BR112019012883B1 - ANTIBODY-DRUG CONJUGATES, THEIR USE AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

The invention relates to novel ligand-drug conjugates (ADCs) having improved properties, to activate metabolites of these ADCs and to processes for their preparation. Furthermore, the present invention relates to the use of these conjugates for the treatment and/or prevention of diseases and to the use of these conjugates to prepare medicaments for the treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders, such as, for example, cancerous diseases.

Description

Introduction and state of the art

[0001] The invention relates to novel ligand-drug conjugates (ADCs) having improved properties, for activating metabolites of these ADCs and to processes for their preparation. Furthermore, the present invention relates to the use of these conjugates for the treatment and/or prevention of diseases and to the use of these conjugates for preparing medicaments for the treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders, such as, for example, cancerous diseases. Such treatments can be carried out as monotherapy or in combination with other drugs or additional therapeutic measures. According to the invention, the ligand is preferably an antibody.

[0002] Cancers are the result of uncontrolled cell growth in most different tissues. In many cases, the new cells penetrate existing tissue (invasive growth), or metastasize to remote organs. Cancers occur in a wide variety of different organs and often have tissue-specific courses. The term "cancer" as a generic term therefore describes a large group of defined diseases of different organ, tissue and cell types.

[0003] Some early stage tumors can be removed by surgical and radiotherapy measures. Metastatic tumors can usually only be treated palliatively with chemotherapy. The aim here is to achieve the optimal combination of an improvement in quality of life and a longer lifespan.

[0004] Conjugates of ligand proteins with one or more drug molecules are known, in particular, in the form of antibody drug conjugates (ADCs) in which an internalizing antibody directed against a tumor-associated antigen is covalently linked via a linker to a cytotoxic agent. Following introduction of the ADCs into the tumor cell and subsequent dissociation of the conjugate, the cytotoxic agent itself or a cytotoxic metabolite formed therefrom is released within the tumor cell and can develop its action thereon directly or selectively. In this way, in contrast to conventional cancer chemotherapy, damage to normal tissue is contained within significantly narrower limits [see, e.g., J. M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); A. M. Wu and P. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005); P. D. Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B. Stump, Bioconjugate Chem. 21, 5-13 (2010)]. Thus, WO2012/171020 describes ADCs in which a plurality of toxophore molecules are attached via a polymeric binder to an antibody. As possible toxophores, WO2012/171020 mentions, among others, the substances SB 743921, SB 715992 (Ispinesib), MK-0371, AZD8477, AZ3146 and ARRY-520.

[0005] The last mentioned substances are kinesin spindle protein inhibitors. Kinesin spindle protein (KSP, also known as Eg5, HsEg5, KNSL1 or KIF11) is a kinesin-like motor protein that is essential for the bipolar mitotic spindle to function. Inhibition of KSP leads to mitotic catastrophe and, in the relatively long term, to apoptosis (Tao et al., Cancer Cell 2005 Jul 8(1), 39-59). Following the disclosure of the first cell-penetrating KSP inhibitor, Monastrol, KSP inhibitors have established themselves as a class of innovative chemotherapeutics (Mayer et al., Science 286:971974, 1999), and are the subject of numerous patent applications (e.g., WO2006/044825; WO2006/002236; WO2005/051922; WO2006/060737; WO03/060064; WO03/040979; and WO03/049527). However, since KSP is active only for a relatively short period of time during the mitotic phase, KSP inhibitors have to be present at a sufficiently high concentration during this phase. WO2014/151030 discloses ADCs that include certain KSP inhibitors. ADCs with KSP inhibitors that also comprise enzymatically cleavable linkers that, however, do not have an ideal activity profile, have already been disclosed in patent applications WO2015/096982 and WO2016/096610.

[0006] Legumain is a tumor-associated asparaginyl endopeptidase (S. Ishii, Methods Enzymol. 1994, 244,604; J. M. Chen et al. J. Biol. Chem. 1997, 272, 8090) and has been used for the processing of prodrugs of small cytotoxic molecules, e.g., dedoxorubicin and etoposide derivatives, among others (W. Wu et al. Cancer Res. 2006, 66, 970; L. Stern et al. Bioconjugate Chem. 2009, 20, 500; K. M. Bajjuri et al. ChemMedChem 2011, 6, 54).

[0007] Other lysosomal enzymes are, for example, cathepsins or glycosidases, for example, β-glucuronidases, which have also been used for release of the active compounds by enzymatic cleavage of prodrugs. Enzymatically cleavable groups in vivo are especially 2-8-oligopeptide groups or glycosides. Peptide cleavage sites are disclosed in Bioconjugate Chem. 2002, 13, 855-869 and Bioorganic & Medicinal Chemistry Letters 8 (1998) 3341-3346 and also Bioconjugate Chem. 1998, 9, 618-626. These include, for example, valine-alanine, valine-lysine, valine-citrulline, alanine-lysine and phenylalanine-lysine (optionally with additional amino group).

Summary of the invention

[0008] The prior art discloses various antibody-drug conjugates with enzymatically cleavable linkers which, however, do not have an optimal activity profile, e.g. with regard to their broad activity on different cells. It is therefore an aim of the present invention to provide more effective compounds which, after administration at a relatively low concentration, exhibit durable apoptotic action and are therefore of benefit for cancer therapy. Here, on the one hand, the profile of the metabolites released intracellularly from the ADCs plays an important role. Often, the metabolites formed from ADCs are substrates of efflux pumps and/or have high cell membrane permeability. Both phenomena can contribute to a short residence time and thus suboptimal apoptotic action on the tumor cell.

[0009] Accordingly, the present invention provides ligand-drug conjugates (ADCs) having a specific toxophore-binder composition which, in association with antibodies, have an activity profile of particular interest with respect to potency and spectrum of activity. In order to further improve the tumor selectivity of ADCs and the metabolites thereof, the ligand conjugates have been provided with peptide binders which can be released by lysosomal tumor-associated enzymes such as legumain or cathepsin. The tumor selectivity is then determined not only by the choice of antibody, but additionally by the enzymatic cleavage of the peptide derivative, for example by tumor-associated enzymes such as legumain. Furthermore, the metabolites released from the ligand-drug conjugates (ADCs) according to the invention in tumor cells are distinguished by a property profile of particular interest. They exhibit low efflux from the tumor cell and lead to high exposure of active compound in tumors. Thus, high activity is achieved in the tumor cell although, due to poor permeability, only low systemic cytotoxic activity is achieved, which results in lower off-target toxicity.

[0010] The kinesin spindle protein inhibitors used according to the invention have an amino group that is essential for the effect. By modifying this amino group with peptide derivatives, the effect towards the kinesin spindle protein is blocked and thus the development of a cytotoxic effect is also inhibited. These peptide derivatives can also be components of the binder for the antibody. If this peptide residue or peptide binder, however, can be released from the active compound by tumor-associated enzymes such as legumain or cathepsin, the effect can be re-established in a controlled manner in the tumor tissue. The particular property profile of the metabolites formed in the tumor is ensured by an additional modification of the kinesin spindle protein inhibitor at a position other than the amino group in the molecule, which, however, does not adversely affect the high potency on the target.

[0011] Furthermore, the structure of the ADCs according to the invention allows, for certain embodiments, high antibody loading (referred to as DAR, drug to antibody ratio) which, surprisingly, has no negative effect on the physicochemical and pharmacokinetic behavior of the ADCs.

[0012] Surprisingly, it has now been found that the ligand-drug conjugates of formula (I) where X1 represents N, X2 represents N, and X3 represents C; orX1 represents N, X2 represents C, and X3 represents N; orX1 represents CH or CF, X2 represents C, and X3 represents N; orX1 represents NH, X2 represents C, and X3 represents C; orX1 represents CH, X2 represents N, and X3 represents C,R1 represents hydrogen or methyl,R2 represents methyl, ethyl, -CH2-CH(CH3)2, -CH2-C(=O)OH, or isopropyl,R3 represents methyl, ethyl, -CH2-CH(CH3)2 or -CH2-C(=O)-NH2,M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-CH2-NH-C(=O)-CH2-CH(##)-COOH, #-C(=O)-CH(CH3)-NH-C(=O)-CH2-NH-C(=O)-CH(##)-CH2-COOH, #-C(=O)-CH(CH3)-NH-C(=O)-CH2-W,#-C(=O)-CH2-NH-C(=O)-CH2-CH(##)-COOH,#-C(=O)-CH2-NH-C(=O)-CH(##)-CH2-COOH, #-C(=O)-CH2-W,#-C(=O)-CH(CH3)-NH-C(=O)-(CH2)2-8 -C(=O)-###,# -C(=O)- (CH2)3-C(=O)-###,# -C(=O)-CH(CH3)-NH-C(=O)-(CH2)5-W,# -C(=O)-CH(CH3)-NH-C(=O)-(CH2)-## or# -C(=O)-CH(CH3)-NH-C(=O)-(CH2-CH2-O)1-8-(CH2)2-NH-C(=O)- CH2-##,W represents the group n represents a number from 1 to 50, AK represents a ligand or a derivative thereof, preferably an antibody or an antigen-binding fragment, # represents binding to the compound, ## represents binding to a sulfur atom of a cysteine side chain of the ligand, ### represents binding to a nitrogen atom of a lysine side chain of the ligand, and their salts, solvates and salts of these solvates have superior properties compared to known conjugates.

[0013] Preference is given to those ligand-drug conjugates of formula (I) wherein X1 represents CH, X2 represents C, X3 represents N, R1 represents hydrogen or methyl, R2 represents methyl, -CH2-CH(CH3)2, -CH2-C(=O)OH or isopropyl, R3 represents methyl, -CH2-CH(CH3)2 or –CH2-C(=O)-NH2, M represents the group#-C(=O)-CH(CH3)-NH-C(=O)-CH2-NH-C(=O)-CH2-CH(##)-COOH,#-C(=O)-CH(CH3)-NH-C(=O)-CH2-NH-C(=O)-CH(##)-CH2-COOH,#-C(=O)-CH(CH3)-NH-C(=O)-CH2 -W,#-C(=O)-CH2-NH-C(=O)-CH2-CH(##)-COOH,#-C(=O)-CH2-NH-C(=O)-CH(##)-CH 2-COOH,#-C(=O)-CH2-W,#-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###,#-C(=O)- (CH2)3-C(=O)-###,#-C(=O)-CH(CH3)-NH-C(=O)-(CH2)5-W,#-C(=O)-CH(CH3)-NH-C(=O)-(CH2)-## or#-C(=O)-CH(CH3)-NH-C(=O)-(CH2-CH2-O)4-(CH2)2-NH-C(=O)-CH2-##,W represents the group n represents a number from 1 to 50, AK represents a ligand or a derivative thereof, preferably an antibody or an antigen-binding fragment, # represents binding to the compound, ## represents binding to a sulfur atom of a cysteine side chain of the ligand, ### represents binding to a nitrogen atom of a lysine side chain of the ligand, and their salts, solvates and salts of such solvates.

[0014] Particular preference is given to those ligand-drug conjugates of the formula (I), in which R1 represents hydrogen or methyl, R2 represents methyl or isopropyl, R3 represents methyl or -CH2-C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 50, AK represents a ligand or a derivative thereof, preferably an antibody or an antigen-binding fragment, # represents the binding to the compound, ### represents the binding to a nitrogen atom of a lysine side chain of the ligand, and their salts, solvates and salts of such solvates.

[0015] Very particular preference is given to those ligand-drug conjugates of the formula (I) in which R1 represents methyl, R2 represents methyl, R3 represents -CH2 — C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 50, AK represents a ligand or a derivative thereof, preferably an antibody or an antigen-binding fragment, # represents the bond to the compound, ### represents the bond to a nitrogen atom of a lysine side chain of the ligand, and their salts, solvates and salts of such solvates.

[0016] Special preference is given to those ligand-drug conjugates of formula (I) in which R1 represents methyl, R2 represents methyl, R3 represents -CH2-C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 20, AK represents a ligand or a derivative thereof, preferably an antibody or an antigen-binding fragment, # represents the linkage to the compound, ### represents the linkage to a nitrogen atom of a lysine side chain of the ligand, and their salts, solvates and salts of such solvates.

[0017] Those ligand-drug conjugates of formula (I) are selected, wherein R1 represents methyl, R2 represents methyl, R3 represents -CH2 — C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 20 and AK represents an anti-CD123 antibody, an anti-CXCR5 antibody, an anti-B7H3 antibody, an anti-TWEAKR antibody, an anti-Her2 antibody or an anti-EGFR antibody or represents an antigen-binding antibody fragment thereof, # represents binding to the compound, ### represents binding to a nitrogen atom of a lysine side chain of the antibody (AK) or of the antigen-binding antibody fragment thereof, and their salts, solvates and salts of these solvates.

[0018] In particular, those linker-drug conjugates of formula (I) are selected, wherein R1 represents methyl, R2 represents methyl, R3 represents -CH2-C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 20 and AK represents an anti-CD123 antibody selected from the group consisting of TPP-9476, TPP-8988, TPP-8987 and TPP-6013, represents an anti-CXCR5 antibody selected from the group consisting of TPP-9574 and TPP-9580, represents an anti-B7H3 antibody TPP-8382, represents an anti-TWEAKR antibody selected from the group consisting of TPP-7006 and TPP-7007, represents an anti-Her2 antibody TPP-1015 or represents an anti-EGFR antibody TPP-981 or represents an antigen-binding antibody fragment thereof, # represents binding to the compound,### represents binding to a nitrogen atom of a lysine side chain of the antibody (AK) or antigen-binding antibody fragment thereof, and their salts, solvates and salts of these solvates.

[0019] Preference is given to those ligand-drug conjugates of the above-mentioned formulae wherein AK represents a ligand that specifically binds to an extracellular cancer target molecule. In a preferred embodiment, the ligand, after binding to its extracellular target molecule on the target cell, is internalized by the target cell through binding. Preferably, the ligand is an antibody or an antigen-binding fragment.

[0020] In a preferred subject matter of the invention, the extracellular cancer target molecule is selected from the group consisting of the cancer target molecules EGFR, CD123, HER2, B7H3, TWEAKR and CXCR5; Particular preference is given to CD123, CXCR5 and B7H3.

[0021] In a preferred subject matter of the invention, the AK ligand is an anti-CD123 antibody, an anti-CXCR5 antibody, an anti-B7H3 antibody, an anti-TWEAKR antibody, an anti-Her2 antibody or an anti-EGFR antibody or an antigen-binding antibody fragment thereof.

[0022] Particularly preferred are those ligand-drug conjugates of the mentioned formulae wherein AK (AK1, AK2) represents an antibody selected from the group consisting of TPP-8382 (anti B7H3), TPP-6013 (anti-CD123), TPP-8987 (anti-CD123), TPP-8988 (anti-CD123), TPP 9476 (anti-CD123), TPP-9574 (anti-CXCR5) and TPP 9580 (anti-CXCR5) or an antigen-binding fragment thereof. Here, preference is given to the antibodies TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD123). The exact structure (sequence) of these antibodies can be seen in the table: Protein sequences of antibodies, the text that follows this table and the sequence listing.

[0023] Particularly preferred are those ligand-drug conjugates of the formula (I) wherein AK represents an antibody selected from the group consisting of TPP-8382 (anti B7H3), TPP-6013 (anti-CD123), TPP-8987 (anti-CD123), TPP-8988 (anti-CD123), TPP 9476 (anti-CD123), TPP-9574 (anti-CXCR5) and TPP 9580 (anti-CXCR5). Here, preference is given to the antibodies (AK) TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD123). Description of the figures Fig. 1: Annotated sequences of preferred antibodies for ligand-drug conjugates. The protein sequences of the light and heavy chains of IgGs, and the VH and VL regions of these antibodies are shown. Below the sequences, important regions are annotated (VH and VL regions in IgGs, and the CDR regions (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3)). Fig. 2: Listing of sequences of preferred antibodies for ligand-drug conjugates and sequences of target proteins.

Detailed description of the invention

[0024] The invention provides conjugates of a ligand or derivative thereof with one or more drug molecules, wherein the drug molecule is a kinesin spindle protein inhibitor (KSP inhibitor).

[0025] The binders which can be used according to the invention, KSP inhibitors thereof which can be used according to the invention and binders which can be used according to the invention which can be used in combination without any limitation are described below. In particular, the binders represented in each case as preferred or particularly preferred can be used in combination with the KSP inhibitors represented in each case as preferred or particularly preferred, optionally in combination with the binders represented in each case as preferred or particularly preferred.

Particularly preferred KSP inhibitor conjugates (ligand-drug conjugates)

[0026] Particular preference is given according to the invention to the following KSP inhibitor conjugates, wherein AK (AK1, AK2) represents ligands or a derivative thereof (preferably an antibody), and n represents a number from 1 to 20, preferably 1 to 8, and more preferably 4 to 8. AK1 preferably represents an antibody linked via a cysteine residue to the KSP inhibitor; AK2 preferably represents an antibody linked via a lysine residue to the KSP inhibitor. The ligands or antibodies used herein are preferably the ligands and antibodies described as preferred in the description.

[0027] Here, particular preference is given to the following ligand-drug conjugates:

[0028] Preference is given to those ligand-drug conjugates of the mentioned formulae wherein AK (AK1, AK2) represents a ligand that specifically binds to an extracellular cancer target molecule. In a preferred embodiment, the ligand, after binding to its extracellular target molecule on the target cell, is internalized by the target cell through binding.

[0029] In a preferred subject matter of the invention, the extracellular cancer target molecule is selected from the group consisting of the cancer target molecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particular CD123, CXCR5 and B7H3.

[0030] In a preferred subject matter of the invention, the AK (AK1, AK2) ligand is an anti-CD123 antibody, an anti-CXCR5 antibody, an anti-B7H3 antibody, an anti-TWEAKR antibody, an anti-Her2 antibody or an anti-EGFR antibody, or an antigen-binding antibody fragment thereof.

[0031] Particularly preferred are those ligand-drug conjugates of the mentioned formulae wherein AK (AK1, AK2) represents an antibody selected from the group consisting of TPP-8382 (anti B7H3), TPP-6013 (anti-CD123), TPP-8987 (anti-CD123), TPP-8988 (anti-CD123), TPP-9476 (anti-CD123), TPP-9574 (anti-CXCR5) and TPP-9580 (anti-CXCR5), or an antigen-binding fragment thereof. Here, preference is given to the antibodies TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD123). The exact structure (sequence) of these antibodies can be seen in the table: Protein sequences of antibodies, the text that follows this table and the sequence listing.

[0032] KSP inhibitor-binder intermediates and preparation of conjugates

[0033] The conjugates according to the invention are prepared by initially supplying the low molecular weight KSP inhibitor thereof with a binder. The intermediate thus obtained is then reacted with the ligand (preferably antibody).

[0034] For an intermediate that couples to a lysine residue and subsequent coupling with the antibody, the reaction can be illustrated as follows:

[0035] In the above reaction scheme, X1, X2, X3, R1, R2, R3 and AK2 have the meanings given in formula (I) and here, R4 represents methyl and n represents 0 or 1.

[0036] The synthesis of building block A was described in WO2015/096982. Peptide derivatives B and C were prepared by classical peptide chemistry methods. Intermediates C and D were coupled using HATU in DMF in the presence of N,N-diisopropylethylamine at RT. Subsequently, both the benzyloxycarbonyl protecting group and the benzyl ester were hydrogenolytically removed over 10% palladium on activated carbon. The fully deprotected intermediate was then reacted with 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in DMF in the presence of N,N-diisopropylethylamine at RT to generate the ADC precursor molecule E. This activated ester was then coupled with the respective antibodies as described in Chapter B-5.

[0037] For an intermediate that couples to a cysteine residue and subsequent coupling with the antibody, the reaction can be illustrated as follows:

[0038] In the above reaction scheme, X1, X2, X3, R1, R2, R3 and AK1 have the meanings given in formula (I) and here, R4 represents methyl and n represents 1.

[0039] Using an analogous procedure, it is also possible to prepare compounds in which n represents 0.

[0040] The synthesis of building block A was described in WO2015/096982. Peptide derivatives B and C were prepared by classical peptide chemistry methods. Intermediates C and D were coupled using HATU in DMF in the presence of N,N-diisopropylethylamine at RT. Subsequently, both the benzyloxycarbonyl protecting group and the benzyl ester were hydrogenolytically removed over 10% palladium on activated carbon. The fully deprotected intermediate was then reacted with 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione in DMF in the presence of N,N-diisopropylethylamine at RT to generate the ADC precursor molecule E. This maleimide derivative was then coupled with the respective antibodies as described in Chapter B-4.

[0041] Depending on the binder, succinimide-linked ADCs can, upon conjugation, be converted to the open-chain succinimides, which have an advantageous stability profile.

[0042] This reaction (ring opening) can be carried out at pH 7.5 to 9, preferably at pH 8, at a temperature of 25 °C to 37 °C, for example by stirring. The preferred stirring time is 8 to 30 hours.

[0043] For an intermediate that couples to a cysteine residue and subsequent coupling with the antibody followed by ring opening of the succinimide ring, the reaction can be illustrated as follows:

[0044] In the above reaction scheme, X1, X2, X3, R1, R2, R3 and AK1 have the meanings given in formula (I) and here, R4 represents methyl and n represents 0 or 1.

[0045] The synthesis of building block A was described in WO2015/096982. Peptide derivatives B and C were prepared by classical peptide chemistry methods. Intermediates C and D were coupled using HATU in DMF in the presence of N,N-diisopropylethylamine at RT. Subsequently, both the benzyloxycarbonyl protecting group and the benzyl ester were hydrogenolytically removed over 10% palladium on activated carbon. The fully deprotected intermediate was then reacted with 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in the presence of N,N-diisopropylethylamine at RT to generate the ADC precursor molecule E. This maleimide derivative was then coupled with the respective antibodies as described in Chapter B-4 under small scale coupling or medium scale coupling.

Binders

[0046] In the broadest sense, the term "ligand" is understood to mean a molecule that binds to a target molecule present in a certain target cell population to be addressed by the ligand-drug conjugate. The term ligand should be understood in its broadest meaning and also encompasses, for example, lectins, proteins capable of binding to certain sugar chains, or phospholipid-binding proteins. Such ligands include, for example, high molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (e.g., aptamers (US5,270,163) reviewed by Keefe AD., et al., Nat. Rev. Drug Discov. 2010;9:537-550), or vitamins) and all other cell-binding molecules or substances. Binding proteins are, for example, antibodies and antibody fragments or antibody mimetics, e.g., affibodies, adnectins, anticalins, DARPins, avimers, nanobodies (reviewed by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr. Opinion in Pharmacology 2008; 8:608-617). Binding peptides are, for example, ligands of a ligand/receptor pair, e.g., VEGF of the VEGF/KDR ligand/receptor pair, transferrin of the ligand/receptor pair, transferrin/transferrin receptor, or cytokine/cytokine receptor, e.g., TNFalpha of the TNFalpha receptor/TNFalpha ligand/receptor pair.

[0047] The ligand may be a binding protein. Preferred embodiments of the ligands are an antibody, an antigen-binding antibody fragment, a multispecific antibody, or an antibody mimic.

[0048] The literature also discloses various options for covalent coupling (conjugation) of organic molecules to ligands and in particular antibodies. According to the invention, preference is given to conjugation of the toxophores to the antibody via one or more sulfur atoms of cysteine residues of the antibody and/or via one or more NH groups of lysine residues of the antibody. However, it is also possible to link the toxophore to the antibody via free carboxyl groups or via sugar residues of the antibody.

[0049] A "target molecule" in the broadest sense is a molecule that is present in the target cell population and that may be a protein (e.g. a growth factor receptor) or a non-peptide molecule (e.g. a sugar or phospholipid). It is preferably a receptor or an antigen.

[0050] The term “extracellular” target molecule describes a cell-attached target molecule that is located outside a cell, or the part of a target molecule that is located outside a cell, i.e. a ligand can bind to its extracellular target molecule in an intact cell. An extracellular target molecule can be anchored in the cell membrane or be a component of the cell membrane. The person skilled in the art is aware of methods for identifying extracellular target molecules. For proteins, this can be done by determining the transmembrane domain (or domains) and the orientation of the protein in the membrane. Such data are usually deposited in protein databases (e.g., SwissProt).

[0051] The term “cancer targeting molecule” describes a targeting molecule that is more abundantly present in one or more cancer cell species than in non-cancerous cells of the same tissue type. Preferably, the cancer targeting molecule is selectively present in one or more cancer cell species compared to non-cancerous cells of the same tissue type, whereby selectively describes at least a two-fold enrichment in cancer cells compared to non-cancerous cells of the same tissue type (a “selective cancer targeting molecule”). The use of cancer targeting molecules allows for selective therapy of cancer cells using the conjugates according to the invention.

[0052] The ligand can be attached to the binder via a bond. The ligand can be attached via a heteroatom of the ligand. The heteroatoms according to the invention of the ligand that can be used for attachment are sulfur (in one embodiment via a sulfhydryl group of the ligand), oxygen (according to the invention via a carboxyl or hydroxyl group of the ligand) and nitrogen (in one embodiment via a primary or secondary amine group or amide group of the ligand). These heteroatoms can be present in the natural ligand or are introduced by chemical methods or molecular biology methods. According to the invention, the attachment of the ligand to the toxophore has only a minor effect on the binding activity of the ligand towards the target molecule. In a preferred embodiment, the bond has no effect on the binding activity of the ligand towards the target molecule.

[0053] According to the present invention, the term "antibody" is to be understood in its broadest meaning and encompasses immunoglobulin molecules, for example, modified or intact monoclonal antibodies, polyclonal antibodies or multispecific antibodies (e.g., bispecific antibodies). An immunoglobulin molecule preferably comprises a molecule having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains) that are typically linked by disulfide bridges. Each heavy chain comprises a heavy chain variable domain (abbreviated VH) and a heavy chain constant domain. The heavy chain constant domain may, for example, comprise three domains CH1, CH2 and CH3. Each light chain comprises a variable domain (abbreviated VL) and a constant domain. The light chain constant domain comprises a domain (abbreviated CL). The VH and VL domains can be further subdivided into regions that have hypervariability, also referred to as complementarity determining regions (abbreviated CDR), and regions that have low sequence variability (framework region, abbreviated FR). Typically, each VH and VL region is composed of three CDRs and up to four FRs. For example, from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody can be obtained from any suitable species, e.g., rabbit, llama, camel, mouse, or rat. In one embodiment, the antibody is of human or murine origin. An antibody can, for example, be human, humanized, or chimeric.

[0054] The term “monoclonal” antibody refers to antibodies obtained from a population of substantially homogeneous antibodies, that is, individual antibodies in the population are identical except for naturally occurring mutations, of which there may be a small number. Monoclonal antibodies recognize a single antigenic binding site with high specificity. The term monoclonal antibody does not refer to a particular preparation process.

[0055] The term “intact” antibody refers to antibodies that comprise both an antigen-binding domain and the constant domain of the heavy and light chain. The constant domain may be a naturally occurring domain or a variant thereof that has a number of modified amino acid positions, and may also be aglycosylated.

[0056] The term “modified intact” antibody refers to intact antibodies fused through their amino terminus or carboxy terminus via a covalent bond (e.g., a peptide bond) to an additional polypeptide or protein that does not originate from an antibody. Additionally, antibodies may be modified such that, at defined positions, reactive cysteines are introduced to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol. 2008 Aug;26(8):925-32).

[0057] "Amino acid modification" or "mutation" herein means an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. The preferred amino acid modification herein is a substitution. "Amino acid substitution" or "substitution" herein means an exchange of an amino acid at a particular position in a protein sequence for another amino acid. For example, the substitution Y50W describes a variant of a parent polypeptide in which the tyrosine at position 50 has been exchanged for a tryptophan. A "variant" of a polypeptide describes a polypeptide that has an amino acid sequence substantially identical to a reference polypeptide, typically a native or "parent" polypeptide. The polypeptide variant may have one or more amino acid exchanges, deletions, and/or insertions at particular positions in the native amino acid sequence.

[0058] The term "human" antibody refers to antibodies that can be obtained from a human or that are synthetic human antibodies. A "synthetic" human antibody is an antibody that is partially or completely obtainable in silico from synthetic sequences based on analysis of human antibody sequences. A human antibody can be encoded, for example, by a nucleic acid isolated from a library of antibody sequences of human origin. An example of such an antibody can be found in Soderlind et al., Nature Biotech. 2000, 18:853-856. Such "human" and "synthetic" antibodies also include aglycosylated variants that have been produced by deglycosylation by PNGaseF or by mutation of N297 (Kabat numbering) of the heavy chain to any other amino acid.

[0059] The term “humanized” or “chimeric” antibody describes antibodies that consist of a non-human portion and a human portion of the sequence. In such antibodies, part of the sequences of the human immunoglobulin (recipient) is replaced by portions of sequence from a non-human immunoglobulin (donor). In many cases, the donor is a murine immunoglobulin. In the case of humanized antibodies, the CDR amino acids of the recipient are replaced by amino acids from the donor. Sometimes, the framework amino acids are also replaced by corresponding amino acids from the donor. In some cases, the humanized antibody contains amino acids present in neither the recipient nor the donor, which were introduced during optimization of the antibody. In the case of chimeric antibodies, the variable domains of the donor immunoglobulin are fused to the constant regions of a human antibody. Such "humanized" and "chimeric" antibodies also include aglycosylated variants that were produced by deglycosylation by PNGaseF or by mutation of N297 (Kabat numbering) of the heavy chain to any other amino acid.

[0060] The term complementarity determining region (CDR) as used herein refers to those amino acids of an antibody variable domain that are required for antigen binding. Typically, each variable region has three CDR regions designated as CDR1, CDR2, and CDR3. Each CDR region may encompass amino acids according to the Kabate definition/or amino acids of a hypervariable cycle defined according to Chotia. The definition according to Kabat comprises, for example, the region from about amino acid positions 24–34 (CDR1), 50–56 (CDR2) and 89–97 (CDR3) of the variable light domain/chain (VL) and 31–35 (CDR1), 50–65 (CDR2) and 95–102 (CDR3) of the variable heavy domain/chain (VH) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The definition according to Chotia comprises, for example, the region from about amino acid positions 26–32 (CDR1), 50–52 (CDR2) and 91–96 (CDR3) of the variable light chain (VL) and 26–32 (CDR1), 53–55 (CDR2) and 96–101 (CDR3) of the variable heavy chain (VH) (Chothia and Lesk; J Mol Biol 196: 901–917 (1987)). In some cases, a CDR may comprise amino acids from a CDR region defined according to Kabate Chotia.

[0061] Depending on the amino acid sequence of the heavy chain constant domain, antibodies can be categorized into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM and several of these can be divided into additional subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains, which correspond to different classes, are named as [alpha/α], [delta/δ], [epsilon/8], [gamma/Y] and [my/μ]. Both the three-dimensional structure and the subunit structure of antibodies are known.

[0062] The term “functional fragment” or “antigen-binding antibody fragment” of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (e.g., the variable domains of an IgG) that still comprises the antigen-binding domains of the antibody/immunoglobulin. The “antigen-binding domain” of an antibody typically comprises one or more hypervariable regions of an antibody, e.g., the CDR, CDR2, and/or CDR3 region. However, the “framework” or “scaffold” region of an antibody may also play a role during binding of the antibody to antigen. The framework region forms the backbone of the CDRs. Preferably, the antigen binding domain comprises at least amino acids 4 to 103 of the variable light chain and amino acids 5 to 109 of the variable heavy chain, more preferably amino acids 3 to 107 of the variable light chain and 4 to 111 of the variable heavy chain, especially preferably the complete variable heavy and light chains, i.e. amino acids 1 - 109 of the VL and 1 to 113 of the VH (numbering according to WO97/08320).

[0063] The “functional fragments” or “antigen-binding antibody fragments” of the invention encompass, inconclusively, Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, Single Domain Antibodies (DAbs), linear antibodies, single antibody chains (single-chain Fv, abbreviated to scFv); and multispecific antibodies, such as bi- and trispecific antibodies, for example, formed from antibody fragments C. A. K Borrebaeck, Editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag. Antibodies other than “multispecific” or “multifunctional” antibodies are those that have identical binding sites. Multispecific antibodies may be specific for different epitopes of an antigen or may be specific for epitopes of more than one antigen (see, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 14760-69; US Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol. 148, 1547-1553). An F(ab')2 or Fab molecule can be constructed so that the number of intermolecular disulfide interactions occurring between the Ch1 and CL domains can be reduced if not completely prevented.

[0064] “Epitopes” refers to protein determinants capable of specifically binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains or combinations thereof, and usually have specific three-dimensional structural properties and also specific charge properties.

[0065] “Functional fragments” or “antigen-binding antibody fragments” may be fused to another polypeptide or protein, which does not originate from an antibody, via the amino terminus or carboxyl terminus thereof, via a covalent bond (e.g., a peptide bond). Furthermore, antigen-binding fragments and antibodies may be modified by the introduction of reactive cysteines at defined locations in order to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol. 2008 Aug;26(8):925-32).

[0066] Polyclonal antibodies can be prepared by methods known to one of ordinary skill in the art. Monoclonal antibodies can be prepared by methods known to one of ordinary skill in the art (Kohler and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies can be prepared by methods known to one of ordinary skill in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et al of US 4,816,567 or Bosset al of US 4,816,397).

[0067] One of ordinary skill in the art is aware of the various methods for preparing human antibodies and fragments thereof, such as, for example, by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13(1):65-93) or Phage Display Technologien (Clackson et al., Nature. 1991 Aug 15;352(6336):624-8). The antibodies of the invention can be obtained from recombinant antibody libraries consisting, for example, of the amino acid sequences of a multiplicity of antibodies compiled from a large number of healthy volunteers. The antibodies can also be produced by known recombinant DNA technologies. The nucleic acid sequence of an antibody can be obtained by routine sequencing or is available from publicly accessible databases.

[0068] An “isolated” antibody or ligand has been purified to remove other constituents of the cell. Contaminating constituents of a cell that may interfere with a diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptide or non-peptide constituents of a cell. A preferred antibody or ligand is one that has been purified to an extent of greater than 95% by weight, relative to the antibody or ligand (determined, for example, by the Lowry method, UV-Vis spectroscopy, or by SDS capillary gel electrophoresis). Further, an antibody that has been purified to such an extent that at least 15 amino acids of the amino terminus or of an internal amino acid sequence can be determined, or that has been purified to homogeneity, where homogeneity has been determined by SDS-PAGE under reducing or non-reducing conditions (detection may be determined by Coomassie Blue staining or, preferably, by silver staining). However, an antibody is typically prepared by one or more purification steps.

[0069] The term “specific binding” or “specifically binds” refers to an antibody or ligand that binds to a predetermined antigen/target molecule. Specific binding of an antibody or ligand typically describes an antibody or ligand that has an affinity of at least 10-7 M (as a Kd value; i.e., preferably those with Kd values less than 10-7 M), with the antibody or ligand having at least two-fold greater affinity for the predetermined antigen/target molecule than for a non-specific antigen/target molecule (e.g., bovine serum albumin or casein) that is not the predetermined antigen/target molecule or a closely related antigen/target molecule. Specific binding of an antibody or ligand does not exclude the antibody or ligand that binds to a plurality of antigens/target molecules (e.g., orthologs from different species). The antibodies preferably have an affinity of at least 10-7 M (as Kd value; in other words, preferably those with Kd values lower than 10-7 M), preferably at least 10-8 M, more preferably in the range of 10-9 M to 10-11 M. Kd values can be determined, for example, by means of surface plasmon resonance spectrometry.

[0070] The antibody-drug conjugates of the invention likewise exhibit affinities in these ranges. The affinity is preferably not substantially affected by the conjugation of the drugs (in general, the affinity is reduced by less than an order of magnitude, in other words, for example, in most, from 10-8 M to 10-7 M).

[0071] The antibodies used according to the invention are also preferably characterized by high selectivity. High selectivity exists when the antibody of the invention exhibits an affinity for the target protein that is better by a factor of at least 2, preferably by a factor of 5 or more preferably by a factor of 10, than for another independent antigen, for example human serum albumin (the affinity can be determined, for example, by means of surface plasmon resonance spectrometry).

[0072] Furthermore, the antibodies of the invention that are used are preferably cross-reactive. In order to be able to facilitate and better interpret preclinical studies, for example activity or toxicological studies (e.g. in xenograft mice), it is advantageous if the antibody used according to the invention does not only bind to the human target protein, but also binds to the target protein of species in the species used for the studies. In one embodiment, the antibody used according to the invention, in addition to the human target protein, is cross-reactive to the target protein of at least one additional species. For activity and toxicological studies, it is preferred to use species from the rodent, dog and non-human primate families. Preferred rodent species are mouse and rat. Preferred non-human primates are rhesus macaques, chimpanzees and old world monkeys.

[0073] In one embodiment, the antibody used according to the invention, in addition to the human target protein, has cross-reactivity to the target protein of at least one additional species selected from the group of species consisting of mouse, rat and old world monkey (Macaca fascicularis). Particularly preferred are antibodies used according to the invention which, in addition to the human target protein, have at least cross-reactivity to the mouse target protein. Preference is given to cross-reactive antibodies whose affinity for the target protein of the additional non-human species differs by a factor of not more than 50, more particularly by a factor of not more than ten, from the affinity for the human target protein.

Antibodies directed against a cancer target molecule

[0074] The target molecule to which the ligand, e.g. an antibody or an antigen-binding fragment thereof, is directed is preferably a cancer targeting molecule. The term “cancer targeting molecule” describes a target molecule that is more abundantly present in one or more cancer cell species than in non-cancerous cells of the same tissue type. Preferably, the cancer targeting molecule is selectively present in one or more cancer cell species compared to non-cancerous cells of the same tissue type, whereby selectively describes an at least two-fold enrichment in cancer cells compared to non-cancerous cells of the same tissue type (a “selective cancer targeting molecule”). The use of cancer targeting molecules allows for selective therapy of cancer cells using the conjugates according to the invention.

[0075] Antibodies that are specific against an antigen, e.g., cancer cell antigen, can be prepared by a person of ordinary skill in the art by methods with which one is familiar (e.g., recombinant expression) or can be purchased commercially (e.g., from Merck KGaA, Germany). Examples of commercially available antibodies known in cancer therapy are Erbitux® (cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche), and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1kappa type that, in a cell-based assay (Kd = 5 nM), binds to the extracellular domains of the human epidermal growth receptor with high affinity. The antibody is recombinantly produced in CHO cells. All of these antibodies can also be produced as aglycosylated variants of these antibodies, either by deglycosylation via PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

[0076] In a preferred embodiment, the target molecule is a selective cancer target molecule.

[0077] In a particularly preferred embodiment, the target molecule is a protein.

[0078] In one embodiment, the target molecule is an extracellular target molecule. In a preferred embodiment, the extracellular target molecule is a protein.

[0079] Cancer target molecules are known to those skilled in the art. Examples thereof are listed below.

[0080] Examples of cancer targeting molecules are:(1) EGFR (EGF receptor, NCBI Reference Sequence NP_005219.2, NCBI Gene ID: 1956)(2) mesothelin (SwissProt Reference Q13421-3), mesothelin which is encoded by amino acids 296-598. Amino acids 37-286 code for megakaryocyte enhancer factor. Mesothelin is anchored to the cell membrane by a GPI anchor and is located extracellularly.(3) Carbohydrase IX (CA9, SwissProt Reference Q16790), NCBI Gene ID: 768)(4) C4.4a (NCBI Reference Sequence NP_055215.2; synonym LYPD3, NCBI Gene ID: 27076)(5) CD52 (NCBI Reference Sequence NP_001794.2)(6) Her2 (ERBB2; NCBI Reference Sequence NP_004439.2; NCBI Gene ID: 2064)(7) CD20 (NCBI Reference Sequence NP_068769.2)(8) the lymphocyte activation antigen CD30 (SwissProt ID P28908)(9) the lymphocyte adhesion molecule lymphocyte CD22 (SwissProt ID P20273; NCBI Gene ID: 933)(10) the myeloid cell surface antigen CD33 (SwissProt ID P20138; NCBI Gene ID: 945)(11) the transmembrane glycoprotein NMB (GPNMB, SwissProt ID Q14956, NCBI Gene ID: 10457)(12) the adhesion molecule CD56 (SwissProt ID P13591)(13) the surface molecule CD70 (SwissProt ID P32970, NCBI Gene ID: 970) CD19 (SwissProt ID P15391, NCBI Gene ID: 930)(16) Mucin-1 surface protein (MUC1, SwissProt ID P15941, NCBI Gene ID: 4582)(17) CD138 surface protein (SwissProt ID P18827)(18) alphaV integrin (NCBI Reference Sequence: NP_002201.1, NCBI Gene ID: 3685)(19) teratocarcinoma-derived growth factor 1 protein TDGF1 (NCBI Reference Sequence: NP_003203.1, NCBI Gene ID: 6997)(20) prostate-specific membrane antigen PSMA (Swiss Prot ID: Q04609; NCBI Gene ID: 2346)(21) protein tyrosine kinase EPHA2 (Swiss Prot ID: P29317, NCBI Gene ID: 1969)(22) surface protein SLC44A4 (NCBI Reference Sequence: NP_001171515.1, NCBI Gene ID: 80736)(23) surface protein BMPR1B (SwissProt: O00238)(24) transport protein SLC7A5 (SwissProt: Q01650)(25) epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8, Gene ID: 26872)(26) ovarian carcinoma antigen MUC16 (SwissProt: Q8WXI7, Gene ID: 94025)(27) the transport protein SLC34A2 (SwissProt: O95436, Gene ID: 10568)(28) the surface protein SEMA5b (SwissProt: Q9P283)(29) the surface protein LYPD1 (SwissProt: Q8N2G4)(30) the endothelin type B receptor EDNRB (SwissProt: P24530, NCBI Gene ID: 1910) (31) the RING-finger protein RNF43 (SwissProt: Q68DV7)(32) the prostate carcinoma-associated protein STEAP2 (SwissProt: Q8NFT2)(33) the cation channel TRPM4 (SwissProt: Q8TD43)(34) the complement receptor CD21 (SwissProt: P20023)(35) the B-cell antigen receptor complex-associated protein CD79b (SwissProt: P40259, NCBI Gene ID: 974)(36) the cell adhesion antigen CEACAM6 (SwissProt: P40199)(37) the dipeptidase DPEP1 (SwissProt: P16444)(38) the interleukin receptor IL20Ralpha (SwissProt: Q9UHF4, NCBI Gene ID: 3559)(39) the proteoglycan BCAN (SwissProt: Q96GW7)(40) the epherin receptor EPHB2 (SwissProt: P29323)(41) the prostate stem cell-associated protein PSCA (NCBI Reference Sequence: NP_005663.2 )(42) the surface protein LHFPL3 (SwissProt: Q86UP9)(43) the receptor protein TNFRSF13C (SwissProt: Q96RJ3)(44) the B cell antigen receptor complex-associated protein CD79a (SwissProt: P11912)(45) the receptor protein CXCR5 (CD185; SwissProt: P32302;NCBI Gene ID 643, NCBI Reference Sequence: NP_001707.1)(46) the P2X5 ion channel (SwissProt: Q93086)(47) the CD180 lymphocyte antigen (SwissProt: Q99467)(48) the FCRL1 receptor protein (SwissProt: Q96LA6)(49) the FCRL5 receptor protein (SwissProt: Q96RD9)(50) the MHC class II molecule Ia antigen HLA-DOB (NCBI Reference Sequence: NP_002111.1) (51) the VTCN1 T-cell protein (SwissProt: Q7Z7D3)(52) TWEAKR (FN14, TNFRSF12A, NCBI Reference: NP_057723.1, NCBI Gene ID: 51330)(53) lymphocyte antigen CD37 (Swiss Prot: P11049, NCBI Gene ID: 951)(54) FGF receptor 2; FGFR2 (NCBI Gene ID: 2263; Official Symbol: FGFR2). The FGFR2 receptor occurs in different splice variants (alpha, beta, IIIb, IIIc). All splice variants can act as target molecules.(55) transmembrane glycoprotein B7H3 (CD276; NCBI Gene ID: 80381 NCBI Reference Sequence:NP_001019907.1, Swiss Prot: Q5ZPR3-1)(56) B-cell receptor BAFFR (CD268; NCBI Gene ID:115650)(57) ROR receptor protein 1 (NCBI Gene ID: 4919)(58) CD123 surface receptor (IL3RA; NCBI Gene ID: 3563; NCBI Reference Sequence: NP_002174.1;Swiss-Prot: P26951)(59) receptor syncytin protein (NCBI Gene ID 30816)(60) aspartate beta-hydroxylase (ASPH; NCBI Gene ID 444)(61) the cell surface glycoprotein CD44 (NCBI Gene ID: 960)(62) CDH15 (Cadherin 15, NCBI Gene ID: 1013)(63) the cell surface glycoprotein CEACAM5 (NCBI Gene ID: 1048)(64) the cell adhesion L1-like molecule (CHL1, Gene ID NCBI Gene ID: 10752) (65) c-Met receptor tyrosine kinase (NCBI Gene ID: 4233)(66) notch ligand DLL3 (NCBI Gene ID: 10683)(67) epherin A4 (EFNA4, NCBI Gene ID: 1945)(68) ectonucleotide pyrophosphatase/phosphodiesterase 3(ENPP3, NCBI Gene ID: 5169)(69) coagulation factor III (F3, NCBI Gene ID: 2152)(70) FGF receptor 3 (FGFR3, NCBI Gene ID: 2261)(71) folate hydrolase FOLH1 (NCBI Gene ID: 2346)(72) folate receptor 1 (FOLR1; NCBI Gene ID: 2348)(73) guanylate cyclase 2C (GUCY2C, NCBI Gene ID: 2984)(74) KIT proto-oncogene receptor tyrosine kinase (NCBI Gene ID: 3815)(75) lysosome-associated membrane protein 1 (LAMP1, NCBI Gene ID: 3916)(76) lymphocyte antigen complex 6, site E (LY6E, NCBI Gene ID: 4061)(77) NOTCH3 protein (NCBI Gene ID: 4854)(78) protein tyrosine kinase 7 (PTK7, NCBI Gene ID: 5754)(79) nectin cell adhesion molecule 4 (PVRL4, NECTIN4, NCBI Gene ID: 81607)(80) the transmembrane protein syndecan 1 (SDC1, NCBI Gene ID: 6382)(81) the SLAM family member 7 (SLAMF7, NCBI Gene ID:57823)(82) the transport protein SLC39A6 (NCBI Gene ID:25800)(83) the SLIT and NTRK-like family member 6 (SLITRK6, NCBI Gene ID: 84189)(84) the cell surface receptor TACSTD2 (NCBI Gene ID: 4070)(85) the TNFRSF8 receptor protein (NCBI Gene ID: 943)(86) the TNFSF13B receptor protein (NCBI Gene ID: 10673)(87) the TPBG glycoprotein (NCBI Gene ID: 7162)(88) the cell surface receptor TROP2 (TACSTD2, NCBI Gene ID: 4070)(89) the galanin-like G-protein-coupled receptor KISS1R (GPR54, NCBI Gene ID: 84634)(90) the transport protein SLAMF6 (NCBI Gene ID: 114836)

[0081] In a preferred subject matter of the invention, the cancer target molecule is selected from the group consisting of the cancer target molecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particular, CD123, CXCR5 and B7H3.

[0082] In a further and particularly preferred subject matter of the invention, the ligand binds to an extracellular cancer target molecule selected from the group consisting of the cancer target molecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particular, CD123, CXCR5 and B7H3.

[0083] In a further and particularly preferred subject matter of the invention, the ligand specifically binds to an extracellular cancer target molecule selected from the group consisting of the cancer target molecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particular CD123, CXCR5 and B7H3. In a preferred embodiment, the ligand, after binding to its extracellular target molecule on the target cell, is internalized by the target cell through binding. This causes the ligand-drug conjugate, which may be an immunoconjugate or an ADC, to be taken up by the target cell. The ligand is then processed, preferably intracellularly, preferably lysosomally.

[0084] In one embodiment, the ligand is a binding protein. In a preferred embodiment, the ligand is an antibody, an antigen-binding antibody fragment, a multispecific antibody, or an antibody mimic.

[0085] Preferred antibody mimetics are affibodies, adnectins, anticalins, DARPins, avimers, or nanobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies.

[0086] In a preferred embodiment, the ligand is an antibody or an antigen-binding antibody fragment, more preferably an isolated antibody or an isolated antigen-binding antibody fragment.

[0087] Preferred antigen-binding antibody fragments are Fab, Fab', F(ab')2 and Fv fragments, diabodies, DAbs, linear antibodies and scFv. Particularly preferred are Fab, diabodies and scFv.

[0088] In a particularly preferred embodiment, the ligand is an antibody. Particularly preferred are monoclonal antibodies or antigen-binding antibody fragments thereof. Particularly preferred are human, humanized or chimeric antibodies or antigen-binding antibody fragments thereof.

[0089] Antigen-binding antibodies or antibody fragments that bind cancer target molecules can be prepared by one of ordinary skill in the art using known processes, such as, for example, chemical synthesis or recombinant expression. Ligands for cancer target molecules can be purchased commercially or can be prepared by one of ordinary skill in the art using known processes, such as, for example, chemical synthesis or recombinant expression. Additional processes for preparing antigen-binding antibodies or antibody fragments are described in WO 2007/070538 (see page 22 “Antibodies”). The skilled person knows how processes such as phage display libraries (e.g., Morphosys HuCAL Gold) can be compiled and used to reveal antibodies or antigen-binding antibody fragments (see WO 2007/070538, page 24 ff and Example AK 1 on page 70, Example AK 2 on page 72). Additional processes for preparing antibodies using B cell DNA libraries are described, for example, on page 26 (WO 2007/070538). Processes for humanizing antibodies are described on page 30-32 of WO2007070538 and in detail in Queen, et al., Pros. Natl. Acad. Sci. USA 8610029-10033, 1989 or in WO 90/0786. Furthermore, processes for recombinant expression of proteins in general and of antibodies in particular are known to the person skilled in the art (see, for example, in Berger and Kimrnel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al., (Molecular Cloning A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.], Current Protocols, Green Publishing Associates, Inc./John Wiley & Sons, Inc.); Harlow et al., (Monoclonal Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press (19881, Paul [Ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al., (Using Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)). The person skilled in the art knows which corresponding vectors, promoters and signal peptides are required for the expression of a protein/antibody. Common processes are also described in WO 2007/070538 at pages 41-45. Processes for preparing an IgG1 antibody are described, for example, in 2007/070538 in Example 6 on page 74 ff. Processes that allow the determination of the internalization of an antibody after binding to its antigen are known to the person skilled in the art and are described, for example, in 2007/070538 at page 80. The person skilled in the art is able to use the processes described in 2007/070538 that were used to prepare carbonanhydrase IX (Mn) antibodies in analogy for the preparation of antibodies with different target molecule specificity.

Bacterial expression

[0090] The person skilled in the art is aware of the manner in which antibodies, antigen-binding fragments thereof or variants thereof can be produced with the aid of bacterial expression.

[0091] Suitable expression vectors for bacterial expression of desired proteins are constructed by inserting a DNA sequence encoding the desired protein into the functional reading frame together with suitable translation initiation and translation termination signals and a functional promoter. The vector comprises one or more phenotypically selectable markers and an origin of replication to allow retention of the vector and, if desired, amplification thereof within the host. Suitable prokaryotic hosts for transformation include, but are not limited to, E. coli, Bacillus subtilis, Salmonella typhimurium and various species of the genera Pseudomonas, Streptomyces and Staphylococcus. Bacterial vectors may be based on, for example, bacteriophages, plasmids or phagemids. Such vectors may contain selectable markers and a bacterial origin of replication, which are derived from commercially available plasmids. Many commercially available plasmids typically contain elements of the well-known cloning vector pBR322 (ATCC 37017). In bacterial systems, several advantageous expression vectors can be selected based on the intended use of the protein to be expressed.

[0092] After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is again conserved/induced by suitable means (e.g., a change in temperature or chemical induction), and the cells are cultured for an additional period. The cells are typically harvested by centrifugation and, if necessary, digested in a physical or chemical manner, and the resulting crude extract is retained for further purification.

[0093] Therefore, an additional embodiment of the present invention is an expression vector comprising a nucleic acid encoding a novel antibody of the present invention.

[0094] The antibodies of the present invention or antigen-binding fragments thereof include naturally purified products, products that originate from chemical synthesis, and products that are produced by recombinant technologies in prokaryotic hosts, for example, E. coli, Bacillus subtilis, Salmonella typhimurium and various species of the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably E. coli.

Mammalian cell expression

[0095] One skilled in the art is aware of the manner in which antibodies, antigen-binding fragments thereof or variants thereof can be produced with the aid of mammalian cell expression.

[0096] Exemplary regulatory sequences for expression in a mammalian cell host include viral elements that lead to high-level expression in mammalian cells, such as promoters and/or expression enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Expression of the antibodies may be constitutive or regulated (e.g., induced by addition or removal of small molecule inducers, such as tetracycline in combination with the Tet system).

[0097] For further description of viral regulatory elements and sequences thereof, reference is made, for example, to US 5,168,062 to Stinski, US 4,510,245 to Bell et al. and US 4,968,615 to Schaffner et al. Recombinant expression vectors may likewise include an origin of replication and selectable markers (see, for example, US 4,399,216, 4,634,665 and US 5,179,017). Suitable selectable markers include genes that confer resistance to substances such as G418, puromycin, hygromycin, blasticidin, zeocin/bleomycin, or methotrexate, or selectable markers that lead to auxotrophy of a host cell, such as glutamine synthetase (Bebbington et al., Biotechnology (N Y). 1992 Feb;10(2):169-75), when the vector has been introduced into the cell.

[0098] For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the neo gene confers resistance to G418, the Aspergillus terreus bsd gene confers resistance to blasticidin, puromycin N-acetyltransferase confers resistance to puromycin, the Shble gene product confers resistance to zeocin, and resistance to hygromycin is conferred by the E. coli hygromycin resistance gene (hyg or hph). Selectable markers such as DHFR or glutamine synthetase are also useful for amplification techniques in conjunction with MTX and MSX.

[0099] Transfection of an expression vector into a host cell can be performed using standard techniques, including electroporation, nucleofection, calcium phosphate precipitation, lipofection, polycation-based transfection such as polyethyleneimine (PEI)-based transfection, and DEAE-dextran transfection.

[0100] Suitable mammalian host cells for the expression of antibodies, antigen-binding fragments thereof, or variants thereof include Chinese hamster ovary (CHO) cells, such as CHO-K1, CHO-S, CHO-K1SV [which include DHFR-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun;33(2):405-12, used with a DHFR-selectable marker, as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621, and other knockout cells, as detailed in Fan et al., Biotechnol Bioeng. 2012 April;109(4):1007-15), NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

[0101] Expression of antibodies, antigen-binding fragments thereof, or variants thereof can also be carried out in a semi-stable or transient manner in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293 Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7, or CAP-T cells (e.g., similar to Durocher et al., Nucleic Acids Res. 2002 Jan;15;30(2):E9).

[0102] In some embodiments, the expression vector is constructed such that the protein to be expressed is secreted into the cell culture medium in which the host cells grow. Antibodies, antigen-binding fragments thereof, or variants thereof can be obtained from the cell culture medium with the aid of protein purification methods known to those skilled in the art.

Purification

[0103] Antibodies, antigen-binding fragments thereof, or variants thereof may be obtained and purified from recombinant cell cultures using well-known methods, examples of which include ethane and ammonium sulfate precipitation, acid extraction, protein A chromatography, protein G chromatography, cation or anion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography (HIC), affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography ("HPLC") may also be employed for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), for example, Chapters 1, 4, 6, 8, 9, 10.

[0104] The antibodies of the present invention or antigen-binding fragments thereof, or variants thereof include naturally purified products, products from chemical synthesis methods, and products that are produced with the aid of recombinant techniques in prokaryotic or eukaryotic host cells. Eukaryotic hosts include, for example, yeast cells, higher plant cells, insect cells, and mammalian cells. Depending on the host cell chosen for recombinant expression, the expressed protein may be in glycosylated or non-glycosylated form.

[0105] In a preferred embodiment, the antibody is purified (1) to an extent of greater than 95 wt %, as measured, for example, by the Lowry method, by UV-vis spectroscopy, or by SDS capillary gel electrophoresis (e.g., with a LabChip GXII calibrator, GX 90, or Biorad Bioanalyzer instrument), and in more preferred embodiments greater than 99 wt %, (2) to an extent suitable for determination of at least 15 residues of the N-terminal or internal amino acid sequence, or (3) to homogeneity determined by SDS-PAGE under reducing or non-reducing conditions with the aid of Coomassie Blue or, preferably, silver staining.

[0106] Usually, an isolated antibody is obtained with the aid of at least one protein purification step. Anti-CD123 antibodies

[0107] According to the invention, it is possible to use anti-CD123 antibodies.

[0108] The phrase “anti-CD123 antibody” or “an antibody that specifically binds to CD123” refers to an antibody that binds the cancer target molecule CD123 (IL3RA; NCBI Gene ID: 3563; NCBI Reference Sequence: NP_002174.1; Swiss-Prot: P26951; SEQ ID NO:111), preferably with an affinity sufficient for a diagnostic and/or therapeutic application. In particular embodiments, the antibody binds CD123 with a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM.

[0109] Sun et al. (Sun et al., 1996, Blood 87(1)83-92) describes the generation and properties of the monoclonal antibody 7G3, which binds the N-terminal domain of IL-3Rα, CD123. US Patent No. 6,177,078 (Lopez) refers to the anti-CD123 antibody 7G3. A chimeric variant of this antibody (CSL360) is described in 2009/070844, and a humanized version (CSL362) in 2012/021934. The sequence of the 7G3 antibody is disclosed in EP2426148. This sequence constitutes the starting point for humanized antibodies obtained by CDR grafting.

[0110] An antibody that, upon binding of cell surface antigen, is internalized particularly well is the anti-CD123 antibody 12F1 disclosed by Kuo et al. (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82). The 12F1 antibody binds with higher affinity to CD123 than the 7G3 antibody and, upon binding of cell surface antigen, is internalized markedly faster than 7G3. Bispecific scFv immunofusion proteins based on 12F1 are disclosed in 2013/173820. The TPP-6013 antibody is a chimeric variant of 12F1.

[0111] The invention relates in particular to conjugates with antibodies or antigen-binding antibody fragments thereof or variants thereof derived from the antibodies 7G3 (Sun et al., 1996, Blood 87(1):83-92) and 12F1 (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82) which originate from the mouse, or to conjugates with antibodies or antigen-binding antibody fragments thereof or variants thereof derived from the antibody 12F1 (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82) which originate from the mouse.

[0112] Humanized variants of the murine 7G3 antibody and the murine 12F1 antibody were generated by CDR grafting into a subsequent optimization and human framework and are preferred examples in the context of the present invention.

[0113] Particular preference is given in the context of the present invention to the anti-CD123 antibodies TPP-9476, TPP-8988, TPP-8987 and TPP-6013. Anti-CXCR5 antibodies

[0114] According to the invention, it is possible to use anti-CXCR5 antibodies.

[0115] The phrase “anti-CXCR5 antibody” or “an antibody that specifically binds to CXCR5” refers to an antibody that binds the cancer target molecule CXCR5 (NCBI Reference Sequence: NP_001707.1; SEQ ID NO: 112), preferably with an affinity sufficient for a diagnostic and/or therapeutic application. In particular embodiments, the antibody binds CXCR5 with a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM.

[0116] Examples of antibodies and antigen-binding fragments that bind to CXCR5 are known to those skilled in the art and are described, for example, in EP2195023.

[0117] Hybridoma cells for the mouse antibody RF8B2 (ACC2153) were purchased from DSMZ and the antibody sequence was identified by standard methods. This sequence constitutes the starting point for humanized antibodies obtained by CDR grafting.

[0118] Humanized variants of this antibody were generated by CDR grafting onto germline sequences.

[0119] Such antibodies and antigen-binding fragments may be used in the context of this invention.

[0120] Particular preference is given in the context of the present invention to the anti-CXCR5 antibodies TPP-9574 and TPP-9580.Anti-B7H3 antibodies

[0121] According to the invention, it is possible to use anti-B7H3 antibodies.

[0122] The phrase “anti-B7H3 antibody” or “an antibody that specifically binds to B7H3” refers to an antibody that binds the cancer target molecule B7H3 (NCBI Reference Sequence: NP_001019907.1 SEQ ID NO: 113), preferably with an affinity sufficient for a diagnostic and/or therapeutic application. In particular embodiments, the antibody binds B7H3 with a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM.

[0123] Examples of antibodies and antigen-binding fragments that bind to B7H3 are known to those skilled in the art and are described, for example, in WO201109400, EP1773884 and WO2014061277. EP2121008 describes the anti-B7H3 antibody 8H9 and the CDR sequences thereof.

[0124] Such antibodies and antigen-binding fragments may be used in the context of this invention.

[0125] A preferred embodiment of the anti-B7H3 antibodies was obtained by screening an antibody phage display library for cells expressing recombinant mouse B7H3 (mouse CD276; Gene ID: 102657) and human B7H3 (human CD276; Gene ID: 80381). The antibodies obtained were transformed into the human IgG1 format. The anti-B7H3 antibody TPP-8382 is a preferred example.

[0126] Particular preference is given in the context of the present invention to the anti-B7H3 antibody TPP-8382. Anti-TWEAKR antibodies

[0127] According to the invention, it is possible to use anti-TWEAKR antibodies.

[0128] The term “anti-TWEAKR antibody” or “an antibody that specifically binds to TWEAKR” refers to an antibody that binds the cancer target molecule TWEAKR (NCBI Reference Sequence: NP_057723.1 SEQ ID NO: 114), preferably with an affinity sufficient for a diagnostic and/or therapeutic application. In particular embodiments, the antibody binds TWEAKR with a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM.

[0129] Examples of antibodies that bind to TWEAKR are disclosed, for example, in WO2009/020933 (A2), WO2009/140177 (A2), WO 2014/198817 (A1) and WO 2015/189143 (A1). Such antibodies and antigen-binding fragments may be used in the context of this invention.

[0130] ITEM-4 is an anti-TWEAKR antibody that has been described by Nakayama et al. (Nakayama, et al., 2003, Biochem Biophy Res Comm, 306:819-825). Humanized variants of this antibody based on CDR grafting are described by Zhou et al. (Zhou et al., 2013, J Invest Dermatol. 133(4):1052-62) and in 2009/020933. Such antibodies and antigen-binding fragments can be used in the context of this invention.

[0131] Particular preference is given in the context of the present invention to the anti-TWEAKR antibodies TPP-7006 and TPP-7007. These are humanized variants of the ITEM-4 antibody. These antibodies and antigen-binding fragments may be used with preference in the context of this invention. Anti-HER2 antibodies:

[0132] According to the invention, it is possible to use anti-HER2 antibodies.

[0133] The phrase “anti-HER2 antibody” or “an antibody that specifically binds to HER2” refers to an antibody that binds the cancer target molecule HER2 (NCBI Reference Sequence: NP_004439.2 SEQ ID NO: 115), preferably with an affinity sufficient for a diagnostic and/or therapeutic application. In particular embodiments, the antibody binds HER2 with a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM.

[0134] An example of an antibody that binds to the cancer targeting molecule Her2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody used, among others, for the treatment of breast cancer. In a particularly preferred embodiment, the anti-HER2 antibody is TPP-1015 (trastuzumab analog).

[0135] Additional examples of antibodies that bind to HER2 are, in addition to trastuzumab (INN 7637, CAS No. RN: 180288-69-1) and pertuzumab (CAS No. 380610-27-5), the antibodies disclosed in 2009/123894-A2, WO 200/8140603-A2 or in 2011/044368-A2. An example of an anti-HER2 conjugate is trastuzumab-emtansine (INN No. 9295). Such antibodies and antigen-binding fragments can be used in the context of this invention.

[0136] Particular preference is given in the context of this invention to the anti-HER2 antibody TPP-1015 (analogous to trastuzumab). Anti-EGFR antibodies

[0137] According to the invention, it is possible to use anti-EGFR antibodies.

[0138] The term “anti-EGFR antibody” or “an antibody that specifically binds to EGFR” refers to an antibody that binds the cancer target molecule EGFR (NCBI Reference Sequence: NP_005219.2 SEQ ID NO: 116), preferably with an affinity sufficient for a diagnostic and/or therapeutic application. In particular embodiments, the antibody binds EGFR with a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM.

[0139] In a preferred embodiment, the anti-EGFR antibodies are selected from the group consisting of TPP-981, Cetuximab, panitumumab, nimotuzumab. In a particularly preferred embodiment, the anti-EGFR antibody is TPP-981.

[0140] Additional EGFR antibody embodiments are as follows: zalutumumab / 2F8 / HuMax-EGFr, from Genmab A/S (WO 02/100348, WO 2004/056847, INN number 8605) necitumumab / 11F8, ImClone / IMC-11F8, from ImCloneSystems Inc. [Eli Lilly & Co] (WO 2005/090407 (EP 01735348-A1, US 2007/0264253-A1, US 7,598,350, WO 2005/090407-A1), INN number 9083) matuzumab / anti-EGFR MAb, Merck KGaA / anti-EGFR MAb, Takeda / EMD 72000 / EMD-6200 / EMD-72000 and EMD-55900 / MAb 425 / monoclonal antibody 425, from MerckKGaA / Takeda (WO 92/15683, number INN 8103 (matuzumab)) • RG-7160 / GA-201 / GA201 / R-7160 / R7160 / RG7160 /RO-4858696 / RO-5083945 / RO4858696 / RO5083945, deGlycart Biotechnology AG (Roche Holding AG)(documents WO 2010/112413-A1, WO 2010/115554) • GT-MAB 5.2-GEX / CetuGEX, from Glicotope GmbH(WO documents 2008/028686-A2 (EP 01900750-A1, EP 01911766-A1, EP 02073842-A2, US 2010/0028947-A1)• ISU-101, from Isu Abxis Inc (ISU Chemical Co Ltd)/Scancell (WO 2008/004834-A1)• ABT-806/mAb-806/ch-806/anti-EGFR monoclonal antibody 806, from Ludwig Institute for Cancer Research/Abbott/Life Science Pharmaceuticals (WO 02/092771, WO 2005/081854 and WO 2009/023265)• SYM-004 (consist of two chimeric antibody IgG1s (992 and 1024)), from Symphogen A/S (WO 2010/022736-A2)• MR1-1 /MR1-1KDEL, from IVAX Corp (Teva Pharmaceutical Industries Ltd) (Duke University), (patent: WO2001/062931-A2)• Antibody against deletion mutant, EGFRvIII, from Amgen/Abgenix (WO 2005/010151, US7,628,986)• SC-100, from Scancell Ltd (WO 01/088138-A1)• MDX-447 / EMD 82633 / BAB-447 / H 447 / MAb, EGFR, Medarex/Merck KGaA, from Bristol-Myers Squibb (US) / Merck KGaA (DE) / Takeda (JP), (WO 91/05871, WO 92/15683)• anti-EGFR-Mab, from Xencor (WO 2005/056606)• DXL-1218 / anti-EGFR monoclonal antibody (cancer), InNexus, from InNexus Biotechnology Inc, Pharmaprojects PH048638Anti-carbonanhydrase IX antibodies

[0141] Examples of antibodies that bind to the cancer targeting molecule carbonanhydrase IX are described in 2007/070538-A2 (e.g., Claims 1—16). Anti-C4.4a antibodies:

[0142] Examples of C4.4a antibodies and antigen-deleting fragments are described in 2012/143499 A2. The sequences of the antibodies are set out in Table 1 of WO 2012/143499 A2, where each row shows the respective amino acid CDR sequences of the variable light chain or the variable heavy chain of the antibody listed in column 1. Anti-CD20 antibodies:

[0143] An example of an antibody that binds to the cancer targeting molecule CD20 is rituximab (Genentech). Rituximab (CAS Number: 174722-31-7) is a chimeric antibody used for the treatment of non-Hodgkin's lymphoma. Such antibodies and antigen-binding fragments thereof may be used in the context of this invention. Anti-CD52 antibodies:

[0144] An example of an antibody that binds to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS Number: 216503-57-0) is a humanized antibody used for the treatment of chronic lymphocytic leukemia. Such antibodies and antigen-binding fragments thereof may be used in the context of this invention. Anti-mesothelin antibodies:

[0145] Examples of anti-mesothelin antibodies are described, for example, in WO2009/068204. All antibodies and antigen-binding fragments disclosed in WO2009/068204 can be used in the context of the invention disclosed herein. Most preferably, the antibody disclosed in WO2009/068204 is MF-T. Anti-CD30 antibodies

[0146] Examples of antibodies that bind to the cancer target molecule CD30 and can be used for the treatment of cancer, e.g. Hodgkin's lymphoma, are brentuximab, iratumumab and antibodies disclosed in 2008/092117, WO 2008/036688 or WO 2006/089232. An example of an anti-CD30 conjugate is brentuximab-vedotin (INN No. 9144). Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-CD22 antibodies

[0147] Examples of antibodies that bind to the cancer target molecule CD22 and can be used for the treatment of cancer, e.g., lymphoma, are inotuzumab and epratuzumab. Examples of anti-CD22 conjugates are inotuzumab-ozagamycin (INN No. 8574) or anti-CD22-MMAE and anti-CD22-MC-MMAE (CAS No. 139504-50-0 and 474645-27-7, respectively). Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-CD33 Antibodies

[0148] Examples of antibodies that bind to the cancer target molecule CD33 and can be used for the treatment of cancer, e.g., leukemia, are gemtuzumab and lintuzumab (INN 7580). An example of an anti-CD33 conjugate is gemtuzumab-ozagamycin. Such antibodies and antigen-binding fragments can be used in the context of this invention. Anti-NMB Antibodies

[0149] An example of an antibody that binds the cancer target molecule NMB and can be used for the treatment of cancer, e.g., melanoma or breast cancer, is glembatumumab (INN 9199). An example of an anti-NMB conjugate is glembatumumab-vedotin (CAS RN: 474645-27-7). Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-CD56 antibodies

[0150] An example of an antibody that binds the cancer targeting molecule CD56 and can be used for the treatment of cancer, e.g., multiple myeloma, small cell lung carcinoma, MCC, or ovarian carcinoma is lorvotuzumab. An example of an anti-CD57 conjugate is lorvotuzumab-mertansine (CAS RN: 139504-50-0). Such antibodies and antigen-binding fragments can be used in the context of this invention. Anti-CD70 antibodies

[0151] Examples of antibodies that bind to the cancer targeting molecule CD70 and can be used for the treatment of cancer, e.g. non-Hodgkin's lymphoma or renal cell cancer, are disclosed in 2007/038637-A2 and WO 2008/070593-A2. An example of an anti-CD70 conjugate is SGN-75 (CD70 MMAF). Such antibodies and antigen-binding fragments can be used in the context of this invention. Anti-CD74 antibodies

[0152] An example of an antibody that binds the cancer targeting molecule CD74 and can be used for the treatment of cancer, e.g., multiple myeloma, is milatuzumab. An example of an anti-CD74 conjugate is milatuzumab-doxorubicin (CAS RN: 23214-92-8). Such antibodies and antigen-binding fragments can be used in the context of this invention. Anti-CD19 antibodies

[0153] An example of an antibody that binds the cancer target molecule CD19 and can be used for the treatment of cancer, e.g. non-Hodgkin's lymphoma, is disclosed in 2008/031056-A2. Additional antibodies and examples of an anti-CD19 (SAR3419) conjugate are disclosed in 2008/047242-A2. Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-mucin antibodies

[0154] Examples of antibodies that bind to the cancer targeting molecule mucin-1 and can be used for the treatment of cancer, e.g. non-Hodgkin's lymphoma, are clivatuzumab and the antibodies disclosed in 2003/106495-A2, WO 2008/028686-A2. Examples of anti-mucin conjugates are disclosed in 2005/009369-A2. Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-CD138 antibodies

[0155] Examples of antibodies that bind to the cancer targeting molecule CD138 and conjugates thereof, which can be used for the treatment of cancer, e.g. multiple myeloma, are disclosed in WO 2009/080829-A1, WO 2009/080830-A1. Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-alphaV-integrin antibodies

[0156] Examples of antibodies that bind to the cancer targeting molecule integrin alphaV and can be used for the treatment of cancer, e.g. melanoma, sarcoma or carcinoma, are intetumumab (CAS RN: 725735-284), abciximab (CAS RN: 143653-53-6), etaracizumab (CAS RN: 892553-42-3) and the antibodies disclosed in US 7,465,449, EP 719859-A1, WO 2002/012501-A1 and WO2006/062779-A2. Examples of anti-integrin-alphaV conjugates are intetumumab-DM4 and other ADCs disclosed in 2007/024536-A2. Such antibodies and antigen-binding fragments thereof may be used in the context of this invention.Anti-TDGF1 antibodies

[0157] Examples of antibodies that bind to the cancer target molecule TDGF1 and can be used for the treatment of cancer are the antibodies disclosed in 02/077033-A1, US 7,318,924, WO 2003/083041-A2 and WO 2002/088170-A2. Examples of anti-TDGF1 conjugates are disclosed in 2002/088170-A2. Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-PSMA antibodies

[0158] Examples of antibodies that bind to the cancer target molecule PSMA and can be used for the treatment of cancer, e.g. prostate carcinoma, are the antibodies disclosed in 97/35616-A1, WO 99/47554-A1, WO 01/009192-A1 and WO2003/034903. Examples of anti-PSMA conjugates are disclosed in 2009/026274-A1 and WO 2007/002222. Such antibodies and antigen-binding fragments can be used in the context of this invention. Anti-EPHA2 antibodies

[0159] Examples of antibodies that bind to the cancer targeting molecule EPHA2 and can be used to prepare a conjugate and for the treatment of cancer are disclosed in 2004/091375-A2. Such antibodies and antigen-binding fragments can be used in the context of this invention. Anti-SLC44A4 antibodies

[0160] Examples of antibodies that bind to the cancer target molecule SLC44A4 and can be used to prepare a conjugate and for the treatment of cancer, e.g. prostate or pancreatic carcinoma, are disclosed in WO2009/033094-A2 and US2009/0175796-A1. Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-HLA-DOB antibodies

[0161] An example of an antibody that binds to the cancer target molecule HLA-DOB is the Lym-1 antibody (CAS RN: 301344-99-0) which can be used for the treatment of cancer, e.g., non-Hodgkin's lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, for example, in 2005/081711-A2. Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-VTCN1 antibodies

[0162] Examples of antibodies that bind to the cancer target molecule VTCN1 and can be used to prepare a conjugate and for the treatment of cancer, e.g. ovarian carcinoma, pancreatic, lung or breast cancer, are disclosed in 2006/074418-A2. Such antibodies and antigen-binding fragments thereof can be used in the context of this invention. Anti-FGFR2 antibodies

[0163] Examples of anti-FGFR2 antibodies and antigen-binding fragments are described in WO2013076186. The sequences of the antibodies are shown in Table 9 and Table 10 of WO2013076186. Preference is given to antibodies, antigen-binding fragments and antibody variants derived from the antibodies designated as M048-D01 and M047-D08.

Preferred antigen-binding antibodies and antibody fragments for ligand-drug conjugates according to the invention

[0164] In this application, in the context of linker-drug conjugates, reference is made to the following preferred antibodies as shown in the following Table: TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574, and TPP-9580. Table: Protein sequences of the antibodies:

[0165] TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580 are antibodies comprising one or more of the CDR sequences specified in the Table above (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) in the heavy chain variable region (VH) or the light chain variable region (VL). Preferably, the antibodies comprise the heavy chain specific variable region (VH) and/or the light chain variable region (VL). Preferably, the antibodies comprise the heavy chain specific region (IgG heavy chain) and/or the light chain specific region (IgG light chain).

[0166] TPP-981 is an anti-EGFR antibody that comprises a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 2, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 3, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 4, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 6, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 7, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 8.

[0167] TPP-1015 is an anti-HER2 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 12, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 13, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 14, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 16, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 17, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 18.

[0168] TPP-6013 is an anti-CD123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 22, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 23, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 24, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 26, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 27, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 28.

[0169] TPP-7006 is an anti-TWEAKR antibody that comprises a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 32, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 33, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 34, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 36, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 37, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 38.

[0170] TPP-7007 is an anti-TWEAKR antibody that comprises a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 42, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 43, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 44, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 46, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 47, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 48.

[0171] TPP-8382 is an anti-B7H3 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 52, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 53, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 54, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 56, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 57, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 58.

[0172] TPP-8987 is an anti-CD123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 62, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 63, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 64, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 66, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 67, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 68.

[0173] TPP-8988 is an anti-CD123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 72, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 73, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 74, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 76, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 77, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 78.

[0174] TPP-9476 is an anti-CD123 antibody that comprises a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 82, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 83, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 84, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 86, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 87, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 88.

[0175] TPP-9574 is an anti-CXCR5 antibody that comprises a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 92, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 93, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 94, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 96, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 97, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 98.

[0176] TPP-9580 is an anti-CXCR5 antibody that comprises a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 102, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 103, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 104, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 106, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 107, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 108.

[0177] TPP-981 is an anti-EGFR antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 1 and a light chain variable region (VL) as shown in SEQ ID NO: 5.

[0178] TPP-1015 is an anti-HER2 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 11 and a light chain variable region (VL) as shown in SEQ ID NO: 15.

[0179] TPP-6013 is an anti-CD123 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 21 and a light chain variable region (VL) as shown in SEQ ID NO: 25.

[0180] TPP-7006 is an anti-TWEAKR antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 31 and a light chain variable region (VL) as shown in SEQ ID NO: 35.

[0181] TPP-7007 is an anti-TWEAKR antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 41 and a light chain variable region (VL) as shown in SEQ ID NO: 45.

[0182] TPP-8382 is an anti-B7H3 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 51 and a light chain variable region (VL) as shown in SEQ ID NO: 55.

[0183] TPP-8987 is an anti-CD123 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 61 and a light chain variable region (VL) as shown in SEQ ID NO: 65.

[0184] TPP-8988 is an anti-CD123 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 71 and a light chain variable region (VL) as shown in SEQ ID NO: 75.

[0185] TPP-9476 is an anti-CD123 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 81 and a light chain variable region (VL) as shown in SEQ ID NO: 85.

[0186] TPP-9574 is an anti-CXCR5 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 91 and a light chain variable region (VL) as shown in SEQ ID NO: 95.

[0187] TPP-9580 is an anti-CXCR5 antibody that preferably comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 101 and a light chain variable region (VL) as shown in SEQ ID NO: 105.

[0188] TPP-981 is an anti-EGFR antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 9 and a light chain region as shown in SEQ ID NO: 10.

[0189] TPP-1015 is an anti-HER2 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 19 and a light chain region as shown in SEQ ID NO: 20.

[0190] TPP-6013 is an anti-CD123 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 29 and a light chain region as shown in SEQ ID NO: 30.

[0191] TPP-7006 is an anti-TWEAKR antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 39 and a light chain region as shown in SEQ ID NO: 40.

[0192] TPP-7007 is an anti-TWEAKR antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 49 and a light chain region as shown in SEQ ID NO: 50.

[0193] TPP-8382 is an anti-B7H3 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 59 and a light chain region as shown in SEQ ID NO: 60.

[0194] TPP-8987 is an anti-CD123 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 69 and a light chain region as shown in SEQ ID NO: 70.

[0195] TPP-8988 is an anti-CD123 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 79 and a light chain region as shown in SEQ ID NO: 80.

[0196] TPP-9476 is an anti-CD123 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 89 and a light chain region as shown in SEQ ID NO: 90.

[0197] TPP-9574 is an anti-CXCR5 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 99 and a light chain region as shown in SEQ ID NO: 100.

[0198] TPP-9580 is an anti-CXCR5 antibody that preferably comprises a heavy chain region as shown in SEQ ID NO: 109 and a light chain region as shown in SEQ ID NO: 110.

Isotopes, salts, solvates, isotopic variants

[0199] The present invention also encompasses all suitable isotopic variants of the compounds of the invention. An isotopic variant of a compound of the invention is understood herein to mean a compound in which at least one atom within the compound of the invention has been replaced by another atom of the same atomic number but with an atomic mass different from the atomic mass that usually or predominantly occurs in nature. Examples of isotopes that may be incorporated into a compound of the invention are those from hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 13C, 14C, 15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 129I and 131I. Particular isotopic variants of a compound according to the invention, especially those in which one or more radioactive isotopes have been incorporated, may be beneficial, for example, for examining the mechanism of action or the distribution of the active ingredient in the body; Due to the comparatively easy preparation and detection capability, especially compounds labeled with 3H or 14C isotopes are suitable for this purpose. Furthermore, the incorporation of isotopes, for example of deuterium, can lead to particular therapeutic benefits, as a consequence of increased metabolic stability of the compound, for example an extension of the half-life in the body or a reduction in the required active dose; such modifications of the compounds according to the invention can therefore in some cases also constitute a preferred embodiment of the present invention. Isotopic variants of the compounds according to the invention can be prepared by processes known to those skilled in the art, for example by the methods described below and by the procedures described in the working examples, by using corresponding isotopic modifications of the respective reagents and/or starting compounds.

[0200] Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds of the invention. Also included are salts that are not suitable for pharmaceutical applications but can be used, for example, for isolation or purification of the compounds of the invention.

[0201] Physiologically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, for example salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0202] Physiologically acceptable salts of the compounds according to the invention also include salts of conventional bases, by way of example and preferably, alkali metal salts (for example sodium and potassium salts), alkaline earth metal salts (for example calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, by way of example and preferably, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine.

[0203] Solvates in the context of the invention are described as those forms of the compounds of the invention that form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates in which the coordination is with water. The preferred solvates in the context of the present invention are hydrates.

Therapeutic use

[0204] Hyperproliferative diseases, for the treatment of which the compounds according to the invention can be used, include in particular the group of cancerous and tumoral diseases. In the context of the present invention, the following diseases are especially understood to mean, but are not limited to them: breast carcinomas and breast tumors (breast carcinomas including ductal and lobular forms, also in situ), tumors of the respiratory tract (small cell and non-small cell carcinoma, bronchial carcinoma), brain tumors (e.g. brainstem and hypothalamus, astrocytoma, ependymoma, glioblastoma, glioma, medulloblastoma, meningioma and neuroectodermal and pineal tumors), tumors of the digestive organs (carcinomas of the esophagus, stomach, gallbladder, small intestine, large intestine, rectum and anal carcinoma), liver tumors (among others hepatocellular carcinoma, mixed hepatocellular cholangiocarcinoma and echolangiocarcinoma), tumors of the head and neck region (larynx, hypopharynx, nasopharynx, oropharynx, lips and carcinomas of the oral cavity, oral melanomas), skin tumors (basalomas, spinaliomas, squamous cell carcinomas, Kaposi's sarcoma, malignant melanoma, nonmelanomatous skin cancer, Merkel cell skin cancer, mast cell tumors), connective tissue tumors (among others, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, chondrosarcomas, fibrosarcomas, haemangiosarcomas, leiomyosarcomas, liposarcomas, lymphosarcomas, and rhabdomyosarcomas), eye tumors (among others, intraocular melanoma and retinoblastoma), endocrine and exocrine gland tumors (e.g., thyroid and parathyroid glands, pancreatic and salivary gland carcinomas, adenocarcinomas), urinary tract tumors (tumors of the bladder, penis, kidneys, renal pelvis, and ureter), and reproductive organ tumors (carcinomas of the endometrium, cervix, ovary, vagina, vulva and uterus in women and carcinomas of the prostate and testes in men). These also include proliferative diseases of the blood, the lymphatic system and the spinal cord, in solid form and as circulating cells, such as leukaemias, lymphomas and myeloproliferative diseases, e.g. acute myeloid, acute lymphoblastic, chronic lymphocytic, chronic myeloid and capillary cell leukaemia and AIDS-related lymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Burkitt's lymphomas and lymphomas in the central nervous system.

[0205] These well-characterized diseases in humans may also occur with a comparable etiology in other mammals and may likewise be treated herein with the compounds of the present invention.

[0206] The antibody-drug or ligand-drug conjugates (ADCs) described herein and directed against CD123 can preferably be used for the treatment of disorders expressing CD123, such as cancerous diseases expressing it. Typically, such cancerous cells exhibit measurable amounts of CD123 measured at the protein (e.g. using an immunoassay) or RNA level. Some of these cancerous tissues show an elevated level of CD123 compared to non-cancerous tissue of the same type, preferably measured in the same patient. Optionally, the CD123 content is measured before starting cancer treatment with an antibody-drug conjugate (ADC) according to the invention (patient stratification). Ligand-drug conjugates (ADCs) directed against CD123 can preferably be used for the treatment of disorders that express CD123, such as cancerous diseases that express CD123, such as tumors of the hematopoietic and lymphatic tissue or malignant hematopoietic and lymphatic tumors. Examples of cancerous diseases associated with CD123 expression include diseases such as acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Other types of cancer include B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, blastic plasmacytoid dendritic cell neoplasia (BPDCN), Hodgkin's lymphoma, immature T-cell acute lymphoblastic leukemia (immature T-ALL), Burkitt's lymphoma, follicular lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL). The methods of the described invention comprise the treatment of patients suffering from CD123-expressing cancer, wherein the method comprises the administration of an antibody-drug conjugate (ADC) according to the invention.

[0207] The treatment of the above-mentioned cancerous diseases with the compounds according to the invention comprises both a treatment of solid tumors and a treatment of metastasizing or circulating forms thereof.

[0208] In the context of this invention, the term "treatment" or "treating" is used in the conventional sense and means attending to, caring for and looking after a patient with the aim of combating, reducing, attenuating or alleviating a disease or health abnormality, and improving the living conditions impaired by this disease, such as, for example, in the case of cancer.

[0209] The present invention thus further provides the use of the compounds of the invention for the treatment and/or prevention of disorders, in particular the aforementioned disorders.

[0210] The present invention further provides the use of the compounds of the invention for the production of a medicament for the treatment and/or prevention of disorders, especially the disorders mentioned above.

[0211] The present invention further provides the use of the compounds of the invention in a method for the treatment and/or prevention of disorders, especially the disorders mentioned above.

[0212] The present invention further provides a method of treating and/or preventing disorders, especially the disorders mentioned above, using an effective amount of at least one of the compounds of the invention.

[0213] The compounds of the invention can be used separately or, if required, in combination with one or more other pharmacologically active substances, provided that this combination does not lead to undesirable or unacceptable side effects. The present invention therefore further provides medicaments comprising at least one of the compounds of the invention and one or more other drugs, in particular for the treatment and/or prevention of the aforementioned disorders.

[0214] For example, the compounds of the present invention may be combined with known anti-hyperproliferative, cytostatic, cytotoxic or immunotherapeutic substances for the treatment of cancerous diseases. Examples of suitable combination drugs include:

[0215] 131I-chTNT, abarelix, abiraterone, aclarubicin, adalimumab, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, hexyl-5-aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole-dithiolethione, anetumab-ravtansine, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabine, arsenic trioxide, asparaginase, atezolizumab, avelumab, axitinib, azacitidine, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, buserelin, bosutinib, brentuximab-vedotin, busulfan, cabazitaxel, cabozantinib, calcitonin, calcium folinate, calcium levofolinate, capecitabine, capromab, carbomazepine, carboplatin, carboquone, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleucin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, dabrafenib, darolutamide, dasatinib, daunorubicin, decitabine, degarelix, denileukin-diftitox, denosumab, depreotide, deslorelin, dexrazoxane, dibrospdium chloride, dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, durvalumab, edrecolomab, elliptinium acetate, endostatin, enocitabine, enzalutamide, epacadostat, epirubicin, epitiostanol, epoetin-alpha, epoetin-beta, epoetin-zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estramustine, etoposide, ethylenyl oestradiol, everolimus, exemestane, fadrozole, fentanyl, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine salt, gadoversetamide, gadoxetic acid disodium salt (gadoxetic acid disodium salt) -EOB- DTPA), gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, glucarpidase, glutoxim, goserelin, granisetron, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF ), histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds, ibandronic acid, ibritumomab-tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, interferon-alpha, interferon-beta, interferon-gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, lasocoline, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine-sodium, lipegfilgrastim, lisuride, lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metyrosine, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, methyl ester hydrochloride morphine, morphine sulfate, nabilone, nabiximols, nafarelin, naloxone + pentazocine, naltrexone, nartograstim, necitumumab, nedaplatin, nelarabine, neridronic acid, netupitant/palonosetron, nivolumab, nivolumab, pentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicin, p53 gene therapy, paclitaxel, palbociclib , palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, pembrolizumab, PEG-interferon Alfa-2b, pembrolizumab, pemetrexed, pentostatin, peplomycin, perflubutane, perfosfamide, pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, polyglusam, polyoestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer-sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, rogaratinib, rolapitant, romidepsin, romurtid, roniciclib, samarium lexidronam (153Sm), satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, glycididazole sodium, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogen laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99m Tc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]- octreotide, tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide,testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alpha, thioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, treosulfan, tretinoin, trifluridine + tipiracil, trametinib, trilostane, triptorelin, trofosfamide, thrombopoietin, ubenimex, valrubicin, vandetanib, vapreotide, vatalanib, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.

[0216] Furthermore, the compounds of the present invention can be combined, for example, with ligands (e.g. antibodies) which, by way of example, can bind to the following targets: OX-40, CD137/4-1BB, DR3, IDO1/IDO2, LAG-3, CD40.

[0217] Since a cell-non-permeable toxophore metabolite of a ligand-drug conjugate (ADC) would have no detrimental effect on cells of the adaptive immune system, the invention further provides the combination of a ligand-drug conjugate (ADC) according to the invention with a cancer immunotherapy for use in the treatment of cancer or tumors. The intrinsic mechanism of action of cytotoxic ligand-drug conjugates comprises the direct triggering of cell death of tumor cells and then the release of tumor antigens that can stimulate an immune response. Furthermore, there are indications that the toxophore class of KSP inhibitor induces immunogenic cell death (ICD) markers in vitro. Thus, the combination of the ligand-drug conjugates (ADCs) of the present invention with one or more cancer immunotherapy therapeutic approaches or with one or more active compounds, preferably antibodies, directed against a cancer immunotherapy molecular target represents a preferred method for treating cancer or tumors. i) Examples of cancer immunotherapy therapeutic approaches comprise immunomodulatory monoclonal antibodies and low molecular weight substances directed against cancer immunotherapy targets, vaccines, CAR T cells, bispecific T cell recruiting antibodies, oncolytic viruses, cell-based vaccination approaches. ii) Examples of selected cancer immunotherapy targets suitable for immunomodulatory monoclonal antibodies comprise CTLA-4, PD-1/PDL-1, oX-40, CD137, DR3, IDO1, IDO2, TDO2, LAG-3, TIM-3 CD40., ICOS/ICOSL, TIGIT; GITR/GITRL, VISTA, CD70, CD27, HVEM/BTLA, CEACAM1, CEACAM6, ILDR2, CD73, CD47, B7H3, TLRs. Consequently, the combination of a ligand-drug conjugate (ADC) according to the invention with cancer immunotherapy can, on the one hand, generate tumors with weaker immunogenic properties and improve the efficacy of cancer immunotherapy and, in addition, develop durable therapeutic action.

[0218] Furthermore, the compounds according to the invention can also be used in combination with surgical intervention and/or radiotherapy.

[0219] In general, the following objectives can be achieved by combining compounds of the present invention with other cytostatic, cytotoxic or immunotherapeutically active agents: improved efficacy in slowing the growth of a tumor, reducing its size or even completely eliminating it, compared to treatment with a single active ingredient; the possibility of using the chemotherapeutics used in a lower dosage than in the case of monotherapy; the possibility of a more tolerable therapy with fewer side effects compared to individual administration; the possibility of treating a broader spectrum of neoplastic disorders; the achievement of a higher response rate to therapy; a longer patient survival time compared to current standard therapy.

[0220] Furthermore, the compounds according to the invention can also be used in combination with surgical intervention and/or radiotherapy.

[0221] The present invention further provides medicaments comprising at least one compound of the invention, typically together with one or more inert, non-toxic and pharmaceutically acceptable excipients, and for the use thereof for the aforementioned purposes.

[0222] The compounds of the invention can act systemically and/or locally. For this purpose, they can be administered in a suitable manner, for example parenterally, possibly by inhalation or as implants or stents.

[0223] The compounds of the invention may be administered in administration forms suitable for these routes of administration.

[0224] Parenteral administration may skip an absorption step (e.g., occur intravenously, intraarterially, intracardiacally, intraspinally or intralumbarly) or include absorption (e.g., occur via inhalation, intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). Suitable administration forms for parenteral administration include preparations for injection and infusion in the form of solutions, suspensions, emulsions or lyophilizates. Preference is given to parenteral administration, especially intravenous administration.

[0225] In general, it has been found advantageous in the case of parenteral administration to administer amounts of about 0.1 to 20 mg/kg, preferably about 0.3 to 7 mg/kg, of body weight to achieve effective results.

[0226] However, it may be necessary in some cases to deviate from the stated amounts, and specifically as a function of body weight, route of administration, individual response to the active ingredient, nature of the preparation and time or interval during which administration takes place. Thus, in some cases it may be sufficient to control with less than the above-mentioned minimum amount, while in other cases the above-mentioned upper limit should be exceeded. In the case of administration of larger amounts, it may be advisable to divide them into several individual doses throughout the day.

Examples

[0227] The following examples illustrate the invention. The invention is not limited to these examples.

[0228] Unless otherwise stated, percentages in the following tests and examples are percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios, and concentration data for liquid/liquid solutions are in each case on a volume basis.

Synthesis pathways:

[0229] As an example for the working examples, the following schemes show illustrative synthetic pathways leading to the working examples: Scheme 1: Synthesis of lysine-linked ADCs with legumain-cleavable linker

[0230] In the above reaction scheme, X1, X2, X3, n and AK2have the meanings given in formula (I).

[0231] a): HATU, DMF, N,N-diisopropylethylamine, RT; b) H2, 10% Pd-C, methanol 1.5 h, RT; c) 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, N,N-diisopropylethylamine, DMF, stirring at RT overnight; d) AK2 in PBS, under argon addition of 3-5 equiv. of active ester dissolved in DMSO, stirring at RT under argon for 60 min, addition of another 3-5 equiv. of active ester dissolved in DMSO, stirring at RT under argon for 60 min, then purification by means of PD 10 columns equilibrated with PBS buffer (pH 7.2) (Sephadex® G-25, GE Healthcare) and subsequent concentration by means of ultracentrifugation and setting of the desired concentration with PBS buffer (pH 7.2)]. In the case of in vivo batches, this is optionally followed by sterile filtration.Scheme 2: Synthesis of cysteine-linked ADCs

[0232] In the above reaction scheme, X1, X2, X3, n and AK1 have the meanings given in formula (I).

[0233] a): HATU, DMF, N,N-diisopropylethylamine, TA; b) zinc chloride, trifluoroethanol, 50 °C, EDTA c): HATU, DMF, N,N-diisopropylethylamine, TA; d) H2, 10% Pd-C, methanol 1.5 h, RT; e) 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione, N,N-diisopropylethylamine, DMF, stirring to TA; f) AK1 dissolved in PBS, under argon addition of 3-4 equivalents of TCEP in PBS buffer and about 30 min of stirring at RT, then addition of 5-10 equivalents of compound E dissolved in DMSO, about 90 min of agitation at RT, then purification by means of PD 10 columns equilibrated with PBS buffer (pH 7.2) (Sephadex® G-25, GE Healthcare) and subsequent concentration by means of ultracentrifugation and definition of the desired concentration with PBS buffer (pH 7.2)]. In the case of in vivo batches, this is optionally followed by sterile filtration. Scheme 3: Synthesis of cysteine-linked ADCs via ring-opened succinimides

[0234] In the above reaction scheme, X1, X2, X3, n and AK1 have the meanings given in formula (I).

[0235] a): HATU, DMF, N,N-diisopropylethylamine, TA; b) zinc chloride, trifluoroethanol, 50 °C, EDTA c): HATU, DMF, N,N-diisopropylethylamine, TA; d) H2, 10% Pd-C, methanol 1.5 h, RT; e) 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione, N,N-diisopropylethylamine, DMF, stirring at RT ; f) AK1 dissolved in PBS, under argon addition of 3-4 equivalents of TCEP in PBS buffer and about 30 min of stirring at RT, then addition of 5-10 equivalents of compound E dissolved in DMSO, about 90 min of stirring at RT, then rebuffering to pH 8 by means of PD 10 columns equilibrated with PBS buffer (pH 8) (Sephadex G-25, GE Healthcare), then stirring at RT overnight, then optionally purification by means of PD 10 columns equilibrated with PBS buffer (pH 7.2) (Sephadex G-25, GE Healthcare) and subsequent concentration by means of ultracentrifugation and definition of the desired concentration with PBS buffer (pH 7.2)]. In the case of in vivo batches, this is optionally followed by sterile filtration. A. Examples Abbreviations and acronyms: ABCB1 ATP-binding cassette subfamily member 1 (synonymous with P-gp and MDR1) abs. absolute Ac acetyl ACN acetonitrile aq. aqueous, aqueous solution ATP Adenosine triphosphate BCRP cancer resistance protein breast, an efflux transporter BEP 2-Bromo-1-ethylpyridinium tetrafluoroborate Boc tert-butoxycarbonyl l. broad (in NMR) Ex. Example BxPC3 Human tumor cell line C concentration ca. circa, about CI chemical ionization (in MS) DAR drug to antibody ratio d doublet (in NMR) d day(s) TLC thin layer chromatography DCI direct chemical ionization (in MS) DCM dichloromethane dd doublet of doublets (in NMR ) DMAP 4-N,N-dimethylaminopyridine DME 1,2-dimethoxyethane DMEM Dulbecco's Modified Eagle's Medium (standardized nutrient medium for cell culture) DMF N,N-dimethylformamide DMSO dimethyl sulfoxide D/P Dye ratio (fluorescent dye )/protein DPBS, DPBS, Dulbecco's Phosphate Buffered Saline Solution DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) PBS PBS = DPBS = D-PBS, pH 7.4, from Sigma, #D8537 Composition: 0.2 g KCl 0.2 g KH2PO4 (anhydrous) 8.0 g NaCl 1.15 g Na2HPO4 (anhydrous) compound up to 1 l with H2O dt doublet of triplets (in NMR) DTT DL-dithiothreitol EDC Hydrochloride N'-(3-dimethylaminopropyl)-Nethylcarbodiimide EGFR epidermal growth factor receptor EI electron impact ionization (in MS) ELISA enzyme-linked immunosorbent assay equivalent eq.(s) ESI electrospray ionization (in MS) ESIMicroTofq ESI- MicroTofq (name of mass spectrometer with Tof = time of flight and q = quadrupole) FCS fetal calf serum Fmoc (9H-fluoren-9-ylmethoxy)carbonyl sat. saturated GTP guanosine-5'-triphosphate H hour(s) HATU O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HEPES 4-(2-hydroxyethyl)piperazine- 1-ethanesulfonic acid HOAc acetic acid HOAt 1-hydroxy-7-azabenzotriazole HOBt 1-hydroxy-1H-benzotriazole hydrate HOSu N-hydroxysuccinimide HPLC high performance liquid chromatography, high pressure IC50 half maximal inhibitory concentration i.m. intramuscularly, administration in the muscle i.v. Intravenously, administration into the conc. vein. concentrate KPL-4 Human tumor cell line KU-19-19 Human tumor cell line LC-MS liquid chromatography-mass spectrometry LLC-PK1 cells Lewis lung carcinoma pig kidney cell line L-MDR LLC cells -PK1 transfected with human MDR1 LoVo Human tumor cell line M multiplet (in NMR) Me methyl MDR1 Multidrug resistance protein 1 MeCN acetonitrile min minute(s) MOLM-13 Human tumor cell line MS mass spectrometry MTT 3-bromide -(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium MV-4-11 Human tumor cell line NB4 Human tumor cell line NCI-H292 Human tumor cell line NMM N-methylmorpholine NMP N-methyl-2-pyrrolidinone NMR nuclear magnetic resonance spectrometry NMRI mouse strain originating from the Naval Medical Research Institute (RMRI) Hairless mice experimental animals NSCLC non-small cell lung cancer PBS phosphate-buffered saline solution Pd/ C palladium on activated carbon P-gp P-glycoprotein, a carrier protein PNGaseF enzyme for sugar cleavage quant. quantitative (in yield) quart quartet (in NMR) quint quintet (in NMR) Rec-1 Human tumor cell line Rf retention index (in TLC) TA room temperature Rt retention time (in HPLC) s singlet (in NMR) s.c. subcutaneously, administration under the skin SCID mice test mice with severe combined immunodeficiency SK-HEP-1 Human tumor cell line t triplet (in NMR) TBAF tetra-n-butylammonium fluoride TCEP tris(2-carboxyethyl)phosphine TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl Teoc tert-tertiary trimethylsilylethoxycarbonyl TFA trifluoroacetic acid THF tetrahydrofuran T3P® 2,4,6-Trioxide 2,4,6-tripropyl-1,3,5,2,4,6- trioxatrifosphinan U251 Human tumor cell line UV ultraviolet spectrometry v/v volume to volume ratio (of a solution) Z benzyloxycarbonyl HPLC and LC methods MS: Method 1 (LC-MS):

[0236] Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8 µ 50 x 1 mm; mobile phase A: 1 L water + 0.25 ml 99% strength formic acid; mobile phase B: 1 L acetonitrile + 0.25 ml 99% strength formic acid; gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; oven: 50 °C; flow rate: 0.40 ml/min; UV detection: 208–400 nm. Method 2 (LC-MS):

[0237] MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, BEH300, 2.1 x 150 mm, C18 1.7 µm; mobile phase A: 1 L water + 0.01 % formic acid; mobile phase B: 1 L acetonitrile + 0.01 % formic acid; gradient: 0.0 min 2 % B → 1.5 min 2 % B → 8.5 min 95 % B → 10.0 min 95 % B; oven: 50 °C; flow rate: 0.50 ml/min; UV detection: 220 nm Method 3 (LC-MS):

[0238] MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100 series; column: Agilent ZORBAX Extend-C18 3.0 x 50 mm 3.5 micron; mobile phase A: 1 L water + 0.01 mol ammonium carbonate, mobile phase B: 1 L acetonitrile; gradient: 0.0 min 98% A → 0.2 min 98% A → 3.0 min 5% A → 4.5 min 5% A; oven: 40 °C; flow rate: 1.75 ml/min; UV detection: 210 nm Method 4 (LC-MS):

[0239] MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1 x 50 mm, C18 1.8 µm; mobile phase A: 1 L water + 0.01 % formic acid; mobile phase B: 1 L acetonitrile + 0.01 % formic acid; gradient: 0.0 min 10 % B → 0.3 min 10 % B → 1.7 min 95 % B → 2.5 min 95 % B; oven: 50 °C; flow rate: 1.20 ml/min; UV detection: 210 nm Method 5 (LC-MS):

[0240] Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8 μ 50 x 1 mm; mobile phase A: 1 L water + 0.25 ml 99% strength formic acid; mobile phase B: 1 L acetonitrile + 0.25 ml 99% strength formic acid; gradient: 0.0 min 95% A ^ 6.0 min 5% A ^ 7.5 min 5% A; oven: 50 °C; flow rate: 0.35 ml/min; UV detection: 210 - 400 nm. Method 6 (LC-MS):

[0241] Instrument: Micromass Quattro Premier with WatersUPLC Acquity; column: Thermo Hypersil GOLD 1.9 μ 50 x 1 mm; mobile phase A: 1 L water + 0.5 ml 50% strength formic acid; mobile phase B: 1 L acetonitrile + 0.5 ml 50% strength formic acid; gradient: 0.0 min 97% A^0.5 min 97% A^3.2 min 5% A^4.0 min 5% A: 50 °C; flow rate: 0.3 ml/min; UV detection: 210 nm. Method 7 (LC-MS):

[0242] Instrument: Agilent MS Quad 6150; HPLC: Agilent1290; column: Waters Acquity UPLC HSS T3 1.8 μ 50 x 2.1 mm; mobile phase A: 1 L water + 0.25 ml 99% strength formic acid; mobile phase B: 1 L acetonitrile +0.25 ml 99% strength formic acid; gradient: 0.0 min 90% A^0.3 min 90% A^1.7 min 5% A^3.0 min 5% A: 50 °C; flow rate: 1.20 ml/min; UV detection: 205 - 305 nm.Method 8 (LC-MS):

[0243] MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1 x 50 mm, C18 1.8 μm; mobile phase A: 1 L water + 0.01% formic acid; mobile phase B: 1 L acetonitrile + 0.01% formic acid; gradient: 0.0 min2% B^2.0 min2% B^13.0 min90% B^15.0 min90% B; oven: 50 °C; flow rate: 1.20 ml/min; UV detection: 210 nm. Method 9: (LC-MS prep. purification method)

[0244] MS instrument: Waters, HPLC instrument: Waters (Waters X-Bridge C18 column, 19 mm x 50 mm, 5 μm, eluent A: water + 0.05 % ammonia, mobile phase B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection: DAD; 210-400 nm). or

[0245] MS instrument: Waters, HPLC instrument: Waters (Phenomenex Luna 5 μ C18(2) 100A column, AXIA Tech. 50 x21.2 mm, Eluent A: water + 0.05 % formic acid, Eluent B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection: DAD; 210-400 nm). Method 10: (LC-MS analysis method)

[0246] MS instrument: Waters SQD; HPLC instrument: Waters UPLC; column: Zorbax SB-Aq (Agilent), 50 mm x2.1 mm, 1.8 μm; mobile phase A: water + 0.025% formic acid, Eluent B: acetonitrile (ULC) + 0.025% formic acid; gradient: 0.0 min 98% A - 0.9 min 25% A -1.0 min 5% A - 1.4 min 5% A - 1.41 min 98% A -1.5 min 98% A; oven: 40 °C; flow rate: 0.600 ml/min; UV detection: DAD; 210 nm.Method 11 (HPLC):Instrument: HP1100 seriesColumn: Merck Chromolith SpeedROD RP-18e, 50-4.6 mm, Cat. No. 1.51450.0001, Pre-column Chromolith Guard Cartridge kit, RP-18e, 5-4.6 mm, Cat. No. 1.51470.0001Gradient: flow rate 5 ml/mininjection volume 5 μlsolvent A: HClO4 (70 %) in water (4 ml/l)solvent B: acetonitrile Column temperature: 40 °C Wavelength: 210 nm Method 12 (LC-MS):

[0247] MS instrument type: Thermo Scientific FT-MS; UHPLC+ instrument type: Thermo Scientific UltiMate 3000; column: Waters, HSST3, 2.1 x 75 mm, C18 1.8 μm; mobile phase A: 1 L water + 0.01 % formic acid; mobile phase B: 1 L acetonitrile + 0.01 % formic acid; gradient: 0.0 min 10 % B ^ 2.5 min 95 % B ^ 3.5 min 95 % B; oven: 50 °C; flow rate: 0.90 ml/min; UV detection: 210 nm/optimal integration path 210300 nm.Method 13: (LC-MS):

[0248] MS instrument: Waters (Micromass) Quattro Micro; Waters UPLC Acquity instrument; column: Waters BEH C18 1.7 μ 50 x 2.1 mm; mobile phase A: 1 L water + 0.01 mol ammonium formate, mobile phase B: 1 L acetonitrile; gradient: 0.0 min. 95% A^ 0.1 min. 95% A^ 2.0 min. 15% A^ 2.5 min. 15% A^ 2.51 min. 10% A^ 3.0 min. 10% A; oven: 40 °C; flow rate: 0.5 ml/min; UV detection: 210 nm.Method 14: (LC-MS) (MCW-LTQ-POROSHELL-TFA98-10 min)

[0249] MS instrument type: ThermoFisherScientific LTQ-Orbitrap-XL; HPLC instrument type: Agilent 1200SL; column: Agilent, POROSHELL 120, 3 x 150 mm, SB-C18 2.7 μm; mobile phase A: 1 L water + 0.1% trifluoroacetic acid; mobile phase B: 1 L acetonitrile + 0.1% trifluoroacetic acid; gradient: 0.0 min 2% B ^ 0.3 min 2% B ^ 5.0 min 95% B ^ 10.0 min 95% B; oven: 40 °C; flow rate: 0.75 ml/min; UV detection: 210 nm.

[0250] All reagents whose preparation is not explicitly described hereinafter were obtained commercially from generally available sources. For all other reagents whose preparation is similar not described hereinafter and which were not commercially obtainable or were obtained from sources that are not generally available, a reference is given to the published literature in which their preparation is described. Starting compounds and intermediates: Intermediate C52(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrol-2-yl]-2,2-dimethylpropan-1-amine

[0251] 10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate was initially charged into 100.0 mL of DMF, and 20.76 g (63.72 mmol) of cesium carbonate and 9.22 g (53.91 mmol) of benzyl bromide were added. The reaction mixture was stirred at RT overnight. The reaction mixture was partitioned between water and ethyl acetate, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The reaction was repeated with 90.0 g of methyl 4-bromo-1H-pyrrole-2-carboxylate.

[0252] The two combined reactions were purified by preparative RP-HPLC (column: Daiso 300x100; 10 μ, flow rate: 250 ml/min, MeCN/water). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This afforded 125.15 g (87% theory) of the compound methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate.

[0253] LC-MS (Method 1): Rt = 1.18 min; MS (ESIpos): m/z= 295 [M+H]+.

[0254] Under argon, 4.80 g (16.32 mmol) of methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate was initially charged in DMF, and 3.61 g (22.85 mmol) of (2,5-difluorophenyl)boronic acid, 19.20 ml of saturated sodium carbonate solution, and 1.33 g (1.63 mmol) of [1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II):dichloromethane were added. The reaction mixture was stirred at 85 °C overnight. The reaction mixture was filtered through Celite, and the filter cake was washed with ethyl acetate. The organic phase was extracted with water and then washed with saturated NaCl solution. The organic phase was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was purified on silica gel (mobile phase: cyclohexane/ethyl acetate 100:3). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This afforded 3.60 g (67% of theory) of the compound methyl 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate.

[0255] LC-MS (Method 7): Rt = 1.59 min; MS (ESIpos): m/z= 328 [M+H]+.

[0256] 3.60 g (11.00 mmol) of methyl 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate was initially charged into 90.0 mL of THF, and 1.04 g (27.50 mmol) of lithium aluminum hydride (2.4 M in THF) was added at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. Saturated potassium sodium tartrate solution was added at 0 °C, and the reaction mixture was admixed with ethyl acetate. The organic phase was extracted three times with saturated potassium sodium tartrate solution. The organic phase was washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was dissolved in 30.0 mL of dichloromethane. English:3.38 g (32.99 mmol) of manganese(IV) oxide was added, and the mixture was stirred at RT for 48 h. An additional 2.20 g (21.47 mmol) of manganese(IV) oxide was added, and the mixture was stirred at RT overnight. The reaction mixture was filtered through Celite, and the filter cake was washed with dichloromethane. The solvent was evaporated under reduced pressure, and the residue 2.80 g of (1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde) was used without further purification in the next step of the synthesis. LC-MS (Method 7): Rt = 1.48 min; MS (ESIpos): m/z = 298[M+H]+.

[0257] 28.21 g (94.88 mmol) of 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde together with 23.00 g (189.77 mmol) of (R)-2-methylpropane-2-sulfinamide were initially charged into 403.0 ml of absolute THF, and 67.42 g (237.21 mmol) of titanium(IV) isopropoxide was added and the mixture was stirred at RT overnight. 500 ml of saturated NaCl solution and 1000.0 ml of ethyl acetate were added, and the mixture was stirred at RT for 1 h. The mixture was filtered through kieselguhr, and the filtrate was washed twice with saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was purified using Biotage Isolera (silica gel, 1500+340 g SNAP column, flow rate 200 ml/min, ethyl acetate/cyclohexane 1:10).

[0258] LC-MS (Method 7): Rt = 1.63 min; MS (ESIpos): m/z= 401 [M+H]+.

[0259] 25.00 g (62.42 mmol) of (R)-N-{(E/Z)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]methylene}-2-methylpropane-2-sulfinamide was initially charged in absolute THF under argon and cooled to -78 °C. 12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane) was then added at -78 °C and the mixture was stirred at that temperature for 3 h. At -78 °C, 71.4 ml of methanol and 214.3 ml of saturated ammonium chloride solution were then added successively and the reaction mixture was allowed to warm to RT and stirred at RT for 1 h. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue (R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamide was used without further purification in the next step of the synthesis.

[0260] LC-MS (Method 6): Rt = 2.97 min; MS (ESIpos): m/z= 459 [M+H]+.

[0261] 28.00 g (61.05 mmol) of (R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamide was initially charged into 186.7 mL of 1,4-dioxane, and 45.8 mL of HCl in 1,4-dioxane solution (4.0 M) was then added. The reaction mixture was stirred at RT for 2 h and the solvent was evaporated under reduced pressure. The residue was purified by preparative HPLC (column: Kinetix 100x30; flow rate: 60 mL/min, MeCN/water). Acetonitrile was evaporated under reduced pressure and dichloromethane was added to the aqueous residue. The organic phase was washed with sodium bicarbonate solution and dried over magnesium sulfate. The solvent was evaporated under reduced pressure and the residue was dried under high vacuum. This afforded 16.2 g (75% of theory) of the title compound.

[0262] LC-MS (Method 6): Rt = 2.10 min; MS (ESIpos): m/z= 338 [M-NH2]+, 709 [2M+H]+.

[0263] 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 0.87 (s, 9H), 1.53 (s, 2H), 3.59 (s, 1H), 5.24 (d, 2H), 6.56 (s, 1H),6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m, 1H), 7.26 (m, 2H),7.34 (m, 2H), 7.46 (m, 1H).Intermediate C58acid (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]- 2-({[2- (trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid

[0264] 4.3 g (12.2 mmol) of Intermediate C52 was dissolved in 525 ml of DCM, and 3.63 g (17.12 mmol) of sodium triacetoxyborohydride and 8.4 ml of acetic acid were added. After 5 min of stirring at RT, 8.99 g (24.5 mmol) of Intermediate L57 dissolved in 175 ml of DCM was added and the reaction was stirred at RT for an additional 45 min. The reaction was then diluted with 300 ml of DCM and washed twice with 100 ml of sodium bicarbonate solution and once with saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was dried under high vacuum. The residue was then purified by preparative RP-HPLC (column: Chromatorex C18). After combining the appropriate fractions, the solvent was evaporated under reduced pressure and the residue was dried under high vacuum. This afforded 4.6 g (61% theory) of (2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate.

[0265] LC-MS (Method 12): Rt = 1.97 min; MS (ESIpos):m/z = 614 (M+H)+.

[0266] 2.06 g (3.36 mmol) of this intermediate was initially charged into 76 ml of DCM and acylated with 0.81 ml (7.17 mmol) of 2-chloro-2-oxoethyl acetate in the presence of 2.1 ml of triethylamine. After 20 h of stirring at RT, 0.36 ml of 2-chloro-2-oxoethyl acetate and 0.94 ml of triethylamine were added and the reaction was stirred at RT for an additional 15 min. The mixture was then diluted with 500 ml of ethyl acetate and extracted successively twice with 300 ml of 5% citric acid, twice with 300 ml of saturated sodium hydrogen carbonate solution and once with 100 ml of saturated sodium chloride solution and then dried over magnesium sulfate and concentrated. Drying under high vacuum gave 2.17 g (79% of theory) of the protected intermediate.

[0267] LC-MS (Method 1): Rt = 1.48 min; MS (ESIpos): m/z= 714 (M+H)+.

[0268] 2.17 mg (2.64 mmol) of this intermediate was dissolved in 54 ml of THF and 27 ml of water, and 26 ml of a 2 mol lithium hydroxide solution were added. The mixture was stirred at RT for 30 min and then adjusted to a pH between 3 and 4 using 1.4 ml of TFA. The mixture was concentrated under reduced pressure. Once most of the THF had distilled off, the aqueous solution was extracted twice with DCM and then concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC (column: Chromatorex C18). After combining the appropriate fractions, the solvent was evaporated under reduced pressure and the residue was lyophilized from acetonitrile/water. This yielded 1.1 g (63% of theory) of the title compound. LC-MS (Method 1): Rt = 1.34 min; MS (ESIpos): m/z = 656 (MH)-.

[0269] 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 0.03 (s, 9H), 0.58 (m, 1H), 0.74-0.92 (m, 11H) , 1.40 (m, 1H), 3.3 (m,2H), 3.7 (m, 1H), 3.8-4.0 (m, 2H), 4.15 (q, 2H), 4.9 and 5.2(2d, 2H), 5.61 (s, 1H), 6.94 (m, 2H), 7.13- 7.38 (m, 7H),7.48 (s, 1H), 7.60 (m, 1H), 12.35 (s, 1H).Intermediate C61N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl) amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-beta-alanine

[0270] The title compound was prepared by coupling 60 mg (0.091 mmol) of Intermediate C58 with methyl β-alaninate, followed by ester cleavage with 2 M lithium hydroxide solution. This afforded 67 mg (61% of theory) of the title compound in 2 steps.

[0271] LC-MS (Method 1): Rt = 1.29 min; MS (ESIpos): m/z = 729 (M+H)+. Intermediate C102(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-{[(benzyloxy)carbonyl]amino}butanoic acid

[0272] First, intermediate C52 was reductively alkylated with benzyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate analogously to intermediate C2. The secondary amino group was then acylated with 2-chloro-2-oxoethyl acetate, and the two ester groups were then hydrolyzed with 2M lithium hydroxyl solution in methanol.

[0273] LC-MS (Method 1): Rt = 1.31 min; MS (ESIpos): m/z= 646 (MH)-.Intermediate C110(D)N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2 ,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamate dibenzyl

[0274] The title compound was prepared by coupling dibenzyl D-glutamate, which was liberated in advance from its p-toluenesulfonic acid salt by partitioning between ethyl acetate and 5% sodium hydrogencarbonate solution, with Intermediate C61 in the presence of HATU and N,N-diisopropylethylamine and subsequent detachment of the Teoc protecting group via zinc chloride in trifluoroethanol.

[0275] LC-MS (Method 1): Rt = 1.08 min; MS (ESIpos): m/z= 894 [M+H]+.Intermediate C111N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2.5 -difluorophenyl)- 1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D- glutamate di-tert-butyl

[0276] First, the dipeptide derivative, di-tert-butyl beta-alanyl-D-glutamate was prepared by conventional peptide chemistry methods by the coupling of commercially available N-[(benzyloxy)carbonyl]-beta-alanine and di-tert-butyl D-glutamate hydrochloride (1:1) in the presence of HATU and subsequent hydrogenolytic detachment of the Z-protecting group. The title compound was then prepared by coupling this intermediate with Intermediate C102 in the presence of HATU and N,N-diisopropylethylamine and subsequent detachment of the Z-protecting group by hydrogenation over 10% palladium on activated carbon in DCM/methanol 1:1 at RT under standard hydrogen pressure for 1 hour.

[0277] LC-MS (Method 1): Rt = 1.06 min; MS (ESIpos): m/z= 826 [M+H]+.Intermediate C117N-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl- salt 4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2- dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-trifluoroacetic acid dibenzyl glutamate

[0278] Intermediate C110D and 2,5-dioxopyrrolidin-1-yl-N2-(tert-butoxycarbonyl)-L—asparaginate (251 mg, 764 μmol) were dissolved in 21 ml DMF and N,N-diisopropylethylamine (363 μl, 2.01 mmol) was added. The reaction was stirred at RT and then directly purified by prep. RP-HPLC (column: Chromatorex C18-10). The solvents were evaporated under reduced pressure and the residue was lyophilized.

[0279] The obtained intermediate (578 mg, 52 μmol) was dissolved in 20.0 mL of trifluoroethanol. The reaction mixture was admixed with zinc chloride (426 mg, 3.13 mmol) and stirred at 50 °C for 40 min. The mixture was admixed with ethylenediamine-N,N,N',N'-tetraacetic acid (914 mg, 3.13 mmol) and diluted with 20 mL of water, TFA (200 μL) was added and the mixture was shaken briefly. The mixture was filtered and purified by preparative RP-HPLC (column: Chromatorex C18-5; 125x40, flow rate: 100 mL/min, MeCN/water, 0.1% TFA gradient). Lyophilization gave the title compound. Intermediate L57 Methyl (2S)-4-Oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate

[0280] 500.0 mg (2.72 mmol) of methyl L-asparaginate hydrochloride and 706.3 mg (2.72 mmol) of 2-(trimethylsilyl)ethyl 2,5-dioxopyrrolidine-1-carboxylate were initially charged into 5.0 mL of 1,4-dioxane, and 826.8 mg (8.17 mmol) of triethylamine was added. The reaction mixture was stirred at RT overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250x40; 10 μ, flow rate 50 mL/min, MeCN/water, 0.1% TFA). The solvents were then evaporated under reduced pressure, and the residue was dried under high vacuum. This yielded 583.9 mg (74% of theory) of the compound (3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid.

[0281] LC-MS (Method 1): Rt = 0.89 min; MS (ESIneg): m/z= 290 (M-H)-.

[0282] 592.9 mg of (3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid were initially charged into 10.0 ml of 1,2-dimethoxyethane, the mixture was cooled to -15 °C, and 205.8 mg (2.04 mmol) of 4-methylmorpholine and 277.9 mg (2.04 mmol) of isobutyl chloroformate were added. The precipitate was filtered off with suction after 15 min and washed twice with, in each case, 10.0 ml of 1,2-dimethoxyethane. The filtrate was cooled to -10 °C, and 115.5 mg (3.05 mmol) of sodium borohydride dissolved in 10 ml of water were added with vigorous stirring. The phases were separated and the organic phase was washed once each with saturated sodium hydrogencarbonate solution and saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under reduced pressure, and the residue was dried under high vacuum. This afforded 515.9 mg (91% of theory) of the compound methyl N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate.

[0283] LC-MS (Method 1): Rt = 0.87 min; MS (ESIpos): m/z= 278 (M+H)+.

[0284] 554.9 mg (2.00 mmol) of methyl N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate were initially charged in 30.0 ml of dichloromethane, and 1.27 g (3.0 mmol) of Dess-Martin periodinane and 474.7 mg (6.00 mmol) of pyridine were added. The mixture was stirred at RT overnight. After 4 h, the reaction was diluted with dichloromethane and the organic phase was washed in each case three times with 10% strength Na2S2O3 solution, 10% strength citric acid solution and saturated sodium hydrogencarbonate solution. The organic phase was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. This afforded 565.7 mg (97% of theory) of the title compound.

[0285] 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 0.03 (s, 9H),0.91 (m, 2H), 2.70-2.79 (m, 1H) , 2.88 (dd, 1H), 3.63 (s,3H), 4.04 (m, 2H), 4.55 (m, 1H), 7.54 (d, 1H), 9.60 (t,1H). Intermediate L95N-[(Benzyloxy)carbonyl]-L-valyl-L-alanine

[0286] This intermediate was prepared from N-[(benzyloxy)carbonyl]-L-valine and tert-butyl L-alaninate hydrochloride by conventional peptide chemistry methods.

[0287] LC-MS (Method 12): Rt = 1.34 min; MS (ESIpos):m/z = 323.16 (M+H)+.Intermediate L103N-(Pyridin-4-ylacetyl)-L-alanyl-L-alanyl-L-asparagine trifluoroacetate

[0288] The title compound was prepared by conventional peptide chemistry methods that begin with the coupling of 4-pyridineacetic acid with commercially available tert-butyl L-alanyl-L-alaninate in the presence of HATU and N,N-diisopropylethylamine, followed by deprotection with trifluoroacetic acid, coupling to tert-butyl L-asparaginate, and subsequent deprotection of the carbonyl group with trifluoroacetic acid.

[0289] LC-MS (Method 1): Rt = 0.15 min; MS (ESIpos): m/z= 394 (M+H)+.Intermediate L116N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine

[0290] The title compound was prepared from commercially available N-[(benzyloxy)carbonyl]-L-alanine by classical peptide chemistry methods via coupling with tert-butyl N-methyl-L-alaninate hydrochloride salt in the presence of HATU and finally by removal of the tert-butyl ester protecting group with TFA.

[0291] LC-MS (Method 1): Rt = 0.68 min; MS (ESIpos): m/z= 309 [M+H]+Intermediate L117N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-asparagine trifluoroacetic acid salt

[0292] The title compound was prepared from commercially available 4-tert-butyl L-asparaginate by classical peptide chemistry methods via coupling with N-[(benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine (Intermediate L116) in the presence of HATU and finally by removal of the tert-butyl ester protecting group with TFA.

[0293] LC-MS (Method 1): Rt = 0.57 min; MS (ESIneg): m/z= 421 [MH]-Intermediate L118N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-alanine

[0294] The title compound was prepared from commercially available tert-butyl L-alaninate hydrochloride salt by classical peptide chemistry methods via coupling with N-[(benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine (Intermediate L116) in the presence of HATU and finally by removal of the tert-butyl ester protecting group with TFA.

[0295] LC-MS (Method 12): Rt = 1.25 min; MS (ESIneg):m/z = 378 [MH]-Intermediate L121N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-leucine

[0296] The title compound was prepared from commercially available tert-butyl L-leucinate hydrochloride salt by classical peptide chemistry methods via coupling with N-[(benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine (Intermediate L116) in the presence of HATU and finally by removal of the tert-butyl ester protecting group with TFA.

[0297] LC-MS (Method 12): Rt = 0.83 min; MS (ESIneg): m/z = 420 [MH]-Intermediate L122(5S,8S,11S)-11-(2-amino-2-oxoethyl)-8-[2-(benzyloxy)-2-oxoethyl]-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oic acid

[0298] The title compound was prepared from commercially available 4-benzyl-1-tert-butyl-L-aspartate hydrochloride (1:1) by classical peptide chemistry methods via initial coupling with 2,5-dioxopyrrolidin-1-yl-N-[(benzyloxy)carbonyl]-L-alaninate, then removal of the tert-butyl ester protecting group with TFA, then subsequent coupling with 4-tert-butyl L-asparaginate in the presence of HATU, and finally once again removal of the tert-butyl ester protecting group with TFA.

[0299] LC-MS (Method 1): Rt = 0.76 min; MS (ESIpos): m/z= 543 [M+H]+Intermediate L1381-Bromo-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid

[0300] The title compound was prepared by coupling 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid with bromoacetic anhydride in the presence of N,N-diisopropylethylamine.

[0301] LC-MS (Method 5): Rt = 1.05 min; MS (ESIpos): m/z= 386 and 388 (M+H)+. Intermediate Q1N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L - alanyl-N-methyl-L-alanyl-N1-{(2S)-4-[{(1R)-1-[1-benzyl-4- (2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}- 3-oxopropyl)amino]-1-oxobutan-2-yl}- L-aspartamide

[0302] The title compound was prepared from compound C110D by firstly coupling with Intermediate L117 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in DCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in the presence of N,N-diisopropylethylamine.

[0303] LC-MS (Method 12): Rt = 1.66 min; MS (ESIneg):m/z = 1119 (MH)-.Intermediate Q2N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L -alanyl-N1-{(2S)-4-[{(1R)-1-[1-benzyl-4- (2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}- 3-oxopropyl)amino]-1-oxobutan-2-yl}- L-aspartamide

[0304] The title compound was prepared from compound C110D, firstly, by coupling with Intermediate L117 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in DCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

[0305] LC-MS (Method 1): Rt = 0.93 min; MS (ESIpos): m/z = 1195 [M+H]+. Intermediate Q3N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-L-alanyl-N1-{(2S)-4-[{(1R)-1 -[1-benzyl-4-(2,5- difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino ]-1-oxobutan-2-yl}- L-aspartamide

[0306] The title compound was prepared from Compound C117 firstly by coupling with N-(tert-butoxycarbonyl)-L-alanyl-L-alanine in the presence of HATU and N,N-diisopropylethylamine. The intermediate was then taken up in trifluoroethanol and the protected tert-butoxycarbonyl amine was released by stirring at 50 °C in the presence of zinc chloride. In the next step, all benzyl protecting groups were removed by hydrogenation over 10% palladium on activated carbon in DCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

[0307] LC-MS (Method 1): Rt = 0.90 min; MS (ESIneg): m/z= 1181 (MH)-.Intermediate Q4N-[(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-alanyl-N -methyl-L-alanyl-N1-{(2S)-4-[{(1R)-1-[1-benzyl-4- (2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}- 3-oxopropyl)amino]-1-oxobutan-2-yl}- L-aspartamide

[0308] The title compound was prepared from compound C110D, firstly, by coupling with Intermediate L117 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in DCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione in the presence of N,N-diisopropylethylamine.

[0309] LC-MS (Method 1): Rt = 3.2 min; MS (ESIpos): m/z= 1177 [M+H]+.Intermediate Q5N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl -L-alanyl-N-{(2S)-4-[{(1R)-1-[1-benzyl-4- (2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}- 3-oxopropyl)amino]-1-oxobutan-2-yl}- L-alaninamide

[0310] The title compound was prepared from compound C110D, firstly, by coupling with Intermediate L118 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in DCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

[0311] LC-MS (Method 1): Rt = 0.96 min; MS (ESIpos): m/z= 1152 [M+H]+.Intermediate Q6Acid N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl) - 1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-[(N- {5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-valyl- L-alanyl)amino]butanoyl}-beta-alanyl-D-glutamic

[0312] The title compound was prepared from compound C110D, firstly, by coupling with Intermediate L95 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in DCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

[0313] LC-MS (Method 1): Rt = 0.98 min; MS (ESIpos): m/z= 1095 [M+H]+.Intermediate Q7N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl) acid - 1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({N- [(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanyl}amino)butanoyl]-beta-alanyl-D-glutamic

[0314] The title compound was prepared from compound C110D by firstly coupling with Intermediate L95 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in DCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in the presence of N,N-diisopropylethylamine.

[0315] LC-MS (Method 1): Rt = 0.98 min; MS (ESIpos): m/z= 1021 [M+H]+.Intermediate Q8N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl -L-alanyl-N1-{(2S)-4-[{(1R)-1-[1-benzyl-4- (2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}- 3-oxopropyl)amino]-1-oxobutan-2-yl}- L-leucinamide

[0316] The title compound was prepared from compound C110D, firstly, by coupling with Intermediate L121 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in ethanol under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

[0317] LC-MS (Method 1): Rt = 1.02 min; MS (ESIpos): m/z= 1194 [M+H]+.Intermediate Q9N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl -L-alpha-aspartyl-N1-{(2S)-4-[{(1R)-1-[1- benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl ]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}- L-aspartamide

[0318] The title compound was prepared from compound C110D by first coupling with Intermediate L122 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in methanol under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with 3 equiv. of 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine.

[0319] LC-MS (Method 1): Rt = 0.89 min; MS (ESIpos): m/z= 1225 [M+H]+.Intermediate Q10N-(Bromoacetyl)-L-alanyl-N-methyl-L-alanyl-N1-{(2S)-4- [{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3- {[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}- L-aspartamide

[0320] The title compound was prepared from compound C110D by first coupling with Intermediate L117 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by hydrogenation over 10% palladium on activated carbon in 1:1 DCM-methanol under standard hydrogen pressure at RT for 1 hour and the deprotected intermediate was then converted to the title compound by reaction with bromoacetic anhydride in the presence of 3 equiv. of N,N-diisopropylethylamine.

[0321] LC-MS (Method 1): Rt = 0.95 min; MS (ESIpos): m/z= 1104 and 1106 (M+H)+.Intermediate Q11N-(18-Bromo-17-oxo-4,7,10,13-tetraoxa-16-azaoctadecan-1- oil)-L-alanyl-N-methyl-L-alanyl-N1-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole -2-yl]-2,2- dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3- dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}- L-aspartamide

[0322] The synthesis of the title compound was carried out by the initial coupling of Intermediate C111 with Intermediate L117 in DMF in the presence of 1.5 equiv. of HATU and 3 equiv. of N,N-diisopropylethylamine. Subsequently, the Z-protecting group was removed by a 2 h hydrogenation over 10% palladium on activated carbon in ethanol under standard hydrogen pressure at RT. The deprotected intermediate was then reacted with Intermediate L138 in DMF in the presence of 1.5 equiv. of HATU and 3 equiv. of N,N-diisopropylethylamine. In the last step, cleavage of the tert-butyl ester groups by 2 h of stirring at 50 °C with 8 equivalents of zinc chloride in trifluoroethanol afforded the title compound.

[0323] LC-MS (Method 8): Rt = 4.06 min; MS (ESI-pos): m/z = 1353 [M+H]+.B: Preparation of antibody-drug conjugates (ADC) B-1. General method for generation of antibodies

[0324] The protein sequence (amino acid sequence) of the antibodies used, e.g., TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574, and TPP-9580, was transformed into a DNA sequence encoding the corresponding protein by a method known to one skilled in the art and inserted into an expression vector suitable for transient mammalian cell culture (as described by Tom et al., Chapter 12 in Methods Express: Expression Systems, edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007). B-2. General method for expression of antibodies in mammalian cells

[0325] Antibodies, e.g., TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574, and TPP-9580, were produced in transient mammalian cell cultures as described by Tom et al., Chapter 12 in Methods Express: Expression Systems, edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007.B-3. General method for purification of antibodies from cell supernatants

[0326] Antibodies, e.g., TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574, and TPP-9580, were obtained from cell culture supernatants. Cell supernatants were clarified by cell centrifugation. The cell supernatant was then purified by affinity chromatography on a MabSelect Sure chromatography column (GE Healthcare). For this purpose, the column was equilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant was applied, and the column was washed with approximately 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride. Antibodies were eluted in 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride and then purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

[0327] Commercially available antibodies were purified by standard chromatography methods (protein A chromatography, preparative gel filtration chromatography (SEC - size exclusion chromatography)) from commercial products.B-4. General method for coupling to cysteine side chains

[0328] The following antibodies were used in the coupling reactions:Example a: TPP-981 cetuximab (anti-EGFR AK)Example c: TPP-6013 (anti-CD123 AK)TPP-8987 (anti-CD123 AK)TPP-8988 (anti-CD123 AK)TPP-9476 (anti-CD123 AK)Examples h: TPP-8382 (anti-B7H3 AK)Example e: TPP-1015 (anti-Her2 AK)Examples k: TPP-7006 (anti-TWEAKR AK)TPP-7007 (anti-TWEAKR AK)Examples x: TPP-9574 (anti-CXCR5 AK)TPP-9580 (anti-CXCR5 AK)

[0329] Coupling reactions were usually carried out under argon.

[0330] Between 2 and 5 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dissolved in PBS buffer, were added to a solution of the appropriate antibody in PBS buffer in the concentration range between 1 mg/ml and 20 mg/ml, preferably in the range of about 10 mg/ml to 15 mg/ml, and the mixture was stirred at RT for 1 h. For this purpose, the solution of the respective antibody used can be employed in the concentrations indicated in the working examples, or it can also optionally be diluted with PBS buffer to about half the indicated starting concentration in order to achieve the predetermined concentration range. Subsequently, depending on the intended loading, from 2 to 12 equivalents, preferably about 5-10 equivalents, of the maleimide precursor compound or halide precursor compound to be coupled were added as a solution in DMSO. Here, the amount of DMSO should not exceed 10% of the total volume. The mixture was shaken for 60–240 min at RT for maleimide precursors and for 8–24 h at RT for halide precursors and then applied to PD10 columns equilibrated with PBS (Sephadex® G-25, GE Healthcare) and eluted with PBS buffer. In general, unless otherwise indicated, 5 mg of the antibody in question in PBS buffer was used for reduction and subsequent coupling. Purification on the PD10 column then yielded in each case solutions of the respective ADCs in 3.5 ml PBS buffer. The sample was then concentrated by ultracentrifugation and optionally rediluted with PBS buffer. If necessary, for better removal of low molecular weight components, the concentration by ultrafiltration was repeated after redilution with PBS buffer. For biological tests, if necessary, the concentrations of the final ADC samples were optionally adjusted to the range of 0.5–15 mg/ml by redilution. The respective protein concentrations, indicated in the working examples, of the ADC solutions were determined. In addition, the antibody loading (drug/mAb ratio) was determined using the methods described under B-6.

[0331] Depending on the binder, the ADCs shown in the examples may also be present to a lesser or greater degree in the form of the hydrolyzed open-chain succinamides linked to the antibodies.

[0332] In particular, the KSP-I-ADCs linked via the binder substructure

[0333] to the thiol groups of the antibodies can optionally also be selectively prepared by rebuffering after coupling and stirring at pH 8 for about 20-24 h according to Scheme 28 on the ADCs linked via the open chain succunamides.

[0334] #1 represents the sulfur bridge to the antibody, and #2 the binding point to the modified KSP inhibitor

[0335] Such ADCs in which the binder is linked to the antibodies via hydrolyzed open chain succinamides can optionally also be selectively prepared by the small scale and large scale couplings shown here by way of example:

[0336] Between 2 and 7 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dissolved in PBS buffer, were added to a solution of 2-5 mg of the appropriate antibody in PBS buffer in the concentration range between 1 mg/ml and 20 mg/ml, preferably in the range of about 5 mg/ml to 15 mg/ml, and the mixture was stirred at RT for 30 min to 1 h. Subsequently, depending on the intended loading, from 2 to 20 equivalents, preferably about 5-10 equivalents, of the maleimide precursor compound to be coupled were added as a solution in DMSO. To achieve higher DARs, it is also possible to use 15-20 equivalents. Here, the amount of DMSO should not exceed 10% of the total volume. The mixture was stirred at RT for 60-240 min. The eluate was diluted with PBS pH 8 buffer to a concentration of 1–5 mg/ml and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS pH 8 buffer, and eluted with PBS pH 8 buffer. The eluate was shaken at RT under argon overnight. Subsequently, the solution was concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2).

Medium scale coupling:

[0337] Under argon, a solution of 2-7 equivalents, preferably 3 equivalents, of TCEP in PBS buffer (c ~ 0.2-0.8 mg/ml, preferably 0.5 mg/ml) was added to 20-200 mg of the antibody in question in PBS buffer (c ~ 5-15 mg/ml). The mixture was stirred at RT for 30 min, then 2-20, preferably 5-10, equivalents of a maleimide precursor compound dissolved in DMSO were added. To achieve higher DARs, it is also possible to use 15-20 equivalents. After stirring at RT for an additional 1.5 h-2 h, the mixture was diluted with PBS buffer that was adjusted to pH 8 beforehand. This solution was then applied to PD 10 columns (Sephadex® G-25, GEHealthcare) that were equilibrated with PBS buffer pH 8 and eluted with PBS buffer pH 8. The eluate was diluted with PBS buffer pH 8 to a concentration of 2-7 mg/ml. This solution was stirred at RT under argon overnight. If required, the solution was then rebuffered to pH 7.2. The ADC solution was concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2) and then optionally concentrated again to a concentration of about 10 mg/ml.

[0338] In the structural formulae shown, AK1 may have the meaningExample a: TPP-981 cetuximab (partially reduced)-S§1Example c: TPP-6013 (anti-CD123 AK) (partially reduced)- S§1TPP-8987 (anti-CD123 AK) (partially reduced)- S§1TPP-8988 (anti-CD123 AK) (partially reduced)- S§1TPP-9476 (anti-CD123 AK) (partially reduced)- S§1Example e: TPP-1015 (anti-Her2 AK) (partially reduced)- S§1Examples h: TPP-8382 (anti-B7H3 AK) (partially reduced)- S§1Examples k: TPP-7006 (anti-TWEAKR) (partially reduced)- S§1TPP-7007 (anti-TWEAKR) (partially reduced)- S§1Examples x: TPP-9574 (anti-CXCR5 AK) (partially reduced)- S§1TPP-9580 (anti-CXCR5 AK) (partially reduced)- S§1where §1 represents the linkage to the succinimide group or to any isomeric hydrolyzed open-chain succinamides or the resulting alkylene radical thereof, and S represents the sulfur atom of a cysteine residue of the partially reduced antibody.B-5. General procedure for coupling to lysine side chains

[0339] The following antibodies were used for the coupling reactions:Example a: TPP-981 cetuximab (anti-EGFR AK)Example c: TPP-6013 (anti-CD123 AK)TPP-8987 (anti-CD123 AK)TPP-8988 (anti-CD123 AK)TPP-9476 (anti-CD123 AK)Example e: TPP-1015 (anti-Her2 AK)Examples k: TPP-7006 (anti-TWEAKR AK)TPP-7007 (anti-TWEAKR AK)Examples x: TPP-9574 (anti-CXCR5 AK)TPP-9580 (anti-CXCR5 AK)

[0340] Coupling reactions were usually performed under argon.

[0341] From 2 to 8 equivalents of the precursor compound to be coupled were added as a solution in DMSO to a solution of the antibody in question in PBS buffer in a concentration range between 1 mg/ml and 20 mg/ml, preferably about 10 mg/ml, depending on the intended loading. After stirring at RT for 30 min to 6 h, the same amount of precursor compound in DMSO was added again. Here, the amount of DMSO should not exceed 10% of the total volume. After stirring at RT for an additional 30 min to 6 h, the mixture was applied to PD 10 columns (Sephadex® G-25, GE Healthcare) equilibrated with PBS and eluted with PBS buffer. Purification on the PD10 column then in each case provided solutions of the respective ADCs in PBS buffer. The sample was then concentrated by ultracentrifugation and optionally rediluted with PBS buffer. If necessary, for better removal of low molecular weight components, concentration by ultrafiltration was repeated after redilution with PBS buffer. For biological testing, if necessary, the concentrations of the final ADC samples were optionally adjusted to the range of 0.5-15 mg/ml by redilution.

[0342] The respective protein concentrations, indicated in the working examples, of the ADC solutions were determined. In addition, the antibody loading (drug/mAb ratio) was determined using the methods described under B-6.

[0343] In the structural formulas shown, AK2 has the meaningExample a: TPP-981 cetuximab (anti-EGFR AK) - NH§2Example c: TPP-6013 (anti-CD123 AK) - NH§2TPP-8987 (anti-CD123 AK ) - NH§2TPP-8988 (anti-CD123 AK) - NH§2TPP-9476 (anti-CD123 AK) - NH§2Example e: TPP-1015 (anti-Her2 AK) - NH§2Examples k: TPP-7006 (anti-TWEAKR AK) - NH§2TPP-7007 (anti- TWEAKR AK) - NH§2Examples x: TPP-9574 (anti-CXCR5 AK) - NH§2TPP-9580 (anti-CXCR5 AK) - NH§2where §2 represents the bond to the carbonyl group and

[0344] NH represents the side chain amino group of a lysine residue of the antibody.Further purification and characterization of the conjugates according to the invention

[0345] After the reaction, in some cases, the reaction mixture was concentrated, e.g., by ultrafiltration, and then desalted and purified by chromatography, e.g., using a Sephadex® G-25 column. Elution was performed, e.g., with phosphate-buffered saline (PBS). The solution was then sterile filtered and frozen. Alternatively, the conjugate may be lyophilized.B-6. Determination of antibody, toxophore loading, and proportion of open cysteine adducts

[0346] For protein identification in addition to molecular weight determination after deglycosylation and/or denaturation, a tryptic digestion was performed which, after denaturation, reduction and derivatization, confirms the identity of the protein through the tryptic peptides found.

[0347] The toxophore loading (in the tables called DAR, drug to antibody ratio) of the PBS buffer solutions obtained from the conjugates described in the working examples was determined as follows:

[0348] Determination of toxophore loading of lysine-linked ADCs was performed by mass spectrometric determination of the molecular weights of the individual conjugate species. Here, the antibody conjugates were first deglycosylated with PNGaseF and the sample was acidified and, after HPLC separation/desalting, analyzed by mass spectrometry using ESI-MicroTofQ (Bruker Daltonik). All spectra over the TIC (Total Ion Chromatogram) signal were added and the molecular weight of the different conjugate species was calculated based on MaxEnt deconvolution. The DAR (= drug/antibody ratio) was then calculated after signal integration of the different species. For this purpose, the total sum of the integration results for all species weighted by toxophore count was divided by the total sum of the simply weighted integration results for all species.

[0349] Toxophore loading of cysteine-linked conjugates was determined by reversed-phase chromatography of the reduced or denatured ADCs. Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-dithiothreitol (DTT) (500 mM, 3 μL) were added to the ADC solution (1 mg/ml, 50 μL). The mixture was incubated at 55 °C for one hour and analyzed by HPLC.

[0350] HPLC analysis was performed on an Agilent 1260 HPLC system with detection at 220 nm. A Polymer Laboratories PLRP-S polymeric reversed-phase column (catalog number PL1912-3802) (2.1 x 150 mm, 8 μm particle size, 1000 Å) was used at a flow rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent A consisted of 0.05% trifluoroacetic acid (TFA) in water, eluent B of 0.05% trifluoroacetic acid in acetonitrile.

[0351] The detected peaks were assigned by retention time comparison with the light chain (L0) and heavy chain (H0) of the unconjugated antibody. The peaks detected exclusively in the conjugated sample were assigned to the light chain with one toxophore (L1) and to the heavy chain with one, two and three toxophores (H1, H2, H3).

[0352] The average loading of the antibody with toxophores (termed DAR, drug to antibody ratio) was calculated from the peak areas determined by integration as twice the sum of HC loading and LC loading, where LC loading is calculated from the sum of the toxophore number-averaged weighted integration results of all LC peaks divided by the sum of the individually weighted integration results of all LC peaks, and where HC loading is calculated from the sum of the toxophore number-averaged weighted integration results of all HC peaks divided by the sum of the individually weighted integration results of all HC peaks. In individual cases, it was possible that due to co-elution of some peaks, it was not possible to determine the toxophore loading accurately.

[0353] In cases where the light and heavy chains cannot be sufficiently separated by HPLC, determination of toxophore loading of cysteine-linked conjugates was performed by mass spectrometric determination of the molecular weights of the individual light and heavy chain conjugate species.

[0354] For this purpose, guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADC solution (1 mg/ml, 50 μl). The mixture was incubated for one hour at 55 °C and analyzed by mass spectrometry after virtual desalting using ESI-MicroTofQ (Bruker Daltonik).

[0355] For the determination of DAR (drug to antibody ratio), all spectra were added over the signal in the TIC (Total Ion Chromatogram) and the molecular weight of the different heavy and light chain conjugate species was calculated based on MaxEnt deconvolution. The average loading of the antibody with toxophores was determined from the peak areas determined by integration as twice the total sum of the HC loading and the LC loading. In this context, the LC loading is calculated from the total sum of the integration results for all LC peaks weighed by toxophore count, divided by the total sum of the integration results individually weighed for all LC peaks, and the HC loading from the total sum of the integration results for all HC peaks weighed by toxophore count, divided by the total sum of the integration results individually weighed for all HC peaks.

[0356] In the case of open constructs, to determine the proportion of open cysteine adduct, the area ratio of closed to open cysteine adduct molecular weight (delta molecular weight 18 daltons) of all individually conjugated heavy and light chain variants was determined. The average of all variants yielded the proportion of open cysteine adduct.B-7. Verification of Antigen Binding of ADCs

[0357] The ability of the binder to bind to the target molecule was verified after coupling had occurred. The person skilled in the art is familiar with various methods that can be used for this purpose; for example, the affinity of the conjugate can be verified using ELISA technology or surface plasmon resonance analysis (BIAcore™ measurement). The conjugate concentration can be measured by the person skilled in the art using common methods, for example, for antibody conjugates by protein determination. (see also Doronina et al.; Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007; 1102:616-623).Working examples of metabolitesExample M1N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoyl)amino]butanoyl}-beta-alanyl-D-glutamic acid

[0358] Intermediate C110D was converted to the title compound by a 1-hour hydrogenation over 10% palladium on activated carbon in ethanol under standard hydrogen pressure at RT.

[0359] LC-MS (Method 1): Rt = 1.78 min; MS (ESIpos): m/z= 714 [M+H]+.

[0360] The ADCs shown below in an exemplary manner are capable of releasing the preferred metabolite M1, which has preferred pharmacological properties.

Working Example ADCs

[0361] The ADCs shown in the structural formulae of the working examples, which have been coupled to the cysteine side chains of the antibodies via maleimide moieties, are, depending on the linker and the coupling procedure, mainly present in the ring-open or ring-closed forms shown in each case. However, the preparation may comprise a small proportion of the respective other form.

[0362] Coupling reactions were performed under bargon. All larger batches for in vivo testing were sterile filtered at the end of preparation.Examples 1

Example procedure A:

[0363] Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer was added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml). The mixture was stirred at RT for 30 min, and then 0.26 mg (0.00023 mmol) of Intermediate Q1 dissolved in 50 μl of DMSO was added. After stirring at RT for an additional 90 min, the mixture was diluted to a volume of 2.5 ml with PBS buffer that had been adjusted to pH 8 in advance, and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8. The eluate was stirred at RT under argon overnight. This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2).Example procedure B:

[0364] Under argon, a solution of 0.172 mg of TCEP in 0.3 ml of PBS buffer was added to 30 mg of the antibody in question in 3 ml of PBS (c=10 mg/ml). The mixture was stirred at RT for 30 min, and then 1.57 mg (0.0014 mmol) of Intermediate Q1 dissolved in 300 μl of DMSO was added. After stirring at RT for an additional 90 min, the mixture was diluted to a volume of 5 ml with PBS buffer that had been adjusted to pH 8 in advance, and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8. The eluate was diluted with PBS buffer pH 8 to a volume of 7.5 ml and stirred at RT under argon overnight. This solution was then applied to a PD 10 column (Sephadex®G-25, GE Healthcare) that was equilibrated with PBS buffer pH 7.2 and eluted with PBS buffer pH 7.2. The eluate was then concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2) and reconcentrated and sterile filtered again.

[0365] The following ADCs were prepared analogously to these procedures and characterized as indicated in the table: Examples 2 Example procedure A:

[0366] Under argon, 5 eq (0.2 mg) of Intermediate Q2 dissolved in 50 μl of DMSO were added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. The reaction mixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and rediluted with PBS (pH 7.2).Example procedure B:

[0367] Under argon, 4 eq (1 mg) of Intermediate Q2 dissolved in 50 μl of DMSO were added to 30 mg of the antibody in question in 3 ml of PBS buffer (pH 7.2) (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. Then, the mixture was diluted to 5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation, rediluted with PBS (pH 7.2) and reconcentrated and again sterile filtered. Exemplary procedure C:

[0368] Under argon, 2.5 eq (1 mg) of Intermediate Q2 dissolved in 250 μl of DMSO was added to 50 mg of the antibody in question in 5 ml of PBS buffer (pH 7.2) (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. Then, the mixture was diluted to 7.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation, rediluted with PBS (pH 7.2) and reconcentrated and again sterile filtered.Example procedure D:

[0369] Under argon, 4.5 eq (36 mg) of Intermediate Q2 dissolved in 7.5 ml of DMSO were added to 1000 mg of the antibody in question in 150 ml of PBS buffer (pH7.2) (c = 6.7 mg/ml).

[0370] After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. The batch was then purified by cross-flow filtration, concentrated and sterile filtered. Examples 3 Example procedure A:

[0371] Under argon, 5 eq (0.2 mg) of Intermediate Q3 dissolved in 50 μl of DMSO were added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. The reaction mixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and rediluted with PBS (pH 7.2).Example procedure B:

[0372] Under argon, 4 eq (1 mg) of Intermediate Q3 dissolved in 50 μl of DMSO were added to 30 mg of the antibody in question in 3 ml of PBS buffer (pH 7.2) (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. Then, the mixture was diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation, rediluted with PBS (pH 7.2) and reconcentrated and again sterile filtered. Examples 4 Example procedure A:

[0373] Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer was added to 5 mg of the antibody in question in 0.4 ml of PBS buffer (pH 7.2) (c=12.5 mg/ml). The mixture was stirred at RT for 30 min, and then 0.275 mg (0.00023 mmol) of Intermediate Q4 dissolved in 50 μl of DMSO was added. After an additional 90 min of stirring at RT, the reaction was diluted to a total volume of 2.5 ml with PBS buffer. This solution was then applied to a PD 10 column (Sephadex® G-25, GE Healthcare) that was equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2). Examples 5 Example procedure A:

[0374] Under argon, 5 eq (0.2 mg) of Intermediate Q5 dissolved in 50 μl of DMSO were added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. The reaction mixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and rediluted with PBS (pH 7.2). Examples 6 Example procedure A:

[0375] Under argon, 5 eq (0.18 mg) of Intermediate Q6 dissolved in 50 μl of DMSO were added to 5 mg of the antibody in question in 0.4 ml of PBS (c=12.5 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. The reaction mixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and rediluted with PBS (pH 7.2). Examples 7 Example procedure A:

[0376] Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer was added to 5 mg of the antibody in question in 0.4 ml of PBS (c=12.5 mg/ml). The mixture was stirred at RT for 30 min, and then 0.24 mg (0.00023 mmol) of Intermediate Q7 dissolved in 50 μl of DMSO was added. After stirring at RT for an additional 90 min, the mixture was diluted to a volume of 2.5 ml with PBS buffer that had been adjusted to pH 8 in advance, and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8. The eluate was stirred at RT under argon overnight. This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2). Examples 8 Example procedure A:

[0377] Under argon, 5 eq (0.2 mg) of Intermediate Q8 dissolved in 50 μl of DMSO were added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. The reaction mixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and rediluted with PBS (pH 7.2). Examples 9 Example procedure A:

[0378] Under argon, 5 eq (0.2 mg) of Intermediate Q9 dissolved in 50 μl of DMSO were added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml). After stirring at RT for 1 h, the same amount was added again and the mixture was stirred at RT for an additional hour. The reaction mixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and rediluted with PBS (pH 7.2). Examples 10 Example procedure A:

[0379] Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer was added to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 7.2) (c=10 mg/ml). The mixture was stirred at RT for 30 min, and then 0.295 mg (0.00023 mmol) of Intermediate Q10 dissolved in 50 μl of DMSO was added. After another 20 h of stirring at RT, the reaction was diluted to a total volume of 2.5 ml with PBS buffer. This solution was then applied to a PD 10 column (Sephadex® G-25, GE Healthcare) that was equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2).Example procedure C to obtain a higher DAR:

[0380] Under argon, a solution of 0.057 mg of TCEP in 0.05 ml of PBS buffer was added to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 7.2) (c=10 mg/ml). The mixture was stirred at RT for 30 min, and then 0.59 mg (0.00053 mmol) of Intermediate Q10 dissolved in 50 μl of DMSO was added. After an additional 20 h of stirring at RT, the reaction was diluted to a total volume of 2.5 ml with PBS buffer. This solution was then applied to a PD 10 column (Sephadex® G-25, GE Healthcare) that was equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2). Examples 11 Example procedure A:

[0381] Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer was added to 5 mg of the antibody in question in 0.4 ml of PBS buffer (pH 7.2) (c=12.5 mg/ml). The mixture was stirred at RT for 30 min, and then 0.32 mg (0.00023 mmol) of Intermediate Q11 dissolved in 50 μl of DMSO was added. After an additional 20 h of stirring at RT, the reaction was diluted to a total volume of 2.5 ml with PBS buffer. This solution was then applied to a PD 10 column (Sephadex® G-25, GE Healthcare) that was equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2). For comparative purposes, the following ADCs have been prepared:Reference Example R1:

[0382] Such ADCs were disclosed in WO2015/096982 and WO2016/096610 with various antibodies including, for example, cetuximab and trastuzumab. For comparative purposes, the parent Intermediate F194 disclosed therein was additionally also reacted with TPP-6013 (anti-CD123 AK). The following ADCs were used for comparative purposes: Reference Example R2:

[0383] Such ADCs were disclosed in WO2016/096610 with an aglycosylated anti-TWEAKR antibody. For comparative purposes, the precursor Intermediate F291 disclosed therein was additionally also reacted with TPP-9574 (anti-CXCR5 AK), TPP-981 (anti-EGFR) and TPP-1015 (anti-HER2 AK). The following ADCs were used for comparative purposes:

[0384] For Reference Examples R1, WO2015/096982 describes metabolite Example 98 derived therefrom. For Reference Examples R2, WO2016/096610 describes identical metabolite Example M9, which is listed here as Reference Example R3M. Reference Example R3M: N-(3-Aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

[0385] The preparation was described in WO2015/096982 as Example 98.

[0386] The biological data for these reference compounds disclosed in said applications or obtained for the innovative reference compounds are described in Chapter C. C: Assessment of biological efficacy

[0387] The biological activity of the compounds according to the invention can be demonstrated in the assays described below: a. C-1a Determination of the cytotoxic effect of ADCs

[0388] Analysis of the cytotoxic effect of ADCs was performed with several cell lines:

[0389] NCI-H292: Human mucoepidermoid lung carcinoma cells, ATCC-CRL-1848, standard medium: RPMI1640 (Biochrom; #FG1215, stab. glutamine) + 10% FCS(Sigma; #F2442), TWEAKR-positive; EGFR-positive.

[0390] BxPC3: human pancreatic carcinoma cells, ATCC-CRL-1687, standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine) + 10% FCS (Sigma; #F2442), TWEAKR-positive.

[0391] LoVo: Human colorectal cancer cells, ATCC # CCL-229, MTT assay culture: Standard medium: Kaighn's + L-glutamine (Invitrogen 21127) + 10% heat-inactivated FCS (from Gibco, # 10500-064). CTG assay culture: RPMI 1640 (Biochrom; #FG1215, glutamine stab.) + 10% FCS (Sigma #F2442). TWEAKR-positive.

[0392] KPL4: Human breast cancer cell line, Bayer Pharma AG (identity checked and confirmed 19.7.2012 in DSMZ), standard medium: RPMI 1640 (from Gibco; #21875-059, stab. L-glutamine) + 10 % heat-inactivated FCS (Gibco, #10500-064); HER2-positive.

[0393] SK-HEP-1: Human liver cancer cell line, ATCC no. HTB-52, standard medium: MEM with Earle's salts + Glutamax I (Invitrogen 41090) + 10% heat-inactivated FCS (from Gibco, no. 10500-064); EGFR-positive, TWEAKR-positive.

[0394] MOLM-13: Human acute monocytic leukemia (AML-M5a) cells, DSMZ, #ACC 554, standard medium: RPMI 1640 (from Gibco; #21875-059, stab. L-glutamine) + 20% heat-inactivated FCS (Gibco, #10500-064); CD123-positive.

[0395] MV-4-11: Human biphenotypic B myelomonocytic leukemia cells obtained from peripheral blood, ATCC-CRL-9591, standard medium: IMDM (ATCC: 30-2005), + 10% heat-inactivated FCS (Gibco, n° 10500064); CD123-positive.

[0396] NB4: Human acute promyelocytic leukemia cells obtained from bone marrow, DSMZ, n° ACC 207, standard medium: RPMI 1640 + GlutaMAX I (Invitrogen 61870) + 10 % heat-inactivated FCS (Gibco, n° 10500-064) + 2.5 g glucose (20 % glucose solution, Gibco, n° 19002) + 10 mM Hepes (Invitrogen 15630) + 1 mM sodium pyruvate (Invitrogen 11360); CD123-negative.

[0397] Rec-1: Human mantle cell lymphoma (B-cell non-Hodgkin lymphoma) cells ATCC CRL-3004, standard medium: RPMI 1640 + GlutaMAX I (Invitrogen 61870) +10% heat-inactivated FCS (Gibco, # 10500-064) +10 mM) CXCR5-positive.

[0398] U251: human glioblastoma cells, standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine) +10% FCS (Biochrom; #S0415), B7H3-positive.

[0399] HBL-1: human B-cell lymphoma cells (diffuse large B-cell lymphoma) ATT CRL-RRID (Resource Identification Initiative): CVCL_4213, first described in Abe et al. Cancer 61:483- 490(1988), obtained from Prof. Lenz, Universitat Münster; standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine) + 10% FCS (Biochrom; #S0415), culture analogous to Rec-1 cells; CXCR5-positive.

[0400] Cells were cultured by the standard method as indicated by American Tissue Culture Collection (ATCC) or Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) for the cell lines in question.

CTG assay

[0401] Cells were cultured by the standard method with the growth media specified under C-1. The test was performed by detaching the cells with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG#L2143), pelleting, resuspending in culture medium, counting and seeding in a 96-well culture plate with a white bottom (Costar #3610) (at 75 μL/well, the following cell numbers per well are: NCI-H292: 2500 cells/well, BxPC3 2500 cells/well, LoVo 3000 cells/well) and incubating in an incubator at 37 °C and 5% carbon dioxide. Suspension cells were counted and seeded into a white bottom 96-well culture plate (Costar #3610) (at 75 μl/well, the following cell numbers per well: Rec-1: 3000 cells/well, HBL-1: 6000 cells/well). After 24 h, antibody-drug conjugates were added in 25 μl of culture medium (fourfold concentrated) to the cells to generate final antibody-drug conjugate concentrations of 3 × 10-7 M to 3 × 10-11 M in the cells (triplicates). The cells were then incubated in a 37 °C, 5% carbon dioxide incubator. In a parallel plate, cell activity at the beginning of drug treatment (day 0) was determined using the CellTiter Glow (CTG) luminescent cell viability assay (Promega #G7573 and #G7571). For this purpose, per cell batch, 100 μL of substrate was added, the plates were then covered with aluminum foil, shaken on a plate shaker at 180 rpm for 2 min, allowed to rest on the laboratory bench for 8 min, and then measured using a luminometer (Victor X2, Perkin Elmer). The substrate detects the ATP content of living cells that generate a luminescence signal whose intensity is directly proportional to cell viability. After 72 h of incubation with the antibody-drug conjugates, the viability of these cells was then also determined using the CellTiter Glow luminescent cell viability assay as described above. From the measured data, the IC50 of growth inhibition was calculated compared to day 0 using the DRC (Dose Response Curve) analysis spreadsheets and a parameter setting of 4. The DRC analysis spreadsheet is a biobook spreadsheet developed by Bayer Pharma AG and BayerBusiness Services on the IDBS E-WorkBook Suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).

MTT assay

[0402] Cells were cultured by the standard method with the growth media specified under C-1. The test was performed by detaching the cells with an Accutase solution in PBS (from Biochrom AG no. L2143), pelleting, resuspending in culture medium, counting and seeding in a 96-well culture plate with a white bottom (from Costar no. 3610) (NCI H292: 2500 cells/well; SK-HEP-1: 1000 cells/well; KPL-4: 1200 cells/well; in total volume 100 μl). The cells were then incubated in an incubator at 37 °C and 5 % carbon dioxide. After 48 h, the medium was replaced. Antibody-drug conjugates in 10 μl of culture medium at concentrations of 10-5M to 10-13M were then pipetted onto the cells (in triplicate), and the assay was then incubated in a 37 °C 5% carbon dioxide incubator. The suspension cells were counted and seeded into a 96-well white-bottom culture plate (from Costar no. 3610) (no. 3610) (MOLM-13: 2000 cells/well; NB4: 7000 cells/well; MV-4-11: 5000 cells/well in a total volume of 100 μl). After 6 h of incubation at 37 °C and 5% carbon dioxide, the medium was changed and the antibody-drug conjugates or metabolites were added by pipette in 10 μl of culture medium at concentrations of 10-5 M to 10-13 M to the cells (triplicates) in 90 μl. The batch was incubated in an incubator at 37 °C and 5% carbon dioxide. After 96 h, cell proliferation was detected using the MTT assay (ATCC, Manassas, Virginia, USA, catalog no. 30-1010K). For this purpose, the MTT reagent was incubated with the cells for 4 h, followed by lysis of the cells overnight by the addition of detergent. The dye formed was detected at 570 nm (Infinite M1000 pro, Tecan). The measured data were used to calculate the IC50 of growth inhibition using the DRC (dose response curve). The proliferation of cells that were not treated with test substance but were otherwise identically treated was defined as the figure 100%.

[0403] Tables 1a and 1b below set forth the IC50 values for representative working examples of these tests:Table 1a Table 1b

[0404] Table 1c below lists the IC50 values for the reference examples of these assays.Table 1c

[0405] The reported activity data refer to the working examples described in the present experimental section, with the indicated drug/mAB ratios. The values may possibly deviate for different drug/mAB ratios. The IC50 values are means of several independent experiments or individual values. The action of the antibody-drug conjugates was selective for the respective isotype control comprising the respective binder and toxophore. For the ADCs directed against CD123, the target specificity was additionally demonstrated by testing with a CD123-negative cell.

[0406] In general, the ADCs according to the invention exhibit significantly improved cytotoxic potency compared to corresponding reference examples.C-1b Determination of inhibition of the kinesin spindle protein KSP/Eg5 by selected examples

[0407] The motor domain of the human kinesin spindle protein KSP/Eg5 (tebu-bio/Cytoskeleton Inc, #027EG01-XL) was incubated at a concentration of 10 nM with microtubuli (bovine or porcine, tebu-bio/Cytoskeleton Inc) stabilized with 50 μg/ml taxol (Sigma #T7191-5MG) for 5 min at RT in 15 mM PIPES, pH 6.8 (5 mM MgCl2 and 10 mM DTT, Sigma). The freshly prepared mixture was aliquoted into a MTP 384 (from Corning). Inhibitors to be examined at concentrations of 1.0 x 10-6 M to 1.0 x 10-13 M and ATP (final concentration 500 μM, Sigma) were then added. Incubation was at RT for 2 h. ATPase activity was detected by detection of the inorganic phosphate formed using malachite green (Biomol). After addition of the reagent, the assay was incubated at RT for 50 min before detection of absorbance at a wavelength of 620 nm. The positive controls used were monastrol (Sigma, M8515-1 mg) and ispinesib (AdooQ Bioscience A10486). Individual data from the dose-activity curve are eight-fold determinations. IC 50 values are means of two independent experiments. The 100% control was the sample that was not treated with inhibitors.

[0408] Table 2 below lists the IC50 values of representative working examples from the described assay and summarizes the corresponding cytotoxicity data (MTT assay):Table 2

[0409] The activity data reported refer to the working examples described in this experimental section.C-1c Enzyme Assaysa: Cathepsin B Assay

[0410] For each cathepsin B-cleavable prodrug to be examined, a mixture was made in a microreaction vessel (0.5 ml, from Eppendorf). The enzyme used here was obtained from human liver tissue. 2 μg of cathepsin B (Sigma C8571 25 μg) was initially loaded and completed to a total volume of 200 μl with 50 mM Na phosphate buffer, pH 6.0, 2 mM DTT. Then, 50 μl of the substrate solution to be examined was pipetted. The mixture was incubated in a thermoblock (from Thermo Fisher Scientific) at 40 °C under constant stirring at 300 rpm. The enzymatic reaction was kinetically controlled. For this purpose, a sample of 10 μl was taken at different times. The sample taken was immediately mixed with 20 μl of ice-cold methanol in order to stop the enzymatic reaction and then frozen at -20 °C. The times selected for sampling were after 10 min, 2 h, 4 h and 24 h. The samples were examined by RP-HPLC analysis (reverse phase HPLC, Agilent Technologies Series 1200). The determination of the released toxophore allowed the determination of the half-life t1/2 of the enzymatic reaction.b: Legumain assay

[0411] The legumain assay was conducted with recombinant human enzyme. The legumain enzyme solution (catalog no. 2199-CY, R&D Systems) was diluted in 50 mM Na acetate/100 mM NaCl buffer, pH 4.0 to the desired concentration and preincubated at 37 °C for 2 h. Legumain was then adjusted to a final concentration of 1 ng/μL in 50 mM MES buffer, 250 mM NaCl, pH 5.0. For each legumain-cleavable prodrug to be examined, a mixture was made in a microreaction vessel (0.5 ml, from Eppendorf). For this purpose, the substrate solution was adjusted to the desired concentration (double the concentration) with 50 mM MES buffer, 250 mM NaCl, pH 5.0. For the legumain solution enzyme reaction, 250 μl of the legumain solution was first loaded and the enzyme reaction was initiated by the addition of 250 μl of the substrate solution (final concentration: single concentration; 3 μM). At various time points, 50 μl samples were taken. Immediately, 100 μl of ice-cold methanol was added to this sample in order to stop the enzyme reaction, and the sample was then frozen at −20 °C. The selected sampling times were after 0.5 h, 1 h, 3 h and 24 h. The samples were then analyzed by RP-HPLC analysis and by LC-MS analysis. The determination of the released toxophore allowed the determination of the half-life t1/2 of the enzyme reaction.

[0412] As representative examples to show legumain-mediated cleavage, the substrates produced in the legumain assay were model compounds A and B. Reference example model compound AN-(Pyridin-4-ylacetyl)-L-alanyl-L-alanyl-N1-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-aspartamide

[0413] First, (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-methylbutanamide trifluoroacetic acid was prepared as described in 2015096982 A1. Subsequently, this intermediate was used to prepare the title compound by coupling Intermediate L103 in DMF in the presence of HATU and N,N-diisopropylethylamine.

[0414] LC-MS (Method 1): Rt = 0.86 min; MS (ESIpos): m/z= 902 [M+H]+. Reference example model compound BN-(Pyridin-4-ylacetyl)-L-alanyl-N-methyl-L-alanyl-N1- [(2S )-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole- 2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-aspartamide

[0415] Firstly, (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-methylbutanamide trifluoroacetic acid was prepared as described in 2015096982 A1. Subsequently, this intermediate was used to prepare the title compound by coupling Intermediate L118 in DMF in the presence of HATU and N,N-diisopropylethylamine.

[0416] LC-MS (Method 1): Rt = 0.83 min; MS (ESIpos): m/z = 916 [M+H]+.

[0417] Model compound A was cleaved under the conditions described above for legumain to the target compound with a half-life of 0.4 h.

[0418] Model compound B was cleaved under the conditions described above for legumain to the target compound with a half-life of 0.5 h. C-2 Internalization assay

[0419] Internalization is a key process that allows efficient and specific prediction of cytotoxic cargo in antigen-expressing cancer cells through antibody drug conjugates (ADC). This process is monitored by fluorescent identification of specific antibodies and an isotype control antibody. First, fluorescent dye was conjugated to lysines of the antibody. Conjugation was performed using a two-fold to ten-fold molar excess of CypHer 5E mono NHS ester (Batch 357392, GE Healthcare) at pH 8.3. After coupling, the reaction mixture was purified by gel chromatography (Zeba Spin Desalting Columns, 40K, Thermo Scientific, #87768; elution buffer: DULBECCO'S PBS, Sigma-Aldrich, #D8537) to remove excess dye and to adjust the pH. The protein solution was concentrated using VIVASPIN 500 columns (Sartorius stedim biotec). The antibody dye loading was determined by spectrophotometric analysis (from NanoDrop) and subsequent calculation (D/P = Dye εprotein : (A280 0.1 6Dye)ε dye).

[0420] The dye loading of the antibodies examined here and the isotype control were of a comparable order of magnitude. In cell binding assays, it was confirmed that coupling did not lead to any change in antibody affinity.

[0421] The identified antibodies were used for the internalization assay. Before starting the treatment, cells (2 x 104/well) were seeded in 100 μl of medium in a 96-well MTP (flat bottom, dark, clean #4308776, from Applied Biosystems). After 18 h of incubation at 37 °C/5% CO2, the medium was replaced and the identified antibodies were added at different concentrations (10, 5, 2.5, 1, 0.1 μg/ml). The same treatment protocol was applied to the identified isotype control (negative control). The incubation times chosen were 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6 h and 24 h. Fluorescence measurement was performed using the InCellAnalyzer 1000 (from GE Healthcare). This was followed by kinetic evaluation by measuring cell/granule counts parameters and total cell/granule intensity.

[0422] After binding to the receptor, the antibodies were examined for their internalization ability. For this purpose, cells with different levels of receptor expression were chosen. A specific target-mediated internalization was observed with the antibodies, while the isotype control showed no internalization. C-2b Internalization assay with suspended cells

[0423] Fluorescent dye coupling was performed as described under C2. The antigen to be examined is expressed by hematopoietic suspension cells; accordingly, internalization was examined in a FACS-based internalization assay.

[0424] Cells having different target expression levels were examined- Cells (5x104/well) were seeded in a 96-MTP (Greiner bio-one, CELLSTAR, 650 180, U-bottom) in a total volume of 100 μl. After addition of the target-specific antibody at a final concentration of 10 μg/ml, the batches were incubated at 37 °C for different periods of time (1 h, 2 h, 6 h, in triplicate). The isotype control was treated under identical conditions. A parallel batch was treated and incubated constantly at 4 °C (negative control). FACS analysis was performed using the Guava flow cytometer (Millipore). Kinetic evaluation was performed by measuring the fluorescence intensity, and evaluation occurs using the guavaSoft 2.6 software (Millipore). For the target-specific and target-specific antibodies described here, significant and specific internalization was detected in several cells; isotope controls showed no internalization.C-2c Colocalization assays of anti-CD123 antibodies

[0425] Due to the binder, the active metabolite of the antibody-drug conjugate is generated by lysosomal degradation. Consequently, intracellular trafficking after internalization has occurred is of essential importance. Studies on antibody co-localization using lysosomal organelle-specific labels (e.g. small GTPases or surface molecules) allow the selection of antibodies that have the desired profile. For this purpose, target-positive cells (5x104/well) in a total volume of 100 μl were seeded in a 96-MTP (Greiner bio-one, CELLSTAR, 650 x 180, U-bottom). After addition of the CypHer5E-labeled anti-target antibody (final concentration 20 μg/ml), batches (duplicates per time point) were incubated at 37 °C for 30 min, 2 h and 6 h in an incubator (5% CO2). 30 min before the end of the chosen incubation time, the lysosome-specific label was added to the batches to be examined. Lysosomes were stained with CytoPainter LysoGreen indicator reagent (final concentration 1:2000; abcam, ab176826). After incubation, 200 μl of ice-cold FACS buffer (DULBECCO'S PBS, Sigma-Aldrich, #D8537 + 3% heat-inactivated FBS, Gibco, #10500-064) was added and the cell suspension was centrifuged at 400 x g and 4 °C for 5 min. The cell pellet was resuspended in 300 μl of ice-cold FACS buffer and centrifuged again (4 min, 400 x g at 4 °C). After centrifugation, the supernatant was discarded and the cell pellet was placed in 30 μl of ice-cold FACS buffer. Samples were then immediately subjected to FACS/imaging analysis (FlowSight amnis, Millipore). Colocalization was assessed using special software (IDEAS Application v6.1 colocalization software). Table 3 summarizes the results of this assay in an exemplary manner for anti-CD123 antibodies.Table 3

[0426] The humanized antibodies TPP-9476 and TPP-8987 exhibit a markedly improved profile compared to the parental murine antibody.C-3 In vitro tests to determine cell permeability

[0427] The cellular permeability of a substance can be investigated by in vitro testing in a flow assay using Caco-2 cells [M.D. Troutman and D.R. Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. For this purpose, cells were cultured for 15-16 days in 24-well filter plates. For the determination of permeation, the respective test substance was applied in a HEPES buffer to the cells apically (A) or basally (B) and incubated for 2 hours. After 0 hours and after 2 hours, samples were taken from the cis- and trans-compartments. The samples were separated by HPLC (Agilent 1200, Boblingen, Germany) using reversed-phase columns. The HPLC system was coupled via a Turbo Ion Spray Interface to an API 4000 triple quadrupole mass spectrometer (AB SCIEX Deutschland GmbH, Darmstadt, Germany). Permeability was assessed based on a Papp value, which was calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716–1725 (2003)]. A substance was classified as actively transported when the ratio of Papp (B–A) to Papp (A–B) (efflux ratio) was >2 or <0.5.

[0428] Of critical importance for toxophores that are released intracellularly is the permeability of B to A [Papp(B-A)] and the ratio of Papp(B-A) to Papp(A-B) (efflux ratio): The lower this permeability, the slower are the passive and active processes of the substance through the Caco-2 cell monolayer, so that the substance, following intracellular release, remains in the cell longer. As a consequence of the metabolite remaining in the cell longer, the probability of interaction with the biochemical target (here: kinesin spindle protein KSP/Eg5) is increased, resulting in an enhanced cytotoxic action.

[0429] Table 4 below sets forth the permeability data for representative working examples from this test:Table 4

[0430] Metabolite M1, which can be formed from the ligand-drug conjugates according to the invention, exhibits both reduced transport from the cell and a reduced efflux ratio compared to the reference metabolite R3M, which can be formed from the ligand-drug conjugates of Reference Examples 2. C-4 In vitro tests to determine substrate properties for P-glycoprotein (P-gp)

[0431] Many tumor cells express drug transport proteins and this is often accompanied by the development of resistance to cytostatics. Substances that are not substrates of such transport proteins, such as P-glycoprotein (P-gp) or BCRP, for example, may therefore exhibit an enhanced activity profile.

[0432] The substrate properties of a substance for P-gp (ABCB1) were determined by means of a flow assay using LLC-PK1 cells overexpressing P-gp (L-MDR1 cells) [A.H. Schinkel et al., J. Clin. Invest. 96, 1698-1705 (1995)]. For this purpose, LLC-PK1 cells or L-MDR1 cells were cultured in 96-well filter plates for 3-4 days. For determination of permeation, the respective test substance, alone or in the presence of an inhibitor (such as ivermectin or verapamil, for example), was applied in a HEPES buffer to the cells apically (a) or basally (b) and incubated for 2 hours. After 0 hours and after 2 hours, samples were taken from the cis- and trans-compartments. The samples were separated by HPLC using reversed-phase columns. The HPLC system was coupled via a Turbo Ion Spray Interface to an API 3000 triple quadrupole mass spectrometer (Applied Biosystems Applera, Darmstadt, Germany). Permeability was assessed based on a Papp value, which was calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716–1725 (2003)]. A substance was classified as a P-gp substrate when the efflux ratio between Papp (B–A) and Papp (A–B) was >2.

[0433] As additional criteria for the evaluation of P-gp substrate properties, the efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in the presence or absence of an inhibitor can be compared. If these values differ by a factor greater than 2, the substance in question is a P-gp substrate.C-5 Pharmacokinetics

[0434] After i.v. administration of 5 mg/kg of Example 2c-9476 (DAR 6.3) and Example 2c-9476 (DAR 3.4) to male Wistar rats, plasma concentrations of the ADCs were measured by ELISA and pharmacokinetic parameters such as clearance (CL), area under the curve (AUC) and half-life (t1/2) were calculated.

[0435] Table 5 summarizes the pharmacokinetic parameters of Example 2c-9476 with DAR 6.3 and DAR 3.4.Table 5

[0436] In this exploratory pharmacokinetic study in rats, for both examples, a typical IgG profile was observed following i.v. administration. No significant differences were observed between Example 2c-9476 with DAR 6.3 and Example 2c-9476 with DAR 3.4.

Analysis for quantification of the ADCs used

[0437] The antibody moiety of the ADCs was determined using a ligand binding assay (ELISA) as the total IgG concentration in plasma samples and tumor lysates. Here, the sandwich ELISA format was used. This ELISA has been qualified and validated for determination in plasma and tumor samples. ELISA plates were coated with goat anti-human IgG Fc antibodies. After incubation with the sample, the plates were washed and incubated with a detector conjugate of simian anti-human IgG(H+L) antibody and horseradish peroxidase (HRP). After the additional washing step, the HRP substrate was added to OPD and color development was monitored by absorption at 490 nm. Standard samples having a known IgG concentration were adjusted using a 4-parameter equation. Within the lower (LLOQ) and upper (ULOQ) limits of quantification, unknown concentrations were determined by interpolation.C5a: Identification of ADC metabolites after in vitro internalizationMethod description:

[0438] Internalization studies with immunoconjugates are performed to analyze metabolites formed intracellularly. For this purpose, NCI H292 human lung tumor cells (3x105/well) are seeded in 6-well plates and incubated overnight (37 °C, 5% CO2). The cells are treated with 10 μg/ml (66 nM) of the ADC to be examined. Internalization is performed at 37 °C and 5% CO2. Cell samples are taken for further analysis at various times (0, 4, 24, 48, 72 h). First of all, the supernatants (about 5 ml) are collected and, after centrifugation (2 min, RT, 1000 rpm Heraeus Variofuge 3.0R), stored at -80 °C. The cells are washed with PBS and detached with Accutase, and the cell number is determined. After another wash, a defined number of cells (2 x 105) are treated with 100 ml of lysis buffer (Mammalian Cell Lysis (Sigma MCL1)) and incubated with continuous shaking (Thermomixer, 15 min, 4 °C, 650 rpm) in Protein LoBind tubes (Eppendorf Cat. No. 0030 108.116). After incubation, the lysate is centrifuged (10 min, 4 °C, 12000 g, Eppendorf 5415R) and the supernatant is collected. The supernatant obtained is stored at -80 °C. All samples are then analyzed as follows.

[0439] For the 50 μl cell lysate/culture supernatant procedure, 150 μl of precipitation reagent (methanol) is added and the mixture is shaken for 10 seconds. The precipitation reagent contains an internal standard (ISTD) at a suitable concentration (usually in the range of 20-100 μg/l). After centrifugation at 1881 g for 10 min, the supernatant is transferred into an autosampler vial, completed with 300 μl of a buffer matched to the eluent and shaken again and centrifuged at 1881 g for 10 min.

[0440] The cell lysate and supernatant samples are finally analyzed using the API6500 triple quadrupole mass spectrometer coupled to HPLC from AB SCIEX Deutschland GmbH.

[0441] For calibration, blank lysate or blank supernatant is admixed with appropriate concentrations (0.1-1000 μg/L). The limit of detection (LLOQ) is about 0.2 μg/L.

[0442] Quality controls for testing validity contain 4 to 40 μg/l.C5b: Identification of ADC metabolites in vivo

[0443] After i.v. administration of 3-30 mg/kg of different ADCs, plasma and tumor concentrations of the ADCs and any occurrence of metabolites can be measured, and pharmacokinetic parameters such as clearance (CL), area under the curve (AUC) and time halves (t1/2) can be calculated.

Analysis for quantification of potential metabolites

[0444] Analysis of the compounds in plasma, tumor, liver and kidney follows after precipitation of the proteins with usually methanol by high pressure liquid chromatography (HPLC) coupled to a triple quadrupole mass spectrometer (MS).

[0445] For the 50 μl plasma procedure, 150 μl of precipitation reagent (usually methanol) is added and the mixture is shaken for 10 s. The precipitation reagent contains an internal standard (ISTD) at a suitable concentration (usually in the range 20-100 μg/l). After centrifugation at 1881 g for 10 min, the supernatant is transferred into an autosampler vial, completed with 300 μl of a buffer matched to the eluent and shaken again.

[0446] In the tumor or organ material procedure, the particular material is mixed 3-20 times with the amount of extraction buffer. The extraction buffer contains 50 ml of Tissue Protein Extraction Reagent (Pierce, Rockford, IL), two pellets of Complete Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO, USA) at a final concentration of 1 mM. According to the tissue type (hard: tumor; soft: liver, kidney), the homogenization and lysis program of the Prescellys 24 Homogenization and Lysis System (Bertin Technologies) is selected (www.prescellys.com). The homogenized samples are left to stand at 4 °C overnight. 50 μl of the homogenate is transferred into an autosampler vial and topped up with 150 μl of methanol including ISTD, shaken for 10 s and then left to stand for 5 min. After the addition of 300 μl of ammonium acetate buffer (pH 6.8) and brief shaking, the sample is centrifuged at 1881 g for 10 min.

[0447] For calibration, plasma for plasma samples and corresponding blank matrix for tissue samples are admixed with concentrations of 0.6-1000 μg/L. Depending on the sample type or tissue type, the limit of detection (LOQ) is between 1 and 20 μg/L.

[0448] The matrix and plasma samples are finally analyzed using the API4500 triple quadrupole mass spectrometer coupled to HPLC from AB SCIEX Deutschland GmbH.

[0449] Quality controls for testing validity contain 4, 40 and 400 μg/l.

[0450] Table 6 shows metabolite concentrations in the MOLM-13 xenograft mouse model in -tumor, -liver, -kidney, and plasma 24 h after administration of 5 mg/kg of the ADC of Example 2c-9476 (n=3). The metabolite measured was: metabolite M1. nc=not calculated; LOQ: Limit of quantification Table 6:

[0451] Administration of ADC Example 2c-9476 according to the invention having a legumain-cleavable binder leads to a markedly selective enrichment of the active compound in the target tissue (tumor) compared to other healthy organs/tissues.C-6 In vivo activity test

[0452] The activity of the conjugates according to the invention was tested in vivo, for example using xenograft models. The person skilled in the art is familiar with methods in the prior art that allow the activity of the compounds according to the invention to be tested (see, for example, WO 2005/081711; Polson et al., Cancer Res. 2009 Mar 15;69(6):2358-64). For this purpose, a tumor cell line expressing the target molecule of the ligand was inoculated into rodents (e.g. mice). A conjugate according to the invention, an isotype antibody control conjugate, a control antibody or isotopic saline was then administered to the inoculated animals. The administration takes place once or more than once. After an incubation time of several days, the tumor size was determined by comparing conjugate-treated animals and the control group. Conjugate-treated animals exhibited smaller tumor size.C-6a.Growth inhibition/regression of experimental tumors in the mouse

[0453] Human tumor cells expressing the antigen for the antibody-drug conjugate are inoculated subcutaneously into the vial of immunosuppressed mice, e.g., SCID or NMRi nude mice. 1-10 million cells are detached from the cell culture, centrifuged and resuspended in medium or medium/matrigel. The cell suspension is injected under the skin of the mouse.

[0454] Within a few days, a tumor develops. Treatment is started after the tumor is established, at a tumor size of approximately 40 mm2. To examine the effect on large tumors, treatment can be started only at a tumor size of 50-100 mm2.

[0455] Treatment with APDCs and ADCs is performed intravenously (i.v.) into the tail vane of the mouse. The ADC is administered in a volume of 5 ml/kg.

[0456] The treatment protocol depends on the pharmacokinetics of the antibody. With the conjugates according to the invention, treatment is carried out once a week for 2 to 3 weeks as standard. For a rapid assessment, a protocol with a single treatment may also be suitable. However, the treatment can also be continued, or a second three-day treatment cycle can follow at a later time.

[0457] As standard, 8 animals are used per treatment group. In addition to the groups to which the active substances are administered, one group is treated as a control group with the buffer alone, according to the same protocol.

[0458] During the experiment, the tumor area is measured regularly in two dimensions (length/width) using a caliper. The tumor area is determined as length x width. The ratio of the mean tumor area of the treatment group to that of the control group is indicated as T/C area.

[0459] When, after the end of treatment, all groups of the experiment are terminated at the same time, the tumors can be removed and weighed. The ratio between the mean tumor weights of the treatment group and those of the control group is indicated as T/C weight.C-6b. Efficacy of ADCs according to the invention in various tumor models

[0460] Tumor cells (e.g., NCI-H292, REC-1, MOLM-13, and MV-4-11) are inoculated subcutaneously into the vial of female NMRI nude mice (Janvier). At a tumor size of ~40 mm2, intravenous treatment is performed with the antibody-drug conjugate. After treatment, monitoring of tumor growth continues if appropriate.

[0461] Treatment with the ADCs according to the invention leads to a distinct and in some cases long-lasting inhibition of tumor growth compared to the control group and the conjugated isotype control antibody. Table 7 shows the T/C values determined for the tumor area on the respective day of the end of the experiment, calculated from the start of treatment. Table 7:

Claims (30)

1. Antibody-drug conjugates of formula (I) characterized by the fact that X1 represents CH, X2 represents C, X3 represents N; R1 represents hydrogen or methyl, R2 represents methyl, -CH2-CH(CH3)2, -CH2-C(=O)OH or isopropyl, R3 represents methyl, -CH2-CH(CH3)2 or -CH2-C(=O)-NH2,M represents the group # -C(=O)-CH(CH3)-NH-C(=O)-CH2-NH-C (=O)-CH2-CH(##)-COOH, #-C(=O)-CH(CH3)-NH-C(=O)-CH2-NH-C(=O)-CH(## )-CH2-COOH, #-C(=O)-CH(CH3)-NH-C(=O)-CH2-W,# -C(=O)-CH2-NH-C(=O)-CH2 -CH(##)-COOH,# -C(=O)-CH2-NH-C(=O)-CH(##)-CH2-COOH,# -C(=O)-CH2-W,# -C(=O)-CH(CH3 )-NH-C(=O)-(CH2)3-C(=O)-###,# -C(=O)-(CH2)3-C(=O)-###,# -C(=O)-CH(CH3)-NH-C(=O)-(CH2)5-W, #-C(=O)-CH(CH3)-NH-C(=O)-(CH2 )-## or# -C(=O)-CH(CH3)-NH-C(=O)-(CH2-CH2-O)4-(CH2)2-NH-C(=O)- CH2- ##,W represents the group n represents a number from 1 to 50, AK represents an antigen-binding antibody or antibody fragment, # represents binding to the compound, # # represents binding to a sulfur atom of a cysteine side chain of the antibody or fragment of antigen-binding antibody, and ## represents the bond to a nitrogen atom of a lysine side chain of the antigen-binding antibody or antibody fragment, or a pharmaceutically acceptable salt thereof. 2. Antibody-drug conjugates of formula (I) according to claim 1, characterized in that: R1 represents hydrogen or methyl, R2 represents methyl or isopropyl, R3 represents methyl or -CH2-C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 50, AK represents an antibody or an antigen-binding antibody fragment, # represents the bond to the compound, and ### represents the bond to a nitrogen atom of a lysine side chain of the antibody or antigen-binding antibody fragment, or a pharmaceutically acceptable salt thereof. 3. Antibody-drug conjugates of formula (I) according to claim 1 or 2, characterized in that: R1 represents methyl, R2 represents methyl, R3 represents -CH2-C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 50, AK represents an antibody or an antigen-binding antibody fragment, # represents the bond to the compound, and ### represents the bond to a nitrogen atom of a lysine side chain of the antibody or antigen-binding antibody fragment, or a pharmaceutically acceptable salt thereof. 4. Antibody-drug conjugates of formula (I) according to any one of claims 1 to 3, characterized in that: R1 represents methyl, R2 represents methyl, R3 represents -CH2-C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 20, AK represents an antigen-binding antibody or antibody fragment, # represents the binding to the compound, and ### represents the binding to a nitrogen atom of a lysine side chain of an antibody or antigen-binding antibody fragment, or a pharmaceutically acceptable salt thereof. 5. Antibody-drug conjugates of formula (I) according to any one of claims 1 to 4, characterized in that: R1 represents methyl, R2 represents methyl, R3 represents -CH2 — C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 20 and AK represents an anti-CD123 antibody, an anti-CXCR5 antibody, an anti-B7H3 antibody, an anti-TWEAKR antibody, an anti-Her2 antibody or an anti-EGFR antibody or represents an antigen-binding antibody fragment thereof, # represents binding to the compound, ### represents binding to a nitrogen atom of a lysine side chain of the antibody (AK) or of the antigen-binding antibody fragment thereof, or a pharmaceutically acceptable salt thereof. 6. Antibody-drug conjugates of formula (I) according to any one of claims 1 to 5, characterized in that: R1 represents methyl, R2 represents methyl, R3 represents -CH2-C(=O)-NH2, M represents the group #-C(=O)-CH(CH3)-NH-C(=O)-(CH2)3-C(=O)-###, n represents a number from 1 to 20, AK represents an anti-CD123 antibody selected from the group consisting of TPP-9476, TPP-8988, TPP-8987 and TPP-6013, or represents an anti-CXCR5 antibody selected from the group consisting of TPP-9574 and TPP-9580, or represents an anti-B7H3 TPP-8382 antibody, or represents an anti-TWEAKR antibody selected from the group consisting of TPP-7006 and TPP-7007, or represents an anti-Her2 TPP-1015 antibody, or represents an anti-EGFR TPP-981 antibody, or represents an antigen-binding antibody fragment thereof,# represents binding to the compound,### represents binding to a nitrogen atom of a lysine side chain of the antibody (AK) or antigen-binding antibody fragment thereof, or a pharmaceutically acceptable salt thereof. 7. Antibody-drug conjugates of formula (I), according to any one of claims 1 to 6, characterized in that the antibody-drug conjugates have a structure selected from the group consisting of: wherein AK1 represents an antibody that is attached through a sulfur atom of a cysteine side chain, AK2 represents an antibody that is attached through a nitrogen atom of a lysine side chain, and n represents a number from 1 to 50, or a pharmaceutically acceptable salt thereof. 8. Antibody-drug conjugates according to claim 7, characterized in that: n represents a number from 1 to 20, or a pharmaceutically acceptable salt thereof. 9. Antibody-drug conjugates according to claim 7 or 8, characterized in that: n represents a number from 1 to 8, or a pharmaceutically acceptable salt thereof. 10. Antibody-drug conjugates according to any one of claims 7 to 9, characterized in that: n represents a number from 4 to 8, or a pharmaceutically acceptable salt thereof. 11. Antibody-drug conjugates according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, characterized in that: AK represents an antibody selected from the group consisting of TPP-8382 (anti B7H3), TPP-6013 (anti-CD123), TPP-8987 (anti-CD123), TPP-8988 (anti-CD123), TPP9476 (anti-CD123), TPP-9580 (anti-CXCR5) and TPP 9574 (anti-CXCR5) or an antigen-binding fragment thereof. 12. Antibody-drug conjugates according to claim 11, or a pharmaceutically acceptable salt thereof, characterized in that AK (AK1, AK2) represents TPP 9476 (anti-CD123). 13. Antibody-drug conjugates according to claim 11, or a pharmaceutically acceptable salt thereof, characterized in that AK (AK1, AK2) represents TPP 9574 (anti-CXCR5). 14. Antibody-drug conjugates according to claim 11, characterized in that AK represents a TPP-6013 anti-CD123 antibody, represents a TPP-8987 anti-CD123 antibody, represents a TPP-8988 anti-CD123 antibody or represents a TPP-9476 anti-CD123 antibody, or an antigen-binding fragment thereof. 15. The antibody-drug conjugates of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2)(i) represents an anti-B7H3 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 52, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 53, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 54, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 56, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 57, and the light chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 58, and the light chain variable CDR4 sequence (L-CDR4) as shown by SEQ ID NO: 59, and the light chain variable CDR5 sequence (L-CDR5) as shown by SEQ ID NO: 60. Light chain variable CDR3 (L-CDR3) as shown by SEQ ID NO: 58,(ii) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 22, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 23 and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 24, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 26, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 27 and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 28,(iii) represents an antibody anti-CD123 comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 62, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 63 and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 64, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 66, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 67 and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 68,(iv) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 72, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 73 and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 74, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 76, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 77 and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 78,(v) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 82, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 83 and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 84, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 86, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 87 and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 88, (vi) represents an anti-CXCR5 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 92, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 93 and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 94, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 96, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 97, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 98, or (vii) represents an anti-CXCR5 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 102, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 103, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 104, and a heavy chain variable region light chain (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 106, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 107, and the light chain variable CDR3 sequence (L-CDR3) as shown by SEQ ID NO: 108, or represents an antigen-binding fragment of such antibodies. 16. The antibody-drug conjugates of claim 15, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 82, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 83, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 84, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 86, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 87, and the light chain variable CDR3 sequence. (L-CDR3) as shown by SEQ ID NO: 88. 17. The antibody-drug conjugates of claim 15, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2) represents an anti-CXCR5 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence (H-CDR1) as shown by SEQ ID NO: 92, the heavy chain variable CDR2 sequence (H-CDR2) as shown by SEQ ID NO: 93, and the heavy chain variable CDR3 sequence (H-CDR3) as shown by SEQ ID NO: 94, and a light chain variable region (VL) comprising the light chain variable CDR1 sequence (L-CDR1) as shown by SEQ ID NO: 96, the light chain variable CDR2 sequence (L-CDR2) as shown by SEQ ID NO: 97, and the light chain variable CDR3 sequence. (L-CDR3) as shown by SEQ ID NO: 98. 18. The antibody-drug conjugates of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2)(i) represents an anti-B7H3 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 51 and a light chain variable region (VL) as set forth in SEQ ID NO: 55,(ii) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 21 and a light chain variable region (VL) as set forth in SEQ ID NO: 25,(iii) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 61 and a light chain variable region (VL) as set forth in SEQ ID NO: 65,(iv) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 66,(v) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 67,(vi) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 68,(vii) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 69,(viii) represents an anti-CD123 antibody comprising a heavy chain variable region (VL) as set forth in SEQ ID NO: 70,(viii) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 71,(viii) represents an anti-CD123 antibody comprising a heavy chain variable region (VL) as set forth in SEQ ID NO: 72,(viii) represents an anti-CD123 antibody comprising a heavy chain variable region (VL) as set forth in SEQ ID NO: 73,(viii) represents an anti-CD1 comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 71 and a light chain variable region (VL) as shown in SEQ ID NO: 75, (v) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as shown in SEQ ID NO: 81 and a light chain variable region (VL) as shown in SEQ ID NO: 85, (vi) represents an anti-CXCR5 antibody comprising a heavy chain variable region (VH) as shown in SEQ ID NO: 91 and a light chain variable region (VL) as shown in SEQ ID NO: 95, (vii) represents an anti-CXCR5 antibody comprising a heavy chain variable region (VH) as shown in SEQ ID NO: 101 and a light chain variable region (VL) as shown in SEQ ID NO: 105, or represents an antigen-binding fragment of such antibodies. 19. The antibody-drug conjugates of claim 18, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2) represents an anti-CD123 antibody comprising a heavy chain variable region (VH) as shown in SEQ ID NO: 81 and a light chain variable region (VL) as shown in SEQ ID NO: 85. 20. The antibody-drug conjugates of claim 18, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2) represents an anti-CXCR5 antibody comprising a heavy chain variable region (VH) as shown in SEQ ID NO: 91 and a light chain variable region (VL) as shown in SEQ ID NO: 95. 21. The antibody-drug conjugates of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2)(i) represents an anti-B7H3 antibody comprising a heavy chain region as shown in SEQ ID NO: 59 and a light chain region as shown in SEQ ID NO: 60,(ii) represents an anti-CD123 antibody comprising a heavy chain region as shown in SEQ ID NO: 29 and a light chain region as shown in SEQ ID NO: 30,(iii) represents an anti-CD123 antibody comprising a heavy chain region as shown in SEQ ID NO: 69 and a light chain region as shown in SEQ ID NO: 70,(iv) represents an anti-CD123 antibody comprising a heavy chain region as shown in SEQ ID NO: 79 and a light chain region as shown in SEQ ID NO: 80, (v) represents an anti-CD123 antibody comprising a heavy chain region as shown in SEQ ID NO: 89 and a light chain region as shown in SEQ ID NO: 90, (vi) represents an anti-CXCR5 antibody comprising a heavy chain region as shown in SEQ ID NO: 99 and a light chain region as shown in SEQ ID NO: 100, or (vii) represents an anti-CXCR5 antibody comprising a heavy chain region as shown in SEQ ID NO: 109 and a light chain region as shown in SEQ ID NO: 110, or represents an antigen-binding fragment of such antibodies. 22. The antibody-drug conjugates of claim 21, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2) represents an anti-CD123 antibody comprising a heavy chain region as shown in SEQ ID NO: 89 and a light chain region as shown in SEQ ID NO: 90. 23. The antibody-drug conjugates of claim 21, or a pharmaceutically acceptable salt thereof, wherein AK (AK1, AK2) represents an anti-CXCR5 antibody comprising a heavy chain region as shown in SEQ ID NO: 99 and a light chain region as shown in SEQ ID NO: 100. 24. Antibody-drug conjugates according to any one of claims 1 to 23, or a pharmaceutically acceptable salt thereof, characterized in that the AK is an antibody or an antigen-binding antibody fragment thereof, and wherein the antibody or antigen-binding antibody fragment binds to an extracellular target molecule. 25. Antibody-drug conjugates according to any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, characterized in that the antibody or antigen-binding antibody fragment binds to an extracellular cancer target molecule. 26. Antibody-drug conjugates according to any one of claims 1 to 25, characterized in that the antibody or antigen-binding antibody fragment, after binding to its extracellular target molecule on the target cell, is internalized by the target cell through the binding. 27. Pharmaceutical composition characterized by comprising an antibody-drug conjugate, as defined in claim 1, or a pharmaceutically acceptable salt thereof, in combination with an inert, non-toxic, pharmaceutically suitable auxiliary. 28. Use of antibody-drug conjugates as defined in any one of claims 1 to 27, characterized in that it is in the manufacture of a medicament for the treatment and/or prophylaxis of hyperproliferative and/or angiogenic disorders. 29. Use of antibody-drug conjugates as defined in any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, characterized in that it is in the manufacture of a medicament for the treatment and/or prophylaxis of cancer and tumors. 30. Use according to claim 29, characterized in that said treatment of cancer and tumors is in combination with a therapeutic approach for cancer immunotherapy or with an active compound directed against a molecular target of cancer immunotherapy.