same molecule. For example, the first ADC and the second ADC of the method of the present invention can be identical in terms of their composition (e.g., antibody, linker, payload), and can, for example, be formulated into a pharmaceutical composition having the same composition. As used herein, the term "pharmaceutical composition" means a product comprising pharmaceutical excipients such as buffers, preservatives and tension regulators and active compounds such as, for example, ADCs based on amanita toxins as disclosed herein. The term "administering" or any grammatical equivalent thereof refers to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing a compound, such as, for example, an ADC based on amanita toxins of the present invention as disclosed herein, or a corresponding pharmaceutical composition comprising the ADC, preferably, administration refers to injection, more preferably intravenous (iv) or subcutaneous (sc) injection of the first ADC based on amanita toxins and / or the second ADC based on amanita toxins as disclosed herein.

[0036] In some cases, for example, it may be advantageous if the first amanita toxin-based ADC and the second amanita toxin-based ADC used in the methods of the invention as disclosed herein are the same to reduce the risk of adverse effects since only one ADC is administered.

[0037] The term "cancer" as used in conjunction with the methods of the present invention refers to breast cancer, including triple negative breast cancer; biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; gastrointestinal stromal tumor (GIST), appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, colorectal cancer or metastatic colorectal cancer, Hematologic malignancies, including acute lymphoblastic leukemia and myeloid leukemia; T-cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myeloid leukemia, multiple myeloma; AIDS-related leukemia and adult T-cell leukemia-lymphoma; non-Hodgkin lymphoma (NHL), follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma (DBNHL), subtypes of non-Hodgkin lymphoma, including mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), Richter syndrome (Richter syndrome), and leukemia-associated lymphoma. syndrome, primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis, and pemphigus vulgaris; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer; lung cancer, including non-small cell lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; glioblastoma; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; colorectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, merkel cell carcinoma, Kaposi's sarcoma, sarcoma), basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including embryonal tumors such as seminoma, non-seminoma (teratoma, choriocarcinoma), stromal tumors, and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and renal cancer, including adenocarcinoma and Wilms tumor.

[0038] In one embodiment of the present invention, the first ADC and the second ADC may be different. For example, the first ADC according to the present invention may bind to a target on a tumor cell, and the second ADC according to the present invention may include an antibody portion that does not specifically bind to a target in a patient administering the ADC. The term "specific binding" of an antibody / antigen or other binding pair or any grammatical equivalent thereof indicates a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological products. Therefore, a given antibody of the contemplated method or a binding composition derived from an antigen binding site of an antibody binds to its antigen with an affinity that is at least 2 times greater, preferably at least 10 times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity with an unrelated antigen. For example, an ADC that does not specifically bind to a target in a patient to whom the ADC is administered may be directed to a non-human target antigen, such as, for example, digoxin (anti-DIG, e.g., as disclosed in WO2022 / 045433A1), hen egg lysozyme (anti-HEL), green fluorescent protein (GFP) and related proteins, such as those disclosed in US7,906,636B2, or any human antibody or humanized antibody that does not specifically bind to a cell surface antigen in a patient. In a preferred embodiment, the first ADC and / or the second ADC comprises a humanized antibody portion or a human antibody portion, which may also be referred to as an antibody. It is advantageous to use a humanized antibody portion or a human antibody portion (antibody) in the first ADC and / or the second ADC because it reduces the immunogenicity of the ADC and the possibility of antibody-drug-antibody.

[0039] According to one embodiment, the first ADC and the second ADC according to the present invention comprise an RNA polymerase II inhibitor. According to a preferred embodiment, the RNA polymerase II inhibitor of the present invention is selected from the group of amanita toxins. In the context of the present invention, the term "amanitin toxins" includes all cyclic peptides containing 8 amino acids isolated from Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260), as well as all chemical derivatives thereof; and all semi-synthetic analogs thereof; and all synthetic analogs thereof, constructed from structural units according to the main structure of the natural compound (cyclic, 8 amino acids), and all synthetic or semi-synthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids; and all synthetic or semi-synthetic analogs, wherein the sulfoxide part is replaced by a sulfone, a thioether or an atom other than sulfur, such as a carbon atom in a carbon analog of amanita nitinate.

[0040] As used herein, a "derivative" of a compound refers to a substance having a chemical structure similar to that of the compound, but containing at least one chemical group not present in the compound and / or lacking at least one chemical group present in the compound. The compound to which the derivative is compared is referred to as the "parent" compound. Typically, a "derivative" can be produced from a parent compound in one or more chemical reaction steps.

[0041] As used herein, an "analog" of a compound is structurally related to the compound but not identical, and exhibits at least one activity of the compound. The compound compared with the analog is called the "parent" compound. The above activities include, but are not limited to: binding activity with another compound; inhibitory activity, such as enzyme inhibitory activity; toxic effects; activation activity, such as enzyme activation activity. It is not required that the analog exhibit the same degree of such activity as the parent compound. In the context of the present application, if a compound exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40% and more preferably at least 50%) of the activity of the parent compound, the compound is considered an analog. Thus, as used herein, an "analog of amanita toxin" refers to a compound that is structurally related to any of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin and monohydroxyamanitin carboxylic acid and exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40% and more preferably at least 50%) of the inhibitory activity against mammalian RNA polymerase II as compared to at least one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin and monohydroxyamanitin carboxylic acid. The "analogs of amanita toxicity" suitable for use in the present invention may even exhibit greater inhibitory activity against mammalian RNA polymerase II than any of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin or monohydroxyamanitin carboxylic acid. The inhibitory activity can be determined by determining the concentration at which 50% inhibition occurs (IC 50 The inhibitory activity on mammalian RNA polymerase II can be indirectly determined by measuring the inhibitory activity on cell proliferation, as disclosed in, for example, Voss et al. BMC Molecular Biology 2014, 15:7.

[0042] "Semisynthetic analogues" refer to analogues obtained by chemical synthesis using compounds from natural sources (e.g. plant material, bacterial cultures, fungal cultures or cell cultures) as starting materials. Typically, the "semisynthetic analogues" of the present invention are synthesized starting from compounds isolated from mushrooms of the Amanitaceae family. In contrast, "synthetic analogues" refer to analogues synthesized from small (usually petrochemical) building blocks by so-called total synthesis. Typically, this total synthesis is performed without the aid of biological processes.

[0043] According to some embodiments, the amanita toxin of the first and / or second ADC used according to the present invention is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin, monohydroxyamanitin carboxylic acid and analogs, derivatives and salts thereof.

[0044] Functionally, amanita toxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amanita toxins are those having a functional group (e.g., a carboxyl group, an amino group, a hydroxyl group, a thiol or a thiol-trapping group) that can react with a linker molecule or a target binding moiety as defined below.

[0045] In the context of the present invention, the term "amanitin" refers in particular to a bicyclic structure based on an aspartic acid or asparagine residue at position 1, a proline residue (in particular a hydroxyproline residue) at position 2, an isoleucine, hydroxyisoleucine or dihydroxyisoleucine at position 3, a tryptophan or hydroxytryptophan residue at position 4, a glycine residue at positions 5 and 7, an isoleucine residue at position 6 and a cysteine ​​residue at position 8, in particular a derivative of cysteine ​​oxidized to a sulfoxide or sulfone derivative (for numbering and representative examples of amanitin, see Figure 1 ), and also includes all chemical derivatives thereof; and all semisynthetic analogs thereof; and all synthetic analogs thereof built from structural units according to the main structure of the natural compound (cyclic, 8 amino acids); and all synthetic or semisynthetic analogs that contain non-hydroxylated amino acids instead of hydroxylated amino acids; and all synthetic or semisynthetic analogs, in each case, wherein any such derivative or analog has functional activity by inhibiting mammalian RNA polymerase II.

[0046] In some embodiments of the methods of the invention as disclosed herein, the amanita toxin is conjugated to the first antibody and / or the second antibody, whereby the amanita toxin may be covalently linked to the antibody directly, or whereby the amanita toxin may be covalently linked to the antibody via a linker, preferably, the amanita toxin is covalently linked to the antibody via a linker.

[0047] According to a preferred embodiment, the first antibody-drug conjugate (ADC) and / or the second antibody-drug conjugate (ADC) in the method of the present invention has the following structure

[0048] Ab-(ZL-Ama) n ,

[0049] wherein Ab is an antibody or antigen-binding fragment thereof, conjugated (covalently linked) to a linker (L) via a chemical moiety (Z), conjugated to an Amanita phalloides toxin moiety (Ama), and wherein n is from about 1, 2, 3, 4 to about 5, 6, 7, 8, or from about 5, 6, 7 to about 9, 10, or n is 1, 2, 3, 4, 5, 6, 7, 8, preferably n is between 1, 2 to 3, 3.5 or 4, more preferably n is about 2.

[0050] According to some embodiments, the first ADC and / or the second ADC used according to the present invention comprises an Amanita phalloides toxin (Ama) of formula (A)

[0051]

[0052] or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, diastereomer or enantiomer thereof, wherein:

[0053] Y is -S-, -S(=O)- or -SO2-;

[0054] R is H, -OH or -OLZ-Ab; and

[0055] X is H or -LZ-Ab, wherein:

[0056] L is the connector;

[0057] Ab is an antibody or antigen-binding fragment thereof as described herein;

[0058] Z is a chemical moiety formed by a coupling reaction between a first reactive substituent previously bound to L and a second reactive substituent previously present in the antibody or antigen-binding fragment thereof;

[0059] The conditions are:

[0060] If X is H, then R is -OLZ-AB, and

[0061] If X is -LZ-Ab, then R is H or -OH.

[0062] As used herein, the term "antibody" shall refer to a protein consisting of one or more polypeptide chains encoded by immunoglobulin genes or immunoglobulin gene fragments or cDNAs derived therefrom. The immunoglobulin genes include light chain kappa, lambda and heavy chain alpha, delta, epsilon, gamma and mu constant region genes and any of a number of different variable region genes.

[0063] The basic immunoglobulin (antibody) structural unit is usually a tetramer containing two pairs of identical polypeptide chains: a light chain (L, with a molecular weight of about 25 kDa) and a heavy chain (H, with a molecular weight of about 50 kDa-70 kDa). Each heavy chain contains a heavy chain variable region (abbreviated as VH or V H ) and the heavy chain constant region (abbreviated as CH or C H The heavy chain constant region consists of three domains, namely CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated as VL or V L ) and the light chain constant region (abbreviated as CL or C L ). The VH and VL regions can be further subdivided into hypervariable regions, which are also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region contains three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains form a binding domain that interacts with an antigen.

[0064] CDR is the most important for the binding of the antibody or its antigen binding portion. FR can be replaced by other sequences, provided that the three-dimensional structure required for antigen binding is retained. Structural changes in the construct most often result in the loss of sufficient binding to the antigen.

[0065] The term "antigen-binding portion" of a (monoclonal) antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the CD20 antigen in its native form. Examples of antigen-binding portions of antibodies include Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; and dAb fragments consisting of a VH domain and an isolated complementarity determining region (CDR).

[0066] The antibody or antibody fragment or antibody derivative according to the present invention may be a monoclonal antibody. The antibody may be an antibody of the IgA, IgD, IgE, IgG or IgM isotype.

[0067] As used herein, the term "monoclonal antibody" ("mAb") refers to a preparation of an antibody molecule of a single specificity. A monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having a variable region and a constant region derived from or based on a human germline immunoglobulin sequence or derived from a completely synthetic sequence. The method for preparing the monoclonal antibody is independent of the binding specificity. Preferably, such an antibody is selected from the group consisting of IgG, IgD, IgE, IgA and / or IgM, or a fragment or derivative thereof, more preferably, such an antibody is an IgG-type antibody or a fragment or derivative thereof.

[0068] As used herein, the term "fragment" shall refer to fragments of such antibodies that retain target binding ability, such as CDR (complementarity determining region), hypervariable region, variable domain (Fv), IgG heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions), IgG light chain (consisting of VL and CL regions) and / or Fab and / or F(ab)2.

[0069] As used herein, the term "antigen binding fragment" shall refer to a protein construct that is structurally different from the common antibody concept but still has some structural relationship with the common antibody concept, such as a protein comprising a peptide scaffold and at least one CDR of the original antibody derived therefrom. Examples include, for example, scFv, Fab and / or F(ab)2, and bispecific, trispecific or higher specific antibody constructs. All of these items are explained below.

[0070] In some embodiments, the antibody of the first ADC and / or the second ADC used according to the present invention can be, for example, a diabody, a camelid antibody, a domain antibody, a bivalent homodimer having two chains consisting of scFv, IgA (two IgG structures connected by a J chain and a secretory component), a shark antibody, or an antibody consisting of a New World primate framework plus a non-New World primate CDR, a dimerization construct comprising CH3+VL+VH, or other scaffold protein forms comprising CDRs, and antibody conjugates (e.g., antibodies or fragments or derivatives thereof, linked to drugs, toxins, cytokines, aptamers, nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), therapeutic polypeptides, radioisotopes or labels). The scaffold protein form can include, for example, antibody-like proteins such as ankyrin and affilin proteins, and others, which are conjugated, for example, to at least 1, 2, 3, 4, 5, 6, 7 or 8 Amanita toxin molecules or Amanita toxin-linker molecules as disclosed herein. For example, the corresponding ADC used according to the present invention as disclosed above may comprise about 1, 2, 3 to about 4, 5, 6, 7, 8, or about 4, 5, 6 to about 8, 9, 10, or about 8 to about 10 Amanita toxicity molecules, preferably linked to said ADC via a linker (L) as disclosed herein.

[0071] Antibodies are usually expressed by their cognate antigen (K D )10 -5 M to 10 -11 A dissociation constant of M or less reflects high affinity binding. D Approximately 10 -4 M is generally considered to refer to non-specific binding. As used herein, an ADC or the antibody portion of a corresponding ADC that "specifically binds" to an antigen is 10 -7 M or less, preferably 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 The specific binding of the antibody or antibody-portion contained in the ADC used in the method of the present invention can be determined, for example, by surface plasmon resonance analysis (Malmqvist M., "Surface plasmonresonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr Opin Immunol. 1993 April; 5(2): 282-6).

[0072] In the context of the present application, the term "linker" (L) refers to a molecule that increases the distance between the two components, for example, to mitigate spatial interference between the target binding moiety and the amanita toxin, which otherwise may reduce the ability of the amanita toxin to interact with RNA polymerase II. The linker can be used for another purpose because it can promote the specific release of the amanita toxin in cells targeted by the target binding moiety. Preferably, the linker and the bond between the linker and the amanita toxin on one side and the bond between the linker and the target binding moiety or antibody on the other side are stable under physiological conditions outside the cell (e.g., blood), while it can be intracellular, particularly cleaved in target cells (e.g., cancer cells). In order to provide this selective stability, the linker can include a preferably pH-sensitive or protease-sensitive functional group. Alternatively, the bond connecting the linker to the target binding moiety can provide selective stability. Preferably, the linker has a length of at least 1 atom, preferably 1-30 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms), wherein one side of the linker has been reacted with the Amanita phalloidin toxin and the other side has been reacted with the target binding moiety. In the context of the present invention, the linker is preferably C 1-30 -alkyl, C 1-30 -heteroalkyl, C 2-30 -Alkenyl, C 2-30 -heteroalkenyl, C 2-30 -Alkynyl, C 2-30 -heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl groups, which are optionally substituted. The joint may include one or more structural elements, such as amides, esters, ethers, thioethers, disulfides, hydrocarbon moieties, etc. The joint may also include a combination of two or more of these structural elements. Each of these structural elements may be present in the joint more than once, for example twice, three times, four times, five times or six times. In some embodiments, the joint may include a disulfide bond. It should be understood that the joint must be attached to the amanita toxin and the target binding moiety in a single step or in two or more subsequent steps. For this reason, the joint will carry two groups, preferably two groups at the proximal and distal ends, which can (i) form a covalent bond with a group on the amanita toxin or target binding peptide, preferably an activated group, or (ii) be activated or can be activated to form a covalent bond with a group on the amanita toxin. Thus, if a linker is present, it is preferred that chemical groups are located at the distal and proximal ends of the linker which are the result of such a coupling reaction, such as esters, ethers, carbamates, peptide bonds, etc. In some embodiments disclosed above, the presence of a "linker" is optional, i.e., the Amanita toxin can be directly linked to a residue of an antibody or antibody fragment.

[0073] Covalent attachment of antibodies and Amanita toxins according to the invention requires that the linker has two reactive functional groups, i.e., bivalency in the sense of the reaction. Bivalent linker reagents useful for attaching two or more functional or biologically active moieties (such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups) are known, and methods for their production of conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242).

[0074] Thus, the linker herein has two reactive ends, one for conjugation to the antibody and the other for conjugation to the amanita ani toxin. The antibody conjugation reactive end of the linker (the reactive moiety, defined herein as Z') is typically a chemical moiety capable of conjugation to the antibody through, for example, a cysteine ​​thiol or lysine amine group on the antibody, and is thus typically a thiol-reactive group such as a Michael acceptor (as in maleimide), a leaving group such as a chloro, bromo, iodo or R-sulfanyl group, or an amine-reactive group such as a carboxyl group.

[0075] The amanita toxin conjugation reactive end of the linker is generally a chemical moiety capable of conjugating to the amanita toxin by forming a bond with a reactive substituent within the amanita toxin molecule. Non-limiting examples include, for example, forming an amide bond with a basic amine or carboxyl group on the amanita toxin via a carboxyl or basic amine group on the linker, respectively, or forming an ether via alkylation of an OH group on the amanita toxin (e.g., via a leaving group on the linker), etc.

[0076] When the term "linker" (L) is used to describe the linker in a conjugated form, due to the formation of bonds between the linker and / or amanita toxicity toxin and between the linker and / or antibody or antigen-binding fragment thereof, one or both reactive termini will be absent (such as a reactive moiety Z' that has been converted to a chemical moiety Z, as described below) or incomplete (such as only a carbonyl of a carboxylic acid).

[0077] A variety of linkers can be used to conjugate the described antibodies, antigen binding fragments and ligands to cytotoxic molecules. Generally, linkers suitable for the present disclosure should be stable in circulation, but allow the release of amanita toxicants in or immediately adjacent to target cells. Linkers suitable for the present disclosure can be broadly classified as non-cleavable or cleavable, each of which is further described below.

[0078] The linker of the amanita toxin-ADC that can be used in the present invention is preferably stable outside the cell, prevents the aggregation of the ADC molecules, and keeps the ADC soluble and monomeric in aqueous media. Prior to transport or delivery into the cell, the ADC is preferably stable and remains intact (i.e., the antibody remains linked to the amanita toxin outside the target cell and can be cleaved at a certain effective rate inside the cell).

[0079] In some embodiments, the first ADC and / or the second ADC used according to the method of the present invention comprises a cleavable linker or a non-cleavable linker. The term "non-cleavable linker" refers to a linker comprising a chemical bond that is resistant to degradation (e.g., proteolysis). Typically, non-cleavable linkers require proteolytic degradation in target cells and exhibit high extracellular stability. Non-cleavable linkers suitable for use herein may also include one or more groups selected from the following: bond, -(C=O)-, C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene and combinations thereof, each of which may be optionally substituted and / or may include one or more heteroatoms (e.g., S, N or O) to replace one or more carbon atoms. Non-limiting examples of such groups include (CH2) p 、(C=O)(CH2) p and polyethylene glycol (PEG; (CH2CH2O) p ) units, wherein p is an integer from 1 to 6, selected independently in each case.

[0080] In some embodiments, the non-cleavable linker L comprises one or more of the following: a bond, -(C=O)-, a -C(O)NH- group, a -OC(O)NH- group, a C1-C6 alkylene, a C1-C6 heteroalkylene, a C2-C6 alkenylene, a C2-C6 heteroalkenylene, a C2-C6 alkynylene, a C2-C6 heteroalkynylene, a C3-C6 cycloalkylene, a heterocycloalkylene, an arylene, a heteroarylene, -(CH2CH2O) p-group, wherein p is an integer from 1 to 6, or a solubility enhancing group; wherein each C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene or heteroarylene can be optionally substituted with 1 to 5 substituents, each of which is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, thiol and nitro.

[0081] In some embodiments, each C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene or heteroarylene of a non-cleavable linker as disclosed herein can be optionally interrupted by one or more heteroatoms selected from O, S and N.

[0082] In some embodiments, each C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene or heteroarylene of a non-cleavable linker as disclosed herein may be optionally interrupted by one or more heteroatoms selected from O, S and N, and may be optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, thiol and nitro.

[0083] In some embodiments, a non-cleavable linker according to the invention comprises -(CH2) n -unit, wherein n is an integer from 2 to 6, 7, 8, 9, 10, 11, 12, preferably n is an integer from 2 to 6, 7, 8, more preferably n is an integer from about 2 to 6, such as 2, 3, 4, 5, 6, and even more preferably, the non-cleavable linker comprises -(CH2) n -, wherein n is 6. In some embodiments, the non-cleavable linker is -(CH2) n -, where n is 6, is represented by the following formula:

[0084]

[0085] In some embodiments, the non-cleavable linker of the first ADC and / or the second ADC used according to the present invention as disclosed herein further comprises a thiol-reactive group before being conjugated to an antibody. The thiol-reactive group of the non-cleavable linker as disclosed above can be, for example, selected from bromoacetamide, iodoacetamide, methanesulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazine-2-ylamino group, methanesulfonylphenyltetrazolyl or methanesulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate or maleimide, preferably the thiol-reactive group is maleimide (maleimido moiety). For example, the non-cleavable linker comprising the maleimide can have the following structure before being conjugated to the corresponding antibody, wherein the wavy line at the end of the linker indicates the attachment point with Amanita phalloidin (Ama):

[0086]

[0087] wherein n is an integer of 2-12, for example n is 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably wherein n is an integer of 2-6, for example n is 1, 2, 3, 4, 5 or 6, more preferably n is 6.

[0088] Upon conjugation to a reactive thiol group on an antibody or a reactive disulfide bond (e.g., a naturally occurring cysteine ​​residue) of an antibody or, e.g., an interchain disulfide bond or an engineered cysteine ​​residue, the maleimido moiety, e.g., a cleavable linker or a non-cleavable linker as disclosed herein, comprises the following structure:

[0089]

[0090] Wherein the wavy line indicates the attachment site of a cleavable linker or a non-cleavable linker (L) as disclosed herein and the sulfur atom is part of a reactive cysteine ​​of the antibody.

[0091] According to a preferred embodiment, the first ADC and / or the second ADC used according to the invention comprises a cleavable linker or a non-cleavable linker as disclosed herein and it further comprises a thiol-reactive group as disclosed herein which can be coupled to a thiol moiety or a disulfide bond naturally present in an antibody, or said cleavable linker or the non-cleavable linker of the first ADC and / or the second ADC used according to the invention comprises a thiol-reactive group which can be coupled to a thiol moiety which has been introduced into an antibody by genetic engineering as described in, e.g., Nat Biotechnol. 2008 Aug; 26(8):925-32, or WO2006 / 034488A2. Preferably, the cleavable linker or non-cleavable linker of the first and / or second ADC used as disclosed herein comprises a thiol-reactive group coupled to a thiol moiety which has been introduced by genetic engineering such as, for example, D265C (according to EU numbering, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)) into the Fc region of the corresponding antibody of the first and / or second ADC used according to the invention.

[0092] According to some embodiments, the linker of the first ADC and / or the second ADC used according to the methods of the invention as disclosed herein is attached to at least one amatoxin via (i) the γC atom of amino acid 1 of amatoxin, or (ii) the δC atom of amino acid 3 of amatoxin, or (iii) the 6′C atom of amino acid 4 of amatoxin.

[0093] In one embodiment, the first ADC and / or the second ADC used according to the invention comprises a conjugate as described herein comprising an Amanita toxin comprising (i) amino acid 4 having a 6'-deoxy position and (ii) amino acid 8 having an S-deoxy position.

[0094] In some embodiments, the linker of the first ADC and / or the second ADC used in the methods of the present invention is a cleavable linker. The term "cleavable linker" is understood to include at least one cleavage site. As used herein, the term "cleavage site" shall refer to a portion that is susceptible to specific cleavage at a defined position under specific conditions. The conditions are, for example, specific enzymes or reducing environments in specific body or cell compartments.

[0095] According to some embodiments, the cleavage site of the linker is an enzyme cleavable portion comprising two or more amino acids. Preferably, the enzyme cleavable portion comprises valine-alanine (Val-Ala), valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg) dipeptide, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or alanine-alanine-proline-valine (Ala Ala Pro Val) peptide, or β-glucuronide or β-galactoside.

[0096] According to some embodiments, the cleavage site is cleavable by at least one protease selected from the group consisting of a cysteine ​​protease, a metalloprotease, a serine protease, a threonine protease, and an aspartic protease.

[0097] Cysteine ​​proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism involving nucleophilic cysteine ​​thiols in a catalytic triad or dyad.

[0098] Metalloproteinases are proteases whose catalytic mechanism involves a metal. Most metalloproteinases require zinc, but some use cobalt. The metal ion is coordinated to the protein via three ligands. The ligands that coordinate the metal ion can vary from histidine, glutamic acid, aspartic acid, lysine, and arginine. The fourth coordination position is occupied by a labile water molecule.

[0099] Serine proteases are enzymes that cleave peptide bonds in proteins; serine serves as the nucleophilic amino acid at the active site of the enzyme. Serine proteases are divided into two major classes based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

[0100] Threonine proteases are a family of proteolytic enzymes that contain a threonine (Thr) residue within the active site. The prototypical member of this class of enzymes is the catalytic subunit of the proteasome, however, acyltransferases have convergently evolved the same active site geometry and mechanism.

[0101] Aspartic proteases are a type of catalytic protease that uses activated water molecules bound to one or more aspartic acid residues to catalyze its peptide substrate. Typically, they have two highly conserved aspartic acid residues in the active site and are optimally active at acidic pH. Almost all known aspartyl proteases are inhibited by pepsin inhibitors.

[0102] In some embodiments of the invention, the cleavable site is cleavable by at least one agent selected from the group consisting of cathepsin A or B, matrix metalloproteinases (MMPs), elastases, β-glucuronidases, and β-galactosidases.

[0103] In some embodiments of the invention, the cleavage site is a disulfide bond and the specific cleavage is performed by a reducing environment, e.g., an intracellular reducing environment, such as, e.g., acidic pH conditions.

[0104] Suitable cleavable linkers include those that can be cleaved by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference when it relates to linkers suitable for covalent conjugation). Suitable cleavable linkers can include, for example, chemical moieties such as hydrazines, disulfides, thioethers, or dipeptides.

[0105] Linkers that can be hydrolyzed under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, and the like. See, for example, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661, the disclosure of each of which is incorporated herein by reference in its entirety when it relates to a linker suitable for covalent conjugation. Such a linker is relatively stable under neutral pH conditions (such as under neutral pH conditions in blood), but is unstable below pH 5.5 or 5.0 (a suitable pH for lysosomes).

[0106] Linkers that can be cleaved under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene), SPDB and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47: 5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer CW Vogel, ed., Oxford U. Press, 1987). See also US Pat. No. 4,880,935, the disclosure of each of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation.

[0107] The linker that is susceptible to enzymatic hydrolysis can be, for example, a peptide-containing linker that is cleaved by an intracellular peptidase or protease (including but not limited to lysosomal or endosomal proteases). An advantage of using intracellular proteolytic release of therapeutic agents is that, when conjugated, the agent is usually weakened, and the serum stability of the conjugate is usually high. In some embodiments, the length of the peptidyl linker is at least two amino acids or at least three amino acids. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides or pentapeptides. Examples of suitable peptides include peptides containing the following amino acids: such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine and glycine. The amino acid residues that constitute the amino acid linker component include naturally occurring amino acid residues, as well as a small amount of amino acids and non-naturally occurring amino acid analogs, such as citrulline. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker includes a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit. Linkers comprising dipeptides such as Val-Cit or Phe-Lys are disclosed in, for example, U.S. Pat. No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation. In some embodiments, the linker includes a dipeptide selected from Val-Ala and Val-Cit.

[0108] Linkers suitable for conjugating the antibody portion or antigen-binding fragment of the first ADC and / or the second ADC of the present invention to a cytotoxic molecule include linkers capable of releasing amanita phalloidin by a 1,6-elimination process. Chemical moieties capable of such an elimination process include p-aminobenzyl (PAB) groups, 6-maleimidocaproic acid, pH-sensitive carbonates, and other agents as described in Jain et al., Pharm. Res. 32: 3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation.

[0109] In some embodiments, the cleavable linker disclosed herein includes a "self-immolative" group, such as the aforementioned PAB or PABC (p-aminobenzyloxycarbonyl), which is disclosed in, for example, Carl et al., J. Med. Chem. (1981) 24: 479-480; Chakravarty et al. (1983) J. Med. Chem. 26: 638-644; US Other such chemical moieties capable of undergoing this process ("self-immolative linkers") include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed in, for example, U.S. Patent Publication Nos. 20160303254 and 20150079114 and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al. (2001) J. Org. Chem. 66:8815-8830; and US 7223837. In some embodiments, a dipeptide is used in combination with a self-immolative linker.

[0110] In some embodiments, the linker L comprises one or more of the following: hydrazine, disulfide, thioether, amino acid, peptide consisting of up to 10 amino acids, p-aminobenzyl (PAB) group, heterocyclic self-immolative group, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -(C=O)- group, -C(O)NH- group, -OC(O)NH- group, -(CH2CH2O) p-group, wherein p is an integer from 1 to 6, or a solubility enhancing group; wherein each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be optionally substituted with 1 to 5 substituents, each of which is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, thiol and nitro.

[0111] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be optionally interrupted by one or more heteroatoms selected from O, S and N.

[0112] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S and N, and may be optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, thiol and nitro.

[0113] In some embodiments, the linker includes a p-aminobenzyl group (PAB). In one embodiment, the p-aminobenzyl group is disposed between the cytotoxic drug and the protease cleavage site in the linker. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzylamido unit.

[0114] According to a preferred embodiment, the cleavable linker of the first ADC and / or the second ADC as disclosed herein comprises a dipeptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit and Val-Arg.

[0115] According to particularly preferred embodiments, the linker comprises one or more of the following: PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB or Ala-PAB.

[0116] In a particularly preferred embodiment, the cleavable linker according to the invention comprises a dipeptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit and Val-Cit, in particular wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and the amanita ani toxin:

[0117]

[0118]

[0119] Thus, the first ADC and / or the second ADC of the method of the invention as disclosed herein may comprise a cleavable linker comprising any of the dipeptide-PAB moieties Phe-Lys-PAB, Val-Lys-PAB, Phe-Ala-PAB, Val-Ala-PAB, Phe-Cit-PAB or Val-Cit-PAB as disclosed above, preferably, the cleavable linker of the first ADC and / or the second ADC of the method of the invention comprises the dipeptide-PAB moiety Val-Ala-PAB:

[0120]

[0121] The PAB part is connected to Amanita phalloides toxin.

[0122] According to some embodiments, the cleavable linker as disclosed herein comprises a thiol-reactive group prior to conjugation to the antibody, the thiol-reactive group being selected from bromoacetamide, iodoacetamide, methanesulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazin-2-ylamino group, methanesulfonylphenyltetrazole or methanesulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate or maleimide.

[0123] According to a preferred embodiment, the thiol-reactive group is a maleimide (maleimido moiety) as depicted below:

[0124]

[0125] Linkers (e.g., cleavable linkers and / or non-cleavable linkers) comprising such thiol-reactive groups are particularly useful for covalently coupling linker-amanitin conjugates as disclosed herein to antibodies comprising a reactive thiol, such as, for example, cysteine-engineered antibodies comprising at least one reactive cysteine ​​residue for coupling, as disclosed in Junutula et al. (2008) Nat Biotechnology Vol. 26: 925-932.

[0126] According to a preferred embodiment, the cleavable linker of the present invention comprises the following structure (i) before coupling or (ii) after coupling with an antibody (Ab) as disclosed herein, wherein (ama) indicates the attachment site to Amanita toxin and Ab indicates the attachment site to the antibody:

[0127]

[0128] According to a particularly preferred embodiment, the non-cleavable linker of the first ADC and / or the second ADC used in the method of the invention comprises the following structure prior to conjugation to the antibody of the first ADC and / or the second ADC used in the method of the invention ("ncl-1"):

[0129]

[0130] According to a particularly preferred embodiment, the non-cleavable linker of the first ADC and / or the second ADC of the present invention comprises structure (Z), wherein (ama) indicates the side of the linker attached to the amanita ani toxin, and Ab indicates the side of the linker attached to the antibody, wherein the sulfur atom is part of the antibody, such as a genetically introduced cysteine ​​residue, or a naturally occurring sulfur atom, such as those involved in an interchain disulfide bond:

[0131]

[0132] In some embodiments, the first ADC and / or the second ADC used in the methods of the invention comprises Amanita phalloides toxin (Ama) according to formula (A1):

[0133]

[0134] or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, diastereomer or enantiomer thereof, wherein R is H or OH, and wherein Y is -S-, -S(=O)-, L is a cleavable linker or a non-cleavable linker as disclosed herein, and Z is as disclosed herein.

[0135] In some embodiments, the first ADC and / or the second ADC of formula (A) for use in the methods of the present invention has formula (A2):

[0136]

[0137] or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, diastereomer or enantiomer thereof, wherein Y, L, Z' and Ab are as defined above, preferably, Y is -S- or -(S=O)-.

[0138] In some embodiments, the first ADC and / or the second ADC of formula (A) used according to the present invention has formula (A2):

[0139]

[0140] or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, diastereomer or enantiomer thereof, wherein L, Z and Ab are as defined above, and Y is -S-.

[0141] In some embodiments, the amanita anitoxin-linker conjugate Ama-L used in the first ADC and / or the second ADC of the method of the invention is selected from the group comprising compounds of Formula (I) to Formula (XI):

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] For example, the amanita toxicity-linker conjugate Ama-L used in the first ADC of the method of the present invention is selected from the above compounds (I)-(XI), and the second ADC used in the method of the present invention is selected from the above compounds (I)-(XI). In one example, the amanita toxicity-linker conjugate Ama-L used in the first ADC of the method of the present invention is selected from the compound (I), (II) or (III), and the second ADC used in the method of the present invention is selected from the compound (I), (II) or (III).

[0148] According to some embodiments, the amanita toxicity-linker conjugate Ama-L used in the first ADC of the method of the present invention is the same as the amanita toxicity-linker conjugate Ama-L used in the second ADC of the method of the present invention, and both are selected from any one of the above compounds (I)-(XII). For example, the amanita toxicity-linker conjugate Ama-L used in the first ADC and the second ADC of the method of the present invention is selected from compound (I), (II) or (III).

[0149] According to some embodiments, the amanita ani toxin-linker conjugate Ama-L of the first ADC used according to the method of the invention is the same as the amanita ani toxin-linker conjugate Ama-L of the second ADC used according to the method of the invention, both of which are selected from compounds (I), (II) or (III) as disclosed above.

[0150] In some embodiments, a first ADC used in the methods of the invention as disclosed herein may, for example, include a different amanita anitoxin-linker conjugate AMA-L than a second ADC used in the methods of the invention.

[0151] For example, a first ADC used in the methods of the invention may include one of the AMA-L conjugates or compounds listed on the left, while a second ADC used in the methods of the invention includes an AMA-L conjugate or compound selected from the right column:

[0152] AMA-L included in the first ADC AMA-L included in the second ADC (I) (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) (II) (I), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) (III) (I), (II), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) (IV) (I), (II), (III), (V), (VI), (VII), (VIII), (IX), (X), (XI) (V) (I), (II), (III), (IV), (VI), (VII), (VIII), (IX), (X), (XI) (VI) (I), (II), (III), (IV), (V), (VII), (VIII), (IX), (X), (XI) (VII) (I), (II), (III), (IV), (V), (VI), (VIII), (IX), (X), (XI) (VIII) (I), (II), (III), (IV), (V), (VI), (VII), (IX), (X), (XI) (IX) (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (X), (XI) (X) (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (XI) (XI) (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X)

[0153] According to a preferred embodiment, the first ADC used in the method of the present invention comprises an amanita toxin-linker conjugate Ama-L according to formula (I), and the second ADC used in the method of the present invention comprises an amanita toxin-linker conjugate Ama-L according to any one of formulas (I), (II), (III) or (IX).

[0154] According to a preferred embodiment, the first ADC used in the method of the present invention comprises an amanita toxin-linker conjugate Ama-L according to formula (II), and the second ADC used in the method of the present invention comprises an amanita toxin-linker conjugate Ama-L according to any one of formulas (I), (II), (III) or (IX).

[0155] According to a preferred embodiment, the first ADC used in the method of the present invention comprises an amanita toxin-linker conjugate Ama-L according to formula (III), and the second ADC used in the method of the present invention comprises an amanita toxin-linker conjugate Ama-L according to any one of formulas (I), (II), (III) or (IX).

[0156] According to a more preferred embodiment, the first ADC used in the method of the invention comprises an amanita toxin-linker conjugate Ama-L according to formula (I), and the second ADC used in the method of the invention comprises an amanita toxin-linker conjugate Ama-L according to one of formulas (I) or (II).

[0157] According to a more preferred embodiment, the first ADC used in the method of the invention comprises an amanita toxin-linker conjugate Ama-L according to formula (II), and the second ADC used in the method of the invention comprises an amanita toxin-linker conjugate Ama-L according to one of formulas (I) or (II).

[0158] According to a more preferred embodiment, the first ADC used in the method of the invention comprises an amanita toxin-linker conjugate Ama-L according to formula (III), and the second ADC used in the method of the invention comprises an amanita toxin-linker conjugate Ama-L according to one of formulas (I) or (II).

[0159] According to a preferred embodiment, the first ADC and / or the second ADC used in the method of the invention as disclosed herein comprises a compound according to any one of formulae (XII) to (XXII) as disclosed below, wherein "antibody" refers to the antibody portion of the first ADC and / or the second ADC of the method of the invention:

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] n is about 1, 2, 3, 4 to about 5, 6, 7, 8, or about 5, 6, 7 to about 9, 10, or n is 1, 2, 3, 4, 5, 6, 7, 8, preferably n is between 1 and 4, such as 1, 2, 3, 4, more preferably n is about 1.5, 2.5 to about 3, 3.5, more preferably n is about 1.5 to about 2.5, or preferably n is about 2. The term "antibody" as used in any of formulas (XII) to (XXII) shall indicate conjugation with a suitable antibody, such as an antibody that binds to the desired antigen (cell surface antigen, tumor-associated antigen or tumor-specific antigen). It should be understood that the inventive method as disclosed herein is applicable to any Amanita phalloides toxin-based ADC comprising an Amanita phalloides toxin linker moiety according to any of formulas (I) to (XI), which Amanita phalloides toxin linker moiety has a corresponding structure as depicted in formulas (XII) to (XXII) after conjugation.

[0166] According to one embodiment, the first ADC used in the method of the present invention is selected from an ADC according to any one of formulas (XII) to (XXII), and the second ADC used in the method of the present invention is selected from an ADC according to any one of formulas (XII) to (XXII). For example, the first ADC and the second ADC used in the method of the present invention may include the following combination:

[0167]

[0168] According to some embodiments, the first ADC and the second ADC used in the method of the present invention may be the same ADC, or may be different ADCs, however, preferably, the first ADC and the second ADC used in the method of the present invention are the same ADC.

[0169] According to a preferred embodiment, the first ADC used in the method of the invention comprises an ADC according to formula (XII), and the second ADC used in the method of the invention comprises an ADC according to one of formulas (XII), (XIV), (XV) or (XX).

[0170] According to a preferred embodiment, the first ADC used in the method of the invention comprises an ADC according to formula (XIV), and the second ADC used in the method of the invention comprises an ADC according to one of formulas (XII), (XIV), (XV) or (XX).

[0171] According to a preferred embodiment, the first ADC used in the method of the invention comprises an ADC according to formula (XV), and the second ADC used in the method of the invention comprises an ADC according to one of formulas (XII), (XIV), (XV) or (XX).

[0172] According to a preferred embodiment, the first ADC used in the method of the invention comprises an ADC according to formula (XX), and the second ADC used in the method of the invention comprises an ADC according to one of formulas (XII), (XIV), (XV) or (XX).

[0173] According to a preferred embodiment, the first ADC used in the method of the invention comprises an ADC according to formula (XX), and the second ADC used in the method of the invention comprises an ADC according to formula (XII).

[0174] According to a preferred embodiment, the first ADC used in the method of the invention comprises an ADC according to formula (XX), and the second ADC used in the method of the invention comprises an ADC according to formula (XIV), (XV) or (XX).

[0175] According to a preferred embodiment, the first ADC used in the method of the invention comprises an ADC according to formula (XX), and the second ADC used in the method of the invention comprises an ADC according to formula (XV) or (XX).

[0176] According to a preferred embodiment, the first ADC used in the method of the present invention comprises an ADC according to formula (XX), and the second ADC used in the method of the present invention comprises an ADC according to formula (XX).

[0177] According to some embodiments, the first dose of ADC of the method of the invention as disclosed herein does not specifically bind to a cell surface antigen, a tumor associated antigen or a tumor specific antigen of the patient. As used herein, the term "cell surface antigen" refers to a glycan, peptide or protein on the surface of a cell.

[0178] The term "tumor-associated antigen" (TAA) as used in the present invention refers to a protein or polypeptide antigen expressed by a tumor cell. For example, a TAA can be one or more surface proteins or polypeptides, nucleoproteins or glycoproteins or fragments thereof expressed by a tumor cell. For example, human tumor-associated antigens include differentiation antigens (such as melanocyte differentiation antigens), mutant antigens (such as p53), overexpressed cellular antigens (such as HER2), viral antigens (such as human papillomavirus proteins), and cancer / testis (CT) antigens, which are expressed in germ cells of the testis and ovary, but are silent in normal somatic cells (such as MAGE and NY-ESO-1). Many TAAs are not cancer or tumor specific and can also be found on normal tissues and include, for example, CEA, MAGE, MUC1, Survivin, WT1, RNF43, TOMM34, VEGFR-1, VEGFR-2, KOC1, ART4, KRas, EpCAM, HER-2, COA-1SAP, TGF-βRII, p53, ASCL2, IL13Rα2, ASCL2, NY-ESO-1, MAGE-A3, PRAME and SART 1-3, CD37, CD20, CD19, BCMA (CD269) or PSMA.

[0179] The term "tumor-specific antigen" (TSA) as used in the present invention refers to a peptide library displayed on the surface of tumor cells and can be specifically recognized by a new antigen-specific T cell receptor (TCR) in the context of a major histocompatibility complex (MHC) molecule. TSA can also be referred to as "tumor neoantigens" in the context of the present invention. From an immunological point of view, tumor neoantigens are truly exogenous proteins and are completely absent in normal human organs / tissues. For most human tumors without viral etiology, tumor neoantigens can, for example, be derived from a variety of non-synonymous genetic changes, including single nucleotide variants (SNVs), insertions and deletions (indels), gene fusions, frameshift mutations, and structural variants (SVs). The term "tumor-specific antigen" (TSA) as used in accordance with the present invention also includes tumor virus antigens, such as antigens of human papillomavirus or Merkel cell polyomavirus (MCPyV). Typically, tumor virus antigens are only found to be expressed on cells infected by the corresponding virus. Tumor neoantigens can be identified using computer prediction tools known in the art, such as Trends in Molecular Medicine, November 2019, disclosed in pages 980-992.

[0180] According to some embodiments, the second ADC of the methods of the invention as disclosed herein administered to said patient suffering from a cancer as disclosed herein specifically binds to a cell surface antigen, a tumor associated antigen or a tumor specific antigen as disclosed herein of said patient.

[0181] According to one embodiment, the first antibody-drug conjugate and / or the second antibody-drug conjugate used according to the present invention (e.g., as disclosed herein) comprises a chimeric antibody. The term "chimeric antibody" refers to a portion of each amino acid sequence of the heavy chain and the light chain that is homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific category, and the remaining segment of the chain is homologous to the corresponding sequence in another species or category. Those antibodies. Typically, the variable regions of both the light chain and the heavy chain simulate the variable regions of antibodies derived from a mammalian species, while the constant portion is homologous to the antibody sequence derived from another mammalian species. An obvious advantage of such chimeric forms is that the variable region can be conveniently derived from currently known sources, using readily available B cells or hybridomas from non-human host organisms, combined with constant regions derived from, for example, human cell products. Although the variable region has the advantage of being easy to prepare and the specificity is not affected by the source, when the antibody is injected, the constant region of the human being is less likely to induce an immune response from a human subject than the constant region from a non-human source.

[0182] According to a preferred embodiment, the first antibody-drug conjugate and / or the second antibody-drug conjugate for the method of the present invention comprises a humanized antibody. A "humanized" antibody refers to an antibody containing a sequence derived from a non-human immunoglobulin to a minimum extent. Therefore, a "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a sequence derived from a non-human antibody to a minimum extent. All or substantially all of the framework (FW) regions may also be those with human immunoglobulin sequences. Humanized antibodies may also contain at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art and have been described in, for example, Riechmann et al., Nature 332:323-7, 1988; U.S. Patent No. 5,530,101; No. 5,585,089; No. 5,693,761; No. 5,693,762 and No. 6,180,370.

[0183] According to a preferred embodiment, the first antibody-drug conjugate and / or the second antibody-drug conjugate of the present invention comprise human antibodies. The term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutations in vitro or site-specific mutagenesis or during gene rearrangement or by somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species (such as mice) have been grafted onto human framework sequences. Human antibodies can be produced in human cells (e.g., by recombinant expression) or by non-human animals or prokaryotic cells or eukaryotic cells that can express functionally rearranged human immunoglobulin (such as heavy chain and / or light chain) genes. When a human antibody is a single-chain antibody, it may include a linker peptide that is not present in a natural human antibody. For example, Fv may contain a linker peptide connecting the variable region of a heavy chain and the variable region of a light chain, such as two to about eight glycine or other amino acid residues. Such a linker peptide is considered to be of human origin. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes (see, e.g., PCT Publication No. WO1998 / 24893; No. WO1992 / 01047; No. WO1996 / 34096; No. WO1996 / 33735; U.S. Patent No. 5,413,923; No. 5,625,126; No. 5,633,425; No. 5,569,825; No. 5,661,016; No. 5,545,806; No. 5,814,318; No. 5,885,793; No. 5,916,771; and No. 5,939,598). In some embodiments, the human antibody of the first antibody-drug conjugate and / or the second antibody-drug conjugate is selected from IgG1, IgG2 or IgG4 isotypes, preferably, the human antibody of the first antibody-drug conjugate and / or the second antibody-drug conjugate is selected from IgG1 isotypes. Therefore, the antibody of the first ADC and / or the second ADC of the method of the present invention is preferably a monoclonal antibody. Monoclonal antibodies can be obtained, for example, by recombinant expression in eukaryotic organisms (preferably yeast) or plant cells, more preferably human cells. The method for making recombinant antibodies can be carried out as described in Frenzel et al. Front Immunol. 2013; 4: 217, or, for example, Nat Protoc. 2018 January; 13 (1): 99-117.

[0184] According to some embodiments, the antibody of the first ADC and / or the second ADC used in the method of the invention comprises at least one amino acid substitution in its Fc region. For example, the antibodies of the first ADC and / or the second ADC of the method of the invention comprise in their Fc region at least one of the following amino acid substitutions from the following group: L234A, L234G, L234T, L234Q, L234H, L235A, L235D, L235G, L235H, L235V, L236I, L236N, L236P, L236R, L236G, G237R, G237S, G237T, G237D, G237I, P238A, P238G, P238S, P238T, D265C, P329A, P329G, P329I, P329L, P329M, P329T (according to the EU numbering system, which may also be referred to as the "EU index as in Kabat" and refers to human IgG1 EU antibody numbering, which refers to the EU antibody numbering of Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85). The corresponding amino acid substitution can be accomplished by genetic engineering, which involves the modification of the amino acid sequence or part thereof of a given or natural polypeptide or protein in the sense of nucleotide and / or amino acid substitution, insertion, deletion or reversal or any combination thereof by gene technology methods, such as, for example, site-directed mutagenesis as described in Biochem. J. (1986) 237, 1-7, or J Biol Chem. 2015 Jan 30; 290 (5): 2577–2592. As used herein, the term "amino acid substitution" refers to the modification of the amino acid sequence of a protein, in which one or more amino acids are replaced by the same number of different amino acids, resulting in a protein containing an amino acid sequence different from the original protein. Conservative amino acid substitutions are understood to involve substitutions that do not significantly affect the structure and function of the protein due to similar size, charge, polarity and / or conformation. The conservative group of amino acids in this sense represents, for example, the nonpolar amino acids Gly, Ala, Val, Ile and Leu; the aromatic amino acids Phe, Trp and Tyr; the positively charged amino acids Lys, Arg and His; and the negatively charged amino acids Asp and Glu.

[0185] According to a preferred embodiment, the antibody of the first ADC and / or the second ADC used in the method of the invention comprises at least two amino acid substitutions in its Fc region selected from the group comprising: L234A / L235A, L234A / L235D, L234A / L235G, L234A / L235H, L234A / L235V, L234G / L235A, L234G / L235D, L234G / L235G, L234G / L235H, L234G / L235V, L234T / L235A, L234T / L235D, L234T / L235G, L234T / L235H, L234T / L235V. 235V, L234A / G237R, L234A / G237S, L234A / G237T, L234A / G237D, L234A / G2 37I, L234G / G237R, L234A / G237S, L234A / G237T, L234A / G237D, L234A / G23 7I, L234T / G237R, L234T / G237S, L234T / G237T, L234T / G237D, L234T / G237 I. L234A / P238A, L234A / P238G, L234G / P238A, L234G / P238G, L234T / P238A , L234T / P238G, L234A / D265C, L234A / D265C, L234A / D265C, L234A / D265C, L234G / D265C, L234G / D265C, L234T / D265C, L234T / D265C, L234T / D265C, L234T / D265C, L235A / D265C, L235G / D265C, L235V / D265C (according to EU numbering), more preferably, at least two amino acid substitutions in the Fc region of the antibody of the first ADC and / or the second ADC of the method of the invention are selected from the group comprising: L234A / L235A / D265C, L234A / L235C, L234A / L235C, L234G / D265C, L234G / D265C, L234T / D265C, L234T / D265C, L234T / D265C, L235A / D265C, L235G / D265C, L235V / D265C. 35D / D265C, L234A / L235G / D265C, L234A / L235H / D265C, L234A / L235V / D26 5C, L234G / L235A / D265C, L234G / L235D / D265C, L234G / L235G / D265C, L234 G / L235H / D265C, L234G / L235V / D265C, L234T / L235A / D265C, L234T / L235D / D265C, L234T / L235G / D265C, L234T / L235H / D265C, L234T / L235V / D265C,More preferably, it is selected from the group consisting of L234A / L235A / D265C, L234A / L235D / D265C, L234A / L235G / D265C, L234G / L235A / D265C, L234G / L235D / D265C, L234G / L235G / D265C, L234T / L235A / D265C, L234T / L235D / D265C, L234T / L235G / D265C.

[0186] According to a more preferred embodiment, the Fc region of the antibody of the first ADC and / or the second ADC used in the method of the invention comprises at least three amino acid substitutions selected from the following group: L234A / L235A / D265C, L234G / L235A / D265C or L234T / L235A / D265C (according to the EU numbering system), most preferably, the Fc region of the antibody of the first ADC and / or the second ADC of the method of the invention as disclosed herein comprises the amino acid substitutions L234A / L235A / D265C.

[0187] In the method of the invention, an ADC comprising a cysteine ​​engineered Fc region and comprising, for example, an amino acid substitution D265C (numbered according to the EU system) is particularly useful for coupling an amanita phalloidins-linker conjugate comprising a thiol-reactive moiety Z' for coupling the linker-amanitin toxin conjugate to an antibody, wherein Z' may be selected from bromoacetamide, iodoacetamide, methanesulfonylbenzothiazole, 4,6-dichloro-1,3,5-triazine-2-ylamino groups, methanesulfonylphenyltetrazolyl or methanesulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonate, or preferably maleimide. The coupling of a construct comprising Z'-L-amanitin toxin may be performed, for example, as disclosed in WO2016 / 142049A1. The use of an antibody comprising a cysteine ​​engineered Fc region (e.g., comprising an amino acid substitution D265C) to prepare the first ADC and / or the second ADC of the method of the present invention is advantageous for obtaining site-specific antibody-drug conjugates with high yield and homogeneity. In addition, amino acid substitutions such as L234A / L235A, L234G / L235A, L234T / L235A reduce the binding of the first ADC and / or the second ADC of the method of the present invention to FcγRI, II, II, thereby reducing the effector function of the ADC, which is antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). The higher purity of the resulting ADC (e.g., a conjugate with a controlled drug to antibody ratio (DAR) of about DAR=2) and the reduction of the effector function both further improve the therapeutic index (TI) of the ADC that can be obtained by the method of the present invention.

[0188] Including additional amino acid substitutions in addition to D265C in the Fc region of the antibody of the first ADC and / or the second ADC (such as, for example, L234A / L235A, L234G / L235A, L234T / L235A) can be particularly advantageous because these mutations reduce the binding of the first ADC and / or the second ADC of the method of the invention to FcγRI, II, II, thereby reducing the effector function of the first ADC and / or the second ADC used. As used herein, the term "effector function" refers here to antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), as well as complement-dependent cytotoxicity (CDC).

[0189] It will be appreciated that where the first ADC is different from the second ADC (e.g. does not bind a tumor antigen, or a cell surface antigen, or a tumor specific antigen), the methods of the invention to increase the therapeutic index of the ADC are advantageous for increasing the TI of the second ADC used in the methods of the invention. It will also be appreciated that the methods of the invention are particularly useful for increasing the TI of ADCs based on amanita phalloides toxin.

[0190] According to one embodiment, the first ADC according to the method of the invention as disclosed above is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 to about 17, 16, 15 days prior to administration of the second dose of the second antibody-drug conjugate, preferably, between about 23 days, 21 days to about 20 days, 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days prior to administration of the second dose of the second antibody-drug conjugate.

[0191] In some embodiments, the second ADC can be administered more than once, for example, according to the treatment plan. For example, after treatment according to the method of the invention, the second ADC can be administered once a week, or once every 10, 12, 14, 16, 18, 20, 21, 28 days, or once a month, twice a month, etc.

[0192] According to one embodiment, the first dose of the first antibody-drug conjugate of the method of the present invention can be administered at about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second antibody-drug conjugate, preferably, the first antibody-drug conjugate is administered at about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100% of the therapeutically effective dose of the second antibody-drug conjugate, or about 85%, 90% to about 95%, 100%. As used herein, the term "therapeutically effective dose" or "therapeutic dose" is an amount sufficient to achieve the desired clinical outcome (i.e., to achieve therapeutic efficacy). The therapeutically effective dose can be administered in one or more administrations. For the purposes of the present invention, the first ADC and / or the second ADC of the method of the present invention, preferably the therapeutically effective dose of the second ADC of the method of the present invention is an amount sufficient to alleviate, improve, stabilize, reverse, prevent, slow down or delay the progression of a disease state (e.g., cancer).

[0193] According to one aspect, the present invention relates to the general structure Ab-(ZL-Ama) as disclosed herein nThe antibody-drug conjugate of the present invention for use in the method of the present invention as disclosed herein. Therefore, the present invention relates to an antibody-drug conjugate according to any one of formula (XII)-(XXII) for use in the method of the present invention, wherein n is about 1, 2, 3, 4 to about 5, 6, 7, 8, or about 5, 6, 7 to about 9, 10, or n is 1, 2, 3, 4, 5, 6, 7, 8, preferably n is between 1 and 4, such as 1, 2, 3, 4, more preferably n is about 1.5, 2.5 to about 3, 3.5, more preferably n is about 1.5 to about 2.5, or preferably n is about 2, as disclosed herein for use in the method of the present invention.

[0194] In one aspect, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC comprises an amanita tin-linker according to any one of formulae (I) to (XI), and wherein the second ADC comprises an amanita tin-linker according to any one of formulae (I) to (XI) as disclosed above, and wherein the first ADC used in accordance with the method of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used in accordance with the method of the invention, preferably between about 15, 28, 26, 24, 23 days to about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used in accordance with the method of the invention. The method of the present invention is to administer the first ADC of the present invention at about 23 days, 21 days to about 20 days, 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days before the administration of the second dose of the second antibody-drug conjugate, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC.

[0195] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC comprises an amanita toxic toxin-linker according to formula (I), and wherein the second ADC comprises an amanita toxic toxin-linker according to any one of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) or (XI) as disclosed above, and wherein the first ADC used in accordance with the methods of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18, 19, 18 days to about 21, 20 ... The method of the present invention is to administer the first ADC of the present invention to a subject comprising administering ...

[0196] In one embodiment, the first ADC and the second ADC for treating cancer are provided in two separate doses, wherein the first dose of the first ADC is a non-therapeutic dose, and wherein the dose of the second ADC is a therapeutic dose, preferably, the first dose of the first ADC used according to the present invention is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first and second doses of the first and second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0197] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC comprises an amanita ani toxin-linker according to formula (II), and wherein the second ADC comprises an amanita ani toxin-linker according to any one of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) or (XI) as disclosed above, and wherein the first ADC used in accordance with the methods of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18, 19, 18 days prior to administration of the second dose of the second antibody-drug conjugate used in accordance with the methods of the invention. The method of the present invention is to administer the first ADC to about 17, 16, 15 days, preferably between about 23, 21 to about 20, 19, 18 days, or 23, 22, 21, 20, 19, 18 days prior to the administration of the second dose of the second antibody-drug conjugate, wherein the first dose of the first ADC used in accordance with the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC.

[0198] In one embodiment, the first ADC and the second ADC for treating cancer are provided in two separate doses, wherein the first dose of the first ADC is a non-therapeutic dose, and wherein the dose of the second ADC is a therapeutic dose, preferably, the first dose of the first ADC used according to the present invention is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first and second doses of the first and second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0199] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC comprises an amanita ani toxin-linker according to formula (III), and wherein the second ADC comprises an amanita ani toxin-linker according to any one of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) or (XI) as disclosed above, and wherein the first ADC used in accordance with the methods of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18, 19, 18 days prior to administration of the second dose of the second antibody-drug conjugate used in accordance with the methods of the invention. The method of the present invention is to administer the first ADC to about 17, 16, 15 days, preferably between about 23, 21 to about 20, 19, 18 days, or 23, 22, 21, 20, 19, 18 days prior to the administration of the second dose of the second antibody-drug conjugate, wherein the first dose of the first ADC used in accordance with the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC.

[0200] In one embodiment, the first ADC and the second ADC for treating cancer are provided in two separate doses, wherein the first dose of the first ADC is a non-therapeutic dose, and wherein the dose of the second ADC is a therapeutic dose, preferably, the first dose of the first ADC used according to the present invention is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first and second doses of the first and second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0201] In one aspect, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to any one of formulae (XII) to (XXII), e.g., (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), and wherein the second ADC is according to any one of formulae (XII) to (XXII) as disclosed above, e.g., (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), and wherein the first ADC used according to the method of the invention is administered from about 30, 28, 26, 24, 23 days to about 21 days prior to administration of the second dose of the second antibody-drug conjugate used according to the method of the invention. The invention relates to a method of administering a first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC of the invention to a subject comprising administering the first ADC

[0202] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XII), and wherein the second ADC is according to any one of formulas (XII) to (XXII) as disclosed above, e.g., (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), and wherein the first ADC used in accordance with the methods of the invention is between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or between about 21, 20, 19, 18, 17, 16, 15 days prior to administration of the second dose of the second antibody-drug conjugate used in accordance with the methods of the invention. The method of the present invention is to administer the first ADC to a patient of the invention within 15 days of administration, preferably between about 23 days, 21 days to about 20 days, 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days prior to administration of the second dose of the second antibody-drug conjugate, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0203] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XIV), and wherein the second ADC is according to any one of formulas (XII) to (XXII) as disclosed above, e.g., (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), and wherein the first ADC used in accordance with the methods of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or between about 21, 20, 19, 18, 17, 16, 15 days prior to administration of the second dose of the second antibody-drug conjugate used in accordance with the methods of the invention. The method of the present invention is to administer the first ADC to a patient of the invention within 15 days of administration, preferably between about 23 days, 21 days to about 20 days, 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days prior to administration of the second dose of the second antibody-drug conjugate, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0204] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XX), and wherein the second ADC is according to any one of formulas (XII) to (XXII) as disclosed above, e.g., (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), and wherein the first ADC used in the methods of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or between about 21, 20, 19, 18, 17, 16, 15 days prior to administration of the second dose of the second antibody-drug conjugate used in the methods of the invention. The method of the present invention is to administer the first ADC to a patient of the invention within 15 days, preferably between about 23 days, 21 days to about 20 days, 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days prior to administration of the second dose of the second antibody-drug conjugate, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0205] For example, the present invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XX), and wherein the second ADC is according to formula (XII) as disclosed above, and wherein the first ADC used according to the method of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used according to the method of the invention, preferably about 23 days, 21 days to about 20 days prior to administration of the second dose of the second antibody-drug conjugate , 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0206] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XX), and wherein the second ADC is according to formula (XIV) as disclosed above, and wherein the first ADC used according to the methods of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used according to the methods of the invention, preferably about 23 days, 21 days to about 20 days prior to administration of the second dose of the second antibody-drug conjugate , 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0207] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XX), and wherein the second ADC is according to formula (XX) as disclosed above, and wherein the first ADC used according to the method of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used according to the method of the invention, preferably about 23 days, 21 days to about 20 days prior to administration of the second dose of the second antibody-drug conjugate. The invention relates to a method of administering the first ADC to a patient of the invention within 1 to 4 days, 19 days, 18 days, or between 23 days, 22 days, 21 days, 20 days, 19 days, or 18 days, wherein the first dose of the first ADC used according to the method of the invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0208] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XII), and wherein the second ADC is according to formula (XII) as disclosed above, and wherein the first ADC used according to the method of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used according to the method of the invention, preferably about 23 days, 21 days to about 20 days prior to administration of the second dose of the second antibody-drug conjugate. , 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0209] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XII), and wherein the second ADC is according to formula (XX) as disclosed above, and wherein the first ADC used according to the method of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used according to the method of the invention, preferably about 23 days, 21 days to about 20 days prior to administration of the second dose of the second antibody-drug conjugate , 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0210] For example, the present invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XII), and wherein the second ADC is according to formula (XIV) as disclosed above, and wherein the first ADC used according to the method of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used according to the method of the invention, preferably about 23 days, 21 days to about 20 days prior to administration of the second dose of the second antibody-drug conjugate , 19 days, 18 days, or 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, wherein the first dose of the first ADC used according to the method of the present invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably, the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0211] For example, the invention relates to a first ADC and a second ADC for use in cancer treatment, wherein the first ADC is according to formula (XIV), and wherein the second ADC is according to formula (XIV) as disclosed above, and wherein the first ADC used according to the methods of the invention is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of a second dose of a second antibody-drug conjugate used according to the methods of the invention, preferably about 23 days, 21 days to about 20 days prior to administration of the second dose of the second antibody-drug conjugate. The invention relates to a method for treating a tumor in a patient with a tumor-associated antigen (TAA) comprising administering the first ADC to a patient on day 1, day 2, day 19, day 18, or day 23, day 22, day 21, day 20, day 19, day 18, wherein the first dose of the first ADC used according to the method of the invention is about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second ADC, preferably the dose of the first ADC is about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the second dose of the second ADC, wherein the antibody binds to any tumor associated antigen (TAA) as disclosed above.

[0212] According to a preferred embodiment, the first ADC and the second ADC for treating cancer comprise an ADC according to formula (I) as disclosed in WO2018 / 115466A1, wherein the antibody "J22.9-ISY-D265C" represents an anti-BCMA antibody comprising a heavy chain amino acid sequence according to SEQ ID NO: 1 and a light chain amino acid sequence according to SEQ ID NO: 2, as disclosed in WO2018 / 115466:

[0213]

[0214] Wherein the cancer is selected from the group comprising: multiple myeloma, diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), in particular multiple myeloma. Thus, the first ADC and the second ADC used according to the present invention are the same ADC, wherein the first dose of the ADC is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first dose and the second dose of the first ADC and the second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0215] According to a preferred embodiment, the first ADC and the second ADC for treating cancer comprise an anti-CD37 ADC as disclosed in WO 2022 / 194988A2, which consists of an anti-CD37 antibody chHH1-HDPLALA-D266C comprising a heavy chain amino acid sequence according to SEQ ID NO: 11 and a light chain amino acid sequence according to SEQ ID NO: 12, wherein the ADC is one of formula (XII), (XIV), (XV) or (XX), and wherein the cancer is selected from non-Hodgkin lymphoma (NHL), follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma (DBNHL), a subtype of non-Hodgkin lymphoma including mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), Richter syndrome (Richter syndrome), and the like. syndrome), primary cutaneous marginal zone lymphoma (PCMZL), hairy cell leukemia, acute myeloid leukemia (AML), rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis, and pemphigus vulgaris. Thus, the first ADC and the second ADC for use in treating cancer according to the present invention are the same ADC, wherein the first dose of the ADC is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first dose and the second dose of the first ADC and the second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days, or 35 days apart from each other.

[0216] According to a preferred embodiment, the first ADC and the second ADC for treating cancer comprise an anti-PSMA ADC as disclosed in WO 2020 / 025564, wherein the antibody of the ADC is one of 3-F11-var1, 3-F11-var2, 3-F11-var3, 3-F11-var4, 3-F11-var5, 3-F11-var6, 3-F11-var7, 3-F11-var8, 3-F11-var9, 3-F11-var10, 3-F11-var11, 3-F11-var12, 3-F11-var13, 3-F11-var14, 3-F11-var115 or 3-F11-var16, and wherein the anti-PSMA ADC has a structure according to one of formula (XII), (XIV), (XV) or (XX), wherein the cancer is selected from the group comprising prostate cancer, castration-resistant prostate cancer. Thus, the first ADC and the second ADC for use in treating cancer according to the present invention are the same ADC, wherein the first dose of the ADC is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first and second doses of the first and second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0217] For example, the first ADC and the second ADC for treating cancer include an anti-PSMA ADC as disclosed in WO 2020 / 025564, wherein the antibody of the ADC is one of 3-F11-var1, 3-F11-var2, 3-F11-var3, 3-F11-var4, 3-F11-var5, 3-F11-var6, 3-F11-var7, 3-F11-var8, 3-F11-var9, 3-F11-var10, 3-F11-var11, 3-F11-var12, 3-F11-var13, 3-F11-var14, 3-F11-var115 or 3-F11-var16, and wherein the anti-PSMA ADC has a structure according to formula (XIV), and wherein the cancer is selected from the group including prostate cancer, castration-resistant prostate cancer. Thus, the first ADC and the second ADC for use in treating cancer according to the present invention are the same ADC, wherein the first dose of the ADC is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first and second doses of the first and second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0218] For example, the first ADC and the second ADC for treating cancer include an anti-PSMA ADC as disclosed in WO 2020 / 025564, wherein the antibody of the ADC is one of 3-F11-var1, 3-F11-var2, 3-F11-var3, 3-F11-var4, 3-F11-var5, 3-F11-var6, 3-F11-var7, 3-F11-var8, 3-F11-var9, 3-F11-var10, 3-F11-var11, 3-F11-var12, 3-F11-var13, 3-F11-var14, 3-F11-var115 or 3-F11-var16, and wherein the anti-PSMA ADC has a structure according to formula (XX), and wherein the cancer is selected from the group including prostate cancer, castration-resistant prostate cancer. Thus, the first ADC and the second ADC for use in treating cancer according to the present invention are the same ADC, wherein the first dose of the ADC is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first and second doses of the first and second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0219] For example, the first ADC and the second ADC for treating cancer include an anti-PSMA ADC as disclosed in WO 2020 / 025564, wherein the antibody of the ADC is one of 3-F11-var1, 3-F11-var2, 3-F11-var3, 3-F11-var4, 3-F11-var5, 3-F11-var6, 3-F11-var7, 3-F11-var8, 3-F11-var9, 3-F11-var10, 3-F11-var11, 3-F11-var12, 3-F11-var13, 3-F11-var14, 3-F11-var115 or 3-F11-var16, and wherein the anti-PSMA ADC has a structure according to formula (XII), and wherein the cancer is selected from the group including prostate cancer, castration-resistant prostate cancer. Thus, the first ADC and the second ADC for use in treating cancer according to the present invention are the same ADC, wherein the first dose of the ADC is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 50%, 55%, 60% to about 65%, 75%, 80%, or about 65%, 70%, 75% to about 80%, 85% of the amount of the second dose, wherein the first and second doses of the first and second ADC used according to the present invention are administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days or 35 days apart from each other.

[0220] In some aspects, the methods of the invention as disclosed herein comprise repeat administration of a second Amanita ani toxin-based ADC as disclosed herein (e.g., a second Amanita ani toxin-based ADC for use in the methods of the invention as disclosed herein) after the second dose, wherein any subsequent therapeutic dose is administered at least 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 28 days, or 35 days after the second or any subsequent dose. Example

[0221] Although the invention has been described and illustrated in detail in the drawings and the foregoing description, such illustration and description are to be considered illustrative or exemplary rather than restrictive; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments may be understood and effected by those skilled in the art in practicing the claimed invention by studying the drawings, the present disclosure and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude more than one. The mere fact that certain means are recited in mutually different dependent claims does not indicate that a combination of these means cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0222] Example 1: Mouse studies

[0223] Synthesis of antibody-drug conjugates based on amanita phalloides toxin

[0224] Anti-BCMA, anti-PSMA and anti-digoxin (DIG) antibodies were produced by Heidelberg Pharma Research GmbH (HDP) as disclosed in WO2018 / 115466A1 and WO2020 / 216947A1. Cysteine-reactive amanitin-linker constructs synthesized at HDP were site-specifically conjugated to cysteine-engineered antibodies as described in WO2016 / 142049A1. Based on LC-MS analysis, the drug-antibody ratio (DAR) was ~2.0 amanitin / IgG.

[0225] Mouse tolerance study

[0226] Female CB17 SCID mice were treated with 10 mg / kg or 25 mg / kg of anti-DIG ATAC or PBS as a single intravenous (iv) dose on day 0. On day 21, all mice were treated with 40 mg / kg or 50 mg / kg of the same amanita phalloides-based ADC.

[0227] Efficacy studies in the LNCaP prostate xenograft model

[0228] 2.5×10 6 LNCaP cells (Horoszewicz et al., Cancer Res. 1983 Apr;43(4):1809-18) were implanted sc into male CB17 SCID mice. 3 When the mean tumor volume reached 1.34 μg / mL, mice were randomized into groups of 15 mice and treated iv with PBS or amanita phalloides-based anti-PSMA ADC as a single dose or in a q7dx4 treatment schedule.

[0229] The effect of repeated dosing on the tolerability of an Amanita phalloidin-based ADC was investigated in immunocompromised mice. Previously, an anti-digoxin (DIG) Amanita phalloidin-based ADC conjugate was found to be tolerated up to 30 mg / kg as a single dose in naive mice treated iv. Based on this finding, mice were pretreated with a tolerable dose of 10 mg / kg or 25 mg / kg of an anti-DIG Amanita phalloidin-based ADC and then 21 days later with a second, higher dose of 40 mg / kg or 50 mg / kg of the same ADC. The mice were analyzed for survival and macroscopic liver changes ( Figure 2 ).

[0230] result

[0231] Pretreatment with a tolerating dose of an anti-DIG amanita anitoxin-based ADC improved the tolerability of a higher dose of the same conjugate, which is toxic if applied as a single dose in naive animals, as evidenced by fewer macroscopic liver changes and improved survival in pretreated animals.

[0232] Example 2: Hepatotoxicity study in cynomolgus monkeys

[0233] Nonhuman primates are the most relevant species for toxicity testing of Amanita phalloides toxin-based ADCs accepted by US and European authorities. Therefore, toxicity was assessed in cynomolgus monkeys.

[0234] Studies in cynomolgus monkeys were conducted at LPT or AltaSciences. Figure 3 Monkeys aged 2 to 4 years were treated with a single dose, multiple doses or ascending dose treatment regimens of amatoxin (referred to as "Amanita phalloides toxin-ADC" in the literature) via 30-minute iv infusion. Blood was collected before dosing, 3 days, 7 days, 14 days and 21 days after treatment. LDH, AST and ALT levels in serum were measured using the method recommended by IFCC.

[0235] result

[0236] Treatment with amanita phalloides toxin-ADC resulted in decreased levels of liver injury markers ALT, AST, and LDH in cynomolgus monkeys if applied after the second, third, or fourth round of dose escalation treatments compared to the single treatment and the first treatment, respectively. Figure 3 Depicted in D. The data suggest that multiple dose treatment also increases the tolerability of the amanita phalloides toxin-based ADC in cynomolgus monkeys.

[0237] Example 3: Multiple dosing increases the anti-tumor efficacy of Amanita phalloides toxin-based ADCs

[0238] In view of the results of Examples 1 and 2 (indicating that repeated treatments improved the tolerability of Amanita cerebroside toxin-based ADCs in mice and cynomolgus monkeys), it was investigated whether multiple administrations affected the anti-tumor efficacy of Amanita cerebroside toxin-based ADCs.

[0239] according to Figure 4 Mice bearing subcutaneous (sc) human prostate adenocarcinoma LNCaP tumors were treated with a single or repeated dose (q7dx4) of an amanita ani toxin-based anti-PSMA ADC according to the treatment schedule depicted in A. Figure 4 Tumor growth and survival were analyzed as shown in B.

[0240] The results showed that multiple dosing (q7dx4) according to the method of the present invention significantly improved the anti-tumor effect of the Amanita phalloides toxin-based anti-PSMA ADC in the sc human prostate adenocarcinoma LNCaP CDX model compared to single-dose treatment, as demonstrated by reduced tumor growth and improved mouse survival.

[0241] Example 4: Conjugation of Amanita Toxin-Linker Payload

[0242] The antibody was conjugated to an amanita phalloidin linker conjugate by the so-called Thiomab technology. In this method, the conjugation occurs via the conjugation of the maleimide residue of the toxin linker construct to the free SH group of a cysteine ​​residue in the antibody, as shown in the following reaction scheme:

[0243]

[0244] The principle of this conjugation method is disclosed in Junutula et al. (2008) Nat Biotechnology Vol. 26: 925-932.

[0245] The antibody used in this experiment contains a D265C substitution in both Fc domains to provide a cysteine ​​residue with such a free SH group. The corresponding technology is disclosed in WO2016 / 142049A1, the contents of which are incorporated herein by reference, and produces a homogeneous product with a fixed drug to antibody ratio ("DAR") of 2 and site-specific conjugation.

[0246] References

[0247] Carl et al., J. Med. Chem. (1981) 24: 479-480

[0248] Chakravarty et al (1983) J. Med. Chem. 26: 638-644

[0249] "Compendium of Chemical Terminology" published by the International Union of Pure and Applied Chemistry, ISBN: 0-9678550-9-8.

[0250] de Groot et al (2001) J. Org. Chem. 66: 8815-8830

[0251] Dubowchik and Walker,1999,Pharm.Therapeutics 83:67-123

[0252] Edelman,G.M.et al.,Proc.Natl.Acad.USA,63,78-85(1969)

[0253] Hay et al.(1999)Bioorg.Med.Chem.Lett.9:2237

[0254] Hermanson,G.T.(1996)Bioconjugate Techniques;Academic Press:New York,p.234-242)

[0255] Jain et al.,Pharm.Res.32:3526-3540,2015

[0256] Junutula et al(2008)Nat Biotechnology Vol.26:925-932.

[0257] Leriche et al.,Bioorg.Med.Chem.,20:571-582,2012

[0258] Malmqvist M.,Curr Opin Immunol.1993 Apr;5(2):282-6

[0259] Neville et al.,1989,Biol.Chem.264:14653-14661

[0260] Thorpe et al.,1987,Cancer Res.47:5924-5931

[0261] Voss et al.BMC Molecular Biology 2014,15:7

[0262] Wawrzynczak et al.,In Immunoconjugates:Antibody Conjugates inRadioimagery and

[0263] Therapy of Cancer(C.W.Vogel ed.,Oxford U.Press,1987

[0264] Wieland,T.and Faulstich H.(Wieland T,Faulstich H.,CRC Crit RevBiochem 5(1978)185-260)。

Claims

1. A method for improving the therapeutic index of an antibody-drug conjugate, wherein the method comprises the following steps: A first dose of a first antibody-drug conjugate is administered to a patient suffering from cancer, wherein the first dose of the first antibody-drug conjugate is administered between about 30 days to about 15 days prior to administration of a second dose of a second antibody-drug conjugate.

2. The method of claim 1, wherein the first dose and the second dose comprise the same antibody-drug conjugate.

3. The method of claim 1, wherein the first dose and the second dose comprise different antibody-drug conjugates.

4. The method of any one of claims 1-3, wherein the first dose and the second dose of the ADC comprise an RNA polymerase II inhibitor.

5. The method according to claim 4, wherein the RNA polymerase II inhibitor is selected from the group of Amanita toxins.

6. The method according to claim 5, wherein the first antibody-drug conjugate and / or the second antibody-drug conjugate has the structure <h2 style=";text-align:left;direction:ltr">Ab-(ZL-Ama)<h2 style=";text-align:left;direction:ltr"> n <h2 style=";text-align:left;direction:ltr"> , wherein the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently linked) to a linker (L) via a chemical moiety (Z), conjugated to an Amanita phalloides toxin moiety (Ama), and n is from about 1, 2, 3, 4 to about 5, 6, 7, 8, or from about 5, 6, 7 to about 9, 10, or n is 1, 2, 3, 4, 5, 6, 7, 8, preferably n is between 1 and 4, more preferably n is about 2. The method according to claim 6 , wherein the linker is a cleavable linker or a non-cleavable linker.

8. The method according to claim 6 or claim 7, wherein the amanita ani toxin-linker conjugate Ama-L is selected from the group comprising conjugates (I) to (XI):

9. The method of claim 8, wherein the first ADC and / or the second ADC comprises a compound according to any one of formula (XII) to formula (XXII): wherein n is from about 1, 2, 3, 4 to about 5, 6, 7, 8, or from about 5, 6, 7 to about 9, 10, or n is 1, 2, 3, 4, 5, 6, 7, 8, preferably n is between 1 and 4, such as 1, 2, 3, 4, more preferably n is from about 1.5, 2.5 to about 3, 3.5, more preferably n is from about 1.5 to about 2.5, or preferably n is about 2.

10. The method of any one of claims 1-9, wherein the first dose of the antibody-drug conjugate does not specifically bind to a cell surface antigen, a tumor associated antigen, or a tumor antigen of the patient.

11. The method of any one of claims 1-10, wherein the second dose of the ADC specifically binds to a cell surface antigen, a tumor associated antigen, or a tumor antigen of the patient.

12. The method of any one of claims 1-11, wherein the first antibody-drug conjugate and / or the second antibody-drug conjugate comprises a chimeric antibody.

13. The method of any one of claims 1-11, wherein the first antibody-drug conjugate and / or the second antibody-drug conjugate comprises a humanized antibody.

14. The method of any one of claims 1-11, wherein the first antibody-drug conjugate and / or the second antibody-drug conjugate comprises a human antibody.

15. The method of claim 14, wherein the first antibody and / or the second antibody is selected from the IgG1, IgG2 or IgG4 isotype.

16. The method of claim 15, wherein the first antibody and / or the second antibody is of IgGl isotype. The method according to claim 16 , wherein the first antibody and / or the second antibody is a recombinant antibody.

18. The method of claim 17, wherein the first antibody and / or the second antibody comprises at least one amino acid substitution in the Fc region thereof.

19. The method of claim 18, wherein the first antibody and / or the second antibody comprises at least one amino acid substitution selected from the group consisting of L234A, L234G, L234T, L234Q, L234H, L235A, L235D, L235G, L235H, L235V, L236I, L236N, L236P, L236R, L236G, G237R, G237S, G237T, G237D, G237I, P238A, P238G, P238S, P238T, D265C, P329A, P329G, P329I, P329L, P329M, P329T (according to EU numbering).

20. The method of claim 19, wherein the first antibody and / or the second antibody comprises at least two amino acid substitutions selected from the group consisting of: L234A / L235A, L234A / L235D, L234A / L235G, L234A / L235H, L234A / L235V, L234G / L235A, L234G / L235D, L234G / L235G, L234G / L235H , L234G / L235V, L234T / L235A, L234T / L235D, L234T / L235G, L234T / L235H, L234T / L235V, L234A / G237R, L234A / G237S, L234A / G237T, L234A / G237D, L234A / G237I, L234G / G237R, L234A / G237S, L2 34A / G237T, L234A / G237D, L234A / G237I, L234T / G237R, L234T / G237S, L234T / G237T, L234T / G23 7D, L234T / G237I, L234A / P238A, L234A / P238G, L234G / P238A, L234G / P238G, L234T / P238A, L234T / P238G、L234A / D265C、L234A / D265C、L234A / D265C、L234G / D265C、L234G / D265C、L234G / D265C、 L234T / D265C, L234T / D265C, L234T / D265C, L235A / D265C, L235G / D265C, L235V / D265C (according to EU number).

21. The method of claim 20, wherein the first antibody and / or the second antibody comprises at least three amino acid substitutions selected from the group consisting of: L234A / L235A / D265C, L234A / L235D / D265C, L234A / L235G / D265C, L234A / L235H / D265C, L234A / L235V / D265C, L234G / L235A ...D / D265C, L234A / L235G / D265C, L234A / L235H / D265C, L234A / L235V / D265C, L234G / L235A / D265C, L234A / L235 234G / L235D / D265C, L234G / L235G / D265C, L234G / L235H / D265C, L234G / L235V / D265C, L234T / L23 5A / D265C, L234T / L235D / D265C, L234T / L235G / D265C, L234T / L235H / D265C, L234T / L235V / D265C.

22. The method of claim 21, wherein the first antibody and / or the second antibody comprises at least three amino acid substitutions selected from the group consisting of L234A / L235A / D265C, L234A / L235D / D265C, L234A / L235G / D265C, L234G / L235A / D265C, L234G / L235D / D265C, L234G / L235G / D265C, L234T / L235A / D265C, L234T / L235D / D265C, L234T / L235G / D265C.

23. The method of claim 22, wherein the first antibody and / or the second antibody comprises the amino acid substitutions L234A / L235A / D265C, L234G / L235A / D265C or L234T / L235A / D265C (according to EU numbering).

24. The method of claim 23, wherein the first antibody and / or the second antibody comprises the amino acid substitutions L234A / L235A / D265C (according to EU numbering).

25. The method of any one of claims 1-24, wherein the first antibody-drug conjugate is administered between about 30, 28, 26, 24, 23 days to about 21, 20, 19, 18, 17, 16, 15 days, or about 21, 20, 19, 18 days to about 17, 16, 15 days prior to administration of the second dose of the second antibody-drug conjugate, preferably, between about 23, 21 to about 20, 19, 18 days, or 23, 22, 21, 20, 19, 18 days prior to administration of the second dose of the second antibody-drug conjugate.

26. The method of any one of claims 1-25, wherein the first antibody-drug conjugate is administered at about 60%, 65%, 70%, 75% to about 80%, 85%, 90%, 100% of the therapeutically effective dose of the second antibody-drug conjugate, preferably, the first antibody-drug conjugate is administered at about 65%, 70% to about 75%, 80%, 85%, 90%, 95%, 100%, or about 85%, 90% to about 95%, 100% of the therapeutically effective dose of the second antibody-drug conjugate.

27. An antibody-drug conjugate according to any one of formula (XII) to formula (XXII) for use in a method for increasing the therapeutic index according to any one of claims 1 to 26 wherein n is from about 1, 2, 3, 4 to about 5, 6, 7, 8, or from about 5, 6, 7 to about 9, 10, or n is 1, 2, 3, 4, 5, 6, 7, 8, preferably n is between 1 and 4, such as 1, 2, 3, 4, more preferably n is from about 1.5, 2.5 to about 3, 3.5, more preferably n is from about 1.5 to about 2.5, or preferably n is about 2.

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