B-lymphocyte specific amatoxin antibody conjugates

Abstract

To provide a conjugate using an anti-CD20 antibody for use in the treatment of B-cell and / or lymphoma associated diseases and / or malignancies.SOLUTION: A conjugate is provided which includes (i) a target-binding moiety, (ii) at least one toxin, and (iii) at least one linker linking the target-binding moiety and the at least one toxin. The target-binding moiety binds to CD20. The at least one toxin is an amatoxin. The target-binding moiety is an antibody which is rituximab or obinutuzumab.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present application relates to a conjugate comprising amatoxin, a target-binding moiety whose target is CD20, i.e., a CD20-binding moiety, and optionally a linker that links the amatoxin and the CD20-binding moiety. The present invention further relates to the synthesis of the conjugate. Furthermore, the present invention particularly relates to a pharmaceutical composition comprising such a conjugate for use in the treatment of B cell and / or lymphoma-related diseases and / or malignancies.

Background Art

[0002] CD20 is a 35-37 kDa non-glycosylated phosphorylated protein embedded in the cell surface membrane, characteristic of certain B cell precursors (pre-B lymphocytes) and mature B lymphocytes. The CD20 antigen is not expressed on plasma cells, hematopoietic stem cells, or early pro-B lymphocytes. No natural CD20 ligand has been identified to date. CD20 plays a role in the development, growth, and differentiation of B cells into plasma cells, and enables an optimal B cell immune response, particularly against T-independent antigens. Some data have shown that CD20 can function as a Ca 2+ concentration-maintaining and B cell-activating Ca 2+ membrane channel (Winiarska et al, 2007).

[0003] The CD20 antigen is a member of the transmembrane 4A protein family. Its structure consists of four transmembrane domains with both the amino and carboxyl termini located intracellularly (thus, CD20 is also called MS4A1 - membrane spanning 4 domain subfamily A, membrane 1). CD20 has two extracellular loops. The smaller one (a 7 - amino - acid segment between the first and second transmembrane regions) probably does not extend beyond the cell membrane. This loop is identical in all members of the MS4A family. The larger loop, a 43 - amino - acid segment between the third and fourth transmembrane regions, has disulfide bonds and is recognized by most anti - CD20 antibodies. Although no tyrosine residues or recognized signal - transduction motifs occur in either cytoplasmic region of the CD20 molecule, there are many consensus sites for phosphorylation of serine and threonine.

[0004] CD20 can exist as dimers and tetramers in complex with at least one additional protein component. The CD20 protein has been reported to be closely associated with the transmembrane adaptor protein p75 / 80 (also called C - terminal src kinase - binding protein Cbp), CD40, and major histocompatibility complex class II proteins (MHC II). The CD20 antigen undergoes conformational changes during B - lymphocyte differentiation, and there are at least two conformational isoforms of CD20 (Winiarska et al, 2007).

[0005] Due to certain characteristics, the CD20 antigen has become an attractive target for monoclonal antibody (mAb) therapy. The CD20 antigen is considered one of the most stable lymphocyte antigens. It does not circulate in plasma as a free protein that can competitively inhibit monoclonal antibodies from binding to lymphoma cells, nor is it released from the surface of CD20 - positive cells after antibody binding. In most in - vivo and in - vitro tests, internalization of the CD20 surface molecule or down - regulation of CD20 expression was not detected.

[0006] The anti-CD20, B cell-specific chimeric monoclonal antibody rituximab was the first monoclonal antibody approved by regulatory authorities for the treatment of various cancers and autoimmune diseases such as non-Hodgkin B cell lymphoma, chronic lymphocytic leukemia, and rheumatoid arthritis. Rituximab has been shown to promote antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of human lymphoid cell lines expressing CD20 (Taylor and Lindorfer, 2008), as well as to directly affect the cell signaling pathway and cell membrane after CD20 binding. A number of events affected by rituximab binding have been identified, including lipid raft modification, activation of kinases and caspases, and effects on transcription factors (Weiner 2010; Bezombes et al, 2011).

[0007] In addition to rituximab, other anti-CD20 antibodies including ibritumomab, tositumomab (both conjugated to radioisotopes), ofatumumab, ocrelizumab, obinutuzumab, and ublituximab have been reported, and all of these are active agents aimed at the treatment of B cell lymphoma, leukemia, and B cell autoimmune diseases (Falchi et al, 2018).

[0008] Rituximab is approved for adults in Europe under the trade name MabThera (Roche) for the following indications: non-Hodgkin lymphoma, NHL (treatment of untreated stage III-IV follicular lymphoma patients in combination with chemotherapy; maintenance therapy for the treatment of follicular lymphoma patients responsive to induction therapy; monotherapy for the treatment of stage III-IV follicular lymphoma patients resistant to chemotherapy or relapsing for the second time or later after chemotherapy; treatment of CD20-positive diffuse large B-cell non-Hodgkin lymphoma patients in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy; chronic lymphocytic leukemia CLL (in combination with chemotherapy for untreated and relapsed / refractory CLL patients), rheumatoid arthritis (in combination with methotrexate for adult patients with severe active rheumatoid arthritis showing insufficient response or intolerance to one or more other disease-modifying anti-rheumatic drugs (DMARDs) including tumor necrosis factor (TNF) inhibitor therapy); granulomatosis with polyangiitis and microscopic polyangiitis (in combination with glucocorticoids for the treatment of adult patients with severe and active granulomatosis with polyangiitis (Wegener's) (GPA) and microscopic polyangiitis (MPA)); and pemphigus vulgaris (for the treatment of moderate to severe pemphigus vulgaris patients) (MabThera, Summary of Product Characteristics).

[0009] However, attempts to use anti-CD20 antibodies for the construction of antibody-drug conjugates (ADCs) have had limited success.

[0010] Negative results using toxin conjugates of anti-CD20 antibodies were reported by Lambert et al. (1985), and it was found that an immunotoxin containing the anti-B1 antibody to CD20 did not show cytotoxicity, in contrast to immunotoxins containing various other monoclonal antibodies of the IgG class that were reactive with three other antigens on human lymphoid cells. The immunotoxins used in this study contained the ribosome-inactivating protein gelonin or three known pokeweed antiviral proteins.

[0011] As disclosed by A.G. Polson of Phillips (2013), CD20 differs from other B cell surface proteins used as targets in that it does not translocate very well internally upon antibody binding and is thus not very suitable as an ADC target. In early studies, anti-CD20 antibodies were considered non-internalizing, and it had already been concluded that such conjugates were not promising. The finding that CD20 is a non-internalizing or poorly internalizing antigen has been widely confirmed in the literature (Press et al, 1989; Vangeepuram et al, 1997; Winiarska et al, 2007; Kim and Kim, 2015; Staudacher and Brown, 2017).

[0012] DiJoseph et al (2007) concluded from studies on non-Hodgkin B cell lymphoma cells that the amide bond conjugate of rituximab does not deliver the toxin calicheamicin, which has cytotoxic activity, to intracellular compartments because there is no intracellular translocation of antibody-bound CD20.

[0013] Other studies have disclosed that an ADC containing an anti-CD20 antibody as the targeting moiety, N(2’)-deacetyl-N(2’)-(3-mercapto-1-oxopropyl)-maytansine (DM1) as the toxin, and a non-cleavable linker was ineffective against CD20-positive target cells, but the corresponding construct with a cleavable linker produced some cytotoxic effect against such cells (Polson et al, 2009). Law et al (2004) found different effects of CD20-targeted ADCs depending on the toxin used: an anti-CD20 antibody conjugated to doxorubicin (Dox) did not deliver the drug or demonstrate antitumor activity, but an anti-CD20 antibody-drug conjugate using the anti-mitotic agent monomethyl auristatin E (MMAE) exhibited potent antitumor activity.

[0014] Furthermore, in addition to the nature of the toxins used and the nature of the linkers used in conjugates of target-binding moieties to such toxins, the specific properties of particular anti-CD20 antibodies appeared to be relevant for determining the therapeutic efficacy of such antibody conjugates by recent studies. So-called "type II" CD20-specific antibodies have been shown to be poorly internalized by CD20-positive target cells, whereas other so-called "type I" CD20-specific antibodies have been found to be internalized and degraded to some extent depending on the expression levels of activating and inhibitory FcγRs on the target cells with which they interact. In particular, the inhibitory FcR, FcγRIIb, expressed on certain types of B-cell malignancies has been described as playing an important role in this context. The mechanisms underlying the differential FcγR-mediated internalization responses of type I and type II anti-CD20 monoclonal antibodies have not yet been defined in detail (Boross and Leussen, 2012; Dransfield 2014; Vaughan et al. 2014).

[0015] Internalization of the target is highly desirable in the context of using antibody-drug conjugates (ADCs) for cytotoxic cancer therapy (e.g., Boross and Leussen, 2012). Since the cytotoxic payload typically acts on intracellular targets, internalization of the ADC upon binding to the target is often necessary for the optimal efficacy of the ADC (Kim and Kim, 2015). This can be considered particularly true for amatoxin-based ADCs that contain amanitin and their derivatives (“amatoxins”), as these amatoxins are less hydrophobic than other toxin molecules that have been used for the production of ADCs and thus have a lower ability to penetrate cell membranes compared to diffusible drugs such as MMAE (Staudacher and Brown, 2017). As described above, considering each bias in the literature and the conflicting results of different studies regarding the internalization and cytotoxic effects of anti-CD20 antibodies and ADCs using them, the inventors of the present application did not have an expectation of success regarding significant cytotoxic effects when using anti-CD20 antibodies in the context of antibody-amatoxin conjugates. Amanitin or amatoxin analogs or derivatives have not been used to date for the synthesis and evaluation of ADCs containing a CD20 target-binding moiety.

[0016] Surprisingly, the inventors have found that amatoxin-based ADCs containing anti-CD20 antibodies, or antibody fragments or antibody derivatives linked to amatoxins via non-cleavable or cleavable linkers, exhibit significant cytotoxic effects against CD20-positive target cells in vitro and in vivo. These results were unexpected, especially for amatoxin-based ADCs containing non-cleavable linkers, as it was thought that intracellular degradation within the lysosomal compartment would be required for the release of the active toxin molecule. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0017] Accordingly, in view of the prior art, one object of the present invention is to provide a conjugate comprising a target binding moiety that binds to CD20, at least one amatoxin, and optionally at least one linker that links the target binding moiety to the at least one toxin, which mediates a cytotoxic effect in target cells, as described in the present application.

[0018] One further object of the present invention is to provide a conjugate comprising a target binding moiety that binds to CD20, at least one amatoxin, and optionally at least one linker, wherein the target binding moiety is an antibody that specifically binds to CD20, or an antigen-binding fragment thereof, or an antigen-binding derivative thereof, or an antibody-like protein.

[0019] One further object of the present invention was to provide a pharmaceutical composition comprising such a conjugate.

[0020] A further object of the present invention was to provide a compound for use in a method for the treatment of cancer and autoimmune diseases.

[0021] One further object of the present invention is to provide a conjugate comprising a target binding moiety that binds to CD20, at least one amatoxin, and optionally at least one linker for use in the treatment of B-lymphocyte-related malignancies or B-cell-mediated autoimmune diseases, particularly non-Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, Richter syndrome, rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis and pemphigus vulgaris.

Means for Solving the Problems

[0022] Surprisingly, conjugates comprising a target binding moiety that binds to CD20, at least one amatoxin, and optionally at least one linker, in particular amatoxin-based ADCs comprising an anti-CD20 antibody and a non-cleavable or cleavable linker that links the anti-CD20 antibody or antibody fragment or antibody derivative to the amatoxin, have been found to have a significant cytotoxic effect on CD20-positive target cells in vitro and in vivo. These results were unexpected, particularly for amatoxin-based ADCs comprising non-cleavable linkers, as they are thought to require intracellular degradation within the lysosomal compartment for release of the active toxin molecule.

[0023] These and further objects are achieved by the methods and means described in the independent claims of the present invention. The dependent claims relate to specific embodiments.

[0024] The present invention and its general advantages of the features will be described in detail below.

Brief Description of the Drawings

[0025]

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Mode for Carrying Out the Invention

[0026] Detailed Description of the Invention Before explaining the present invention in detail, it should be understood that since the devices and methods can vary, the present invention is not limited to the specific component parts of the devices described or the process steps of the methods described. Also, it should be understood that the terms used in this specification are for the purpose of describing only specific embodiments and are not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" should be noted to include the singular and / or plural objects unless the context clearly dictates otherwise. Further, it should be understood that when a parameter range separated by numerical values is given, that range is considered to include these limiting values.

[0027] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate and unrelated embodiments. Features discussed in one embodiment are intended to be disclosed in relation to other embodiments shown herein as well. In one case, if a particular feature is not disclosed in one embodiment and is disclosed in another embodiment, one of ordinary skill in the art will understand that this does not necessarily mean that the feature is not intended to be disclosed in the other embodiment. One of ordinary skill in the art will understand that it is the gist of this application to disclose the feature for other embodiments as well, but this has not been done for the purpose of clarity and to keep the specification at a manageable volume.

[0028] Furthermore, the content of prior art documents referred to herein is incorporated by reference. This refers in particular to prior art documents that disclose standard or conventional methods. In that case, the incorporation by reference mainly has the purpose of providing a sufficient disclosure possible and avoiding long repetitions.

[0029] According to a first aspect of the present invention, the present invention relates to a conjugate comprising (i) a target binding moiety, (ii) at least one toxin, and (iii) optionally at least one linker linking the target binding moiety to the at least one toxin, wherein the target binding moiety binds to CD20 and the at least one toxin is an amatoxin.

[0030] Amatoxins are cyclic peptides composed of eight amino acids that are found in Amanita phalloides mushrooms (see Figure 1). Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby also inhibiting transcription and protein synthesis in affected cells. Inhibition of transcription within the cell causes growth and proliferation to cease. Although not covalent, the complex between amatoxin and RNA polymerase II is very tight (KD = 3 nM). Dissociation of amatoxin from the enzyme is a very slow process, and thus recovery of affected cells is unlikely. If inhibition of transcription continues long enough, the cells will undergo programmed cell death (apoptosis).

[0031] In the context of the present invention, the term "amatoxin" includes all cyclic peptides composed of eight amino acids isolated from the genus Amanita and as described by Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5(1978)185-260); all of their chemical derivatives; all of their semi-synthetic analogs; all synthetic analogs constructed from building blocks constructed according to the master structure of the natural compound (cyclic, eight amino acids); all synthetic or semi-synthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids; and all synthetic or semi-synthetic analogs in which the sulfoxide moiety is replaced by a sulfone, thioether, or an atom different from sulfur, such as a carbon atom as in the carboanalog of amatoxin.

[0032] As used herein, a "derivative" of a compound refers to a species having a chemical structure similar to the above compound, but containing at least one chemical group not present in the above compound and / or lacking at least one chemical group present in the above compound. The compound to which the derivative is compared is known as the "parent" compound. Typically, a "derivative" can be produced from the parent compound in one or more chemical reaction steps.

[0033] As used herein, an "analog" of a compound is structurally related but not identical to the compound and exhibits at least one activity of the compound. The compound to which the analog is compared is known as the "parent" compound. The aforementioned activities include, but are not limited to, binding activity to another compound; inhibitory activity (e.g., enzyme inhibitory activity); toxic effects; activation activity (e.g., enzyme activation activity). The analog does not have to exhibit such activity to the same extent as the parent compound. A compound is considered an analog within the context of this application if it exhibits related activity to 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%, more preferably at least 50%) of the activity of the parent compound. Thus, an "analog of amatoxin" as used herein refers to a compound that is structurally related to any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, and amanullinic 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%, 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, amanine, amanine amide, amanullin, and amanullinic acid. An "analog of amatoxin" suitable for use in the present invention may even exhibit greater inhibitory activity against mammalian RNA polymerase II than any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, or amanullinic acid. The inhibitory activity can be measured by measuring the concentration at which 50% inhibition occurs (IC 50 ). The inhibitory activity against mammalian RNA polymerase II can be measured indirectly by measuring the inhibitory activity against cell proliferation.

[0034] "Semi-synthetic analog" refers to an analog obtained by chemical synthesis using a compound from a natural source (e.g., plant material, bacterial culture, fungal culture or cell culture) as a starting material. Typically, the "semi-synthetic analog" of the present invention is synthesized starting from a compound isolated from a mushroom of the Amanitacea family. In contrast, a "synthetic analog" refers to an analog synthesized by so-called total synthesis from small (typically petrochemical) building blocks. Usually, this total synthesis is carried out without the aid of biological processes.

[0035] According to some embodiments of the present invention, the amatoxin can be selected from the group consisting of α-amanitin, β-amanitin, amanin, amanin amide and their analogs, derivatives and salts.

[0036] Functionally, amatoxin is defined as a peptide or depsipeptide that inhibits mammalian RNA polymerase II. Preferred amatoxins have a functional group (e.g., carboxyl group, amino group, hydroxy group, thiol or thiol trapping group) that can react with the linker molecule or target binding moiety defined below.

[0037] In the context of the present invention, the term "amanitin" refers, in particular, to a bicyclic structure based on a derivative of cysteine oxidized to a sulfoxide or sulfone derivative, a cysteine derivative having an aspartic acid or asparagine residue at position 1, a proline residue at position 2, in particular a hydroxyproline residue, an isoleucine, hydroxyisoleucine, or dihydroxyisoleucine at position 3, a tryptophan or hydroxytryptophan residue at position 4, glycine residues at positions 5 and 7, an isoleucine residue at position 6, and a cysteine residue at position 8 (see FIG. 1 for the numbering and representative example of amanitin), all its chemical derivatives; all its further semi-synthetic analogs; all its further synthetic analogs made from building blocks according to the master structure of the natural compound (cyclic, 8 amino acids), all its further synthetic or semi-synthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids, and all its further synthetic or semi-synthetic analogs, and in each case, any such derivative or analog is functionally active by inhibiting mammalian RNA polymerase II.

[0038] The term "target-binding moiety", as used herein, refers to any molecule or part of a molecule that can specifically bind to a target molecule or target epitope. Preferred target-binding moieties in the context of the present application are (i) an antibody or an antigen-binding fragment thereof; (ii) an antibody-like protein; and (iii) a nucleic acid aptamer. A "target-binding moiety" suitable for use in the present invention typically has a molecular weight of 40,000 Da (40 kDa) or more.

[0039] As used in the context of the present application, a "linker" refers to, for example, a molecule that increases the distance between two components in order to alleviate steric hindrance between a target binding moiety and an amatoxin, without which the amatoxin may reduce its ability to interact with RNA polymerase II. The linker can serve another purpose as it can specifically promote the release of the amatoxin in the cells targeted by the target binding moiety. The linker and preferably the bond between the linker on one side and the amatoxin, and the bond between the linker on the other side and the target binding moiety or antibody are stable under physiological conditions outside the cell, such as in blood, but are preferably cleavable inside the cell, particularly inside the target cell, such as a cancer cell. To provide this selective stability, the linker may preferably contain a functional group that is pH-sensitive or protease-sensitive. Alternatively, the bond linking the linker to the target binding moiety may provide selective stability. Preferably, the linker has a length of at least 1, preferably 1 to 30 atoms in length (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), where one side of the linker reacts with the amatoxin and the other side reacts 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- A heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group, which is optionally substituted. The linker can include one or more structural elements such as amide, ester, ether, thioether, disulfide, hydrocarbon moiety, etc. The linker can also include a combination of two or more of these structural elements. Each of these structural elements can be present two or more times, for example, 2 times, 3 times, 4 times, 5 times, or 6 times in the linker. In some embodiments, the linker may contain a disulfide bond. It is understood that the linker must be attached to the amatoxin and the target-binding moiety either in a single step or in two or more subsequent steps. For that purpose, the linker preferably has, at its proximal and distal ends, (i) a group that can form a covalent bond to a group, preferably an activating group on the amatoxin or the target-binding peptide, or (ii) two groups that can be activated to form a covalent bond to a group on the amatoxin. Thus, when a linker is present, the chemical groups are preferably at the distal and proximal ends of the linker, which is the result of such coupling reactions, such as ester, ether, urethane, peptide bond, etc. The presence of a "linker" is optional, i.e., in some embodiments of the target-binding moiety-toxin conjugate, the toxin may be directly linked to the residue of the target-binding moiety.

[0040] The present invention further relates to a conjugate comprising a target-binding moiety that binds to CD20, at least one amatoxin, and optionally a linker, wherein the target-binding moiety is each binding to CD20 (i) an antibody, preferably a monoclonal antibody, (ii) an antigen-binding fragment thereof, preferably a variable region (Fv), Fab fragment or F(ab)2 fragment, (iii) an antigen-binding derivative thereof, preferably a single-chain Fv (scFv), and (iv) an antibody-like protein selected from the group consisting of.

[0041] As used herein, the term "antibody" refers to a protein consisting of one or more polypeptide chains encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene or cDNA derived therefrom. The immunoglobulin gene includes any of the light chain κ, λ and heavy chain α, δ, ε, γ and μ constant region genes, as well as many different variable region genes.

[0042] The basic immunoglobulin (antibody) structural unit is usually a tetramer composed of two identical pairs of polypeptide chains, a light chain (L, having a molecular weight of about 25 kDa) and a heavy chain (H, having a molecular weight of about 50 - 70 kDa). Each heavy chain is composed of a heavy chain variable region (VH or V H abbreviated as) and a heavy chain constant region (CH or C H abbreviated as). The heavy chain constant region is composed of three domains, namely CH1, CH2 and CH3. Each light chain contains a light chain variable region (VL or V L abbreviated as) and a light chain constant region (CL or C L abbreviated as). The VH and VL regions can be further subdivided into hypervariable regions, also called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains form a binding domain that interacts with the antigen.

[0043] The CDRs are most important for the binding of the antibody or its antigen-binding portion. The FRs can be replaced with other sequences if the three-dimensional structure necessary for antigen binding is maintained. Structural changes in the construct will most often result in a loss of sufficient binding to the antigen.

[0044] The term "antigen-binding portion" of a (monoclonal) antibody refers to one or more fragments of an antibody that, in their native form, retain the ability to specifically bind to the CD20 antigen. Said CD20 antigen can be a mammalian, non-primate, primate, and especially a human CD20 antigen. "CD20" herein refers to a protein comprising, or consisting of, an amino acid sequence according to SEQ ID NO: 6, or a sequence that is at least 90%, 92.5%, 95%, or at least 97% identical to SEQ ID NO: 6. Examples of antigen-binding portions of an antibody include Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains, F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region, Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of 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).

[0045] The sequence identity according to the present invention is determined, for example, over the entire length of each sequence being compared to a respective reference sequence (so-called "global alignment") that is particularly suitable for sequences of the same or similar length, or over a shorter defined length (so-called "local alignment") that is more suitable for sequences of unequal length. In the above context, an amino acid sequence having at least, for example, 95% "sequence identity" to a query amino acid sequence is intended to mean that the sequence of the target amino acid sequence is identical to the query sequence, except that the target amino acid sequence may contain up to 5 amino acid changes per 100 amino acids of the query amino acid sequence. In other words, up to 5% (5 out of 100) of the amino acid residues in the target sequence can be inserted or substituted with another amino acid, or deleted, in order to obtain an amino acid sequence having at least 95% identity to the query amino acid sequence. Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage by which two sequences are identical can be determined, for example, by using a mathematical algorithm. A preferred example of a mathematical algorithm that can be used is the algorithm of Karlin et al (1993), PNAS USA, 90:5873-5877, but is not limited thereto. Such algorithms are incorporated into the BLAST family of programs (see also Altschul et al.1990, J. Mol. Biol.215,403-410 or Altschul et al.(1997), Nucleic Acids Res,25:3389-3402), which are accessible via the NCBI homepage at the world wide web site ncbi.nlm.nih.gov and FASTA (Pearson(1990), Methods Enzymol. 83, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U. S. A 85, 2444-2448). Sequences that are identical to other sequences to some extent can be identified by these programs.Furthermore, the % identity between two polypeptide sequences may be determined using programs available in the Wisconsin Sequence Analysis Package (Devereux et al. 1984, Nucleic Acids Res., 387-395; Womble Methods Mol Biol. 2000; 132:3-22), such as the programs BESTFIT and GAP.

[0046] The antibodies, or antibody fragments or antibody derivatives according to the invention can be monoclonal antibodies. The antibodies can be of the IgA, IgD, IgE, IgG or IgM isotype.

[0047] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody composition having a homogeneous population of antibodies (i.e., a homogeneous population consisting of all immunoglobulins, or fragments or derivatives thereof). Particularly preferably, such antibodies are selected from the group consisting of IgG, IgD, IgE, IgA and / or IgM, or fragments or derivatives thereof. Monoclonal antibodies can be produced, for example, by the hybridoma method first described by Kohler and Milstein, Nature 256:495 (1975); Eur. J. Immunol. 6:511 (1976), by recombinant DNA technology, or isolated from phage antibody libraries.

[0048] As used herein, the term "fragment" or "antibody fragment" refers to fragments of such antibodies that retain target binding ability, such as CDRs (complementary determining regions), hypervariable regions, variable domains (Fv), IgG heavy chains (consisting of VH, CH1, hinge, CH2 and CH3 regions), IgG light chains (consisting of VL and CL regions), and / or Fab and / or F(ab)2).

[0049] As used herein, the term "derivative" refers to a protein construct that is structurally different from common antibody concepts such as scFv, Fab and / or F(ab)2, and bispecific, trispecific or higher-order specific antibody constructs, but still has some structural relatedness. These matters will be described below.

[0050] Other antibody derivatives known to those skilled in the art include bispecific antibodies, camelid antibodies, domain antibodies, bivalent homodimers having two chains consisting of scFvs, IgAs (two IgG structures linked by a J chain and a secretory component), shark antibodies, antibodies consisting of a New World primate framework and non-New World primate CDRs, dimerization constructs containing CH3 + VL + VH, other scaffold protein formats containing CDRs, and antibody conjugates (e.g., an antibody, or a fragment or derivative thereof, linked to a drug, toxin, cytokine, aptamer, nucleic acid such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), therapeutic polypeptide, radioisotope or label). The scaffold protein formats can include, for example, antibody-like proteins such as ankyrin and affilin proteins.

[0051] As used herein, the term "antibody-like protein" refers to a protein engineered to specifically bind to a target molecule (e.g., by mutagenesis of Ig loops). Typically, such an antibody-like protein contains at least one variable peptide loop with both ends attached to a protein scaffold. This dual structural constraint significantly increases the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The length of the variable peptide loop typically consists of 10-20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Examples of antibody-like proteins include, but are not limited to, affibodies, anticalins, and designed ankyrin repeat proteins (Binz et al., 2005). Antibody-like proteins can be derived, for example, by panning from large phage display libraries and can be isolated in the same manner as normal antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.

[0052] As used herein, the term "Fab" relates to an IgG fragment containing an antigen-binding region, which fragment is composed of one constant domain and one variable domain from each of the heavy and light chains of an antibody.

[0053] The term "F(ab)2" as used herein relates to an IgG fragment consisting of two Fab fragments linked to each other by disulfide bonds.

[0054] As used herein, the term "scFv" relates to a single-chain variable fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short linker (usually containing serine (S) and / or glycine (G) residues). This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker peptide.

[0055] The modified antibody forms are, for example, bispecific or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, and the like.

[0056] IgG, scFv, Fab and / or F(ab)2 are forms well known to those skilled in the art. The related enabling technologies are available from respective textbooks.

[0057] According to a preferred embodiment of the present invention, the antibody, or its antigen-binding fragment or antigen-binding derivative, is respectively a mouse, chimeric, humanized or human antibody, or its antigen-binding fragment or antigen-binding derivative.

[0058] Mouse-derived monoclonal antibodies (mAbs) can cause undesirable immunological side effects due to the fact that they contain proteins from another species that can induce antibodies. To overcome this problem, antibody humanization and maturation methods have been designed to generate antibody molecules that ideally retain the specificity and affinity of the non-human parental antibody while having minimal immunogenicity when applied to humans (see Almagro and Fransson 2008 for a review). Using these methods, for example, the framework regions of mouse mAbs are replaced by the corresponding human framework regions (so-called CDR grafting). WO200907861 discloses the generation of humanized forms of mouse antibodies by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA technology. US6548640 by the Medical Research Council describes CDR grafting technology, and US5859205 by Celltech describes the production of humanized antibodies.

[0059] As used herein, the term "humanized antibody" relates to an antibody, its fragment or derivative, where at least a part of the constant region and / or framework region of the antibody, and optionally a part of the CDR region, is derived from or regulated by a human immunoglobulin sequence.

[0060] The antibodies, antibody fragments or antibody derivatives disclosed in this specification may comprise humanized sequences, in particular, preferred VH- and VL-based antigen-binding regions that maintain appropriate ligand affinity. Amino acid sequence modifications for obtaining the humanized sequences may occur in the CDR regions and / or framework regions and / or antibody constant region sequences of the original antibody.

[0061] The antibody, or antibody fragment or antibody derivative thereof, can be glycosylated. The glycan can be an N-linked oligosaccharide at asparagine 297 of the heavy chain.

[0062] The antibodies or fragments or derivatives of the present invention can be produced by transfecting host cells with an expression vector containing the coding sequence of the antibody according to the present invention. The expression vector or recombinant plasmid is produced by placing the coding antibody sequence under the control of appropriate regulatory gene elements including promoter and enhancer sequences (e.g., CMV promoter, etc.). The heavy and light chain sequences can be expressed from individual expression vectors co-transfected or from a dual expression vector. The transfection may be transient transfection or stable transfection. The transfected cells are then cultured to produce the transfected antibody construct. If stable transfection is performed, then stable clones secreting antibodies with appropriately associated heavy and light chains are selected by screening with an appropriate assay (e.g., ELISA), subcloned, and grown for future production.

[0063] According to a preferred embodiment of the present invention, the antibody, or antigen-binding fragment thereof, or antigen-binding derivative thereof, is selected from the group consisting of rituximab, obinutuzumab, ibritumomab, tositumomab, ofatumumab, ocrelizumab, and ublituximab, respectively. As used herein, the International Non-Proprietary Name (INN) also means to include all biosimilar antibodies having an amino acid sequence and / or glycosylation pattern identical or substantially identical to the originator antibody as disclosed above, in accordance with 42 USC §262 subsection (i) or equivalent rules in other jurisdictions.

[0064] According to some embodiments of the present invention, the antibodies disclosed herein are genetically engineered to contain heavy chain 118Cys, heavy chain 239Cys, or heavy chain 265Cys according to the EU numbering system (see, for example, Proc. Natl. Acad. Sci. USA 1969, 63, 78-85), preferably heavy chain 265Cys according to the EU numbering system, wherein, when present, the linker, or the amatoxin, is linked to the antibody via the heavy chain 118Cys, or the heavy chain 239Cys, or heavy chain 265Cys residue, respectively. For example, WO2006 / 034488 A2 discloses a corresponding method for producing antibodies engineered with cysteine.

[0065] According to one embodiment of the present invention, the antibody is rituximab genetically engineered to contain heavy chain 265Cys according to the EU numbering system.

[0066] As used herein, the term "genetically engineered" or "genetic engineering" relates to the modification of the amino acid sequence of a given or natural polypeptide or protein, or a part thereof, in the sense of nucleotide and / or amino acid substitutions, insertions, deletions or revertants, or any combination thereof, by genetic technology methods.

[0067] As used herein, the term "amino acid substitution" relates to the modification of the amino acid sequence of a protein, where one or more amino acids are replaced with the same number of different amino acids, resulting in a protein that contains a different amino acid sequence from the original protein. Conservative amino acid substitutions are understood to relate to substitutions that do not significantly affect the structure and function of the protein due to similar size, charge, polarity and / or conformation. Conservative amino acid groups in that sense represent, for example, non-polar amino acids Gly, Ala, Val, Ile and Leu; aromatic amino acids Phe, Trp and Tyr; positively charged amino acids Lys, Arg and His; and negatively charged amino acids Asp and Glu.

[0068] According to a preferred embodiment of the present invention, the linker (if present), or the amatoxin, or the amatoxin conjugated to the linker is bound to the antibody via any of the naturally occurring Cys residues of the antibody, preferably via a disulfide bond. As used herein, the term "naturally occurring Cys residue" is present in a native antibody such as rituximab and forms an intrachain disulfide bond in the light and heavy chains of the antibody, or an interchain disulfide bond between the heavy and light chains and / or between the heavy chains of the antibody (e.g., the disulfide bond in the hinge region of an IgG immunoglobulin). A preferred naturally occurring Cys residue for linking or coupling the linker (if present), or the amatoxin conjugated to the linker disclosed herein, or the amatoxin is a cysteine residue that forms an interchain disulfide bond linking both heavy chains in the hinge region of a native IgG immunoglobulin. Thus, an amatoxin, linker, or amatoxin conjugated to a linker as disclosed herein is bound to an antibody via, for example, a cysteine residue that forms an interchain disulfide bond. Coupling to a cysteine residue that contributes to an interchain disulfide bond in a native antibody can be performed, for example, according to the methods disclosed in mAbs 6:1,46-53 (2014), or Clinical Cancer Research Vol.10,7063-7070, October 15,2004.

[0069] According to a particularly preferred embodiment of the present invention, the antibody is rituximab.

[0070] In a preferred embodiment, the antibody of the conjugate, or an antibody fragment or antibody derivative thereof, binds to the extracellular domain of the CD20 molecule.

[0071] In a preferred embodiment, the present invention relates to a conjugate comprising the above antibody, or an antibody fragment or antibody derivative thereof, which binds to the extracellular domain of CD20.

[0072] Furthermore, the conjugate according to the present invention has a cytotoxicity of 10×10 -9 M, 9×10 -9 M, 8×10 -9 M, 7×10 -9 M, 6×10 -9 M, 5×10 -9 M, 4×10 -9 M, 3×10 -9 M, 2×10 -9 M or more, preferably 10×10 -10 M, 9×10 -10 M, 8×10 -10 M, 7×10 -10 M, 6×10 -10 M, 5×10 -10 M, 4×10 -10 M, 3×10 -10 M, 2×10 -10 M or more, more preferably 10×10 -11 M, 9×10 -11 M, 8×10 -11 M, 7×10 -11 M, 6×10 -11 M, 5×10 -11 M, 4×10 -11 M, 3×10 -11 M, 2×10 -11 M, or 1×10 -11 M or more and may have cytotoxicity.

[0073] In a preferred embodiment of the present invention, the conjugate described above comprises an amatoxin comprising (i) amino acid 4 having a 6'-deoxy position and (ii) amino acid 8 having an S-deoxy position.

[0074] According to a preferred embodiment of the present invention, the conjugate described above comprises a linker, and the linker is a non-cleavable or cleavable linker.

[0075] The cleavable linker can be selected from the group consisting of enzymatically cleavable linkers, preferably proteolytically cleavable linkers, and chemically cleavable linkers, preferably linkers containing disulfide bridges.

[0076] The "cleavable linker" is understood to include at least one cleavage site. As used herein, the term "cleavage site" refers to a portion that is sensitive to specific cleavage at a defined position under specific conditions. The conditions are, for example, a specific enzyme or reducing environment in a specific body or cell compartment.

[0077] According to an embodiment of the present invention, the cleavage site is an enzymatically cleavable portion containing two or more amino acids. Preferably, the enzymatically cleavable portion contains a 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 a β-glucuronide or β-galactoside.

[0078] According to some embodiments, the cleavage site may be cleavable by at least one protease selected from the group consisting of cysteine proteases, metalloproteases, serine proteases, threonine proteases, and aspartic proteases.

[0079] Cysteine proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism involving a nucleophilic cysteine thiol in the catalytic triad or dyad.

[0080] Metalloproteases are proteases whose catalytic mechanism involves a metal. Most metalloproteases require zinc, although some use cobalt. The metal ion coordinates to the protein via three ligands. The ligands coordinating the metal ion can vary depending on histidine, glutamic acid, aspartic acid, lysine, and arginine. The fourth coordination position is occupied by a labile water molecule.

[0081] Serine proteases are enzymes that cleave peptide bonds in proteins, and serine acts as a nucleophilic amino acid at the active site of the enzyme. Serine proteases are broadly classified into two categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

[0082] Threonine proteases are a family of proteolytic enzymes that have a threonine (Thr) residue within their active site. The archetypal member of this class of enzymes is the catalytic subunit of the proteasome, although acyltransferases have convergently evolved the same active site geometry and mechanism.

[0083] Aspartic proteases are catalytic proteolytic enzymes that use an activated water molecule bound to one or more aspartic acid residues for the catalysis of their peptide substrates. Generally, they have two highly conserved aspartic acids in their active site and are optimally active at acidic pH. Almost all known aspartyl proteases are inhibited by pepstatin.

[0084] In certain embodiments of the present invention, the cleavage site is cleavable by at least one agent selected from the group consisting of cathepsin A or B, matrix metalloproteinase (MMP), elastase, β-glucuronidase, and β-galactosidase.

[0085] In certain embodiments of the present invention, the cleavage site is a disulfide bond and specific cleavage is effected in a reducing environment, such as an intracellular reducing environment, such as acidic pH conditions.

[0086] According to a preferred embodiment of the present invention, in the conjugate described above, the linker (if present) or the target binding moiety is linked to the amatoxin via (i) the γC atom of amatoxin amino acid 1, or (ii) the δC atom of amatoxin amino acid 3, or (iii) the 6'-C atom of amatoxin amino acid 4.

[0087] According to a particularly preferred embodiment of the present invention, the conjugate comprises, as a linker - amatoxin moiety, any one of the following compounds of formulas (I) - (XII):

[0088]

Chemical formula

[0089]

Chemical formula

[0090]

Chemical formula

[0091]

Chemical formula

[0092] Furthermore, according to a particularly preferred embodiment of the present invention, the conjugate comprises, as a target binding moiety obtained by conjugating an antibody to an amatoxin linker moiety, any one according to formulas XIII - XXII.

[0093]

Chemical formula

[0094]

Chemical formula

[0095] [Chemistry]

[0096] [Chemistry]

[0097] In the formula, the amatoxin linker moiety is bonded to the ε-amino group of a naturally occurring lysine residue of the antibody, and n is preferably 1 to 7.

[0098] Furthermore, according to a particularly preferred embodiment of the present invention, the conjugate comprises, as a target-binding moiety, an antibody conjugated to an amatoxin linker moiety according to any one of formulas XXIII and XXIV.

[0099] [Chemistry]

[0100] Here, the amatoxin linker moiety is bonded to the thiol group of a cysteine residue of the antibody, and n is preferably 1 to 7.

[0101] According to an even more particularly preferred embodiment of the present invention, the conjugate is selected from the group consisting of: (i) A conjugate according to formula XXV, comprising, as a target-binding moiety, an antibody rituximab conjugated via a thioether bond to at least one amatoxin-linker moiety of formula (XI) to at least one naturally occurring Cys residue of rituximab, for example, at least one Cys residue contributing to the interchain disulfide bond of rituximab;

[0102] [Chemistry]

[0103] (ii) An antibody rituximab genetically engineered to contain a heavy chain 265Cys by the EU numbering system, conjugated via a thioether bond to the heavy chain 265Cys residue of the genetically engineered rituximab to an amatoxin linker moiety of formula (XI) as a target-binding moiety, a conjugate according to formula XXVI;

[0104] [Chemical formula]

[0105] (iii) An antibody rituximab conjugated via a thioether bond to at least one amatoxin-linker moiety of formula (XII), or to at least one naturally occurring Cys residue of rituximab, for example, at least one Cys residue contributing to the interchain disulfide bond of rituximab according to formula XXVII, as a target-binding moiety, a conjugate according to formula XXVII;

[0106] [Chemical formula]

[0107] (iv) An antibody rituximab genetically engineered to contain a heavy chain 265Cys by the EU numbering system, conjugated via a thioether bond to the heavy chain 265Cys residue of the genetically engineered rituximab to an amatoxin linker moiety of formula (XII) as a target-binding moiety, a conjugate according to formula XXVIII;

[0108] [Chemical formula] Wherein, for (i) and (iii), n is from 1 to 7, and for (ii) and (iv), n is from 1 to 2.

[0109] According to another aspect of the present invention, the present invention relates to a pharmaceutical composition comprising the conjugate described above.

[0110] The pharmaceutical composition may include one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives.

[0111] In the aqueous form, the pharmaceutical preparation is ready for administration, while in the lyophilized form, the preparation can be converted to the liquid form before administration by adding, for example, water for injection, which may or may not contain preservatives such as antioxidants like benzyl alcohol, vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium, the amino acids cysteine and methionine, citric acid and sodium citrate, and synthetic preservatives such as methylparaben and propylparaben of parabens.

[0112] The pharmaceutical preparation may further include one or more stabilizers which can be, for example, amino acids, sugar polyols, disaccharides, and / or polysaccharides. The pharmaceutical preparation may further include one or more surfactants, one or more isotonic agents, and / or one or more metal ion chelating agents, and / or one or more preservatives.

[0113] The pharmaceutical preparation described herein may be suitable for at least intravenous, intramuscular, or subcutaneous administration. Alternatively, the conjugate according to the present invention may be provided as a depot preparation that enables sustained release of the biologically active agent over a period of time.

[0114] In yet another aspect of the present invention, there is provided a primary package such as a pre-filled syringe or pen, vial, or infusion bag containing the preparation according to the previous aspect of the present invention.

[0115] The pre-filled syringe or pen may contain the formulation either in lyophilized form (which then has to be dissolved prior to administration, for example with water for injection), or in aqueous form. The syringe or pen is often a disposable article for single use only and may have a volume of 0.1 to 20 ml. However, the syringe or pen may also be a multi-use or multi-dose syringe or pen.

[0116] The vial may also contain the formulation in lyophilized or aqueous form and may be used as a single-use or multi-use device. As a multi-use device, the vial can have a larger volume. The infusion bag usually contains the formulation in aqueous form and may have a volume of 20 to 5000 ml.

[0117] According to another aspect of the invention, the invention relates to the use of the conjugate or pharmaceutical composition described for the treatment of B-lymphocyte related malignancies or B-cell mediated autoimmune diseases, in particular for the treatment of non-Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, Richter's syndrome, rheumatoid arthritis, polyangiitis and granulomatosis with microscopic polyangiitis and pemphigus vulgaris.

[0118] The invention relates to the use of the conjugate or pharmaceutical composition for the treatment of B-lymphocyte related malignancies or B-cell mediated autoimmune diseases, in particular for the treatment of non-Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, Richter's syndrome, rheumatoid arthritis, polyangiitis and granulomatosis with microscopic polyangiitis and pemphigus vulgaris.

[0119] Richter's syndrome is defined as the transformation of chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). It occurs in approximately 2% - 10% of CLL patients. Richter's syndrome is very aggressive, often resistant to treatment, and has a poor treatment outcome of approximately 8 - 14 months. Approximately 80% of cases are clonally related to the underlying CLL, while the remaining 20% of patients have clonally unrelated DLBCL with a favorable prognosis similar to de novo DLBCL (Vaisitti et al 2018). The combination of germline genetic characteristics, clinical characteristics, biological and somatic genetic characteristics of CLL B cells and specific CLL therapies is associated with a higher risk of Richter's syndrome.

[0120] The present invention also relates to a method of treating a patient suffering from a B-lymphocyte related malignancy or a B-cell mediated autoimmune disease, comprising administering to the patient an effective amount of said conjugate or pharmaceutical composition. For example, a method of treating a patient suffering from a B-lymphocyte related malignancy or a B-cell mediated autoimmune disease as disclosed herein comprises administering to the patient from about 0.1 mg / kg body weight to about 25 mg / kg body weight of said conjugate or pharmaceutical composition, whereby said conjugate or pharmaceutical composition is administered to the patient at least once. Preferred routes of administration of said conjugate or pharmaceutical composition may include, for example, intravenous (i.v.) administration or subcutaneous (s.c.) administration at a therapeutically effective amount.

[0121] Sequence [Table 1] TIFF2025109708000016.tif107156 [Example]

[0122] Although the present invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description should be considered as exemplary or illustrative and not restrictive, and the present invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the 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 a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be advantageously used. Any reference signs in the claims should not be construed as limiting the scope.

[0123] All amino acid sequences disclosed herein are shown from the N-terminus to the C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0124] Example 1: Binding of Rituximab to Lymphoma Cell Lines The binding of the anti-CD20 monoclonal antibody rituximab to the CD20-positive human chronic B-cell leukemia cell lines MEC-1 and MEC-2 and the human Burkitt lymphoma cell line Raji was examined by FACS analysis. Rituximab was shown to bind strongly to the CD20-positive MEC-1, MEC-2, and Raji cell lines (Figure 2).

[0125] Example 2: Anti-CD20 Amanitoxin Conjugate with a Cleavable Linker Example 2.1: Synthesis of Anti-CD20 Amanitoxin Conjugate with a Non-Cleavable Linker

[0126] Step 1: 6’-O-(6-Boc-Aminohexyl)-α-Amanitin (HDP 30.0132)

Chemical Formula

[0127] A solution of α-amanitin (105 mg, 114 μmol) and 6-(Boc-amino)-hexyl bromide (128 mg, 457 μmol) in DMSO (3.5 mL) was treated with 2 M lithium hydroxide (LiOH) solution (68.6 μl, 137.1 μmol) under an argon atmosphere. After stirring at ambient temperature for 40 minutes, the reaction mixture was acidified by the addition of AcOH (7.84 μl), and then the mixture was added dropwise to a flask containing MTBE (40 mL) to precipitate the desired ether intermediate. The supernatant was decanted and discarded. The precipitate was purified by preparative RP-HPLC [λ = 305 nm; concentration gradient: 5% B from 0 to 5 minutes; 100% B from 20 to 25 minutes; 5% B from 27 to 35 minutes; A = water; B = methanol] to give HDP 30.0132 (84.37 mg, 66%) as a white powder. MS (ESI+): m / z found: 1118.5 calculated: 1119.29 [M+H] +

[0128] Step 2: 6’-O-(6-Aminohexyl)-α-Amanitin (HDP 30.0134)

Chemical formula

[0129] TFA (5 mL) was added to HDP 30.0132 (152 mg, 136 μmol), and the reaction mixture was stirred at ambient temperature for 2 minutes. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by preparative RP-HPLC [λ = 305 nm; concentration gradient: 5% B at 0 minute; 30% B from 0 to 1 minute; 39% B from 1 to 10 minutes; 100% B from 10 to 13 minutes; 5% B from 13 to 18 minutes; A = water containing 0.05% TFA; B = methanol containing 0.05% TFA]. The fractions containing the product were combined, concentrated, and lyophilized to give derivative HDP 30.0134 (118.67 mg, 86%). MS (ESI+): m / z found: 1018.5 calculated: 1019.17 [M+H] +

[0130] Step 3: 6’-O-(6-Aminohexyl)-α-Amanitin N-Succinimidyl Carbamate HDP 30.0643

Chemical formula

[0131] The product of Project 2, 207 mg (183 μmol) of HDP 30.0134, was dissolved and transferred to a 50 ml conical centrifuge tube containing 4000 μl of dry dimethylformamide (DMF).

[0132] A 0.2 M solution of N,N'-disuccinimidyl carbonate was prepared by dissolving 128 mg (500 μmol) of DSC in 2500 μl of DMF, and 1828 μl (366 μmol = 2 equivalents) of the resulting solution was added to HDP 30.0134, followed by the addition of 50.7 μl (366 μmol = 2 equivalents) of triethylamine.

[0133] After vortexing, the centrifuge tube was placed on an orbital shaker at ambient temperature. TLC control after 5 minutes showed complete consumption of the starting material. Subsequently, 40 ml of ice-cold MTBE and 20 μl of TFA were added to the centrifuge tube. After vigorous vortexing, the centrifuge tube was placed in an ice bath for 10 minutes, and the precipitate was centrifuged at 4000×g for 3 minutes. The supernatant was decanted, and the pellet was washed with 10 mL of ice-cold 0.05% TFA / MTBE by means of resuspension and sedimentation. The solid was dried in vacuo and redissolved in 2400 μl of water / methanol 5:95 + 0.05% TFA purified by preparative HPLC.

[0134] The product-containing fractions were combined and evaporated. The residue was dissolved in 10 ml of a mixture of tert-butanol and water 4:1 containing 0.05% TFA. The solution was passed through a syringe filter (nylon 0.2 μm, 30 mm) and lyophilized: 170 mg (80%) colorless powder MS (ESI+): 1159.42; calc for C50H71N12O18S (MH+): 1159.47.

[0135] Step 4a: Conjugate of 6’-O-(6-Aminohexyl)-α-Amanitin to Rituximab

Chemical Structure

[0136] Variant A: In situ Activation Using DSC and HDP 30.0134 The anti-CD20 monoclonal antibody rituximab was conjugated to Compound I by using the coupling reagent DSC (N,N'-disuccinimidyl carbonate), and a rituximab-amatoxin conjugate (Rtx-DSC-30.0134) having a non-cleavable linker that links the indole-based 6'-position of amino acid 4 of amatoxin to the lysine residue of rituximab was obtained as follows:

[0137] 0.66 mg of 6'-(-6-aminohexyl)-α-amanitin HDP 30.0134 was dissolved in 72 μl of dry dimethylformamide (DMF). Under argon, while stirring at room temperature, 6.7 μl of a solution of dihydroxysuccinimidyl carbonate (DSC) in DMF (2.56 mg in 100 μl of DMF) and 1.3 μl of triethylamine were added all at once. The reaction mixture was stirred at room temperature. After 12 hours, 30 ml of cold diethyl ether was added. The precipitate of α-amanitin-6'-(-6-aminohexyl-6-hydroxysuccinimidyl carbonate) was collected, washed several times with diethyl ether, and dried in vacuo. The remaining solid was dissolved in 100 μl of DMF.

[0138] 4.0 μl of the DMF solution prepared above was added to 225 μl of a rituximab (Roche) solution (2.0 mg / ml in phosphate-buffered saline (PBS)). The mixture was shaken at 4 °C for 14 hours and separated by Sephadex G25 gel filtration on a PD-10 column. The protein fraction was detected by UV absorption and concentrated with a Vivaspin concentrator centrifuge (3000 g). The protein concentration was determined by the RotiQuant-Assay (Carl Roth; Germany) and adjusted to 2.0 mg / ml. The amatoxin payload of rituximab was determined by UV absorption at 280 nm and 310 nm using the extinction coefficients of the antibody and α-amanitin, and a drug-to-antibody ratio (DAR) of 2.6 was obtained.

[0139] Variant B: Use of pre-activated HDP 30.0643 0.90 mg of 6’-O-(6-aminohexyl)-α-amanitin N-succinimidyl carbamate HDP 30.0643 was dissolved in 180 μl of dry dimethyl sulfoxide (DMSO), and 165 μl of the resulting solution was immediately added to 2 ml of rituximab (6 mg / ml in PBS).

[0140] The mixture was shaken overnight at 4 °C and subsequently separated by Sephadex G25 gel filtration on a PD-10 column. Protein fractions were detected by UV absorption and concentrated using an Amicon spin concentrator (2000 g). Protein concentration was measured by Bradford-Assay and adjusted to 3.0 mg / ml.

[0141] The amantitin payload of rituximab was determined by UV absorption at 280 nm and 310 nm using the extinction coefficients of the antibody and α-amanitin, and a drug-antibody ratio (DAR) of 4.6 was obtained.

[0142] Step 4b: Conjugate of 6’-O-(6-Aminohexyl)-α-Amanitin with a Reductive DSP Linker to Rituximab

Chemical formula

[0143] The anti-CD20 monoclonal antibody rituximab was also conjugated to the product of step 2 (HDP 30.0134) by use of the coupling reagent DSP (dithiobis-succinimidyl-propionate) to obtain a rituximab-amatoxin conjugate (Rtx-DSP-30.0134) having a disulfide-containing linker that links the indole-based 6'-position of amino acid 4 of rituximab to a lysine residue of rituximab as follows:

[0144] 1.0 mg of 6’-(-6-aminohexyl)-α-amanitin HDP 30.0134 was dissolved in 56.6 μl of dry dimethylformamide (DMF). Under argon, while stirring at room temperature, 12 μl of a DMF solution of dithiobis(succinimidyl propionate) (DSP) (3.7 mg in 100 μl of DMF) and 2.8 μl of a solution of triethylamine were added all at once. The reaction mixture was stirred at room temperature. After 18 hours, 30 ml of cold diethyl ether was added. The precipitate was collected, washed several times with diethyl ether, and dried in vacuo. The remaining solid was dissolved in 100 μl of DMF.

[0145] Three samples of 500 μl of a rituximab (Roche) solution (2.0 mg / ml) in phosphate-buffered saline (PBS) were treated with 4.2, 14.7, and 29.4 μl of the DMF solution prepared above corresponding to 1-fold, 3.5-fold, and 7.0-fold molar excesses of the toxin linker.

[0146] The mixtures were shaken overnight at 4 °C and separated by Sephadex G25 gel filtration chromatography (XK-16 column; 2 ml / min) respectively. The conjugate fractions were detected by UV absorption and concentrated with a Vivaspin concentrator centrifuge (3000 g). The protein concentration was determined by RotiQuant-Assay (Carl Roth; Germany) and adjusted to 3.0 mg / ml.

[0147] The amanitin payload of rituximab was determined by UV absorption at 280 nm and 310 nm using the extinction coefficients of the antibody and α-amanitin, and drug-antibody ratios (DAR) of 0.9, 4.4, and 7.0 were obtained.

[0148] Example 2.2: In Vitro Cytotoxicity of Anti-CD20 Amanitoxin Conjugate with a Non-Cleavable Linker The cytotoxic activities of rituximab - amatoxin conjugates Rtx - DSC - 30.0134 and Rtx - DSP - 30.0134 were evaluated in vitro against the human chronic B - cell leukemia cell line MEC - 1 using a chemiluminescent BrdU - ELISA incorporation assay according to the manufacturer's (Roche) protocol. Unconjugated rituximab was used as a control. The results are illustrated in Figure 3.

[0149] Both rituximab - amatoxin conjugates Rtx - DSC - 30.0134 and Rtx - DSP - 30.0134 (non - cleavable linker and disulfide - containing linker, respectively) showed significant cytotoxic activity against CD20 - positive cells in vitro.

[0150] Example 2.3: In Vitro Cytotoxicity of Anti-CD20 Amanitoxin Conjugate with a Non-Cleavable Linker Compared with Anti-EGF-R Amanitoxin Conjugate The anti - CD20 monoclonal antibody rituximab was conjugated to Compound I by using the coupling reagent DSC (N,N’ - disuccinimidyl carbonate) (see Example 2.2) to obtain a rituximab - amatoxin conjugate (Rtx - 30.0643) with a non - cleavable linker that links the indole - based 6’ - position of amino acid 4 of amatoxin to the lysine residue of rituximab. This conjugate showed no significant cytotoxicity when examined in the CD20 - negative cell line SK - Hep - 1, a hepatoma cell line derived from sinusoidal endothelium (Figure 4).

[0151] Furthermore, the anti - epidermal growth factor receptor (EGF - R) monoclonal antibodies trastuzumab and panitumumab were each conjugated to Compound I (see Example 2.2) using the coupling reagent DSC (N,N’ - disuccinimidyl carbonate) to obtain antibody - amatoxin conjugates (Her - 30.0643 and Pan - 30.0643, respectively) with non - cleavable linkers that link the indole - based 6’ - position of amino acid 4 of amatoxin to the lysine residues of trastuzumab and panitumumab, respectively.

[0152] The cytotoxic activities of rituximab, trastuzumab, and panitumumab - amatoxin conjugates were evaluated in vitro against unstimulated peripheral blood mononuclear cells (PBMC) using the WST - 1 assay. All three conjugates (Rtx - 30.0643, Her - 30.0643, and Pan - 30.0643) showed cytotoxic effects in the WST - 1 assay, and the dose - response curves for all three conjugates were found to cover a fairly wide concentration range (Figure 5, upper panel). Her - 30.0643 was found to have the strongest cytotoxicity.

[0153] When the cytotoxic activities of the various conjugates were evaluated in CD20 - enriched unstimulated PBMC (Figure 5, lower panel), the CD20 - specific conjugate Rtx - 30.0643 had an IC -7 of about 2×10 50 M, which was about 2×10 -10 M lower than the IC 50 of the two other conjugates, Her - 30.0643 and Pan - 30.0643, and was considerably more cytotoxic.

[0154] Example 2.4: Anti-CD20-F(ab’) 2 In Vitro Cytotoxicity of Anti-CD20 Amanitoxin Conjugate with a Non-Cleavable Linker Compared with Fragment Amanitoxin Conjugate In addition to the rituximab - amatoxin conjugate Rtx - 30.0643, a rituximab - F(ab’)2 fragment amatoxin conjugate (RtxF(ab’)2 - 30.0643) was generated as described in step 4 of Example 2.2. Both the conjugate and unconjugated rituximab were evaluated in vitro for cytotoxicity against CD20 - positive MEC - 1 cells using a chemiluminescent BrdU - ELISA incorporation assay. The results are shown in Figure 6. Both conjugates showed significant cytotoxic effects against MEC - 1 cells, with an IC -10 of 4.6×10 -9 M (Rtx - 30.0643) and 4×10 50 M (RtxF(ab’)2 - 30.0643).

[0155] Tests using the WST-1 cytotoxicity assay against MEC-1 cells yielded similar results, with approximately 4.1x10 -10 M(Rtx-30.0643) and 2.9x10 -9 The IC 50 was shown.

[0156] Example 2.5: Anti-Tumor Activity of Anti-CD20 Amanitoxin Conjugate with a Non-Cleavable Linker In Vivo Cytotoxicity of the non-cleavable linker-containing rituximab - amatoxin conjugate Rtx-30.0643 was also investigated in vivo in a SCID beige-based mouse tumor model (Figure 7). The doses used were 28 mg / kg of the rituximab - amatoxin conjugate, related to a dose of 600 μg / kg of amanitin. The conjugate showed a cytotoxic effect that prevented any significant increase in tumor volume over the test period.

[0157] Example 3: Exploratory Toxicity Study of Anti-CD20 Amanitoxin Conjugate with a Non-Cleavable Linker in Cynomolgus Monkeys The non-cleavable linker-containing rituximab - amatoxin conjugate Rtx-30.0643 (having a payload of 3.2 amanitin moieties per IgG molecule) was investigated in a pilot toxicity study in cynomolgus monkeys (Macaca fascicularis); unconjugated rituximab was used as a reference (control). Six male animals, 3.6 - 4.2 years old and weighing 3.6 - 4.2 kg at the first administration, were used. Parameters evaluated in the study included local tolerance, mortality, clinical signs, body weight, hematological tests (HGB, RBC, WBC, differential cell count (rel, abs.), Reti, PCT, HCT, MCV, MCH, MCHC), coagulation (TPT, aPTT, ESR), and clinical chemistry tests (albumin, globulin, albumin / globulin ratio, cholesterol (total), bilirubin (total), creatinine, glucose, protein (total), urea, triglyceride, electrolytes, ALAT, aP, ASAT, LDH, CK, γ-GT, GLDH).

[0158]

Table 2

[0159]

Table 3

[0160] In Test Group 2, which was administered Rituximab-Amatoxin conjugate Rtx-30.0643 and not Test Group 1 that was administered Rituximab, significant B cell depletion was observed, and then the B cell count recovered after the final treatment. The results of the test are shown in Fig. 8.

[0161] The body weight remained constant in both test groups during the test period (Fig. 9). No findings were observed during the test period for organ weights, histopathological examinations (heart, liver, spleen, kidney, ureter), and gross autopsy findings.

[0162] In the hematological examination, an increase in aPTT, monocytes, and basophils was observed 5 days after treatment at a dose of 3 μg / kg on Day 13 of the test; the aPTT returned to normal by the end of the test; monocytes and basophils increased in Test Group 2 compared to Test Group 1.

[0163] In the clinical biochemical examination, an increase in enzyme activities (ALAT, ASAT, LDH, CK, GGT, GLDH) was observed 5 days after treatment at a dose of 3 μg / kg on Day 13 of the test, on Day 20 of the test for ALAT, and 5 days after treatment at a dose of 9 μg / kg of amanitin. These parameters returned to normal by the end of the study.

[0164] Example 4: Anti-CD20 Amanitoxin Conjugate with a Disulfide Linker Example 4.1: Synthesis of Anti-CD20 Amanitoxin Conjugate with a Disulfide Linker A. In-situ Binding Method Step 1:

Chemical formula

[0165] Similar to Step 1 of Example 2.1, 5.67 mmol of α-amanitin was converted with tert-butyl 2-(2-bromo-ethyldisulfanyl)-ethyl]-carbamate to give 1.29 mg (15%) of HDP 30.0341 as a white powder. MS(ESI + ): m / z measured value: 1155.2 calculated value: 1154.3.5[M+H] +

[0166]

Chemical formula

[0167] Similar to Step 1 of Example 2.1, 5.67 mmol of α-amanitin was converted with tert-butyl [2-(3-bromo-propylsulfanyl)-ethyl]-carbamate to give 4.83 (67%) of HDP 30.0349 as a white powder. MS(ESI + ): m / z measured value: 1168.6 calculated value: 1168.5[M+H] +

[0168]

Chemical formula

[0169] Similar to Step 1 of Example 2.1, 5.67 mmol of α-amanitin was converted with tert-butyl [2-(3-bromo-1,1-dimethyl-propylsulfanyl)-ethyl]-carbamate to give 0.51 mg (7%) of HDP 30.0350 as a white powder. MS(ESI + ): m / z measured value: 1196.7 calculated value: 1196.5[M+H] +

[0170] Step 2: Similar to Step 2 of Example 2.1, the product of Step 1 was deprotected to the free amine.

[0171]

Table 4

[0172] Step 3: The amanitin linker amine HDP 30.0353-5 was pre-activated in situ and conjugated with rituximab according to the method described in Example 2, Step 4 Variant A to obtain conjugates Rtx-30.0353[1.6], Rtx-30.0355[0.7] and Rtx-30.0355[0.2].

[0173] B. Branched Linker Step 1: 6’-O-(3-S-Tritylsulfanylpropyl)-α-Amanitin (HDP 30.0517)

Chemical formula

[0174] Under argon, 46 mg (50 μmol) of vacuum-dried α-amanitin was dissolved in 2500 μl of dry DMSO. 3-(S-Trityl)-mercapto-propyl-1-bromide (159 mg, 8 equivalents) was added, followed by 60 μl of 1 M sodium hydroxide (NaOH) solution. After 1.5 hours at room temperature, the reaction mixture was acidified to pH 5 with 50 μl of 1 M AcOH in DMSO and the solvent was evaporated. The residue was dissolved in 200 μl of MeOH and added dropwise to a centrifuge tube filled with 10 ml of MTBE. The resulting precipitate was cooled to 0 °C for 10 minutes, isolated by centrifugation (4000×g), and then washed with 10 ml of MTBE. The supernatant was discarded, the pellet was dissolved in 750 μl of MeOH, and purified by preparative HPLC on a C18 column (250×21.2 mm, Luna RP-18, 10 μm, 100 Å) in three portions [concentration gradient: 5% B at 0 min; 5% B at 5 min, 100% B at 20 min; 100% B at 25 min; 5% B at 27 min, 5% B at 35 min; flow rate 30 ml / min]. Fractions with a retention time of 21.1 - 21.8 min were collected and the solvent was evaporated to give 36.5 mg (59%) of HDP 30.0517 as a colorless solid. MS(ESI + ): m / z Observed: 1234.8 Calculated: 1236.45 [M+H]+ ; Measured value: 1257.3 Calculated value: 1258.45 [M+Na] +

[0175] 6’-O-(3-S-Tritylsulfanylbutyl)-α-Amanitin (HDP 30.1168)

Chem.

[0176] By repeating the above procedure using 3-(S-trityl)-mercaptobutyl-1-bromide, the title product was obtained in a yield of 64%. MS(ESI + ): m / z Measured value: 1271.5 Calculated value: 1271.5 [M+Na] +

[0177] Step 2: 6’-O-(3-(3-Amino-1-methylpropyldisulfanyl)-propyl)-α-amanitin (HDP 30.1214)

Chem.

[0178] The product of Step 1 (10 mg) was weighed into a 15 ml centrifuge tube and dissolved in 0.5 M DTNP solution in TFA (80.94 μl, 5 equivalents). The reaction mixture was stirred at room temperature for 4 minutes. Next, the reaction mixture was diluted with MTBE / n - hexane (1:1, 10 ml). The precipitate was cooled to 0 °C for 10 minutes, isolated by centrifugation (4000×g), and subsequently washed with MTBE (10 ml). The supernatant was discarded, and the pellet was dissolved in 500 μl of MeOH. 4 - Amino - thiol HDP 30.1157 (17 mg, 9 equivalents) was added. After 1 hour, the mixture was triturated with MTBE (10 ml) containing 0.05% TFA, the ether was decanted, and replaced with fresh MTBE (10 ml) containing 0.05% TFA. The resulting precipitate was dissolved in MeOH (200 μl) and purified by preparative HPLC on a C18 column (250×21.2 mm, Luna RP - 18, 10 μm, 100 Å) [λ = 305 nm; concentration gradient: 0 - 5 minutes 5% B; 20 - 25 minutes 100% B; 27 - 35 minutes 5% B; A = water containing 0.05% TFA; B = methanol containing 0.05% TFA]. The fractions corresponding to the product were collected, and the solvent was evaporated to obtain 8.05 mg (81%) of HDP 30.1172 as a white powder. MS(ESI + ): m / z found: 1110.39 calculated: 1110.44 [M + H] +

[0179] By repeating the above procedure with the products HDP 30.0517 and HDP 30.1168 of Step 1 in combination with appropriate thiols, the following additional compounds were obtained:

[0180]

Table 5

[0181] Step 3: 6’-O-(3-(3-Amino-1-methylpropyldisulfanyl)-propyl)-α-amanitin N-succinimidyl carbamate HDP 30.1214

Chemical formula

[0182] The product HDP 30.1171, 7.60 mg (6.28 μmol) from Step 2 was dissolved and transferred to a 15 ml conical centrifuge tube containing 200 μl of dry dimethylformamide (DMF). A 0.2 M solution of N,N'-disuccinimidyl carbonate was prepared by dissolving 128 mg (500 μmol) of DSC in 2500 μl of DMF, and 314 μl (10 equivalents) of the resulting solution was added to HDP 30.0134, followed by the addition of 12.56 μl (366 μmol = 2 equivalents) of 1 M triethylamine in DMF. After vortexing, the centrifuge tube was placed on an orbital shaker at ambient temperature. TLC control after 5 minutes showed complete consumption of the starting material. Subsequently, 10 ml of ice-cold MTBE and 5 μl of TFA were added to the centrifuge tube. After vigorous vortexing, the centrifuge tube was placed in an ice bath for 10 minutes, and the precipitate was centrifuged at 4000 × g for 3 minutes. The supernatant was decanted, and the pellet was washed with 10 mL of ice-cold 0.05% TFA / MTBE by means of resuspension and sedimentation. The solid was dried in vacuo, redissolved in 2400 μl of water / methanol 5:95 + 0.05% TFA, and purified by preparative HPLC. The product-containing fractions were combined and evaporated. The residue was dissolved in 3 ml of a mixture of tert-butanol and water 4:1 containing 0.05% TFA. The solution was passed through a syringe filter (nylon 0.2 μm, 13 mm) and lyophilized: 4.68 mg (60%) of a colorless powder MS (ESI + ): 1237.25; MH + (C 50 H 71 N 12 O 18 S) calculated value for: 1237.43 (C 51 H 73 N 12 O 18 S3)

[0183] By repeating the above procedure using the modified method of Step 2, the following additional compounds were obtained:

[0184]

Table 6

[0185] Step 4: Synthesis of Rituximab-amanitin derivative with branched disulfide linker 1.00 mg of each of the succinimidyl carbonate derivatives from Project 3 was dissolved in 100 μl of dry dimethyl sulfoxide (DMSO), and 30 μl (10-fold excess) of each of the resulting solutions was immediately added to 394 μl of rituximab solution (9.5 mg / ml in PBS). The mixture was shaken overnight at 4 °C and subsequently separated by Sephadex G25 gel filtration on a PD-10 column. The protein fraction was detected by ultraviolet absorbance, and for 1 L of PBS (pH 7.4), Slide-A-Lyzer TM dialysis cassette (MWCO 20,000) was dialyzed overnight at 4 °C. The protein concentration was determined by RotiQuant-Assay (Carl Roth; Germany), concentrated with an Amicon spin concentrator at 2000 g, and adjusted to 3.0 mg / ml. The amatoxin payload of rituximab was determined by UV absorption at 280 nm and 310 nm using the extinction coefficients of the antibody and α-amanitin, and the following conjugates were obtained.

[0186] [Table 7]

[0187] Example 4.2: In vitro cytotoxicity of anti-CD20 amatoxin conjugate with disulfide linker The anti-CD20 monoclonal antibody rituximab was conjugated to Compounds III, IV, and V, respectively, using the coupling reagent DSC (N,N'-disuccinimidyl carbonate) to obtain rituximab-amanitin conjugates having a disulfide linker that links the 6'-position of the indole moiety of amino acid 4 of amatoxin to the lysine residue of rituximab (Rtx-DSC-30.0353, Rtx-DSC-30.0354, and Rtx-DSC-30.0355).

[0188] [Chemical formula]

[0189] The cytotoxic activities of rituximab - amatoxin conjugates Rtx - DSC - 30.0353, Rtx - DSC - 30.0354, and Rtx - DSC - 30.0355 were evaluated in vitro against the human chronic B - cell leukemia cell line MEC - 1 using a chemiluminescent BrdU - ELISA incorporation assay according to the manufacturer's (Roche) protocol. The results are shown in Figure 10.

[0190] The rituximab - amatoxin conjugate Rtx - DSC - 30.0353 showed the highest cytotoxic activity against CD20 - positive cells in vitro.

[0191] Furthermore, the anti - CD20 monoclonal antibody rituximab was conjugated to the DSC pre - activated compound from Example 4.1 to obtain rituximab - amatoxin conjugates having a disulfide linker that links the 6' - position of the indole moiety of amino acid 4 of amatoxin to the lysine residue of rituximab (Rtx - 30.0748, Rtx - 30.1214, Rtx - 30.1215, Rtx - 30.1216, Rtx - 30.1217, and Rtx - 30.1218).

[0192]

Chemical formula

[0193] The cytotoxic activities of rituximab - amatoxin conjugates Rtx - 30.0748, Rtx - 30.1214, Rtx - 30.1215, Rtx - 30.1216, Rtx - 30.1217, and Rtx - 30.1218 were evaluated in vitro against the human chronic B - cell leukemia cell line MEC - 1 using a WST assay. The results are shown in Figure 11.

[0194] In the WST assay using MEC-1 cells, cytotoxicity could be shown for all conjugates used. The EC 50 of conjugates Rtx-30.1214, Rtx-30.1215, Rtx-30.1216, Rtx-30.1217 and Rtx-30.1218 was in approximately the same range as the two reference compounds Rtx-30.0643 and Rtx-30.0748, where all conjugates were more cytotoxic than Rtx-30.0643 (see Table 3).

[0195] Less stabilized disulfides with one or fewer shielding methyl groups on each side (i.e., Rtx‐30.0748, Rtx‐30.1217, Rtx‐30.1216 and Rtx‐30.1214) showed the highest cytotoxicity, while the highly stabilized Rtx‐30.1215 and Rtx‐30.1218 were slightly more cytotoxic than Rtx‐30.0643 with a non-cleavable linker and showed limited reductive cleavage of these compounds.

[0196] [Table 8]

[0197] Example 5: Rituximab-amanitin conjugate with enzymatically cleavable linker Example 5.1: Synthesis of Rituximab-amanitin conjugate with enzymatically cleavable linker A: 6’-[(3-Maleimidopropanamido)-Val-Ala-PAB]-α-amanitin (HDP 30.1699)

[0198] Step 1: 6’-[Boc-Val-Ala(SEM)-PAB]-α-amanitin (HDP 30.1698) [Chemical Structure]

[0199] Under argon, at room temperature, 57 mg (62.02 μmol) of vacuum-dried α-amanitin was dissolved in 3000 μl of dry dimethylacetamide (DMA). Boc-Val-Ala(SEM)-4-aminobenzyl bromide (disclosed in EP 17192686) (145.5 mg, 248.1 μmol) and 0.2 M cesium carbonate (Cs2CO3) (372.2 μl, 74.43 μmol) were added. After 4 hours at room temperature, the reaction mixture was acidified to pH = 5 with 10 μl of AcOH. The solvent was removed in vacuo, and the residue was purified by preparative HPLC on a C18 column [λ = 305 nm; concentration gradient: 0 - 5 min 5% B; 20 - 25 min 100% B; 27 - 35 min 5% B; A = water; B = methanol]. The fractions containing the product were evaporated to give 54.46 mg (62%) of HDP 30.1698. MS(ESI + ): m / z (measured): 1425.23 calculated: 1424.6

[0200] Step 2: 6’-[H-Val-Ala-PAB]-α-amanitin (HDP 30.1702)

Chemical Structure

[0201] The Boc- and SEM-protected product of step 5 (134.29 mg, 94.25 μmol) was dissolved in 5 ml of TFA. After 2 minutes, the mixture was evaporated to dryness at room temperature, redissolved in 5 ml of water, and adjusted to pH 10 by dropwise addition of 3.2% ammonia. The resulting suspension was lyophilized and applied to RP18-HPLC [λ = 305 nm; concentration gradient: 0 - 2 min 5% B; 2 - 10 min 20% B; 10 - 10.5 min 25% B; 10.5 - 13 min 100% B; 13 - 14 min 5% B; A = water containing 0.05% TFA; B = acetonitrile]. The pure fractions were evaporated and lyophilized to give 68.59 mg (55%) of a colorless powder. MS (ESI + ): m / z measured: 1194.8 calculated: 1194.53 [M + H] + ; measured: 1217.8 calculated: 1216.51 [M + Na] +

[0202] Step 3: 6’-[(3-Maleimidopropanamido)-Val-Ala-PAB]-α-amanitin (HDP 30.1699)

Chem.

[0203] HDP 30.1702 (17.09 mg, 14.3 μmol) was dissolved in dry DMF (350 μl). 3-(Maleimido)-propanoic acid N-hydroxysuccinimide ester (BMPS) (7.62 mg, 28.6 μmol, 2.0 eq), and undiluted DIPEA (9.79 μl, 57.2 μmol, 4.0 eq) dissolved in DMF (350 μl) were added. After stirring at room temperature for 1 hour 30 minutes under argon, the mixture was added dropwise into 40 ml of cold MTBE and centrifuged at 0 °C. The precipitate was collected, washed with 40 ml of MTBE and centrifuged again. The crude product was dried and purified by RP18-HPLC [λ = 305 nm; concentration gradient: 5% B for 0 - 5 min; 100% B for 20 - 25 min; 5% B for 27 - 35 min; A = water containing 0.05% TFA; B = methanol containing 0.05% TFA]. The pure fractions were lyophilized to give 12.51 mg (65%) of the title product 6’-[(3-Maleidopropanamido)-Val-Ala-PAB]-α-Amanitin as a white powder. MS (ESI + ): m / z found: 1367.50 calculated: 1368.45 [M+Na] +

[0204] B: S-Deoxyamanine(3-maleimidopropanamido)-Val-Ala-p-aminobenzylamide (HDP 30.2115)

Chem.

[0205] S-Deoxyamanine (15.0 mg, 16.5 μmol) was treated at room temperature with 429 μl of a 0.1 M solution of (3-maleimidopropanamide)-Val-Ala-p-aminobenzylamine (25.2 μmol, 1.5 eq), 492 μl of 0.1 M TBTU (25.2 μmol, 1.5 eq) and 492 μl of 0.2 M DIEA (49.1 μmol, 3.0 eq). The reaction was monitored by RP-HPLC. After completion, the reaction was quenched with 100 μL of H2O, stirred for 15 minutes and injected into preparative RP-HPLC. Yield: 12.2 mg, 56% Mass spectrometry: 1313.2 [M+H] + , 1335.5 [M+Na] +

[0206] C: Conjugates of HDP 30.1699 and HDP 30.2115 to Rituximab To conjugate the maleimide-amatoxin derivatives HDP 30.1699 and HDP 30.2115 to rituximab, a stock solution of the linker toxin was prepared at 10 mg / ml in DMSO. To 4.4 ml of the antibody solution (9.5 mg / ml in PBS), 44 μl of 1 mM EDTA pH 8.0, and 16.7 μl of 50 mM TCEP solution (3 eq) were added and reduction was carried out at 37 °C for 2 hours. The reduced antibody was aliquoted into 2.2 μl portions and each was treated with 112.5 μl of the HDP 30.1699 or 109.8 μl of the HDP 30.2115 stock solution. After shaking at 4 °C for 30 minutes, 16.7 μl of 100 mM N-ethylmaleimide was added and the remaining thiols were capped by shaking at room temperature for 1 hour. Subsequently, 27.9 μL of 100 mM N-acetyl-L-cysteine was added and shaking was continued for a further 15 minutes. The amatoxin-ADC was purified by gel filtration chromatography using a PD-10 column equilibrated with 1× PBS (pH 7.4). The protein-containing fractions were dialyzed overnight at 4 °C in a Slide-A-Lyzer dialysis cassette (MWCO 20’000) against 4 liters of PBS (pH 7.4). The protein concentration was determined by absorbance measurement at 280 nm and adjusted to 5.0 mg / ml and the sample was sterile filtered (Millex-GV).

[0207]

Table 9

[0208] The integrity of conjugate Rtx-30.1699, which contains an enzymatically cleavable linker (interchain conjugate) that links one of the native cysteine residues of rituximab to the indole-based 6'-position of amino acid 4 of amatoxin, and Rtx-30.2115, which contains an enzymatically cleavable linker (interchain conjugate) that links one of the native cysteine residues of rituximab to amino acid 1 of amatoxin, was confirmed by SDS-PAGE analysis and Western blot developed using anti-amanitin antibody. The results are illustrated in Figure 12. The drug / antibody ratio (DAR) of conjugate Rtx-30.1699 was determined to be 3.70, and the DAR of conjugate Rtx-30.2115 was determined to be 3.75.

[0209] Example 5.2: In vitro cytotoxicity of Rituximab-amanitin conjugate with enzymatically cleavable linker The cytotoxic activities of rituximab-amatoxin conjugates Rtx-30.1699 and Rtx-30.2115 against human chronic B-cell leukemia cell lines MEC-1 and MEC-2 were evaluated in vitro using a 96-hour CTG assay. Unconjugated rituximab was used as a reference compound. The results are illustrated in Figure 14. Both conjugates induced a strong cytotoxic effect against both cell lines, while unconjugated rituximab showed no cytotoxic effect at all.

[0210] Furthermore, the cytotoxic activities of the rituximab amatoxin conjugates Rtx-30.1699 and Rtx-30.2115 were also evaluated in vitro in MEC-1, MEC-2, Raji, Nalm-6, and Ramos cell lines using 96-hour CTG quantification, compared to unconjugated α-amanitin. The results are shown in Figure 15. Both conjugates showed strong cytotoxic effects in the low nanomolar range against all cell lines except the CD20-negative Nalm-6 cells. In contrast, unconjugated #-amanitin showed cytotoxic effects against all cell lines only in the millimolar range due to nonspecific uptake by pinocytosis.

[0211] Example 6: Obinutuzumab-amanitin conjugate with enzymatically cleavable linker Example 6.1: Synthesis of Obinutuzumab-amanitin conjugate with enzymatically cleavable linker Using the method described in Example 5, the following ADCs were obtained with the antibody obinutuzumab:

[0212]

Table 10

[0213] Example 6.2: In vitro cytotoxicity of Obinutuzumab-amanitin conjugate with enzymatically cleavable linker The cytotoxic activities of the obinutuzumab amatoxin conjugates Obi-30.1699 and Obi-30.2115 were evaluated in vitro in MEC-1, MEC-2, Raji, Nalm-6, and Ramos cell lines using 96-hour CTG quantification, compared to unconjugated α-amanitin. The results are shown in Figure 16. Both conjugates showed strong cytotoxic effects against all cell lines except the CD20-negative Nalm-6 cells. In contrast, unconjugated #-amanitin showed cytotoxic effects against all cell lines only in the millimolar range due to nonspecific uptake by pinocytosis.

[0214] Example 7: In vivo cytotoxic activity of anti-CD20 amatoxin conjugate with enzymatically cleavable linker The in vivo cytotoxic effects of the anti-CD20 amatoxin conjugates Rtx-30.2115 and Obi-30.2115 (see Example 6) were evaluated in a Scid mouse xenograft model system. 2.5×10 6 Raji cells were intravenously injected into CB17 Scid mice. Rtx-30.2115 and Obi-30.2115 were used for treatment at doses of 1 mg / kg and 3 mg / kg, respectively. The results are illustrated in Figure 17.

[0215] In the treated test animals, during the 52-day test period, 100% survival was obtained with both conjugates at a dose of 3 mg / kg and 90% survival at a dose of 1 mg / kg. In contrast, in the PBS control, only 10% of the animals survived longer than 28 days.

[0216] Furthermore, the in vivo efficacy of the anti-CD20 amatoxin conjugate Obi-30.1699 was evaluated in two patient-derived tumor xenograft models of Richter's syndrome based on RS9737 and RS1316 cells, respectively. Richter's syndrome xenografts based on these cells are reported to be genetically, morphologically, and phenotypically stable and similar to the corresponding primary tumors (Vaisitti et al 2018).

[0217] The expression of CD20 in RS9737 and RS1316 cells was evaluated by RNA-seq analysis (whole transcriptome shotgun sequencing). The results are shown in Figure 18; the data are plotted as TPM (transcripts per million). RS9737 cells were shown to express a significantly lower level of CD20 than RS1316 cells.

[0218] Cell suspensions of patient-derived tumor xenografts RS1316 and RS9737 cells were injected into the tail veins of female NOG mice, respectively. The animals were treated with a single intravenous administration of the obinutuzumab-amatoxin conjugate Obi-30.1699 on the day of group assignment (day 21 for RS1316, day 10 for RS9737).

[0219]

Table 11

[0220] Treatment of mice with obinutuzumab amatoxin conjugate Obi-30.1699 had a significant effect on overall survival in both of the patient-derived tumor xenograft models tested. The results are illustrated in Figure 19. Percent survival over time is shown for the RS9737-based xenograft model (A) and the RS1316-based xenograft model (B). Corresponding to the different CD20 expression levels in RS9737 and RS1316 cells, an extension of the survival period by Obi-30.1699 compared to the control was observed in the RS9737-based xenograft model (A), and a substantial extension of the survival period was observed in the RS1316-based xenograft model (B); in the latter case, 100% of the anti-CD20-ADC-treated animals survived until day 90, and 50% of the anti-CD20-ADC-treated animals were still alive and disease-free (as shown by FACS analysis) at the end of the 98-day observation period.

[0221]

Table 12

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Claims

**Claim 1** A conjugate comprising: (i) a target binding moiety, (ii) at least one toxin, and (iii) optionally at least one linker that links the target binding moiety to the at least one toxin, wherein the target binding moiety binds to CD20 and the at least one toxin is an amatoxin. **Claim 2** The target binding moiety is each binding to CD20 (i) an antibody, preferably a monoclonal antibody, (ii) its antigen-binding fragment, preferably the variable region (Fv), Fab fragment or F(ab) ２ fragment (iii) an antigen-binding derivative thereof, preferably a single-chain Fv (scFv), and (iv) an antibody-like protein The conjugate according to claim 1, selected from the group consisting of. **Claim 3** The conjugate according to claim 2, wherein the antibody, or an antigen-binding fragment or antigen-binding derivative thereof, is respectively a mouse, chimeric, humanized or human antibody, or an antigen-binding fragment or antigen-binding derivative thereof. **Claim 4** The conjugate according to claim 2, wherein the antibody, or an antigen-binding fragment or antigen-binding derivative thereof, is respectively selected from the group consisting of rituximab, obinutuzumab, ibritumomab, tositumomab, ofatumumab, ocrelizumab, and ublituximab. **Claim 5** The antibody is genetically engineered to contain heavy chain 118Cys, heavy chain 239Cys, or heavy chain 265Cys according to the EU numbering system, preferably heavy chain 265Cys according to the EU numbering system, and when present, the linker or the amatoxin is linked to the antibody via the heavy chain 118Cys, or the heavy chain 239Cys, or heavy chain 265Cys residue, respectively. The conjugate according to any one of claims 2 to 4. **Claim 6** The conjugate according to any one of claims 2 to 4, wherein when present, the linker or the amatoxin is linked to the antibody via any of the natural Cys residues of the antibody, preferably via a disulfide bond. **Claim 7** The conjugate according to any one of claims 4 to 6, wherein the antibody is rituximab or rituximab genetically engineered to contain heavy chain 265Cys according to the EU numbering system. **Claim 8** The conjugate according to any one of claims 4 to 6, wherein the antibody is rituximab, and when present, the linker or the amatoxin is linked to rituximab via any of the naturally occurring Cys residues that form the inter-chain disulfide bond of rituximab.

9. The conjugate according to any one of claims 1 to 8, wherein the linker is a non-cleavable or cleavable linker.

10. The conjugate according to claim 9, wherein the cleavable linker is selected from the group consisting of enzymatically cleavable linkers, preferably proteolytically cleavable linkers, and chemically cleavable linkers, preferably linkers containing disulfide bridges.

11. The conjugate according to any one of claims 1 to 10, wherein the amatoxin comprises (i) amino acid 4 having a 6'-deoxy position and (ii) amino acid 8 having an S-deoxy position.

12. The conjugate according to any one of claims 1 to 11, wherein when present, the linker or the target-binding moiety is attached to the amatoxin via (i) the γC atom of amatoxin amino acid 1, or (ii) the δC atom of amatoxin amino acid 3, or (iii) the 6'-C atom of amatoxin amino acid 4.

13. The conjugate according to any one of claims 1 to 11, wherein the conjugate comprises, as the linker - amatoxin moiety, any of the following compounds of formulas (I) to (XII). 【Chemical Formula 1】 [Chemical Formula 2] 【Chemical Formula 3】 【Chemical 4】

14. The conjugate according to any one of claims 2 to 5, wherein the conjugate comprises, as the target-binding moiety, an antibody conjugated to an amatoxin linker moiety, a conjugate according to any one of formulas XIII to XXII; [Chemical Formula 5] 【Chemical Formula 6】 【Chemical Formula 7】 【Chem.】 wherein the amatoxin linker moiety is attached to the ε-amino group of a naturally occurring lysine residue of the antibody, and n is preferably 1 to 7.

15. The conjugate according to any one of claims 2 to 5, wherein the conjugate comprises, as the target-binding moiety, an antibody conjugated to an amatoxin linker moiety, a conjugate according to any one of formulas XXIII to XXIV; 【Chemical Formula 8】 wherein the amatoxin linker moiety is attached to the thiol group of a cysteine residue of the antibody, where n is preferably 1 to 7.

16. The conjugate according to claim 2, wherein the conjugate is selected from the group consisting of: (i) A conjugate according to formula XXV, comprising a targeting moiety in which the antibody rituximab is conjugated via a thioether bond to at least one amatoxin-linker moiety of formula (XI) to at least one naturally occurring Cys residue of rituximab; 【Chemical Formula 9】 (ii) A conjugate according to formula XXVI, comprising a targeting moiety in which an antibody rituximab genetically engineered to contain heavy chain 265Cys according to the EU numbering system is conjugated via a thioether bond to the amatoxin linker moiety of formula (XI) to the heavy chain 265Cys residue of the genetically engineered rituximab; 【Chemical Formula 10】 (iii) A conjugate according to formula (XXVII), comprising a targeting moiety in which the antibody rituximab is conjugated via a thioether bond to at least one amatoxin-linker moiety of formula (XII) to at least one naturally occurring Cys residue of rituximab; 【Chemical 11】 (iv) A conjugate according to formula (XXVIII), comprising a targeting moiety in which an antibody rituximab genetically engineered to contain heavy chain 265Cys according to the EU numbering system is conjugated via a thioether bond to the amatoxin linker moiety of formula (XII) to the heavy chain 265Cys residue of the genetically engineered rituximab; 【Chemical Formula 12】 wherein, for (i) and (iii), n is from 1 to 7, and for (ii) and (iv), n is from 1 to 2.

17. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 16.

18. The pharmaceutical composition according to claim 17, further comprising one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives.

19. For use in the treatment of B lymphocyte-related malignancies or B cell-mediated autoimmune diseases, particularly for use in the treatment of non-Hodgkin lymphoma, follicular lymphoma, diffuse large B cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, Richter's syndrome, rheumatoid arthritis, polyangiitis and granulomatosis with microscopic polyangiitis and pemphigus vulgaris, the conjugate according to any one of claims 1 to 16, or the pharmaceutical composition according to any one of claims 17 to 18.

20. Use of a conjugate according to any one of claims 1 to 16 or a pharmaceutical composition according to any one of claims 17 to 18 for the treatment of B lymphocyte-related malignancies or B cell-mediated autoimmune diseases, in particular non-Hodgkin lymphoma, follicular lymphoma, diffuse large B cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, Richter's syndrome, rheumatoid arthritis, polyangiitis and granulomatosis with microscopic polyangiitis, and pemphigus vulgaris.

21. A method for treating a patient suffering from a B lymphocyte-related malignancy or a B cell-mediated autoimmune disease, the method comprising administering to the patient an effective amount of a conjugate according to any one of claims 1 to 16 or a pharmaceutical composition according to any one of claims 17 to 18.

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