Antibody drug conjugates (ADCS) having enzymatically cleavable groups

Abstract

The invention relates to novel antibody drug conjugates (ADCs) having improved properties, active metabolites of said ADCs and methods for the production thereof. The present invention further relates to the use of said conjugates for the treatment and / or prevention of diseases and to the use of said conjugates for the production of medicaments for the treatment and / or prevention of diseases, in particular hyperproliferative and / or angiogenic diseases such as, for example, cancer diseases.

Description

[0001] Binder-drug conjugates (ADCs) with enzymatically cleavable groups

[0002] Introduction and Prior Art The invention relates to novel binder-drug conjugates (ADCs) with improved properties, active metabolites of these ADCs, and their preparation methods. Furthermore, the present invention relates to the use of these conjugates for the treatment and / or prevention of diseases, as well as the use of these conjugates for the manufacture of medicaments for the treatment and / or prevention of diseases, in particular hyperproliferative and / or angiogenic diseases such as

[0003] For example, cancer. Such treatments can be administered as monotherapy or in combination with other drugs or further therapeutic measures.

[0004] Measures are taken. According to the invention, the binder is preferably an antibody.

[0005] Cancers result from uncontrolled cell growth in various tissues. In many cases, the new cells invade existing tissues (invasive growth) or metastasize to distant organs. Cancers occur in a wide variety of organs and often have tissue-specific disease courses.

[0006] Therefore, the term cancer, as a general term, describes a large group of defined diseases of various organs, tissues, and cell types. Tumors in early stages can sometimes be treated surgically and

[0007] Radiotherapeutic measures are used to remove the tumor. Metastatic tumors can generally only be treated palliatively with chemotherapy. The goal is to achieve the optimal combination of improved quality of life and extended lifespan. Conjugates of binder proteins with one or more drug molecules are known, particularly in the form of so-called "antibody-drug conjugates" (ADCs), in which an internalizing antibody directed against a tumor-associated antigen is covalently linked to a cytotoxic agent via a linker. After the ADC is introduced into the tumor cell and the conjugate is subsequently cleaved, either the cytotoxic agent itself or another cytotoxic metabolite formed from it is released within the tumor cell, where it can exert its effect directly and selectively.In this way, damage to normal tissue could be kept within significantly narrower limits compared to conventional chemotherapy of the cancer [see e.g. JM Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); AM Wu and PD Senter, Nat. Biotechnol. 23, 1137-1146 (2005); PD Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B. Stump, Bioconjugate Chem. 21, 5-13 (2010)]. WO2012 / 171020 describes ADCs in which several toxophore molecules are linked to an antibody via a polymeric linker. Possible toxophores mentioned in WO2012 / 171020 include the substances SB 743921, SB 715992 (ispinesib), MK-0371, AZD8477, AZ3146, and ARRY-520. The latter two substances are so-called kinesin spindle protein inhibitors.Kinesin spindle protein (KSP, also known as Eg5, HsEg5, KNSL1, or KIF1) is a kinesin-like motor protein essential for the function of the bipolar mitotic spindle. Inhibition of KSP leads to mitotic arrest and, over a longer period, to apoptosis (Tao et al., Cancer Cell 2005 Jul 8(1), 39–59). Following the discovery of the first cell-penetrating KSP inhibitor, monastrol, KSP inhibitors have established themselves as a class of novel chemotherapeutic agents (Mayer et al., Science 286: 971–974, 1999) and are the subject of numerous patent applications (e.g.,

[0008] WO2006 / 044825; WO2006 / 002236; WO2005 / 051922; WO2006 / 060737; WO03 / 060064; WO03 / 040979; and WO03 / 049527). However, since KSP is only active for a short period during the mitotic phase, KSP inhibitors must be present in sufficiently high concentrations during this phase. WO2014 / 151030 discloses ADCs with specific KSP inhibitors. Patent applications WO2015 / 096982 and WO2016 / 096610 already disclose ADCs with KSP inhibitors that also contain enzymatically cleavable linkers, but which do not exhibit an optimal activity profile. Legumain is a tumor-associated asparaginyl endopeptidase (S. Ishii, Methods).

[0009] Enzymol. 1994, 244, 604; JM Chen et al. J. Biol. Chem. 1997, 272, 8090) and has been used for the processing of prodrugs of small cytotoxic molecules such as doxorubicin and etoposide derivatives (W. Wu et al. Cancer Res. 2006, 66, 970; L. Stern et al. Bioconjugate Chem. 2009, 20, 500; KM Bajjuri et al.

[0010] ChemMedChem 201 1 , 6, 54).

[0011] Other lysosomal enzymes include cathepsin and glycosidases such as β-glucuronidases, which have also been used to release active substances through the enzymatic cleavage of prodrugs. Groups that can be cleaved enzymatically in vivo include, in particular, 2-8-oligopeptide groups and glycosides. Peptide cleavage sites are disclosed in Bioconjugate Chem. 2002, 13, 855-869, as well as Bioorganic & Medicinal Chemistry Letters 8 (1998) 3341-3346 and Bioconjugate Chem. 1998, 9, 618-626. These include, for example, valine-alanine, valine-lysine, valine-citrulline, alanine-lysine, and phenylalanine-lysine (possibly with an additional amide group).

[0012] Summary of the invention

[0013] Various antibody-drug conjugates with enzymatically cleavable linkers are described in the prior art, but these do not exhibit an optimal activity profile, e.g., with regard to their broad efficacy on different cells. Therefore, the object of the present invention is to provide more effective compounds that, according to

[0014] Administered at relatively low concentrations, these drugs exhibit a long-lasting apoptotic effect and are therefore useful for cancer therapy. The profile of the metabolites released intracellularly from the ADCs plays a crucial role in this. Frequently, the metabolites formed from ADCs are substrates of efflux pumps and / or exhibit high permeability through cell membranes. Both of these phenomena can contribute to a short residence time and thus to a suboptimal apoptotic effect in the tumor cell. The present invention therefore relates to binder-drug conjugates (ADCs) with a specific toxophore-linker composition, which, in combination with antibodies, exhibit a particularly interesting efficacy profile with regard to potency and spectrum of activity.To further improve the tumor selectivity of ADCs and their metabolites, binder conjugates were modified with peptide linkers that can be released by lysosomal tumor-associated enzymes, such as legumain or cathepsin. Tumor selectivity is thus determined not only by the choice of antibody but also by the enzymatic cleavage of the peptide derivative, e.g., by tumor-associated enzymes such as legumain. The binder-drug conjugates (ADCs) according to the invention are released in the tumor cells.

[0015] The released metabolites are further characterized by a particularly interesting...

[0016] Property profile. They exhibit low efflux from the tumor cell and lead to high exposure of the active ingredient in tumors. Thus, a high efficacy is achieved within the tumor cell, whereas, due to the poor permeability, only a low systemic cytotoxic effect is present, resulting in lower off-target toxicity.

[0017] The kinesin spindle protein inhibitors used according to the invention possess an amino group essential for their activity. Modifying this amino group with peptide derivatives blocks its action against the kinesin spindle protein and thus also inhibits the development of a cytotoxic effect. These peptide derivatives can also be components of the linker to the antibody. However, if this peptide residue or the peptide linker can be cleaved from the active ingredient by tumor-associated enzymes such as legumain or cathepsin, the effect can be specifically restored in the tumor tissue. The special property profile of the metabolites formed in the tumor is ensured by a further modification of the kinesin spindle protein inhibitor at a position other than the amino group in the molecule, which, however, does not impair its high potency at the target.

[0018] Furthermore, the structure of the ADCs according to the invention enables certain

[0019] In some embodiments, a high antibody loading (called DAR, drug-to-antibody ratio) surprisingly does not negatively affect the physicochemical and pharmacokinetic behavior of the ADCs.

[0020] It was now surprisingly found that binder-active ingredient conjugates of formula (I)

[0021] he

[0022] x2 for N and

[0023] Xs stands for C;

[0024] or

[0025]

[0026] x2 for C and

[0027] Xs stands for N;

[0028] or

[0029]

[0030] X2 stands for C and X3 for N;

[0031] or

[0032] X3 stands for C;

[0033] or

[0034] X3 stands for C.

[0035] R 1 stands for hydrogen or methyl

[0036] R 2 for methyl, ethyl, -CH2-CH(CH3)2, -CH2-C(=0)OH or iso-propyl

[0037] R 3 for Methyl, Ethyl, -CH2-CH(CH3)2 or -CH2-C(=0)-NH2

[0038] M for the group

[0039] #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH,

[0040] #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH,

[0041] #-C(=0)-CH(CH3)-NH-C(=0)-CH2-W,

[0042] #-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH,

[0043] #-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH,

[0044] #-C(=0)-CH2-W,

[0045] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)2-8 -C(=0)-###, #-C(=0)- (CH2)3-C(=0)-###, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)5-W, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)-## or

[0046] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2-CH2-0)i-8-(CH2)2-N HC(=0)-CH2-## stands for the group

[0047] n represents a number from 1 to 50, AK represents a binder or a derivative thereof, preferably a

[0048] Antibody or an antigen-binding fragment is present,

[0049] # stands for the bond at the connection,

[0050] ## stands for the bond to a sulfur atom of a cysteine ​​side chain of the binder,

[0051] ### for the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates, exhibit superior properties compared to the known conjugates.

[0052] Binder-active ingredient conjugates of formula (I) are preferred, in which

[0053] X2 for C

[0054] X3 stands for N;

[0055] R 1 R stands for hydrogen or methyl 2 R stands for methyl, -CH2-CH(CH3)2, -CH2-C(=0)OH or iso-propyl 3 M stands for methyl, -CH2-CH(CH3)2 or -CH2-C(=0)-NH2, and M represents the group

[0056] #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH,

[0057] #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH,

[0058] #-C(=0)-CH(CH3)-NH-C(=0)-CH2-W,

[0059] #-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH,

[0060] #-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH,

[0061] #-C(=0)-CH2-W,

[0062] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-###, #-C(=0)- (CH2)3-C(=0)-###, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)5-W, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)-## or

[0063] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2-CH2-0)4-(CH2)2-NH-C(=0)-CH2-## stands,

[0064] W for the group

[0065] AK stands for a number from 1 to 50, or for a binder or a derivative thereof, preferably for a

[0066] Antibody or an antigen-binding fragment is present,

[0067] # stands for the bond at the connection,

[0068] ## represents the bond to an S atom of a cysteine ​​side chain of the binder, ### represents the bond to an N atom of a lysine side chain of the binder, as well as their salts, solvates and salts of these solvates.

[0069] Binder-active ingredient conjugates of formula (I), in which R, are particularly preferred. 1 stands for hydrogen or methyl

[0070] R 2 stands for methyl or isopropyl,

[0071] R 3 stands for Methyl or -CH2-C(=0)-NH2

[0072] M for the group

[0073] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for, where n is a number from 1 to 50,

[0074] AK for a binder or a derivative thereof, preferably for a

[0075] Antibody or an antigen-binding fragment is present,

[0076] # stands for the bond at the connection,

[0077] ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

[0078] Binder-active ingredient conjugates of formula (I), in which R, are particularly preferred. 1 R stands for methyl 2 stands for methyl,

[0079] R 3 for -CH2-C(=0)-NH2

[0080] M for the group

[0081] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for, where n is a number from 1 to 50,

[0082] AK for a binder or a derivative thereof, preferably for a

[0083] Antibody or an antigen-binding fragment is present,

[0084] # stands for the bond at the connection,

[0085] ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

[0086] Binder-active ingredient conjugates of formula (I) are particularly preferred, in which

[0087] R 1 R stands for methyl 2 stands for methyl,

[0088] R 3 for -CH2-C(=0)-NH2

[0089] M for the group

[0090] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for, n for a number from 1 to 20, AK for a binder or a derivative thereof, preferably for a

[0091] Antibody or an antigen-binding fragment is present,

[0092] # stands for the bond at the connection,

[0093] ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

[0094] Selected are such binder-active ingredient conjugates of formula (I), in which

[0095] R 1 stands for methyl,

[0096] R 2 stands for methyl,

[0097] R3 for -CH2-C(=0)-NH2

[0098] M stands for the group #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-###, n stands for a number from 1 to 20 and

[0099] Antibodies for an anti-CD123 antibody, an anti-CXCR5 antibody, an anti-

[0100] B7H3 antibody, an anti-TWEAKR antibody, an anti-Her2 antibody

[0101] antibody or an anti-EGFR antibody or an antigen-binding antibody fragment thereof stands for,

[0102] # stands for the bond at the connection,

[0103] ### for the binding to an N atom of a lysine side chain of the antibody (AK) or of the antigen-binding antibody fragment thereof, as well as their salts, solvates and salts of these solvates.

[0104] In particular, binder-active ingredient conjugates of formula (I) in which R are selected are those that are selected. 1 stands for methyl,

[0105] R 2 stands for methyl,

[0106] R 3 for -CH2-C(=0)-NH2

[0107] M for the group

[0108] #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for n, which is a number from 1 to 20 and

[0109] Antibody for an anti-CD123 antibody selected from the group consisting of

[0110] TPP-9476, TPP-8988, TPP-8987 and TPP-6013, for an anti-CXCR5 antibody selected from the group consisting of TPP-9574 and TPP-9580, for an anti-B7H3 antibody TPP-8382, for an anti-TWEAKR antibody selected from the group consisting of TPP-7006 and TPP-7007, for an anti-Her2 antibody TPP-1015, or for an anti-EGFR antibody TPP-981 or for an antigen-binding antibody fragment of these,

[0111] # stands for the bond at the connection,

[0112] ### for the binding to an N atom of a lysine side chain of the antibody (AK) or of the antigen-binding antibody fragment thereof, as well as their salts, solvates and salts of these solvates.

[0113] Preferably, such binder-drug conjugates of the above-mentioned formulas are used in the invention for a binder that binds specifically to an extracellular cancer target molecule. In a preferred embodiment, the binder is internalized by the target cell after binding to its extracellular target molecule. Preferably, the binder is an antibody or an antigen-binding fragment. In a preferred aspect 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; CD123, CXCR5, and B7H3 are particularly preferred.In a preferred aspect of the invention, the binder AK 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. Particularly preferred are such binder-drug conjugates of the aforementioned formulas in the AK (AK1, AK2) for 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. The antibodies TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (each anti-CD123) are preferred. The exact structure (sequence) of these antibodies can be found in the table: Protein sequences of the antibodies, the text following this table, and the sequence listing.

[0114] Particularly preferred are binder-drug conjugates of formula (I) in which 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). The antibodies (AK) TPP-6013, TPP-8987, TPP-8988, and TPP-9476 (each anti-CD123) are particularly preferred.

[0115] Description of the characters

[0116] Annotated sequences of preferred antibodies for binder-drug conjugates. Shown are the protein sequences of the heavy and light chains of IgGs, as well as the VH and VL regions of these antibodies.

[0117] Important regions are annotated below the sequences (VH and VL regions in IgGs, and the CDR regions (H-CDR1 , H-CDR2, H-CDR3, L- CDR1 , L-CDR2, L-CDR3)).

[0118] Fig. 2: Sequence listing of sequences of preferred antibodies for binder-

[0119] Drug conjugates and sequences of the target proteins.

[0120] Detailed description of the invention

[0121] The invention provides conjugates of a binder or derivative thereof with one or more active substance molecules, wherein the active substance molecule is a kinesin spindle protein inhibitor (KSP inhibitor).

[0122] The following describes binders, KSP inhibitors thereof, and linkers that can be used according to the invention and that can be used in combination without restriction.

[0123] In particular, the binders shown to be preferred or particularly preferred can be used in combination with the KSP inhibitors shown to be preferred or particularly preferred, optionally in combination with the linkers shown to be preferred or particularly preferred.

[0124] Particularly preferred KSP inhibitor conjugates (binder-drug conjugates)

[0125] The following KSP inhibitor conjugates are particularly preferred according to the invention, wherein AK (AKi, AK2) represents binder or a derivative thereof (preferably for a

[0126] antibody), and n represents a number from 1 to 50, preferably 1 to 20, preferably 1 to 8, particularly preferably 4 to 8. AKi preferably represents an antibody bound to the KSP inhibitor via a cysteine ​​residue; AK2 preferably represents an antibody bound to the KSP inhibitor via a lysine residue. The binders or antibodies preferably used are those described as preferred in the description.

[0127] The following binder-active ingredient conjugates are particularly preferred: 16

[0128]

[0129] 117

[0130]

[0131] 18

[0132]

[0133] 19

[0134]

[0135] 20

[0136]

[0137]

[0138] Such binder-drug conjugates of the aforementioned formulas are preferred in the AK (AK1, AK2) for a binder that specifically binds to an extracellular cancer target molecule. In a preferred embodiment, after binding to its extracellular target molecule on the target cell, the binder is transported by the target cell.

[0139] internalized.

[0140] In a preferred aspect 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.

[0141] In a preferred aspect of the invention, the binder AK (AKi, AK2) 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. Particularly preferred are such binder-drug conjugates of the aforementioned formulas in the AK (AK1, AK2) for 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. The antibodies TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (each anti-CD123) are preferred. The exact structure (sequence) of these antibodies can be found in the table: Protein sequences of the antibodies, the text following this table, and the sequence listing.KSP inhibitor - Linker intermediate and production of conjugates.

[0142] The conjugates according to the invention are produced by first the

[0143] A low-molecular-weight KSP inhibitor is then coupled with a linker. The resulting intermediate is then reacted with the binder (preferably an antibody).

[0144] For an intermediate coupling to a lysine residue and the subsequent coupling with the antibody, the reaction can be illustrated as follows:

[0145] 

[0146] In the above reaction scheme, Xi, X2, X3, R 1 , R 2 , R 3 and AK2 the meanings given in formula (I) and R 4 Here, stands for methyl and n is either 0 or 1.

[0147] The synthesis of building block A was described in WO2015 / 096982. Peptide derivatives B and C were prepared using classical methods of peptide chemistry.

[0148] Intermediate C and D were converted to DMF by HATU in the presence of N, N-

[0149] Diisopropylethylamine was coupled at room temperature. Subsequently, both the benzyloxycarbonyl protecting group and the

[0150] Benzyl esters were cleaved. The fully deprotected intermediate was then reacted with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in DMF in the presence of N,N-diisopropylethylamine at room temperature to give the ADC precursor molecule E. This active ester was then coupled with the appropriate antibodies as described in Chapter B-5.

[0151] For an intermediate coupling to a cysteine ​​residue and the subsequent coupling with the antibody, the reaction can be illustrated as follows:

[0152] 

[0153] In the above reaction scheme, Xi, X2, X3, R 1 , R 2 , R 3and AK1 the meanings given in formula (I) and R 4 stands for methyl and n is 1.

[0154] Compounds where n represents 0 can also be prepared using an analogous procedure. The synthesis of building block A was described in WO2015 / 096982. Peptide derivatives B and C were prepared using classical methods of peptide chemistry.

[0155] Intermediates C and D were coupled via HATU in DMF in the presence of N,N-diisopropylethylamine at room temperature. Subsequently, both the benzyloxycarbonyl protecting group and the

[0156] Benzyl esters were cleaved. 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 room temperature to give the ADC precursor molecule E. This maleimide derivative was then coupled with the appropriate antibodies as described in Chapter B-4.

[0157] Depending on the linker, succinimide-linked ADCs can be converted to open-chain succinic acid amides after conjugation, which has an advantageous effect.

[0158] exhibit a stability profile.

[0159] This reaction (ring opening) can be carried out at pH 7.5 to 9, preferably at pH 8, at a temperature of 25 0 Temperature changes can be carried out between 37°C and 40°C, e.g., by stirring. The preferred stirring time is 8 to 30 hours.

[0160] For an intermediate coupling to a cysteine ​​residue and the subsequent coupling with the antibody with subsequent ring opening of the succinimide ring, the

[0161] The reaction can be illustrated as follows:

[0162]

[0163] In the above reaction scheme, Xi, X2, X3, R 1 , R 2 , R 3 and AK1 the meanings given in formula (I) and R 4 Here, n stands for methyl and n is either 0 or 1. The synthesis of building block A was described in WO2015 / 096982. Peptide derivatives B and C were prepared using classical methods of peptide chemistry.

[0164] Intermediates C and D were coupled via HATU in DMF in the presence of N,N-diisopropylethylamine at room temperature. Subsequently, both the benzyloxycarbonyl protecting group and the

[0165] Benzyl esters were cleaved. The completely 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 room temperature to give the ADC precursor molecule E.

[0166] The maleimide derivative was then coupled with the corresponding antibodies as described in Chapter B-4 under Small Scale Coupling or under Medium Scale Coupling.

[0167] binder

[0168] The term "binder" is understood in the broadest sense as a molecule that binds to a target molecule located on a specific surface containing the binder-active ingredient conjugate.

[0169] The term binder is used in its broadest sense and includes, for example, lectins, proteins that can bind certain sugar chains, or phospholipid-binding proteins. Such binders include, for example, high-molecular-weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (e.g., aptamers (US5,270, 163) Review article 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 include, for example, antibodies and antibody fragments or antibody mimetics such as affibodies, adnectins, anticalins, DARPins, avimers, and nanobodies (review article by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall SD et al., Curr. Opinion in Pharmacology 2008; 8:608-617). Binding peptides include, for example, ligands of a ligand-receptor pair, such as...VEGF of the ligand-receptor pair VEGF / KDR, such as transferrin of the ligand-receptor pair transferrin / transferrin receptor, or cytokine / cytokine receptor, such as TNF-alpha of the ligand-receptor pair TNF-alpha / TNF-alpha receptor. The binder can be a binding protein. Preferred embodiments of the binder are an antibody, an antigen-binding antibody fragment, a multispecific antibody, or an antibody mimetic. Various methods of covalent coupling (conjugation) of organic molecules to binders, and especially antibodies, are known from the literature.

[0170] According to the invention, 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 is preferred. However, it is also possible to bind the toxophore to the antibody via free carboxyl groups or via sugar residues of the antibody.

[0171] A "target molecule" is understood in the broadest sense as a molecule that is present in the target cell population and can be a protein (e.g., a receptor of a

[0172] It can be a growth factor) or a non-peptide molecule (e.g., a sugar or phospholipid). Preferably, it is a receptor or an antigen.

[0173] The term "extracellular" target molecule describes a molecule bound to the cell.

[0174] A target molecule located on the outside of a cell, or a part of a target molecule located on the outside of a cell, i.e., a binder that can bind to its extracellular target molecule on an intact cell. An extracellular target molecule may be anchored in the cell membrane or be a component of the cell membrane. Those skilled in the art know methods for identifying extracellular target molecules. For proteins, this can be done by determining the transmembrane domain(s) and the

[0175] The orientation of the protein within the membrane occurs. This information is usually stored in protein databases (e.g., SwissProt).

[0176] The term "cancer target molecule" describes a target molecule that is present in increased amounts on one or more types of cancer cells compared to non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more types of cancer cells compared to non-cancer cells of the same tissue type, with "selective" describing an enrichment at least twofold on cancer cells compared to non-cancer cells of the same tissue type (a "selective cancer target molecule"). The use of cancer target molecules allows for the selective therapy of cancer cells with the conjugates according to the invention.

[0177] The binder can be linked to the linker via a bond. This linkage can be effected by means of a heteroatom of the binder. Heteroatoms of the binder that can be used for linkage according to the invention are sulfur (in one embodiment via a sulfhydryl group of the binder), oxygen, and sulfur.

[0178] (according to the invention, by means of a carboxyl or hydroxyl group of the binder) and nitrogen (in one embodiment, via a primary or secondary amine or amide group of the binder). These heteroatoms can be present in the natural binder or introduced by chemical or molecular biological methods. According to the invention, the linkage of the binder to the toxophore has only a minor influence on the binding activity of the binder to the target molecule. In a preferred embodiment

[0179] In this embodiment, the linkage has no influence on the binding activity of the binder to the target molecule.

[0180] According to the present invention, the term "antibody" is understood in its broadest sense and includes immunoglobulin molecules, for example intact or modified monoclonal antibodies, polyclonal antibodies or multispecific antibodies.

[0181] Antibodies (e.g., bispecific antibodies). An immunoglobulin molecule preferably comprises a molecule with four polypeptide chains: two heavy chains (H chains) and two light chains (L chains), which are typically linked by disulfide bonds. Each heavy chain comprises a variable domain (VH) and a constant domain. The constant domain can, for example, contain three domains: CH1, CH2, and CH3. Each light chain comprises a variable domain (VL) and a constant domain. The constant domain contains a domain (CL). The VH and VL domains can be further subdivided into regions of hypervariability, also called complementarity-determining regions (CDR), and regions of lower sequence variability (framework regions, FR).Each VH and VL region typically consists of three CDRs and up to four FRs. For example, from the amino terminus to the...

[0182] Carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody can be obtained from any suitable species, e.g.

[0183] Rabbit, llama, camel, mouse, or rat. In one embodiment, the antibody is of human or murine origin. An antibody can be, for example, human, humanized, or chimeric.

[0184] The term "monoclonal" antibody refers to antibodies obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies of the

[0185] Populations are identical except for naturally occurring mutations, which may occur in minor numbers. Monoclonal antibodies recognize a single antigenic binding site with high specificity. The term monoclonal antibody does not refer to a specific manufacturing process. The term "intact" antibody refers to antibodies that include both an antigen-binding domain and the constant domain of the light and heavy chains. The constant domain can be a naturally occurring domain, a variant thereof in which several amino acid positions have been altered, and it can also be aglycosylated.

[0186] The term "modified intact" antibody refers to intact antibodies that have been fused with another polypeptide or protein, not originating from an antibody, via their amino or carboxy terminus by means of a covalent bond (e.g., a peptide linkage). Furthermore, antibodies can

[0187] modified by introducing reactive cysteines at defined positions to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol. 2008 Aug;26(8):925-32).

[0188] Here, "amino acid modification" or "mutation" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. The preferred amino acid modification is substitution. "Amino acid substitution" or "substitution" here refers to the exchange of one amino acid at a given position in a protein sequence for another amino acid. For example, the substitution Y50W describes a variant of a parental polypeptide in which the tyrosine at position 50 is replaced by tryptophan. A "variant" of a

[0189] A polypeptide describes a polypeptide that has an amino acid sequence substantially identical to a reference polypeptide, typically a native or "parental" polypeptide. The polypeptide variant can have one or more

[0190] Amino acid substitutions, deletions, and / or insertions at specific positions in the native amino acid sequence. The term "human" antibody refers to antibodies that can be obtained from a human or are synthetic human antibodies. A "synthetic" human antibody is an antibody that is obtainable in part or in its entirety from synthetic sequences derived in silico from the analysis of human

[0191] antibody sequences are based on this. A human antibody can, for example, be formed by a

[0192] The nucleic acid may be encoded by an antibody sequence isolated from a library of antibody sequences of human origin. An example of such an antibody can be found in Söderlind et al., Nature Biotech. 2000, 18:853-856. Such “human” and “synthetic” antibodies also include aglycosylated variants produced either by deglycosylation by PNGase F or by mutation of N297 (kabat numbering) of the heavy chain to any other amino acid. The term “humanized” or “chimeric” antibody describes antibodies composed of a non-human and a human sequence component.

[0193] In antibodies, part of the sequences of human immunoglobulin (recipient) is replaced by sequence segments of a non-human immunoglobulin (donor). In many cases, the donor is a murine immunoglobulin. In humanized antibodies,

[0194] Amino acids from the recipient's CDR are replaced by amino acids from the donor.

[0195] Sometimes, amino acids from the framework are also replaced by corresponding amino acids from the donor. In some cases, the humanized antibody contains amino acids that were not present in either the recipient or the donor and that were inserted during antibody optimization. In chimeric antibodies, the variable domains of the donor immunoglobulin are fused with the constant regions of a human antibody. Such "humanized" and "chimeric" antibodies also include aglycosylated variants, which are produced either by deglycosylation by PNGase F or by mutation of N297 (kabat number) of the heavy chain to any other amino acid.

[0196] The term complementarity-determining region (CDR), as used here, refers to those amino acids of a variable antibody domain that are necessary for binding to the antigen. Each variable region typically has three CDR regions, designated CDR1, CDR2, and CDR3. Each CDR region can contain amino acids as defined by Kabat and / or amino acids of a hypervariable loop as defined by Chotia. The definition according to Kabat, for example, includes the region of approximately amino acid positions 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) of the variable light chain / domain (VL) and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) of the variable heavy chain / domain (VH) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).The Chotia definition, for example, includes the region of approximately 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) (Chotia and Lesk; J Mol Biol 196: 901–917 (1987)). In some cases, a CDR may include amino acids from a CDR region defined according to Kabat and Chotia.

[0197] Depending on the amino acid sequence of the heavy chain constant domain, antibodies can be classified into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes are designated [α / α], [δ / δ], [ε / ε], [γ / γ], and [μ / μ]. Both the three-dimensional structure and the subunit structure of antibodies are known.

[0198] The term “functional fragment” or “antigen-binding antibody fragment” of an antibody / immunoglobulin is defined as a fragment of a

[0199] Antibody / immunoglobulin (e.g., the variable domains of an IgG), which also includes the antigen-binding domains of the antibody / immunoglobulin. The "antigen-binding domain" of an antibody typically comprises one or more

[0200] Hypervariable regions of an antibody, e.g., the CDR, CDR2, and / or CDR3 regions. However, the framework region of an antibody can also play a role in binding the antibody to the antigen. The framework region forms the scaffold for 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; particularly preferred are the complete variable light and heavy chains, i.e., amino acids 1 to 109 of the VL and 1 to 113 of the VH (numbering according to WO97 / 08320).“Functional fragments” or “antigen-binding antibody fragments” of the invention include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, diabodies, single domain antibodies (DAbs), linear antibodies, single-chain antibodies (single-chain Fv, abbreviated scFv); and multispecific, such as bi- and tri-specific, antibodies formed from antibody fragments. CA K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S.

[0201] Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). Antibodies other than "multi-specific" or "multi-functional" are those with identical binding sites. Multispecific antibodies can be specific for

[0202] different epitopes of an antigen or specific for epitopes of more than one antigen (see e.g. WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO

[0203] 92 / 05793; Tutt et al., 1991, J. Immunol. 147:60-69; US Pat. 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 engineered to reduce or completely prevent the number of intermolecular disulfide interactions occurring between the Chi and CL domains. "Epitopes" are protein determinants that can bind specifically 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 typically exhibit specific 3-dimensional structural properties as well as specific charge properties.

[0204] “Functional fragments” or “antigen-binding antibody fragments” can be fused with another polypeptide or protein, not derived from an antibody, via their amino or carboxy terminus by means of a covalent bond (e.g., a peptide linkage). Furthermore, antibodies and antigen-binding fragments can be modified by introducing reactive cysteines at defined sites to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol. 2008 Aug; 26(8):925-32).

[0205] Polyclonal antibodies can be produced by methods known to the average professional. Monoclonal antibodies can be produced by methods known to the average professional (Köhler and Milstein, Nature, 256, 495-497, 1975). Human or humanized monoclonal antibodies can be produced by methods known to the average professional.

[0206] The antibodies can be produced using methods known to the average person skilled in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et al. US 4,816,567 or Boss et al. US 4,816,397). The average person skilled in the art is familiar with a variety of methods for producing human antibodies and their fragments, such as using transgenic mice (N. Lonberg and D. Huszar, Int Rev Immunol. 1995; 13(1):65-93) or phage display technologies (Clackson et al., Nature. 1991 Aug 15;352(6336):624-8). Antibodies of the invention can be obtained from recombinant antibody libraries, which are based, for example, on the

[0207] The amino acid sequences of a large number of antibodies, generated from a large number of healthy volunteers, are used. Antibodies can also be produced using known recombinant DNA technologies. The nucleic acid sequence of an antibody can be obtained through routine sequencing or is available from publicly accessible databases. An "isolated" antibody or binder has been purified from other cellular components. Contaminating components of a cell that may interfere with diagnostic or therapeutic use include, for example, enzymes, hormones, or other peptide or non-peptide components of a cell. Preferably, an antibody or binder is purified to more than 95% by weight, based on the antibody or binder (determined, for example, by the Lowry method, UV-Vis spectroscopy, or SDS-capillary gel electrophoresis).Furthermore, an antibody that has been purified to such an extent that at least 15 amino acids of the amino terminus or an internal amino acid sequence can be determined, or has been purified to homogeneity, wherein the homogeneity is determined by SDS-PAGE under reducing or non-reducing conditions (detection can be determined by Coomassie blue staining or preferably by silver staining).

[0208] However, an antibody is usually produced through one or more purification steps. The term "specific binding" or "binds specifically" refers to a

[0209] An antibody or binder that binds to a predetermined antigen / target molecule. Specific binding of an antibody or binder typically describes an antibody or binder with an affinity of at least 10. "7 M (as Kd value; therefore preferably those with smaller Kd values ​​than 10) "^ M), wherein the antibody or binder has an affinity at least twice as high for the predetermined antigen / target molecule as 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 binder does not preclude the antibody or binder from binding to multiple antigens / target molecules (e.g., orthologs from different species). The antibodies preferably have an affinity of at least 10 ~ 7 M (as Kd value; therefore preferably those with smaller Kd values ​​than 10) ~ 7 M), preferably of at least 10 " 8 M, particularly preferably in the range of 10 "9 M to 10 " M. The Kd values ​​can be determined by, for example,

[0210] Surface plasmon resonance spectroscopy can be used to determine the affinities. The antibody-drug conjugates according to the invention also exhibit affinities in these regions. The conjugation of the active substances preferably does not significantly affect the affinity (generally, the affinity is reduced by less than one [unit of measurement]).

[0211] The magnitude is reduced, for example, by a maximum of 10 " 8 meters over 10 " ^ M).

[0212] The antibodies used according to the invention are further preferably characterized by high selectivity. High selectivity is present when the

[0213] The antibody according to the invention has an affinity for the target protein that is at least 2 times, preferably 5 times, or particularly preferably 10 times, greater than that for an independent antigen, e.g., human serum albumin (the affinity can be determined, e.g., by surface plasmon resonance spectroscopy). Furthermore, the antibodies used according to the invention are preferably cross-reactive. To facilitate and better interpret preclinical studies, e.g., toxicological or efficacy studies (e.g., in xenograft mice), it is advantageous if the antibody used according to the invention binds not only the human target protein but also the species-specific target protein in the species used for the studies. In one embodiment, the antibody used according to the invention is cross-reactive to the target protein of at least one other species in addition to the human target protein.For toxicological and efficacy studies, species from the families rodents, dogs, and non-human primates are preferred. Preferred rodent species are mice and rats. Preferred non-human primates are rhesus monkeys, chimpanzees, and long-tailed macaques.

[0214] In one embodiment, the antibody used according to the invention is, in addition to the human target protein, cross-reactive to the target protein of at least one further species selected from the group of species consisting of mouse, rat and

[0215] Long-tailed macaque (Macaca fascicularis). They are particularly favored by

[0216] According to the invention, antibodies used are those that, in addition to the human target protein, are at least cross-reactive with the mouse target protein. Preferably, cross-reactive antibodies are those whose affinity for the target protein of the other non-human species does not differ by more than a factor of 50, and in particular not by more than a factor of ten, from the affinity for the human target protein.

[0217] Antibodies directed against a cancer target molecule

[0218] Preferably, the target molecule against which the binder, e.g., an antibody or an antigen-binding fragment thereof, is directed, is a cancer target molecule. The term "cancer target molecule" describes a target molecule that is present in increased amounts on one or more cancer cell types compared to non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell types compared to non-cancer cells of the same tissue type, where "selective" describes at least a twofold increase on cancer cells compared to non-cancer cells of the same tissue type (a "selective cancer target molecule").

[0219] The use of cancer target molecules allows the selective therapy of cancer cells with the conjugates according to the invention.

[0220] Antibodies specifically directed against an antigen, such as a cancer cell antigen, can be produced by the average practitioner using familiar methods (such as recombinant expression) or purchased commercially (e.g., from Merck KGaA, Germany). Examples of well-known commercially available antibodies in cancer therapy include Erbitux® (cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche), and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgGl kappa type that binds with high affinity to the extracellular domain of the human epidermal growth factor receptor in a cell-based assay (Kd = 5 nM). The antibody is produced recombinantly in CHO cells.All these antibodies can also be produced as aglycosylated variants of these antibodies, either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

[0221] In a preferred embodiment, the target molecule is a selective cancer target molecule.

[0222] In a particularly preferred embodiment, the target molecule is a protein.

[0223] In one embodiment, the target molecule is an extracellular target molecule. In a preferred embodiment, the extracellular target molecule is a protein.

[0224] Cancer target molecules are known to experts. Examples are listed below.

[0225] Examples of cancer target molecules include:

[0226] (1) EGFR (EGF receptor, NCBI reference sequence NP_005219.2, NCBI gene ID: 1956) (2) Mesothelin (SwissProt reference Q13421-3), where mesothelin is encoded by amino acids 296-598. Amino acids 37-286 encode megakaryocyte-potentiating factor. Mesothelin is anchored to the cell membrane by a GPI anchor and is located extracellularly.

[0227] (3) Carbonic anhydrase IX (CA9, SwissProt reference Q16790), NCBI Gene ID: 768) (4) C4.4a (NCBI reference sequence NP_055215.2; synonym LYPD3, NCBI Gene ID: 27076)

[0228] (5) CD52 (NCBI reference sequence NP_001794.2)

[0229] (6) Her2 (ERBB2; NCBI reference sequence NP_004439.2; NCBI Gene ID: 2064)

[0230] (7) CD20 (NCBI reference sequence NP_068769.2) (8) the lymphocyte activation antigen CD30 (SwissProt ID P28908)

[0231] (9) the lymphocyte adhesion molecule CD22 (SwissProt ID P20273; NCBI gene ID: 933)

[0232] (10) the myloid cell surface antigen CD33 (SwissProt ID P20138; NCBI gene ID: 945)

[0233] (1 1 ) the transmembrane glycoprotein NMB (GPNMB, SwissProt ID Q14956, NCBI-Gene ID: 10457)

[0234] (12) the adhesion molecule CD56 (SwissProt ID P13591)

[0235] (13) the surface molecule CD70 (SwissProt ID P32970, NCBI-Gene ID: 970)

[0236] (14) the surface molecule CD74 (SwissProt ID P04233, NCBI-Gene ID: 972)

[0237] (15) the B-lymphocyte antigen CD19 (SwissProt ID P15391 , NCBI-Gene ID: 930)

[0238] (16) the surface protein mucin-1 (MUC1 , SwissProt ID P15941 , NCBI-Gene ID: 4582)

[0239] (17) the surface protein CD138 (SwissProt ID P18827)

[0240] (18) the integrin alphaV (NCBI reference sequence: NP_002201.1, NCBI Gene ID: 3685) (19) the teratocarcinoma-derived growth factor 1 protein TDGF1 (NCBI reference sequence: NP_003203.1, NCBI Gene ID: 6997)

[0241] (20) the prostate-specific membrane antigen PSMA (Swiss Prot ID: Q04609; NCBI- Gene ID: 2346)

[0242] (21) the tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317, NCBI gene ID: 1969) (22) the surface protein SLC44A4 (NCBI reference sequence: NP_001 171515.1 , NCBI gene ID: 80736)

[0243] (23) the surface protein BMPR1 B (SwissProt: 000238)

[0244] (24) the transport protein SLC7A5 (SwissProt: Q01650)

[0245] (25) the epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8, Gene ID: 26872) (26) the ovarian carcinoma antigen MUC16 (SwissProt: Q8WXI7, Gene ID: 94025)

[0246] (27) the transport protein SLC34A2 (SwissProt: 095436, Gene ID: 10568)

[0247] (28) the surface protein SEMA5b (SwissProt: Q9P283)

[0248] (29) the surface protein LYPD1 (SwissProt: Q8N2G4)

[0249] (30) the endothelin receptor type B EDNRB (SwissProt: P24530, NCBI-Gene ID: 1910)

[0250] (31 ) the ring finger protein RNF43 (SwissProt: Q68DV7)

[0251] (32) the prostate cancer-associated protein STEAP2 (SwissProt: Q8NFT2)

[0252] (33) the cation channel TRPM4 (SwissProt: Q8TD43)

[0253] (34) the complement receptor CD21 (SwissProt: P20023)

[0254] (35) the B-cell antigen receptor complex-associated protein CD79b (SwissProt: P40259, NCBI-Gene ID: 974)

[0255] (36) the cell adhesion antigen CEACAM6 (SwissProt: P40199)

[0256] (37) the dipeptidase DPEP1 (SwissProt: P16444)

[0257] (38) the interleukin receptor IL20Ralpha (SwissProt: Q9UHF4, NCBI-Gene ID: 3559)

[0258] (39) the proteoglycan BCAN (SwissProt: Q96GW7)

[0259] (40) the ephrin receptor EPHB2 (SwissProt: P29323)

[0260] (41) the prostate stem cell-associated protein PSCA (NCBI reference sequence:

[0261] NP_005663.2 )

[0262] (42) the surface protein LHFPL3 (SwissProt: Q86UP9)

[0263] (43) the receptor protein TNFRSF13C (SwissProt: Q96RJ3)

[0264] (44) the B-cell antigen receptor complex-associated protein CD79a (SwissProt: P1 1912)

[0265] (45) the receptor protein CXCR5 (CD185; SwissProt: P32302; NCBI gene ID 643, NCBI reference sequence: NP_001707.1 ) (46) the ion channel P2X5 (SwissProt: Q93086)

[0266] (47) the lymphocyte antigen CD180 (SwissProt: Q99467)

[0267] (48) the receptor protein FCRL1 (SwissProt: Q96LA6)

[0268] (49) the receptor protein FCRL5 (SwissProt: Q96RD9) (50) the MHC class II molecule 1a antigen HLA-DOB (NCBI reference sequence:

[0269] NP_0021 1 1.1 )

[0270] (51 ) the T-cell protein VTCN1 (SwissProt: Q7Z7D3)

[0271] (52) TWEAKR (FN14, TNFRSF12A, NCBI reference sequence: NP_057723.1, NCBI Gene ID: 51330) (53) the lymphocyte antigen CD37 (Swiss Prot: P1 1049, NCBI Gene ID: 951)

[0272] (54) The FGF receptor 2; FGFR2 (NCBI gene ID: 2263; Official Symbol: FGFR2). The FGFR2 receptor exists in different splice variants (alpha, beta, 1111, 112c). All splice variants can function as a target molecule.

[0273] (55) the transmembrane glycoprotein B7H3 (CD276; NCBI Gene ID: 80381 NCBI - Reference sequence: NP_001019907.1 , Swiss Prot: Q5ZPR3-1 )

[0274] (56) the B cell receptor BAFFR (CD268; NCBI gene ID: 1 15650)

[0275] (57) the receptor protein ROR 1 (NCBI gene ID: 4919)

[0276] (58) the surface receptor CD123 (IL3RA; NCBI gene ID: 3563; NCBI reference sequence: NP_002174.1; Swiss-Prot: P26951) (59) the receptor protein syncytin (NCBI gene ID 30816)

[0277] (60) the aspartate beta hydroxylase (ASPH; NCBI gene ID 444)

[0278] (61) the cell surface glycoprotein CD44 (NCBI gene ID: 960)

[0279] (62) CDH15 (Cadherin 15, NCBI Gene ID: 1013)

[0280] (63) the cell surface glycoprotein CEACAM5 (NCBI gene ID: 1048) (64) the cell adhesion molecule L1-Iike (CHL1 , NCBI gene ID: 10752)

[0281] (65) the receptor tyrosine kinase c-Met (NCBI gene ID: 4233)

[0282] (66) the Notch ligand DLL3 (NCBI Gene ID: 10683)

[0283] (67) the ephrin A4 (EFNA4, NCBI-Gene ID: 1945)

[0284] (68) the ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3, NCBI Gene ID: 5169)

[0285] (69) the coagulation factor III (F3, NCBI-Gene ID: 2152)

[0286] (70) the FGF receptor 3 (FGFR3, NCBI gene ID: 2261 )

[0287] (71 ) the folate hydrolase FOLH1 (NCBI-Gene ID: 2346)

[0288] (72) the folate receptor 1 (FOLR1 ; NCBI gene ID: 2348)

[0289] (73) the guanylate cyclase 2C (GUCY2C, NCBI-Gene ID: 2984)

[0290] (74) the KIT proto-oncogene receptor tyrosine kinase (NCBI-Gene ID: 3815)

[0291] (75) the lysosomal-associated membrane protein 1 (LAMP1 , NCBI gene ID: 3916)

[0292] (76) the lymphocyte antigen 6 complex, locus E (LY6E, NCBI-Gene ID: 4061)

[0293] (77) the protein NOTCH3 (NCBI Gene ID: 4854)

[0294] (78) protein tyrosine kinase 7 (PTK7, NCBI Gene ID: 5754)

[0295] (79) the Nectin cell adhesion molecule 4 (PVRL4, NECTIN4, NCBI gene ID: 81607)

[0296] (80) the transmembrane protein syndecan 1 (SDC1, NCBI Gene ID: 6382)

[0297] (81 ) the SLAM family member 7 (SLAMF7, NCBI gene ID: 57823)

[0298] (82) the transport protein SLC39A6 (NCBI gene ID: 25800)

[0299] (83) the SLIT and NTRK-like family member 6 (SLITRK6, NCBI gene ID: 84189)

[0300] (84) the cell surface receptor TACSTD2 (NCBI gene ID: 4070) (85) the receptor protein TNFRSF8 (NCBI gene ID: 943)

[0301] (86) the receptor protein TNFSF13B (NCBI gene ID: 10673)

[0302] (87) the glycoprotein TPBG (NCBI gene ID: 7162)

[0303] (88) the cell surface receptor TROP2 (TACSTD2, NCBI gene ID: 4070) (89) the galanin-like G protein-coupled receptor KISS1 R (GPR54, NCBI gene ID: 84634)

[0304] (90) the transport protein SLAMF6 (NCBI-Gene ID: 1 14836)

[0305] In a preferred aspect 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.

[0306] In a further particularly preferred aspect of the invention, the binder binds to an extracellular cancer target molecule, which is selected from the group consisting of the cancer target molecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particular CD123, CXCR5, and B7H3.

[0307] In a further particularly preferred embodiment of the invention, the binder binds specifically to an extracellular cancer target molecule, which is 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

[0308] In one embodiment, after binding to its extracellular target molecule on the target cell, the binder is internalized by the target cell. This causes the binder-drug conjugate, which can be an immunoconjugate or an ADC, to be taken up by the target cell. Subsequently, the binder is preferably processed intracellularly, preferably lysosomally. In one embodiment, the binder is a binding protein. In a preferred embodiment

[0309] In this embodiment, the binder is an antibody, an antigen-binding antibody fragment, a multispecific antibody, or an antibody mimetic. Preferred antibody mimetics are affibodies, adnectins, anticalins, DARPins, avimers, or nanobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies.

[0310] In a preferred embodiment, the binder is an antibody or an antigen-binding antibody fragment; more preferably, an isolated antibody or an isolated antigen-binding antibody fragment is used.

[0311] Preferred antigen-binding antibody fragments are Fab, Fab', F(ab')2 and Fv

[0312] Fragments, diabodies, DAbs, linear antibodies, and scFv. Fabs, diabodies, and scFv are particularly preferred. In a particularly preferred embodiment, the binder is an antibody. Monoclonal antibodies or antigen-binding antibody fragments thereof are particularly preferred. Human, humanized, or chimeric antibodies or antigen-binding antibody fragments thereof are further particularly preferred.

[0313] Antibodies or antigen-binding antibody fragments that bind to cancer target molecules can be produced by the average person using known methods, such as chemical synthesis or recombinant expression. Binders for cancer target molecules can be purchased commercially or can be produced by the average person using known methods, such as chemical synthesis or

[0314] Recombinant expression. Further methods for the production of antibodies or antigen-binding antibody fragments are described in WO 2007 / 070538 (see page 22, "Antibodies"). Those skilled in the art are familiar with methods for creating so-called phage display libraries (e.g., Morphosys HuCAL Gold) and using them to detect antibodies or antigen-binding antibody fragments (see WO 2007 / 070538, page 24 ff. and antibody example 1 on page 70, antibody example 2 on page 72). Further methods for the production of antibodies using DNA libraries from B cells are described, for example, on page 26 (WO 2007 / 070538). Methods for the humanization of antibodies are described on pages 30-32 of WO 2007 / 070538 and in detail in Queen et al., Pros. Natl. Acad. Sci. USA 86:10029-10033, 1989 or described in WO 90 / 0786. Furthermore, methods for the recombinant expression of proteins in general and antibodies in particular are known to those skilled in the art (see, e.g.,in Berger and Kimrnel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vo1. 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 Biolony, (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.

[0315] 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)). Those skilled in the art are familiar with the relevant vectors, promoters, and signal peptides necessary for the expression of a protein / antibody. Common procedures are also described in WO 2007 / 070538 on pages 41–45. For example, procedures for the production of an IgG1 antibody are described in WO

[0316] 2007 / 070538 described in Example 6 on page 74 ff. Procedures with which the

[0317] Methods for determining the internalization of an antibody after binding to its antigen are known to those skilled in the art and are described, for example, in WO 2007 / 070538 on page 80. Those skilled in the art can use the methods described in WO 2007 / 070538, which are used to determine the internalization of an antibody after binding to its antigen.

[0318] The production of carbonic anhydrase IX (Mn) antibodies was used analogously to the production of antibodies with other target molecule specificity.

[0319] Bacterial expression

[0320] Those skilled in the art are aware of how antibodies, antigen-binding fragments thereof, or variants thereof can be produced by bacterial expression. Suitable expression vectors for the bacterial expression of desired proteins are generated by inserting a DNA sequence encoding the desired protein into the functional reading frame together with suitable translation initiation and

[0321] Translation termination signals and constructed with a functional promoter. The vector includes one or more phenotypically selectable markers and a

[0322] Replication origin to conserve the vector and, if desired, the

[0323] to enable amplification of the same within the host. Suitable prokaryotic hosts for transformation include, but are not limited to, E. coli, Bacillus subtilis, Salmonella typhimurium, and various species from the genera Pseudomonas, Streptomyces, and Staphylococcus. Bacterial vectors can be based, for example, on bacteriophages, plasmids, or phagemids. These vectors can contain selectable markers and a bacterial origin of replication derived from commercially available plasmids. Many commercially available plasmids typically contain elements of the well-known cloning vector pBR322 (ATCC 37017). In bacterial systems, a number of advantageous expression vectors can be selected based on the intended use of the protein to be expressed.

[0324] After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed / induced by suitable means (e.g., temperature change or chemical induction), and the cells are cultured for an additional period. The cells are usually harvested by centrifugation, disrupted physically or chemically if necessary, and the resulting crude extract is retained for further purification.

[0325] Therefore, a further embodiment of the present invention is a

[0326] Expression vector comprising a nucleic acid encoding a novel antibody of the present invention.

[0327] Antibodies of the present invention or antigen-binding fragments thereof include naturally purified products, products derived from chemical syntheses, and products produced by recombinant technologies in prokaryotic hosts, such as E. coli, Bacillus subtilis, Salmonella typhimurium, and various species from the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably E. coli. Acid cell expression

[0328] Those skilled in the art know how antibodies, antigen-binding fragments thereof, or variants thereof can be produced using mammalian cell expression.

[0329] Preferred regulatory sequences for expression in mammalian host cells include viral elements that lead to high 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 polyomavirus. Antibody expression can be constitutive or regulated (e.g., induced by the addition or removal of small-molecule inducers such as tetracycline in combination with the Tet system). For further descriptions of viral regulatory elements and their sequences, see, for example, US 5,168,062 by Stinski and US 4,510,245 by Bell et al. and US 4,968,615 by Schaffner et al. The recombinant expression vectors can also include a replication origin and selectable markers (see e.g. US

[0330] 4,399,216, 4,634,665 and US 5,179,017). Suitable selectable markers include genes conferring resistance to substances such as G418, puromycin, hygromycin,

[0331] Blasticidin, zeocin / bleomycin, or methotrexate confer, or selectable markers that lead to auxotrophy of a host cell, such as glutamine synthetase (Bebbington et al., Biotechnology (NY). 1992 Feb; 10(2): 169-75), when the vector enters the cell.

[0332] was introduced.

[0333] For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the neo gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blasticidin, puromycin N-acetyltransferase confers resistance to puromycin, the Sh ble gene product confers resistance to zeocin, and resistance to hygromycin is mediated by the E. coli hygromycin resistance gene (hyg or hph). Selectable markers such as DHFR or glutamine synthetase are also helpful for amplification techniques in conjunction with MTX and MSX.

[0334] The transfection of an expression vector into a host cell can be achieved using

[0335] Standard techniques are performed, including electroporation, nucleofection, calcium phosphate precipitation, lipofection, polycation-based transfection such as polyethyleneimine (PEI)-based transfection and DEAE-dextran transfection.

[0336] 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-K1 SV [including DHFR-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sei. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun;33(2):405-12, used with a DHFR-selectable marker, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621], as well as other knockout cells, as detailed in Fan et al., Biotechnol Bioeng. 2012 Apr; 109(4): 1007-15), NS0 myeloma cells, COS cells, HEK293 cells, HKB1 1 cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

[0337] The expression of antibodies, antigen-binding fragments of these, or

[0338] Variants of these can also be expressed transiently or semi-stably in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB1, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1 SV, CHOEBNALT85, CHOS-XE, CHO-3E7, or CAP-T cells (for example, as described by Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9). In some embodiments, the expression vector is designed such that the protein to be expressed is secreted into the cell culture medium in which the host cells are growing. The antibodies, the antigen-binding fragments thereof, or variants thereof can be extracted from the cell culture medium using the

[0339] Protein purification methods known to experts are used.

[0340] The antibodies, the antigen-binding fragments thereof, or variants thereof can be obtained and purified from recombinant cell cultures using well-known methods, including, for example, ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, and protein G chromatography.

[0341] Chromatography, anion or cation exchange chromatography, phospho-cellulose chromatography, hydrophobic interaction chromatography (HIC),

[0342] Affinity chromatography, hydroxylapatite chromatography and lectin

[0343] Chromatography. High-performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997–2001), e.g., Chapters 1, 4, 6, 8, 9, 10.

[0344] The antibodies of the present invention, or antigen-binding fragments thereof, or variants thereof, comprise naturally purified products, products from chemical synthesis processes, and products produced using 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 can be glycosylated or non-glycosylated.

[0345] In a preferred embodiment, the antibody is purified (1) to more than 95 wt%, measured, for example, by the Lowry method, by UV-Vis spectroscopy, or by SDS-capillary gel electrophoresis (for example, using a Caliper LabChip GXII, GX 90, or Bio-Rad Bioanalyzer device), and in more preferred embodiments to more than 99 wt%, (2) to a degree suitable for determining at least 15 residues of the N-terminal or internal amino acid sequence, or (3) for homogeneity determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining.

[0346] An isolated antibody is usually detected using at least one

[0347] obtained during the protein purification step.

[0348] According to the invention, anti-CD123 antibodies can be used.

[0349] The term “anti-CD123 antibody” or “an antibody that binds specifically 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 diagnostic and / or therapeutic applications. In certain embodiments, the antibody binds CD123 with a dissociation constant (KD). D ) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 ηΜ, < 0.01 ηΜ, or < 0.001 nM.

[0350] The generation and properties of the monoclonal antibody 7G3, which binds the N-terminal domain of IL-3Ra, CD123, are described by Sun et al. (Sun et al., 1996, Blood 87(1): 83-92). US Patent No. 6,177,078 (Lopez) relates to the anti-CD123 antibody 7G3. A chimeric variant of this antibody (CSL360) is listed in WO 2009 / 070844, and a humanized version (CSL362) in WO 2012 / 021934.

[0351] The sequence of the 7G3 antibody was disclosed in EP2426148. This sequence represents the starting point for the humanized antibodies obtained by CDR grafting.

[0352] An antibody that internalizes particularly well after cell surface antigen binding is the anti-CD123 antibody 12F1, disclosed by Kuo et al. (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82). Antibody 12F1 binds to CD123 with a higher affinity than antibody 7G3 and internalizes significantly faster than 7G3 after cell surface antigen binding. Bispecific scFv immunofusion proteins based on 12F1 are disclosed in WO 2013 / 173820. Antibody TPP-6013 is a chimeric variant of 12F1.

[0353] The invention relates in particular to conjugates with antibodies or antigen-binding antibody fragments thereof or variants thereof, which are based on the antigen-binding antibody fragments derived from the mouse.

[0354] derived from murine antibodies 7G3 (Sun et al., 1996, Blood 87(1): 83-92) and 12F1 (Kuo et al., 2009, Bioconjug Chem. 20(10): 1975-82), or conjugates with antibodies or antigen-binding antibody fragments thereof or variants thereof derived from the mouse-derived antibody 12F1 (Kuo et al., 2009, Bioconjug Chem. 20(10): 1975-82). Humanized variants of the murine 7G3 antibody and the murine 12F1 antibody.

[0355] Antibodies were generated based on CDR implementation (“CDR grafting”) into a human framework and subsequent optimization and are preferred examples within the scope of this invention.

[0356] Particularly preferred within the scope of this invention are the anti-CD123 antibodies TPP-9476, TPP-8988, TPP-8987 and TPP-6013, and the anti-CXCR5 antibodies.

[0357] According to the invention, anti-CXCR5 antibodies can be used.

[0358] The term “anti-CXCR5 antibody” or “an antibody that binds specifically 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 diagnostic and / or therapeutic applications. In certain embodiments, the antibody binds CXCR5 with a

[0359] Dissociation constant (K D ) of < 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 ηM, < 0.01 ηM, or < 0.001 nM. 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.

[0360] Hybridoma cells for the rat antibody RF8B2 (ACC2153) were obtained from DSMZ, and the antibody sequence was identified using standard methods. This sequence forms the basis for the humanized antibodies obtained through CDR grafting. Humanized variants of this antibody were generated based on CDR grafting in germline sequences.

[0361] These antibodies and antigen-binding fragments can be used within the scope of this invention. Particularly preferred within the scope of this invention are the anti-XCCR5 antibodies TPP-9574 and TPP-9580.

[0362] anti-B7H3 antibody

[0363] According to the invention, anti-B7H3 antibodies can be used. The term "anti-B7H3 antibody" or "an antibody that binds specifically to B7H3" refers to an antibody that binds the cancer target molecule B7H3 (NCBI - reference sequence: NP_001019907.1 SEQ ID NO: 1 13), preferably with an affinity sufficient for diagnostic and / or therapeutic applications.

[0364] In certain embodiments, the antibody B7H3 binds with a

[0365] Dissociation constant (K D ) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM.

[0366] 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 WO201 109400, EP1773884 and WO2014061277. EP2121008 describes the anti-B7H3 antibody 8H9 and its CDR sequences.

[0367] These antibodies and antigen-binding fragments can be used within the scope of this invention.

[0368] A preferred embodiment of the ant-B7H3 antibodies was obtained by screening an antibody-phage display library on recombinant B7H3-expressing mouse (mouse CD276; gene ID: 102657) and human B7H3 (human CD276; gene ID: 80381) cells. The obtained antibodies were converted into the human IgG1 format. The anti-B7H3 antibody TPP-8382 is a preferred example.

[0369] Particularly preferred within the scope of this invention are the anti-B7H3 antibodies TPP-8382 and anti-TWEAKR antibodies.

[0370] According to the invention, anti-TWEAKR antibodies can be used.

[0371] The term “anti-TWEAKR antibody” or “an antibody that binds specifically 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 diagnostic and / or therapeutic applications. In certain embodiments, the antibody binds TWEAKR with a

[0372] Dissociation constant (K D ) of < 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM. Examples of antibodies that bind to TWEAKR are, for example, in

[0373] WO2009 / 020933(A2), WO2009 / 140177 (A2), WO 2014 / 198817 (A1) and WO

[0374] Patent 2015 / 189143 (A1) discloses these antibodies and antigen-binding fragments, which can be used within the scope of this invention.

[0375] ITEM-4 is an anti-TWEAKR antibody described by Nakayama et al. (Nakayama, et al., 2003, Biochem Biophy Res Comm, 306:819-825). Humanized

[0376] Variants of this antibody based on CDR implementation (“CDR grafting”) are described by Zhou et al. (Zhou et al., 2013, J Invest Dermatol. 133(4):1052-62) and in WO

[0377] Described in 2009 / 020933. These antibodies and antigen-binding fragments can be used within the scope of this invention. Particularly preferred within the scope of this invention are the anti-TWEAKR antibodies TPP-7006 and TPP-7007. These are humanized variants of the antibody ITEM-4. These antibodies and antigen-binding fragments can be preferably used within the scope of this invention. Anti-HER2 antibodies: According to the invention, anti-HER2 antibodies can be used.

[0378] The term “anti-HER2 antibody” or “an antibody that binds specifically to HER2” refers to an antibody that binds to the cancer target molecule HER2 (NCBI reference sequence: NP_004439.2; SEQ ID NO: 115), preferably with an affinity sufficient for diagnostic and / or therapeutic applications. In certain embodiments, the antibody binds to HER2 with a dissociation constant (KD). D ) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM.

[0379] An example of an antibody that binds to the cancer target molecule Her2 is

[0380] Trastuzumab (Genentech). Trastuzumab is a humanized antibody used, among other things, to treat breast cancer. In a particularly preferred embodiment, the anti-HER2 antibody TPP-1015 (analogous to trastuzumab) is used.

[0381] Other examples of antibodies that bind to HER2, besides trastuzumab (INN 7637, CAS No. RN: 180288-69-1) and pertuzumab (CAS No.: 380610-27-5), include antibodies disclosed in WO 2009 / 123894-A2, WO 200 / 8140603-A2, or WO 201 1 / 044368-A2. An example of an anti-HER2 conjugate is trastuzumab emtansine (INN No. 9295). These antibodies and antigen-binding fragments can be used within the scope of this invention.

[0382] Particularly preferred within the scope of this invention is the anti-HER2 antibody TPP-1015 (analogous to trastuzumab).

[0383] anti-EGFR antibodies

[0384] According to the invention, anti-EGFR antibodies can be used.

[0385] The term “anti-EGFR antibody” or “an antibody that binds specifically to EGFR” refers to an antibody that binds to the cancer target molecule EGFR (NCBI reference sequence: NP_005219.2; SEQ ID NO: 116), preferably with an affinity sufficient for diagnostic and / or therapeutic applications. In certain embodiments, the antibody binds to EGFR with a

[0386] Dissociation constant (K D ) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM.

[0387] In a preferred embodiment, the anti-EGFR antibodies are selected from the group consisting of TPP-981, cetuximab, panitumumab, and nimotuzumab. In a particularly preferred embodiment, the anti-EGFR antibody is TPP-981.

[0388] Other formulations of EGFR antibodies are: • Zalutumumab / 2F8 / HuMax-EGFR, Genmab A / S (WO 02 / 100348, WO 2004 / 056847, INN number 8605)

[0389] • Necitumumab / 11F8, ImClone / IMC-11F8, Firm ImClone Systems 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)

[0390] • Matuzumab / anti-EGFR MAb, Merck KGaA / anti-EGFR MAb, Takeda / EMD 72000 / EMD-6200 / EMD-72000 and EMD-55900 / MAb 425 / monoclonal antibody 425, Merck KGaA / Takeda Company (WO 92 / 15683, INN-Number 8103 (Matuzumab)) · RG-7160 / GA-201 / GA201 / R-7160 / R7160 / RG7160 / RO-4858696 / RO-

[0391] 5083945 / R04858696 / RO5083945, The Company Glycart Biotechnology AG (Roche Holding AG) (WO 2010 / 1 12413-A1 , WO 2010 / 1 15554);

[0392] • GT-MAB 5.2-GEX / CetuGEX, Firma Glycotope GmbH (WO 2008 / 028686-A2 (EP 01900750-A1, EP 0191 1766-A1, EP 02073842-A2, US 2010 / 0028947-A1) · SU-101 , Signature Isu Abxis Inc (ISU Chemical Co Ltd) / Scancell (WO 2008 / 004834-

[0393] A1 )

[0394] • ABT-806 / mAb-806 / ch-806 / anti-EGFR monoclonal antibody 806, Firma Ludwig Institute for Cancer Research / Abbott / Life Science Pharmaceuticals (WO 02 / 092771 , WO 2005 / 081854 und WO 2009 / 023265) · SYM-004 (consists of two chimeric lgG1 antibodies (992 and 1024)), Firma

[0395] Symphogen A / S (WO 2010 / 022736-A2)

[0396] • MR1-1 / MR1-1 KDEL, Firma IVAX Corp (Teva Pharmaceutical Industries Ltd) (Duke University), (Patent: WO2001 / 062931 -A2)

[0397] • Antikörper gegen die Deletionsmutante, EGFRvlll, Firma Amgen / Abgenix (WO 2005 / 010151 , US 7,628,986)

[0398] • SC-100, Firma Scancell Ltd (WO 01 / 088138-A1 ) • MDX-447 / EMD 82633 / BAB-447 / H 447 / MAb, EGFR, Medarex / Merck KgaA, Firma Bristol-Myers Squibb (US) / Merck KGaA (DE) / Takeda (JP), (WO

[0399] 91 / 05871 , WO 92 / 15683)

[0400] • anti-EGFR-Mab, company Xencor (WO 2005 / 056606) · DXL-1218 / anti-EGFR monoclonal antibody (cancer), InNexus, company InNexus

[0401] Biotechnology Inc, Pharmaprojects PH048638 anti-carbonic anhydrase IX antibody

[0402] Examples of antibodies that bind to the cancer target molecule carbonic anhydrase IX are described in WO 2007 / 070538-A2 (e.g., claims 1-16). Anti-C4.4a antibodies:

[0403] Examples of C4.4a antibodies and antigen-binding fragments are described in WO 2012 / 143499 A2. The antibody sequences are given in Table 1 of WO 2012 / 143499 A2, with each row representing the respective CDR amino acid sequences of the variable light chain and variable heavy chain of the antibody listed in column 1. Anti-CD20 antibodies:

[0404] An example of an antibody that binds to the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS number: 174722-31-7) is a chimeric antibody used to treat non-Hodgkin lymphoma. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-CD52 antibodies:

[0405] An example of an antibody that binds to the cancer target molecule CD52 is

[0406] Alemtuzumab (Genzyme). Alemtuzumab (CAS number: 216503-57-0) is a

[0407] Humanized antibody used to treat chronic lymphocytic leukemia. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-Mesot hei in antibody:

[0408] 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 within the scope of the invention disclosed herein. The antibody MF-T disclosed in WO2009 / 068204 is particularly preferred.

[0409] anti-CD30 antibodies

[0410] Examples of antibodies that bind to the cancer target molecule CD30 and can be used to treat cancers such as Hodgkin lymphoma include brentuximab,

[0411] Iratumumab and antibodies, as disclosed in WO 2008 / 0921 17, WO 2008 / 036688 or WO 2006 / 089232. An example of an anti-CD30 conjugate is brentuximab vedotin (INN No. 9144). These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. anti-CD22 antibodies

[0412] Examples of antibodies that bind to the cancer target molecule CD22 and can be used to treat cancers such as 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 RN: 139504-50-0 and 474645-27-7, respectively). These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-CD33 antibodies

[0413] Examples of antibodies that bind to the cancer target molecule CD33 and can be used to treat cancer, e.g., leukemia, are gemtuzumab or lintuzumab (INN 7580). An example of an anti-CD33 conjugate is gemtuzumab ozagamycin. These antibodies and antigen-binding fragments can be used within the scope of this invention. Anti-NMB antibodies

[0414] An example of an antibody that binds to the cancer target molecule NMB and leads to

[0415] Glembatumumab (INN 9199) is an anti-NMB conjugate used to treat cancers such as melanoma or breast cancer. Glembatumumab vedotin (CAS RN: 474645-27-7) is another example of an anti-NMB conjugate. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-CD56 antibodies

[0416] An example of an antibody that binds to the cancer target molecule CD56 and leads to

[0417] Lorvotuzumab can be used to treat cancers such as multiple myeloma, small cell lung cancer (MCC), or ovarian cancer. An example of an anti-CD56 conjugate is lorvotuzumab mertansine (CAS RN: 139504-50-0). These antibodies and antigen-binding fragments can be used within the scope of this invention. Anti-CD70 antibodies

[0418] Examples of antibodies that bind to the cancer target molecule CD70 and can be used to treat cancer, e.g., non-Hodgkin lymphoma or renal cell carcinoma, are disclosed in WO 2007 / 038637-A2 or WO 2008 / 070593-A2. An example of an anti-CD70 conjugate is SGN-75 (CD70 MMAF). These antibodies and antigen-binding fragments can be used within the scope of this invention. Anti-CD74 antibodies

[0419] An example of an antibody that binds to the cancer target molecule CD74 and leads to

[0420] Milatuzumab can be used to treat cancers such as multiple myeloma. An example of an anti-CD74 conjugate is milatuzumab doxorubicin (CAS RN: 23214-92-8). These antibodies and antigen-binding fragments can be used in this context.

[0421] The invention can be used. Anti-CD19 antibodies.

[0422] An example of an antibody that binds to the cancer target molecule CD19 and leads to

[0423] The use of this antibody in the treatment of cancer, e.g., non-Hodgkin lymphoma, is disclosed in WO 2008 / 031056-A2. Further antibodies and examples of an anti-CD19 conjugate (SAR3419) are disclosed in WO 2008 / 047242-A2. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-mucin antibodies

[0424] Examples of antibodies that bind the cancer target molecule mucin-1 and can be used to treat cancer, e.g., non-Hodgkin lymphoma, are clivatuzumab or the antibodies disclosed in WO 2003 / 106495-A2 and WO 2008 / 028686-A2. Examples of anti-mucin conjugates are disclosed in WO 2005 / 009369-A2. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-CD138 antibodies

[0425] Examples of antibodies that bind the cancer target molecule CD138 and conjugates thereof, which can be used to treat cancer, e.g., multiple myeloma, are disclosed in WO 2009 / 080829-A1 and WO 2009 / 080830-A1. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-Inteqrin-alphaV antibodies

[0426] Examples of antibodies that bind to the cancer target molecule integrin alphaV and can be used to treat cancers such as melanoma, sarcoma, or carcinoma include intetumumab (CAS RN: 725735-28-4), abciximab (CAS RN: 143653-53-6), etaracizumab (CAS RN: 892553-42-3), or the antibodies disclosed in US 7,465,449, EP 719859-A1, WO 2002 / 012501-A1, or WO 2006 / 062779-A2. Examples of anti-integrin alphaV conjugates include intetumumab-DM4 and other ADCs disclosed in WO 2007 / 024536-A2. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. anti-TDGF1 antibodies

[0427] Examples of antibodies that bind to the cancer target molecule TDGF1 and can be used to treat cancer are those disclosed in WO 02 / 077033-A1, US 7,318,924, WO 2003 / 083041-A2, and WO 2002 / 088170-A2. Examples of anti-TDGF1 conjugates are disclosed in WO 2002 / 088170-A2. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. Anti-PSMA antibodies

[0428] Examples of antibodies that bind to the cancer target molecule PSMA and can be used to treat cancer, e.g., prostate cancer, are the antibodies disclosed in WO 97 / 35616-A1, WO 99 / 47554-A1, WO 01 / 009192-A1 and WO2003 / 034903.

[0429] Examples of anti-PSMA conjugates are disclosed in WO 2009 / 026274-A1 and WO 2007 / 002222. These antibodies and antigen-binding fragments can be used within the scope of this invention. anti-EPHA2 antibodies

[0430] Examples of antibodies that bind to the cancer target molecule EPHA2, which can be used to produce a conjugate and treat cancer, are described in WO

[0431] 2004 / 091375-A2 disclosed. These antibodies and antigen-binding fragments can be used within the scope of this invention. anti-SLC44A4 antibody

[0432] Examples of antibodies that bind the cancer target molecule SLC44A4, which can be used to produce a conjugate and to treat cancer, e.g., pancreatic or prostate cancer, are disclosed in WO2009 / 033094-A2 and US2009 / 0175796-A1. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. anti-HLA-DOB antibodies

[0433] An example of an antibody that binds the cancer target molecule HLA-DOB is the antibody Lym-1 (Cas-RN: 301344-99-0), which can be used to treat cancer, e.g., non-Hodgkin lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, e.g., in WO 2005 / 08171 1-A2. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. anti-VTCN1 antibody

[0434] Examples of antibodies that bind the cancer target molecule VTCN1, which can be used to produce a conjugate and to treat cancer, e.g., ovarian, pancreatic, lung, or breast cancer, are disclosed in WO 2006 / 074418-A2. These antibodies and antigen-binding fragments thereof can be used within the scope of this invention. anti-FGFR2 antibodies

[0435] Examples of anti-FGFR2 antibodies and antigen-binding fragments are found in

[0436] WO2013076186 describes the antibody sequences. These sequences are given in Tables 9 and 10 of WO2013076186. Antibodies, antigen-binding fragments, and antibody variants derived from the antibodies designated M048-D01 and M047-D08 are preferred. Preferred antibodies and antigen-binding antibody fragments for binder-drug conjugates according to the invention.

[0437] In this application, the following preferred antibodies are referred to as binder-drug conjugates, as shown in the table below: TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580.

[0438] Table: Protein levels of antibodies:

[0439] 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 (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) of the variable region of the heavy chain (VH) or the variable region of the light chain (VL) specified in the table above. Preferably, the antibodies comprise the specified variable region of the heavy chain (VH) and / or the variable region of the light chain (VL). Preferably, the antibodies comprise the specified region of the heavy chain (IgG heavy chain) and / or the specified region of the light chain (IgG light chain).

[0440] TPP-981 is an anti-EGFR antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 2, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 3, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 4, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 6, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 7, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 8.

[0441] TPP-1015 is an anti-HER2 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 12, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 13, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 14, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 16, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 17, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 18.

[0442] TPP-6013 is an anti-CD123 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 22, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 23, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 24, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 26, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 27, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 28.

[0443] TPP-7006 is an anti-TWEAKR antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 32, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 33, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 34, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 36, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 37, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 38.

[0444] TPP-7007 is an anti-TWEAKR antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 42, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 43, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 44, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 46, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 47, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 48.

[0445] TPP-8382 is an anti-B7H3 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 52, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 53, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 54, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 56, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 57, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 58.TPP-8987 is an anti-CD123 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 62, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 63, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 64, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 66, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 67, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 68.

[0446] TPP-8988 is an anti-CD123 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 72, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 73, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 74, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 76, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 77, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 78.TPP-9476 is an anti-CD123 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 82, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 83, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 84, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 86, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 87, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 88.

[0447] TPP-9574 is an anti-CXCR5 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 92, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 93, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 94, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 96, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 97, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 98.TPP-9580 is an anti-CXCR5 antibody comprising a variable heavy chain region (VH) including the variable CDR1 heavy chain sequence (H-CDR1), as represented by SEQ ID NO: 102, the variable CDR2 heavy chain sequence (H-CDR2), as represented by SEQ ID NO: 103, and the variable CDR3 heavy chain sequence (H-CDR3), as represented by SEQ ID NO: 104, as well as a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1), as represented by SEQ ID NO: 106, the variable CDR2 light chain sequence (L-CDR2), as represented by SEQ ID NO: 107, and the variable CDR3 light chain sequence (L-CDR3), as represented by SEQ ID NO: 108.

[0448] TPP-981 is an anti-EGFR antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 1 and a variable light chain region (VL) as represented by SEQ ID NO: 5.

[0449] TPP-1015 is an anti-HER2 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 1 1 and a variable light chain region (VL) as represented by SEQ ID NO: 15.

[0450] TPP-6013 is an anti-CD123 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 21 and a variable light chain region (VL) as represented by SEQ ID NO: 25. TPP-7006 is an anti-TWEAKR antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 31 and a variable light chain region (VL) as represented by SEQ ID NO: 35.

[0451] TPP-7007 is an anti-TWEAKR antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 41 and a variable light chain region (VL) as represented by SEQ ID NO: 45.

[0452] TPP-8382 is an anti-B7H3 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 51 and a variable light chain region (VL) as represented by SEQ ID NO: 55. TPP-8987 is an anti-CD123 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 61 and a variable light chain region (VL) as represented by SEQ ID NO: 65.

[0453] TPP-8988 is an anti-CD123 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 71 and a variable light chain region (VL) as represented by SEQ ID NO: 75.

[0454] TPP-9476 is an anti-CD123 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 81 and a variable light chain region (VL) as represented by SEQ ID NO: 85. TPP-9574 is an anti-CXCR5 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 91 and a variable light chain region (VL) as represented by SEQ ID NO: 95.

[0455] TPP-9580 is an anti-CXCR5 antibody comprising preferably a variable heavy chain region (VH) as represented by SEQ ID NO: 101 and a variable light chain region (VL) as represented by SEQ ID NO: 105.

[0456] TPP-981 is an anti-EGFR antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 9 and a light chain region as represented by SEQ ID NO: 10. TPP-1015 is an anti-HER2 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 19 and a light chain region as represented by SEQ ID NO: 20.

[0457] TPP-6013 is an anti-CD123 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 29 and a light chain region as represented by SEQ ID NO: 30.

[0458] TPP-7006 is an anti-TWEAKR antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 39 and a light chain region as represented by SEQ ID NO: 40. TPP-7007 is an anti-TWEAKR antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 49 and a light chain region as represented by SEQ ID NO: 50.

[0459] TPP-8382 is an anti-B7H3 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 59 and a light chain region as represented by SEQ ID NO: 60.

[0460] TPP-8987 is an anti-CD123 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 69 and a light chain region as represented by SEQ ID NO: 70. TPP-8988 is an anti-CD123 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 79 and a light chain region as represented by SEQ ID NO: 80.

[0461] TPP-9476 is an anti-CD123 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 89 and a light chain region as represented by SEQ ID NO: 90.

[0462] TPP-9574 is an anti-CXCR5 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 99 and a light chain region as represented by SEQ ID NO: 100.

[0463] TPP-9580 is an anti-CXCR5 antibody comprising preferably a heavy chain region as represented by SEQ ID NO: 109 and a light chain region as represented by SEQ ID NO: 110.

[0464] Isotopes, salts, solvates, isotopic variants

[0465] The present invention also includes all suitable isotopic variants of the compounds according to the invention. An isotopic variant of a

[0466] The compound according to the invention is understood to be a compound in which at least one atom within the compound according to the invention is replaced by another atom of the same atomic number, but with a different atomic mass than that which usually or predominantly occurs in nature. Examples of isotopes that can be incorporated into a compound according to the invention are those of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, such as 2 H (Deuterium), 3 H (Tritium), 13 C, 14 C, 15 N, 17 0,

[0467] 18 0 j 32 Pi 33 P i 33 Si 34 S i 35 S i 129| u nd 1311 Certain ISO-optic

[0468] Variants of a compound according to the invention, such as those in which one or more radioactive isotopes are incorporated, can be advantageous.

[0469] for example, for investigating the mechanism of action or the distribution of active ingredients in the body; due to their comparatively easy production and detectability, they are particularly suitable for this purpose with 3 H- or 14 C-isotope-labeled compounds are suitable. Furthermore, the incorporation of isotopes, such as deuterium, can lead to certain therapeutic benefits as a result of greater metabolic stability.

[0470] This can lead to changes such as an increase in the half-life in the body or a reduction in the required dose; such modifications of the

[0471] The compounds according to the invention may therefore also represent a preferred embodiment of the present invention. Isotopic variants of the compounds according to the invention can be prepared according to methods known to those skilled in the art, for example, according to the methods described below and the instructions given in the exemplary embodiments, by making appropriate isotopic modifications of the respective reagents and / or

[0472] Starting compounds are used. Within the scope of the present invention, physiologically harmless salts of the compounds according to the invention are preferred. This also includes salts that are not themselves suitable for pharmaceutical applications, but can be used, for example, for the isolation or purification of the compounds according to the invention. Physiologically harmless salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids, and sulfonic acids, e.g., 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.

[0473] Physiologically harmless salts of the compounds according to the invention also include salts of common bases, such as, by way of example and preferably, alkali metal salts (e.g., sodium and potassium salts), alkaline earth salts (e.g., calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines with 1 to 16 carbon atoms, such as, by way of example and preferably, ethylamine, diethylamine, triethylamine, ethyl diisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, α / -methylpiperidine, α / -methylmorpholine, arginine, lysine and 1,2-ethylenediamine.

[0474] Within the scope of the invention, the term solvates refers to those forms of the compounds according to the invention which, in solid or liquid state, form a complex through coordination with solvent molecules. Hydrates are a special form of solvates in which coordination occurs with water. Hydrates are preferred as solvates within the scope of the present invention.

[0475] Therapeutic use

[0476] The hyperproliferative diseases for which the compounds according to the invention can be used include, in particular, the group of cancers and tumor diseases. Within the scope of the present invention, this includes, but is not limited to, the following diseases: breast carcinomas and breast tumors (mammary carcinomas including ductal and lobular forms, also in situ), respiratory tract tumors (small cell and non-small cell carcinoma, bronchial carcinomas), brain tumors (e.g., of the brainstem and hypothalamus, astrocytoma, ependymoma, glioblastoma, gliomas, medulloblastoma, meningiomas, as well as neuroectodermal and pineal tumors), tumors of the digestive organs (esophageal, gastric, gallbladder, small intestine, large intestine, rectal, and anal carcinomas), liver tumors (among others).hepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma), tumors of the head and neck region (laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal, lip and oral cavity carcinomas, oral melanomas), skin tumors (basaliomas, spinaliomas, squamous cell carcinomas, Kaposi's sarcoma, malignant melanomas, non-melanoma skin cancer, Merkel cell skin cancer, mast cell tumors), tumors of the supporting and connective tissue (including soft tissue sarcomas, etc.).

[0477] Osteosarcomas, malignant fibrous histiocytomas, chondrosarcomas, fibrosarcomas,

[0478] Hemangiosarcomas, leiomyosarcomas, liposarcomas, lymphosarcomas and

[0479] Rhabdomyosarcomas), tumors of the eyes (including intraocular melanoma and retinblastoma), tumors of the endocrine and exocrine glands (e.g., of the thyroid and parathyroid glands, pancreatic and salivary gland carcinomas,

[0480] Adenocarcinomas), tumors of the urinary tract (bladder, penis, kidney, renal pelvis and ureter tumors) as well as tumors of the reproductive organs (endometrial, cervical, ovarian, vaginal, vulvar and uterine carcinomas in women as well as prostate and

[0481] Testicular cancer in men). This also includes proliferative disorders of the blood, lymphatic system, and spinal cord, in solid form and as circulating cells, such as leukemias, lymphomas, and myeloproliferative disorders, e.g., acute myeloid, acute lymphoblastic, chronic lymphocytic, chronic myelogenous, and hairy cell leukemia, as well as AI / DS-related lymphomas, Hodgkin lymphomas, non-Hodgkin lymphomas, cutaneous T-cell lymphomas, Burkitt lymphomas, and lymphomas of the central nervous system. These well-characterized human diseases can be treated with comparable

[0482] Etiology also occurs in other mammals and is also associated with the

[0483] The compounds of the present invention are discussed.

[0484] The binder or antibody-drug conjugates (ADCs) directed against CD123 described here can preferably be used to treat CD123-expressing disorders, such as CD123-expressing cancers. Typically, such cancer cells show measurable amounts of CD123 as measured at the protein (e.g., by immunoassay) or RNA level. Some of these cancerous tissues show elevated levels of CD123 compared to non-cancerous tissue of the same type, preferably measured in the same patient. Optionally, the CD123 content is measured before cancer treatment with an antibody-drug conjugate (ADC) according to the invention is initiated (patient stratification). CD123-directed binder-drug conjugates (ADCs) can be preferably used to treat CD123-expressing disorders, such as CD123-expressing cancers, like tumors of hematopoietic and lymphatic tissue or hematopoietic and lymphatic malignant tumors.Examples of cancers associated with CD123 expression include myeloid diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Other cancers include B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), Hodgkin lymphoma, immature T-cell acute lymphoblastic leukemia (immature T-ALL), Burkitt lymphoma, follicular lymphoma, chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL). Methods of the described invention include the treatment of patients with a CD123-expressing cancer, wherein the method comprises the administration of an inventive antibody-drug conjugate (ADC). The treatment of the aforementioned cancers using the compounds according to the invention includes both treatment of solid tumors and treatment of metastatic or circulating forms thereof.

[0485] The term "treatment" or "treat" is used within the scope of this invention.

[0486] Conventionally used, it means the care, treatment, and support of a patient with the aim of combating, reducing, alleviating, or easing an illness or health condition and improving the living conditions affected by this illness, such as in the case of cancer. A further object of the present invention is therefore the use of the compounds according to the invention for the treatment and / or prevention of diseases, in particular the aforementioned diseases.

[0487] A further object of the present invention is the use of the compounds according to the invention for the manufacture of a medicinal product for the treatment and / or prevention of diseases, in particular the aforementioned diseases.

[0488] A further aspect of the present invention is the use of the

[0489] The compounds according to the invention in a method for the treatment and / or prevention of diseases, in particular the aforementioned diseases.

[0490] A further object of the present invention is a method for the treatment and / or prevention of diseases, in particular the aforementioned diseases, using an effective amount of at least one of the compounds according to the invention.

[0491] The compounds according to the invention can be used alone or, if necessary, in combination with one or more other pharmacologically active substances, as long as this combination does not lead to undesirable and unacceptable side effects.

[0492] This leads to side effects. Therefore, a further subject of the present invention is pharmaceuticals containing at least one of the compounds according to the invention and one or more further active ingredients, in particular for the treatment and / or

[0493] Prevention of the aforementioned diseases. For example, the compounds of the present invention can be combined with known anti-hyperproliferative, cytostatic, cytotoxic, or immunotherapeutic substances for the treatment of cancer. Suitable combination agents include, for example:

[0494] 131 1-chTNT, Abarelix, Abiraterone, Aclarubicin, Adalimumab, Ado-Trastuzumab Emtansine, Afatinib, Aflibercept, Aldesleukin, Alemtuzumab, Alendronsäure, Alitretinoin, Altretamine, Amifostine, Aminogluxolehidine, Aminoglycoside-5, Amrubicin, Amsacrine, Anastrozole, Ancestim, Anethole dithiolethione, Anetumab ravtansine, Angiotensin II, Antithrombin III, Aprepitant, Arcitumomab, Arglabine, Arsentrioxide, Asparaginase, Atezolizumab, Avelumab Axitinib, Azaciti, Belotecan, Besindamab, Besindamab, Belinostat, Bevacizumab, Bexaroten, Bicalutamide, Bisantren, Bleomycin, Blinatumomab, Bortezomib, Buserelin, Bosutinib, Brentuximab vedotin, Busulfan, Cabazitaxel, Cabozantinib, Calcitonin,

[0495] Calciumfolinat, Calciumlevofolinat, Capecitabin, Capromab, Carbamazepine, Carboplatin, Carboquon, Carfilzomib, Carmofur, Carmustin, Catumaxomab, Celecoxib, Celmoleukin, Ceritinib, Cetuximab, Chlorambucil, Chlormadinon, Chlormethin, Cidofovir, Cinacalcet, Cisplatin, Cladribin, Clodronsäure, Clofarabin, Cobimetinib, Copanlisib, Crisantaspase, Crizotinib, Cyclophosphamid, Cyproteron, Cytarabin, Dacarbazin, Dactinomycin,

[0496] Daratumumab, Dabrafenib, Darolutamide, Dasatinib, Daunorubicin, Decitabin, Degarelix, Denileukin-Diftitox, Denosumab, Depreotid, Deslorelin, Dexrazoxane,

[0497] Dibrospidiumchlorid, Dianhydrogalactitol, Diclofenac, Docetaxel, Dolasetron, Doxifluridin, Doxorubicin, Doxorubicin + Estron, Dronabinol, Durvalumab Edrecolomab,

[0498] Elliptiniumacetat, Endostatin, Enocitabin, Enzalutamid, Epacadostat, Epirubicin,

[0499] Epitiostanol, Epoetin-alfa, Epoetin-beta, Epoetin-zeta, Eptaplatin, Eribulin, Erlotinib, Esomeprazol, Estramustin, Etoposid, Ethinylestradiol, Everolimus, Exemestan, Fadrozol, Fentanyl, Fluoxymesteron, Floxuridin, Fludarabin, Fluoruracil, Flutamid, Folinsäure, Formestan, Fosaprepitant, Fotemustin, Fulvestrant, Gadobutrol, Gadoteridol,

[0500] Gadoteric acid meglumine salt, gadoversetamide, gadoxetic acid disodium salt (Gd-EOB-DTPA disodium salt), gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, glucarpidase, glutoxime, 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, lobitridol, lobenguane (1231), lomeprol, Ipilimumab, Irinotecan, Itraconazole, Ixabepilone, Ixazomib, Lanreotide, Lansoprazole, Lansoprazole, Lapatinib, Lasocholine, Lenalidomide, Lenvatinib, Lenograstim, Lentinan, Letrozole, Leuprorelin, Levamisole, Levonorgestrel, Levothyroxine sodium, Lipegfilgrastim, Lisuride, Lobaplatin, Lomustine, Lonidamine, Masoprocol, Medroxyprogesterone, Megestrol, Melarsoprol,Melphalan, Mepitiostan, Mercaptopurin, Mesna, Methadon, Methotrexat, Methoxsalen,

[0501] Methylaminolevulinat, Methylprednisolon, Methyltestosteron, Metirosin, Mifamurtid, Miltefosin, Miriplatin, Mitobronitol, Mitoguazon, Mitolactol, Mitomycin, Mitotan,

[0502] Mitoxantron, Mogamulizumab, Molgramostim, Mopidamol, Morphinhydrochlorid,

[0503] Morphinsulfat, Nabilon, Nabiximols, Nafarelin, Naloxon + Pentazocin, Naltrexon,

[0504] Nartograstim, Necitumumab, Nedaplatin, Nelarabin, Neridronsäure,

[0505] Netupitant / palonosetron, Nivolumab, Nivolumabpentetreotid, Nilotinib, Nilutamid,

[0506] Nimorazol, Nimotuzumab, Nimustin, Nintedanib, Nitracrin, Nivolumab, Obinutuzumab, Octreotid, Ofatumumab, Olaparib, Olaratumab, Omacetaxin-Mepesuccinat, Omeprazol, Ondansetron, Orgotein, Orilotimod, Osimertinib, Oxaliplatin, Oxycodon, Oxymetholon, Ozogamicin, p53-Gentherapie, Paclitaxel, Palbociclib, Palifermin, Palladium-103-Seed, Palonosetron, Pamidronsäure, Panitumumab, Panobinostat, Pantoprazol, Pazopanib, Pegaspargase, Pembrolizumab, Peg-interferon-alfa-2b, Pembrolizumab, Pemetrexed, Pentostatin, Peplomycin, Perflubutane, Perfosfamid, Pertuzumab, Picibanil, Pilocarpin, Pirarubicin, Pixantron, Plerixafor, Plicamycin, Poliglusam, Polyestradiolphosphat, Polyvinylpyrrolidone + Natriumhyaluronat, Polysaccharid-K, Pomalidomid, Ponatinib, Porfimer-Natrium, Pralatrexat, Prednimustin, Prednison, Procarbazin, Procodazole, Propranolol, Quinagolid, Rabeprazol, Racotumomab, Radium-223-chlorid, Radotinib, Raloxifen, Raltitrexed, Ramosetron, Ramucirumab, Ranimustin, Rasburicase, Razoxan,Refametinib, Regorafenib, Risedronsäure, Rhenium-186 Etidronat, Rituximab,

[0507] Rogaratinib, Rolapitant, Romidepsin, Romurtid, Roniciclib, Samarium (153Sm) lexidronam, Satumomab, Secretin, Siltuximab, Sipuleucel-T, Sizofiran, Sobuzoxan, Natriumglycididazol, Sonidegib, Sorafenib, Stanozolol, Streptozocin, Sunitinib, Talaporfin, Talimogen Laherparepvec, Tamibaroten, Tamoxifen, Tapentadol, Tasonermin,

[0508] Teceleukin, Technetium (99mTc) Nofetumomab Merpentan, 99mTc-HYNIC-[Tyr3]- octreotid, Tegafur, Tegafur + Gimeracil + Oteracil, Temoporfin, Temozolomid,

[0509] Temsirolimus, Teniposid, Testosteron, Tetrofosmin, Thalidomid, Thiotepa, Thymalfasin, Thyrotropin alfa, Tioguanin, Tocilizumab, Topotecan, Toremifen, Tositumomab,

[0510] Trabectedin, trametinib, tramadol, trastuzumab, treosulfan, tretinoin, trifluridine + tipiracil, trametinib, trilostane, triptorelin, trofosfamide, thrombopoietin, ubenimex, valrubicin, vandetanib, vapreotide, valatinib, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, yttrium-90 glass microspheres, zinostatin, zinostatin stimulamer, zoledronic acid, zorubicin. Furthermore, the compounds of the present invention can, for example, be combined with binders (e.g., antibodies) that can bind to the following targets: OX-40, CD137 / 4-1 BB, DR3, ID01 / ID02, LAG-3, CD40.Since a non-cell-permeable toxophore metabolite of a binder-drug conjugate (ADC) should not have a damaging effect on the cells of the adaptive immune system, the combination of a binder-drug conjugate (ADC) according to the invention with a cancer immunotherapy for use in the treatment of cancer or tumors is a further object of this invention. The intrinsic mechanism of action of cytotoxic binder-drug conjugates involves the direct induction of tumor cell death and thus the release of tumor antigens that can stimulate an immune response. Furthermore, there is evidence that the KSP inhibitor toxophore class induces markers of so-called immunogenic cell death (ICD) in vitro.Thus, the combination of the binder-drug conjugates (ADCs) of the present invention with one or more therapeutic approaches for cancer immunotherapy or with one or more active substances, preferably antibodies, directed against a molecular target from cancer immunotherapy represents a preferred method for the treatment of cancer or tumors. Examples of therapeutic approaches for cancer immunotherapy include immunomodulatory monoclonal agents.

[0511] Antibodies and small molecule substances directed against targets from cancer immunotherapy, vaccines, CAR T cells, bispecific T cell-recruiting antibodies, oncolytic viruses, cell-based vaccination approaches. ii) Examples of selected targets from cancer immunotherapy suitable for immunomodulatory monoclonal

[0512] Antibodies include CTLA-4, PD-1 / PDL-1, OX-40, CD137, DR3, ID01, ID02, TD02, LAG-3, TIM-3, CD40.JCOS / ICOSLJIGIT; GITR / GITRL, VISTA, CD70, CD27.

[0513] HVEM / BTLA, CEACAM1, CEACAM6, ILDR2, CD73, CD47, B7H3, TLR's. The

[0514] The combination of a binder-drug conjugate (ADC) according to the invention with a cancer immunotherapy could therefore, on the one hand, make tumors with weakly immunogenic properties more immunogenic and enhance the efficacy of a cancer immunotherapy, and on the other hand, develop a long-lasting therapeutic effect.

[0515] Furthermore, the compounds according to the invention can also be used in combination with radiotherapy and / or surgical intervention. In general, the following objectives can be pursued by combining compounds of the present invention with other cytostatic, cytotoxic, or immunotherapeutic agents:

[0516] • improved efficacy in slowing tumor growth, reducing its size, or even completely eliminating it

[0517] Comparison to treatment with a single active ingredient;

[0518] • the possibility of using the chemotherapeutic agents in lower doses than in monotherapy;

[0519] • the possibility of a more tolerable therapy with fewer side effects in

[0520] Comparison to a single dose;

[0521] • the possibility to treat a wider range of tumor diseases;

[0522] • achieving a higher response rate to therapy;

[0523] • longer patient survival time compared to today

[0524] Standard therapy. In addition, the compounds according to the invention can also be used in combination with radiotherapy and / or surgical intervention.

[0525] Further subject matter of the present invention is pharmaceuticals containing at least one compound according to the invention, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the aforementioned purposes.

[0526] The compounds according to the invention can act systemically and / or locally. For this purpose, they can be administered in a suitable manner, such as parenterally, possibly by inhalation, or as an implant or stent.

[0527] For these application routes, the compounds according to the invention can be administered in suitable application forms.

[0528] Parenteral administration can bypass a absorption step (e.g., intravenously, intra-arterially, intracardiac, intraspinal, or intralumbarly) or involve absorption (e.g., intramuscularly, subcutaneously, intradermally, percutaneously, or intraperitoneally). Suitable routes of administration for parenteral therapy include, among others, injection and infusion preparations in the form of solutions and suspensions.

[0529] Emulsions or lyophilisates. Parenteral administration, particularly intravenous administration, is preferred. In general, it has proven advantageous to administer amounts of approximately 0.1 to 20 mg / kg, preferably approximately 0.3 to 7 mg / kg body weight, to achieve effective results when administered parenterally.

[0530] Nevertheless, it may be necessary to deviate from the stated amounts, depending on body weight and route of administration.

[0531] Individual tolerance to the active ingredient, the method of preparation, and the time or interval of administration all play a role. In some cases, less than the aforementioned minimum amount may be sufficient, while in others, the stated upper limit must be exceeded. When administering larger quantities, it may be advisable to divide them into several doses throughout the day.

[0532] Examples

[0533] The following examples illustrate the invention. The invention is not limited to these examples. Unless otherwise stated, percentages in the following tests and examples are percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios, and concentrations of liquid / liquid solutions refer to volume.

[0534] Synthesis pathways:

[0535] The following diagrams illustrate exemplary synthesis routes for the embodiments. Diagram 1: Synthesis of lysine-linked ADCs with a legume-cleavable linker

[0536]

[0537] In the reaction scheme above, Xi, X2, X3, n, and AK2 have the meanings given in formula (I). 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, stir overnight at RT; d) AK2 in PBS, under argon, add 3-5 equiv. of active ester dissolved in DMSO, stir at RT under argon for 60 min, add again 3-5 equiv. Add the active ester dissolved in DMSO, stir for 60 min at room temperature under argon, then purify using PD 10 columns (Sephadex) equilibrated with PBS buffer (pH 7.2). ®G-25, GE Healthcare) and subsequent concentration by ultracentrifugation and adjustment to the desired concentration with PBS buffer (pH 7.2)]. Sterile filtration may follow for batches.

[0538] Scheme 2: Synthesis of cysteine-linked ADCs

[0539]

[0540] In the reaction scheme above, Xi, X2, X3, n, and AKi have the meanings given in formula (I). a) HATU, DMF, N,N-Diisopropylethylamine, RT; b) Zinc chloride, trifluoroethanol, 50°C, EDTA; c) HATU, DMF, N,N-Diisopropylethylamine, RT; 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 at RT; f) Dissolve AK1 in PBS, add 3-4 equivalents of TCEP to the PBS buffer under argon and stir at room temperature for approximately 30 minutes. Then add 5-10 equivalents of compound E dissolved in DMSO and stir at room temperature for approximately 90 minutes. Purify using PD-10 columns (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer (pH 7.2) and subsequently concentrate by ultracentrifugation and adjust to the desired concentration using PBS buffer (pH 7.2). Sterile filtration may be performed afterward for in vivo batches.

[0541] Scheme 3: Synthesis of cysteine-linked ADCs via ring-opened succinimides

[0542] In the reaction scheme above, Xi, X2, X3, n, and AK1 have the meanings given in formula (I). a) HATU, DMF, N,N-Diisopropylethylamine, RT; b) Zinc chloride, trifluoroethanol, 50°C, EDTA; c) HATU, DMF, N,N-Diisopropylethylamine, RT; 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) Dissolve AK1 in PBS, add 3-4 equivalents of TCEP to the PBS buffer under argon and stir at room temperature for approximately 30 minutes, then add 5-10 equivalents of compound E dissolved in DMSO, stir at room temperature for approximately 90 minutes, then buffer to pH 8 using PD-10 columns equilibrated with PBS buffer (pH 8).

[0543] (Sephadex® G-25, GE Healthcare), then stir overnight at room temperature; then, if necessary, clean using PD 10 columns (Sephadex® G-25, GE) equalized with PBS buffer (pH 7.2).

[0544] Healthcare) and subsequent concentration by ultracentrifugation and

[0545] Adjustment of the desired concentration using PBS buffer (pH 7.2). Sterile filtration may be performed afterward for in vivo batches.

[0546] A. Examples

[0547] Abbreviations and acronyms:

[0548] ABCB1 ATP-binding cassette sub-family B member 1 (synonym for P-gp and MDR1)

[0549] abs. Absolute

[0550] Ac Acetyl

[0551] ACN Acetonitril

[0552] aq. aqueous, aqueous solution

[0553] ATP Adenosine Triphosphate

[0554] BCRP breast cancer resistance protein, an efflux transporter

[0555] BEP 2-bromo-1-ethylpyridinium tetrafluoroborate

[0556] Boc te / t-Butoxycarbonyl

[0557] br. broad (in NMR)

[0558] Example Example

[0559] BxPC3 human tumor cell line

[0560] C concentration

[0561] approximately, about

[0562] Cl chemical ionization (in MS)

[0563] DAR drug-to-antibody ratio

[0564] D doublet (in NMR)

[0565] D day(s)

[0566] TLC thin-layer chromatography

[0567] DCI direct chemical ionization (in MS)

[0568] DCM Dichloromethane

[0569] Dd doublet of doublet (at NMR)

[0570] DMAP 4- / V, / V-dimethylaminopyridine

[0571] DME 1,2-Dimethoxyethane

[0572] DMEM Dulbecco's Modified Eagle Medium (standardized)

[0573] nutrient medium for cell culture)

[0574] DMF Λ / JV-Dimethylformamide

[0575] DMSO Dimethyl sulfoxide

[0576] D / P Dye (fluorescent dye) / Protein ratio DPBS, D-PBS, Dulbecco's phosphate-buffered saline solution

[0577] DSMZ German Collection of Microorganisms and Cell Cultures

[0578] PBS PBS = DPBS = D-PBS, pH7.4, Sigma, No D8537

[0579] Composition:

[0580] 0.2 g KCl

[0581] 0.2 g KH2PO4 (anhydrous)

[0582] 8.0 g NaCl

[0583] 1.15 g Na2HP04(anhydrous)

[0584] Add 1 I with H2O

[0585] Dt doublet of triplet (in NMR)

[0586] DTT DL-Dithiothreitol

[0587] d. Th. of the theory (with chemical yield)

[0588] EDC / V'-(3-Dimethylaminopropyl)-A / -ethylcarbodiimide hydrochloride EGFR Epidermal growth factor receptor = epidermal

[0589] Growth factor receptor

[0590] Electron impact ionization (in MS)

[0591] ELISA Enzyme-Iinked Immunosorbent Assay

[0592] eq. Equivalent(s)

[0593] ESI Electrospray Ionization (at MS)

[0594] ESI-MicroTofq ESI-MicroTofq (name of the mass spectrometer with Tof =

[0595] Time Of Flight and q = quadrupole)

[0596] FCS fetal calf serum

[0597] Fmoc (9 / - -Fluoren-9-ylmethoxy)carbonyl

[0598] total saturated

[0599] GTP Guanosine-5'-triphosphate

[0600] H hour(s)

[0601] HATU 0-(7-Azabenzotriazol-1-yl)- / V, / V, / V',A / '-tetramethyluronium hexafluorophosphate

[0602] HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

[0603] HOAc acetic acid

[0604] HOAt 1 -Hydroxy-7-azabenzotriazole

[0605] HOBt 1-Hydroxy-1 H-benzotriazole hydrate

[0606] HOSu A / -Hydroxysuccinimid

[0607] HPLC High-Pressure, High-Performance Liquid Chromatography

[0608] IC50 is half-maximal inhibition concentration in the intramuscular system, applied to the muscle.

[0609] IV (intravenous), application into the vein

[0610] conc. concentrated

[0611] KPL-4 human tumor cell line

[0612] KU-19-19 human tumor cell line

[0613] LC-MS Liquid chromatography-coupled mass spectrometry

[0614] LLC-PK1 cells Lewis lung carcinoma pork kidney cell line

[0615] L-MDR Human MDR1 transfected LLC-PK1 cells

[0616] LoVo human tumor cell line

[0617] M Multiple (in NMR)

[0618] Me Methyl

[0619] MDR1 multidrug resistance protein 1

[0620] MeCN Acetonitril

[0621] Minute(s)

[0622] MOLM-13 human tumor cell line

[0623] MS Mass Spectrometry

[0624] MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

[0625] MV-4-1 1 human tumor cell line

[0626] NB4 human tumor cell line

[0627] NCI-H292 human tumor cell line

[0628] NMM A / -Methylmorpholine

[0629] NMP A / -Methyl-2-pyrrolidone

[0630] NMR nuclear magnetic resonance spectrometry

[0631] NMRI mouse strain, originating from Naval Medical Research

[0632] Institute (NMRI)

[0633] Nude mice (laboratory animals)

[0634] NSCLC Non-small cell lung cancer

[0635] Bronchial carcinoma)

[0636] PBS phosphate-buffered saline solution

[0637] Pd / C Palladium on activated carbon

[0638] P-gp P-glycoprotein, a transporter protein

[0639] PNGaseF enzyme for sugar cleavage

[0640] quant. quantitative (in terms of yield)

[0641] Quart Quartett (at NMR) Quintett (at NMR)

[0642] Rec-1 human tumor cell line

[0643] RF Retention Index (at DC)

[0644] RT room temperature

[0645] Rt retention time (in HPLC)

[0646] S singlet (in NMR)

[0647] subcutaneous application, application under the skin

[0648] SCI D mice, laboratory mice with a severe combined

[0649] Immunodeficiency (severe combined immunodeficiency)

[0650] SK-HEP-1 human tumor cell line

[0651] t triplet (in NMR)

[0652] TBAF tetra-n-butylammonium fluoride

[0653] TCEP Tris(2-carboxyethyl)phosphine

[0654] TEMPO (2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl

[0655] Teoc Trimethylsilylethoxycarbonyl

[0656] tert. Tertiary

[0657] TFA Trifluoroacetic acid

[0658] TH F Tetrahydrofuran

[0659] T3P ® 2,4, 6-Tripropyl-1, 3,5,2, 4,6-trioxatriphosphinane-2,4,6-trioxide

[0660] U251 human tumor cell line

[0661] UV ultraviolet spectrometry

[0662] v / v volume to volume ratio (of a solution)

[0663] Z Benzyloxycarbonyl

[0664] HPLC and LC-MS methods:

[0665] Method 1 (LC-MS): Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 μm 50 x 1 mm; Eluent A: 1 L water + 0.25 mL 99% formic acid, Eluent B: 1 L acetonitrile + 0.25 mL 99% 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):

[0666] Instrument type MS: Waters Synapt G2S; Instrument type UPLC: Waters Acquity l-CLASS; Column: Waters, BEH300, 2.1 x 150 mm, C18 1.7 m; Eluent A: 1 l water + 0.01%

[0667] Formic acid; Eluent 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

[0668] Method 3 (LC-MS): Instrument MS: Waters (Micromass) QM; Instrument HPLC: Agilent 1 100 series; Column: Agient ZORBAX Extend-C18 3.0x50mm 3.5-Micron; Eluent A: 1 l water + 0.01 mol ammonium carbonate, eluent B: 1 l acetonitrile; Gradient: 0.0 min 98% A ->■ 0.2min 98% A - 3.0 min 5% A^ 4.5 min 5% A ; Oven: 40°C; Flow: 1.75 mL / min; UV detection: 210 nm Method 4 (LC-MS):

[0669] Instrument type MS: Waters Synapt G2S; Instrument type UPLC: Waters Acquity l-CLASS; Column: Waters, HSST3, 2.1 x 50 mm, C18 1.8 μηι; Eluent A: 1 L water + 0.01% formic acid; Eluent 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

[0670] Method 5 (LC-MS):

[0671] Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 μm 50 x 1 mm; Eluent A: 1 L water + 0.25 mL 99% formic acid, Eluent B: 1 L acetonitrile + 0.25 mL 99% 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.

[0672] Method 6 (LC-MS):

[0673] Instrument: Micromass Quattro Premier with Waters UPLC Acquity; Column: Thermo Hypersil GOLD 1.9 μm 50 x 1 mm; Eluent A: 1 L water + 0.5 mL 50% formic acid, Eluent B: 1 L acetonitrile + 0.5 mL 50% formic acid; Gradient: 0.0 min 97% A -> 0.5 min 97% A 3.2 min 5% A 4.0 min 5% A; Oven: 50°C; Flow rate: 0.3 mL / min; UV detection: 210 nm. Method 7 (LC-MS):

[0674] Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; Column: Waters Acquity UPLC HSS T3 1.8 μm 50 x 2.1 mm; Eluent A: 1 L water + 0.25 mL 99% formic acid, Eluent B: 1 L acetonitrile + 0.25 mL 99% formic acid; Gradient: 0.0 min 90% A -> ■ 0.3 min 90% A -> 1.7 min 5% A 3.0 min 5% A; Oven: 50°C; Flow rate: 1.20 mL / min; UV detection: 205–305 nm.

[0675] Method 8 (LC-MS):

[0676] Instrument type MS: Waters Synapt G2S; Instrument type UPLC: Waters Acquity l-CLASS; Column: Waters, HSST3, 2.1 x 50 mm, C18 1.8 μηι; Eluent A: 1 L water + 0.01% formic acid; Eluent B: 1 L acetonitrile + 0.01% formic acid; Gradient: 0.0 min 2% B -> ■ 2.0 min 2% B -> 13.0 min 90% B -> 15.0 min 90% B; Oven: 50°C; Flow rate: 1.20 ml / min; UV detection: 210 nm.

[0677] Method 9: (LC-MS prep purification method) MS instrument: Waters, HPLC instrument: Waters (Waters X-Bridge C18 column, 19 mm x 50 mm, 5 μηη, Eluent A: Water + 0.05% ammonia, Eluent B: Acetonitrile (ULC) with gradient; Flow rate: 40 ml / min; UV detection: DAD; 210–400 nm).

[0678] Instrument MS: Waters, Instrument HPLC: Waters (column Phenomenex Luna 5μ C18(2) 100A, AXIA Tech. 50 x 21 .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).

[0679] Method 10: (LC-MS analysis method)

[0680] Instrument MS: Waters SQD; Instrument HPLC: Waters UPLC; Column: Zorbax SB-Aq (Agilent), 50 mm x 2.1 mm, 1.8 μηη; Eluent 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.

[0681] Method 1 1 (HPLC):

[0682] Device: HP1 100 series Merck Chromolith SpeedROD RP-18e, 50-4.6mm, best

[0683] No.1 .51450.0001, guard column Chromolith Guard Cartridge Kit, RP-18e, 5-4.6mm, order no. 1 .51470.0001

[0684] Gradient: Flow 5 ml / min

[0685] Injection Volume 5μI

[0686] Solvent A: HCL04 (70%) in water (4m UI)

[0687] Solvent B: Acetonitril

[0688] Start 20% B

[0689] 0.50 min 20% B

[0690] 3.00 min 90% B

[0691] 3.50 min 90% B

[0692] 3.51 min 20% B

[0693] 4.00 min 20% B

[0694] Column temperature: 40°C

[0695] Wavelength: 210nm

[0696] Method 12 (LC-MS):

[0697] Instrument type MS: Thermo Scientific FT-MS; Instrument type UHPLC+: Thermo Scientific UltiMate 3000; Column: Waters, HSST3, 2.1 x 75 mm, C18 1.8 μηι; Eluent A: 1 L water + 0.01% formic acid; Eluent 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 / Optimum integration path 210-300 nm.

[0698] Method 13: (LC-MS):

[0699] Instrument MS: Waters (Micromass) Quattro Micro; Instrument Waters UPLC Acquity; Column: Waters BEH C18 1.7 μ 50 x 2.1 mm; Eluent A: 1 l water + 0.01 mol

[0700] Ammonium formate, eluent 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: 0.5ml / min; UV detection: 210 nm. Method 14: (LC-MS) (MCW-LTQ-POROSHELL-TFA98-10min)

[0701] Instrument type MS: Thermo Fisher Scientific LTQ-Orbitrap-XL; Instrument type HPLC: Agilent 1200SL; Column: Agilent, POROSHELL 120, 3 x 150 mm, SB - C18 2.7 μι; Eluent A: 1 L water + 0.1% trifluoroacetic acid; Eluent 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

[0702] For all reactants or reagents whose preparation is not explicitly described below, it is assumed that they were obtained commercially from publicly accessible sources. For all other reactants or reagents whose preparation is also not described below and which were not commercially available or were obtained from sources that are not publicly accessible, a reference is provided to the published literature in which their preparation is described.

[0703] Output connections and intermediates:

[0704] Intermediate C52 (1 R)-1-[1 -Benzyl-4-(2,5-difluorohenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropane-1 -amine

[0705]

[0706] 10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate were placed in 100.0 mL of DMF and mixed with 20.76 g (63.72 mmol) of cesium carbonate and 9.22 g (53.91 mmol) of benzyl bromide. The reaction mixture was stirred overnight at room temperature.

[0707] The reaction mixture was divided 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 vacuum. The reaction was repeated with 90.0 g of methyl 4-bromo-1H-pyrrole-2-carboxylate.

[0708] The combined reactions were purified by prep. RP-HPLC (column: Daiso 300x100; 10 μm, flow rate: 250 mL / min, MeCN / water). The solvents were evaporated under vacuum and the residue was dried under high vacuum. 125.15 g (87% of theory) of the compound methyl-1-benzyl-4-bromo-1H-pyrrole-2-carboxylate were obtained.

[0709] LC-MS (Method 1): R t = 1.18 mins; MS (ESIpos): m / z = 295 [M+H] + .

[0710] Under argon, 4.80 g (16.32 mmol) of methyl-1-benzyl-4-bromo-1H-pyrrole-2-carboxylate were placed in DMF and treated with 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. The reaction mixture was stirred overnight at 85 °C. 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 vacuum. The residue was purified using silica gel (eluent: cyclohexane / ethyl acetate 100:3).

[0711] Solvents were evaporated under vacuum and the residue was dried under high vacuum. 3.60 g (67% of theory) of the compound methyl-1-benzyl-4-(2,5-difluorophenyl)-1 H-pyrrole-2-carboxylate were obtained.

[0712] LC-MS (Method 7): R t = 1.59 mins; MS (ESIpos): m / z = 328 [M+H] + .

[0713] 3.60 g (1 1.00 mmol) of methyl 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate were placed in 90.0 mL of THF and, at 0 °C, 1.04 g (27.50 mmol) of lithium aluminum hydride (2.4 M in THF) was added. The reaction mixture was stirred for 30 minutes at 0 °C. Saturated potassium sodium tartrate solution was added at 0 °C, and the reaction mixture was treated with ethyl acetate. The organic phase was treated three times with saturated

[0714] Potassium sodium tartrate solution was extracted. The organic phase was washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum and the residue dissolved in 30.0 mL of dichloromethane. 3.38 g (32.99 mmol) of manganese(IV) oxide were added and the mixture was stirred at room temperature for 48 h. A further 2.20 g (21.47 mmol) of manganese(IV) oxide was added and the mixture was stirred overnight at room temperature. The reaction mixture was filtered through Celite and the filter cake was washed with dichloromethane. The solvent was evaporated under vacuum and the

[0715] Residue 2.80 g (1-Benzyl-4-(2,5-difluorophenyl)-1 H-pyrrole-2-carbaldehyde) was used in the next synthesis step without further purification.

[0716] LC-MS (Method 7): R t = 1.48 mins; MS (ESIpos): m / z = 298 [M+H] +28.21 g (94.88 mmol) of 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde were placed together with 23.00 g (189.77 mmol) of (R)-2-methylpropane-2-sulfinamide in 403.0 mL of absolute THF and treated with 67.42 g (237.21 mmol) of titanium(IV) isopropylate and stirred overnight at room temperature. 500.0 mL of saturated sodium chloride solution and 1000.0 mL of ethyl acetate were added and stirred for 1 h at room temperature. The mixture was filtered over diatomaceous earth and the filtrate washed twice with saturated sodium chloride solution. The organic phase was filtered over

[0717] Magnesium sulfate was dried, the solvent was evaporated under vacuum, and the residue was purified using Biotage Isolera (silica gel, column 1500+340 g SNAP, flow rate 200 mL / min, ethyl acetate / cyclohexane 1:10). LC-MS (Method 7): R t = 1.63 mins; MS (ESIpos): m / z = 401 [M+H] + .

[0718] 25.00 g (62.42 mmol) of (R)-N-{(E / Z)-[1-Benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]methylene}-2-methylpropane-2-sulfinamide were placed in absolute THF under argon and cooled to -78 °C. Then, 12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane) were added at -78 °C and stirred for 3 h at this temperature. Then, at -78 °C, 71.4 mL of methanol and 214.3 mL of saturated ammonium chloride solution were added successively, and the reaction mixture was allowed to cool to room temperature and stirred for 1 h at room temperature. 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 vacuum. The residue (R)-N-{(1 R)-1 -[1-Benzyl-4-(2,5-difluorophenyl)-1 H- pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamide was removed without further

[0719] Purification is used in the next synthesis stage.

[0720] LC-MS (Method 6): R t = 2.97 mins; MS (ESIpos): m / z = 459 [M+H]+ .

[0721] 28.00 g (61.05 mmol) of (R)-N-{(1 R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamide were placed in 186.7 mL of 1,4-dioxane and then treated with 45.8 mL of HCl in 1,4-dioxane solution (4.0 M). The reaction mixture was stirred for 2 h at room temperature and the solvent was evaporated under vacuum. The residue was purified by prep. RP-HPLC (column: Kinetix 100x30; flow rate: 60 mL / min, MeCN / water). The acetonitrile was evaporated under vacuum and the aqueous residue was treated with dichloromethane. The organic phase was treated with

[0722] Sodium bicarbonate solution was washed and dried over magnesium sulfate. The solvent was evaporated under vacuum and the residue was dried under high vacuum. 16.2 g (75% of theory) of the title compound were obtained. LC-MS (Method 6): R t = 2.10 mins; MS (ESIpos): m / z = 338 [M-NH2] + , 709 [2M+H] + .

[0723] 1 H-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 C58

[0724] (2S)-4-[{(1 R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid

[0725]

[0726] 4.3 g (12.2 mmol) of intermediate C52 were dissolved in 525 mL of DCM and mixed with 3.63 g (17.12 mmol) of sodium triacetoxyborohydride and 8.4 mL of acetic acid. After stirring for 5 min at room temperature, 8.99 g (24.5 mmol) of intermediate L57 dissolved in 175 mL of DCM were added, and the mixture was stirred for a further 45 min at room temperature. The mixture 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 removed via

[0727] Magnesium sulfate was dried, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. The residue was then purified by preparative RP-HPLC (column: Chromatorex C18). After combining the respective fractions, the solvent was evaporated under vacuum, and the residue was dried under high vacuum.

[0728] Dried under high vacuum. This yielded 4.6 g (61% of theory) of methyl-(2S)-4-({(1 R)-1 -[1-benzyl- 4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-({[2- (trimethylsilyl)ethoxy]carbonyl}amino)butanoate.

[0729] LC-MS (Method 12): R t = 1.97 min; MS (ESIpos): m / z = 614 (M+H) +2.06 g (3.36 mmol) of this intermediate were placed in 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 stirring at room temperature for 20 h, a further 0.36 mL of 2-chloro-2-oxoethyl acetate and 0.94 mL of triethylamine were added, and the mixture was stirred at room temperature for another 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 bicarbonate solution, and once with 100 mL of saturated sodium chloride solution.

[0730] Magnesium sulfate was dried and concentrated. After drying under high vacuum, 2.17 g (79% of theory) of the protected intermediate were obtained. LC-MS (Method 1): R t = 1.48 mins; MS (ESIpos): m / z = 714 (M+H) + .

[0731] 2.17 g (2.64 mmol) of this intermediate were dissolved in 54 mL of THF and 27 mL of water and mixed with 26 mL of a 2 molar lithium hydroxide solution. The mixture was stirred for 30 min at room temperature and then adjusted to a pH between 3 and 4 with 1.4 mL of TFA. The mixture was concentrated under vacuum. After THF had largely been distilled off, the aqueous solution was extracted twice with DCM and then concentrated to dryness under vacuum. The residue was analyzed by preparative HPLC (column:

[0732] The mixture was purified using Chromatorex C18. After combining the respective fractions, the solvent was evaporated under vacuum and the residue was lyophilized from acetonitrile / water. This yielded 1.1 g (63% of theory) of the title compound. LC-MS (Method 1): R t = 1.34 mins; MS (ESIpos): m / z = 656 (MH)-.

[0733] 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 0.03 (s, 9H), 0.58 (m, 1 H), 0.74-0.92 (m, 1 1 H), 1.40 (m, 1 H), 3.3 (m, 2H), 3.7 (m, 1 H), 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).

[0734] Intermediate C61

[0735] N-[(2S)-4-[{(1 R)-1 -[1-benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-beta-alanine

[0736]

[0737] The title compound was prepared by coupling 60 mg (0.091 mmol) of intermediate C58 with β-alanine methyl ester and subsequent ester cleavage with 2 M lithium hydroxide solution. 67 mg (61% of theory) of the title compound were obtained via two steps. LC-MS (Method 1): R t = 1.29 mins; MS (ESIpos): m / z = 729 (M+H) + .

[0738] Intermediate C102

[0739] (2S)-4-[{(1 R)-1 -[1-Benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}

[0740] (glycoloyl)amino]-2-{[(benzyloxy)carbonyl]amino}butanoic acid

[0741]

[0742] First, intermediate C52 was reductively alkylated with benzyl-(2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate in analogy to intermediate C2. Subsequently, the secondary amino group was acylated with 2-chloro-2-oxoethyl acetate and finally with 2M

[0743] Hydrolysis of the two ester groups was carried out in lithium hydroxide solution in methanol. LC-MS (Method 1): R t = 1.31 mins; MS (ESIpos): m / z = 646 (MH)\Intermediate C110(D)

[0744] Dibenzyl-N-{(2S)-2-amino-4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamate

[0745]

[0746] The title compound was prepared by coupling dibenzyl-D-glutamate, previously released from its p-toluenesulfonic acid salt by partitioning between ethyl acetate and 5% sodium bicarbonate solution, with intermediate C61 in the presence of HATU and N,N-diisopropylethylamine, followed by cleavage of the Teoc protecting group using zinc chloride in trifluoroethanol.

[0747] LC-MS (Method 1): R t = 1.08 min; MS (ESIpos): m / z = 894 [M+H] + Intermediate C111

[0748] Di-tert-butyl-N-{(2S)-2-amino-4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamate

[0749] First, the dipeptide derivative di-tert-butyl-beta-alanyl-D-glutamate was prepared using classical peptide chemistry methods by coupling commercially available N-[(benzyloxy)carbonyl]-beta-alanine and di-tert-butyl D-glutamate hydrochloride (1:1) in the presence of HATU, followed by hydrogenolytic cleavage 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, followed by cleavage of the Z-protecting group via hydrogenation over 10% palladium on activated carbon in a 1:1 DCM / methanol solution at room temperature under atmospheric pressure. LC-MS (Method 1): R t = 1.06 min; MS (ESIpos): m / z = 826 [M+H] + .

[0750] Intermediate C117

[0751] Trifluoressigsäure -dibenzyl-N-{(2S)-2-(L-asparaginylamino)-4-[{(1 R)-1-[1 -benzyl-4-(2,5- difluorphenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D- glutamat Salz

[0752]

[0753] Intermediat C1 10D und 2,5-Dioxopyrrolidin-1 -yl-N 2-(tert-butoxycarbonyl)-L-asparaginate (251 mg, 764 μηηοΙ) was dissolved in 21 mL of DMF and treated with N,N-diisopropylethylamine (363 μΙ, 2.01 mmol). The reaction was stirred at room temperature and then purified directly by preparative reversed-phase high-performance liquid chromatography (column: Chromatorex C18-10). The solvents were evaporated under vacuum and the residue was lyophilized. The resulting intermediate (578 mg, 52 μηηοΙ) was dissolved in 20.0 mL of trifluoroethanol. The reaction mixture was treated with zinc chloride (426 mg, 3.13 mmol) and stirred for 40 min at 50°C. The mixture was treated with ethylenediamine-N,N,N',N'-tetraacetic acid (914 mg, 3.13 mmol), diluted with 20 mL of water, treated with TFA (200 μL), and briefly stirred. The mixture was filtered and purified by preparatory reversed-phase high-performance liquid chromatography (RP-HPLC) (column: Chromatorex C18-5, 125 x 40; flow rate: 100 mL / min, MeCN / water, 0.1% TFA gradient). After lyophilization, the title compound was obtained.

[0754] Intermediate L57 Methyl (2S)-4-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate

[0755]

[0756] 500.0 mg (2.72 mmol) of L-aspartic acid methyl ester hydrochloride and 706.3 mg (2.72 mmol) of 2-(trimethylsilyl)ethyl-2,5-dioxopyrrolidine-1-carboxylate were placed in 5.0 mL of 1,4-dioxane and treated with 826.8 mg (8.17 mmol) of triethylamine. The reaction mixture was stirred overnight at room temperature. The reaction mixture was directly purified by prep. RP-HPLC (column: Reprosil 250x40; 10 μm, flow rate: 50 mL / min, MeCN / water, 0.1% TFA). The solvents were then evaporated under vacuum, and the residue was collected in the

[0757] Dried under high vacuum. 583.9 mg (74% of theory) of the compound (3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid were obtained.

[0758] LC-MS (Method 1): R t = 0.89 min; MS (ESIneg): m / z = 290 (MH)-.

[0759] 592.9 mg of (3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid were placed in 10.0 mL of 1,2-dimethoxyethane and cooled to -15 °C. 205.8 mg (2.04 mmol) of 4-methylmorpholine and 277.9 mg (2.04 mmol) of isobutyl chloroformate were then added. The precipitate was filtered off after 15 min and washed twice with 10.0 mL of 1,2-dimethoxyethane each time. The filtrate was cooled to -10 °C and mixed with 15.5 mg (3.05 mmol) of sodium borohydride dissolved in 10 mL of water with vigorous stirring. The phases were separated, and the organic phase was washed once each with saturated sodium bicarbonate solution and saturated sodium chlorite solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under vacuum, and the residue was dried under high vacuum. 515.9 mg (91% of theory) of the compound methyl-N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate were obtained. LC-MS (Method 1): R t = 0.87 min; MS (ESIpos): m / z = 278 (M+H) + .

[0760] 554.9 mg (2.00 mmol) of methyl N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate were placed in 30.0 mL of dichloromethane and treated with 1.27 g (3.0 mmol) of Dess-Martin periodinan and 474.7 mg (6.00 mmol) of pyridine. The mixture was stirred overnight at room temperature. After 4 hours, the mixture was diluted with dichloromethane, and the organic phase was treated three times each with 10% Na₂S₂C₃ solution, 10% citric acid solution, and saturated hydrochloride.

[0761] The organic phase was washed with sodium bicarbonate solution. It was dried over magnesium sulfate and the solvent was evaporated under vacuum. 565.7 mg (97% of theory) of the title compound was obtained.

[0762] 1 H-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).

[0763] Intermediate L95

[0764] N-[(Benzyloxy)carbonyl]-L-valyl-L-alanine

[0765] This intermediate was prepared from N-[(Benzyloxy)carbonyl]-L-valine and tert-butyl-L-alaninate hydrochloride using classical methods of peptide chemistry.

[0766] LC-MS (Method 12): R t = 1.34 min; MS (ESIpos): m / z = 323.16 (M+H) + Intermediate L103

[0767] N-(pyridin-4-ylacetyl)-L-alanyl-L-alanyl-L-asparagine trifluoroacetate

[0768] The title compound was prepared according to classical methods of peptide chemistry starting 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 with tert-butyl-L-asparaginate and subsequent deprotection of the carboxyl group with trifluoroacetic acid.

[0769] LC-MS (Method 1): R t = 0.15 min; MS (ESIpos): m / z = 394 (M+H) + .

[0770] Intermediate L116

[0771] N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine

[0772]

[0773] The title combination was based on commercially available N-

[0774] [(Benzyloxy)carbonyl]-L-alanine was prepared by classical peptide chemistry methods through coupling with tert-butyl-N-methyl-L-alaninate hydrochloride salt in the presence of HATU, and finally by cleavage of the tert-butyl ester protecting group with TFA. LC-MS (Method 1): R t = 0.68 min; MS (ESIpos): m / z = 309 [M+H] +

[0775] Intermediate L117

[0776] N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-asparagine -trifluoroacetic acid salt

[0777] The title compound was prepared from commercially available 4 tert-butyl-L-asparaginate using classical peptide chemistry methods by coupling with N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine (intermediate L1 16) in the presence of HATU, and finally by cleavage of the tert-butyl ester protecting group with TFA.

[0778] LC-MS (Method 1): R t = 0.57 min; MS (ESIneg): m / z = 421 [MH]-

[0779] Intermediate L1 18

[0780] N-[(Benzyloxy)carbon l]-L-alanyl-N-methyl-L-alanyl-L-alanine

[0781]

[0782] The title compound was prepared from commercially available tert-butyl-L-alaninate hydrochloride salt by classical methods of peptide chemistry through coupling with N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine (intermediate L1 16) in the presence of HATU, and finally by cleavage of the tert-butyl ester protecting group with TFA.

[0783] LC-MS (Method 12): R t = 1.25 min; MS (ESIneg): m / z = 378 [MH]-

[0784] Intermediate L121

[0785] N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-leucine

[0786]

[0787] The title compound was prepared from commercially available tert-butyl-L-leucinate hydrochloride salt using classical methods of peptide chemistry by coupling with N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine (intermediate L1 16) in the presence of HATU, and finally by cleavage of the tert-butyl ester protecting group with TFA.

[0788] LC-MS (Method 12): R t = 0.83 min; MS (ESIneg): m / z = 420 [MH]-

[0789] Intermediate L122

[0790] (5S,8S,1 1 S)-1 1-(2-Amino-2-oxoethyl)-8-[2-(benzyloxy)-2-oxoethyl]-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7, 10-triazadodecano-12-acid

[0791]

[0792] The title compound was prepared from commercially available 4-benzyl-1-tert-butyl-L-aspartate hydrochloride (1:1) using classical peptide chemistry methods, first by coupling with 2,5-dioxopyrrolidin-1-yl-N-[(benzyloxy)carbonyl]-L-alaninate, then by cleavage of the tert-butyl ester protecting group with TFA, then by subsequent coupling with 4-tert-butyl-L-asparaginate in the presence of HATU, and finally by renewed

[0793] Elimination of the tert-butyl ester protecting group produced with TFA.

[0794] LC-MS (Method 1): R t = 0.76 min; MS (ESIpos): m / z = 543 [M+H] + Intermediate L138

[0795] 1-Bromo-2-oxo-6,9, 12,15-tetraoxa-3-azaoctadecane-18-acid

[0796]

[0797] The title compound was prepared by coupling 1-Amino-3,6,9,12-tetraoxapentadecane-15- acid with bromoacetic anhydride in the presence of N,N-diisopropylethylamine.

[0798] LC-MS (Methode 5): R t = 1.05 min; MS (ESIpos): m / z = 386 und 388 (M+H) + .

[0799] Intermediat Q1

[0800] N-[(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)acetyl]-L-alanyl-N-methyl-L-alanyl- [{(1 R)-1-[1 -benzyl-4-(2,5-difluorphenyl)-1 H-pyrrol-2-yl]-2,2- dimethylpropyl}(glycoloyl)amino]-1 -[(3-{[(1 R)-1 ,3-dicarboxypropyl]amino}-3- oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamid

[0801]

[0802] The title compound was initially prepared from compound C1 10D by coupling with intermediate L1 17 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 a 1:1 ratio of DCM-methanol for 1 hour under normal hydrogen pressure at room temperature. 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.

[0803] LC-MS (Method 12): R t = 1 .66 min; MS (ESIneg): m / z = 1 1 19 [MH]-.

[0804] Intermediate Q2

[0805] N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alanyl-N 1-{(2S)- 4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1 -[(3-{[(1 R)-1,3-dicarboxypropyl]amino}-3- oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

[0806]

[0807] The title compound was initially prepared from compound C1 10D by coupling with intermediate L1 17 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 a 1:1 ratio of DCM-methanol for 1 hour under normal hydrogen pressure at room temperature, and the deprotected intermediate was subsequently 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.

[0808] LC-MS (Method 1): R t = 0.93 min; MS (ESIpos): m / z = 1 195 [M+H] + .

[0809] Intermediate Q3

[0810] N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-L-alanyl-N 1 -{(2S)-4-[{(1 R)-1 - [1 -benzyl-4-(2,5-difluorphenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1 -[(3- {[(1 R)-1 ,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1 -oxobutan-2-yl}-L-aspartamid

[0811]

[0812] The title compound was initially prepared from compound C1 17 by coupling with N-(tert-butoxycarbonyl)-L-alanyl-L-alanine in the presence of HATU and N,N-diisopropylethylamine. The intermediate was then dissolved in trifluoroethanol, and stirring at 50°C in the presence of zinc chloride released the tert-butoxycarbonyl-protected amine. In the next step, all benzyl protecting groups were removed by hydrogenation over 10% palladium on activated carbon in a 1:1 ratio of DCM-methanol for 1 hour under normal hydrogen pressure at room temperature. 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.

[0813] LC-MS (Method 1): R t = 0.90 min; MS (ESIneg): m / z = 1 181 [MH]-.

[0814] Intermediate Q4 N-[6-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanoyl]-L-alanyl-N-methyl-L-alanyl-N1 -{(2S)- 4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1 -[(3-{[(1 R)-1,3-dicarboxypropyl]amino}-3- oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

[0815]

[0816] The title compound was initially prepared from compound C1 10D by coupling with intermediate L1 17 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 a 1:1 ratio of DCM-methanol for 1 hour under normal hydrogen pressure at room temperature. 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.

[0817] LC-MS (Method 1): R t = 3.2 min; MS (ESIpos): m / z = 1 177 [M+H] + .

[0818] Intermediate Q5

[0819] N-{5-[(2,5-Dioxopyrrolidin-1 -yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alanyl-N-{(2S)-4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2- dimethylpropyl}(glycoloyl)amino]-1 -[(3-{[(1 R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1 -oxobutan-2-yl}-L-alaninamide

[0820]

[0821] The title compound was initially prepared from compound C1 10D by coupling with intermediate L1 18 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 a 1:1 ratio of DCM-methanol for 1 hour under normal hydrogen pressure at room temperature. 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.

[0822] LC-MS (Method 1): R t= 0.96 min; MS (ESIpos): m / z = 1 152 [M+H] + .

[0823] Intermediate Q6

[0824] N-{(2S)-4-[{(1 R)-1-[1 -Benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}

[0825] (glycoloyl)amino]-2-[(N-{5-[(2,5-dioxopyrrolidin-1 -yl)oxy]-5-oxopentanoyl}-L-valyl-L-alanyl)amino]butanoyl}-beta-alanyl-D-glutamic acid

[0826]

[0827] The title compound was initially prepared from compound C1 10D by coupling with intermediate L95 in the presence of HATU and N,N-diisopropylethylamine. In the next step, all protecting groups were removed by 1 hour hydrogenation over 10% palladium on activated carbon in a 1:1 ratio of DCM methanol under normal hydrogen pressure at room temperature. 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.

[0828] LC-MS (Method 1): R t = 0.98 min; MS (ESIpos): m / z = 1095 [M+H] + .

[0829] Intermediate Q7

[0830] N-[(2S)-4-[{(1 R)-1 -[1-Benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({N-[(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetyl]-L-valyl-L- alanyl}amino)butanoyl]-beta-alanyl-D-glutamic acid

[0831] The title compound was initially prepared from compound C1 10D 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 a 1:1 ratio of DCM methanol for 1 hour under normal hydrogen pressure at room temperature. 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.

[0832] LC-MS (Methode 1 ): R t = 0.98 min; MS (ESIpos): m / z = 1021 [M+H] + .

[0833] Intermediat Q8

[0834] N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alanyl-N 1 -{(2S)- 4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorphenyl)-1 H-pyrrol-2-yl]-2,2- dimethylpropyl}(glycoloyl)amino]-1 -[(3-{[(1 R)-1 ,3-dicarboxypropyl]amino}-3- oxopropyl)amino]-1-oxobutan-2-yl}-L-leucinamid

[0835]

[0836] The title compound was initially prepared from compound C1 10D 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 for 1 hour under normal hydrogen pressure at room temperature, and the deprotected intermediate was subsequently 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.

[0837] LC-MS (Method 1): R t = 1.02 min; MS (ESIpos): m / z = 1 194 [M+H] + .

[0838] Intermediate Q9

[0839] N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alpha-aspartyl-N 1-{(2S)-4-[{(1 R)-1 -[1-benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl} (glycoloyl) amino]-1 -[(3-{[(1 R)-1 ,3-dicarboxypropyl]amino}-3-oxopropyl) amino]-1 - oxobutan-2-yl}-L-aspartamide

[0840]

[0841] The title compound was initially prepared from compound C1 10D by 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 for 1 hour under normal hydrogen pressure at room temperature, and the deprotected intermediate was subsequently 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 3 equiv. of N,N-diisopropylethylamine.

[0842] LC-MS (Method 1): R t = 0.89 min; MS (ESIpos): m / z = 1225 [M+H] + .

[0843] Intermediate Q10

[0844] N-(Bromoacetyl)-L-alanyl-N-methyl-L-alanyl-N 1 -{(2S)-4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1 -[(3-{[(1 R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1 -oxobutan-2-yl}-L-aspartamide

[0845]

[0846] The title compound was initially prepared from compound C1 10D by coupling with intermediate L1 17 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 a 1:1 dilution of DCM-methanol for 1 hour under normal hydrogen pressure at room temperature. 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.

[0847] LC-MS (Method 1): R t = 0.95 min; MS (ESIpos): m / z = 1 10⁴ and 1 10⁶ [M+H] + .

[0848] Intermediate Q11

[0849] N-(18-Bromo-17-oxo-4,7,10,13-tetraoxa-16-azaoctadecan-1-oyl)-L-alanyl-N-methyl-L-alanyl-N 1 -{(2S)-4-[{(1 R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1 -[(3-{[(1 R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1 -oxobutan-2-yl}-L-aspartamide

[0850]

[0851] The synthesis of the title compound was initially carried out by coupling intermediate C1 1 1 with intermediate L1 17 in DMF in the presence of 1.5 equiv. HATU and 3 equiv. N,N-diisopropylethylamine. Subsequently, the Z-protecting group was removed by 2-hour

[0852] Hydrogenation over 10% palladium on activated carbon in ethanol under atmospheric pressure at room temperature removed the palladium. The deprotected intermediate was then reacted with intermediate L138 in DMF in the presence of 1.5 equiv. HATU and 3 equiv. N,N-diisopropylethylamine. In the final step, the title compound was obtained by cleavage of the tert-butyl ester groups by stirring at 50°C with 8 equivalents of zinc chloride in trifluoroethanol for 2 h.

[0853] LC-MS (Method 8): R t = 4.06 mins; MS (ESI-pos): m / z = 1353 [M+H] + .

[0854] B: Production of antibody-drug conjugates (ADO)

[0855] B-1. General procedure for antibody generation

[0856] The protein sequence (amino acid sequence) of the antibodies used, for example TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580, was converted into a DNA sequence encoding the corresponding protein according to a method known to those 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).

[0857] B-2. General method for antibody expression in mammalian cells

[0858] The antibodies, for example 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:

[0859] Expression Systems, edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007.

[0860] B-3. ​​General procedure for the purification of antibodies from cell supernatants.

[0861] The antibodies, for example TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574, and TPP-9580, were isolated from the cell culture supernatants. The cell supernatants were clarified by centrifugation of cells. Subsequently, the cell supernatant was purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. 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. The antibodies were eluted in 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride and subsequently further purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

[0862] Commercially available antibodies were purified from the commercial products using standard chromatographic methods (protein A chromatography, preparative chromatography).

[0863] Gel filtration chromatography (SEC - size exclusion chromatography). B-4. General method for coupling to Cvstein side chains

[0864] The following antibody was used in the coupling reactions: Example a: TPP-981 Cetuximab (Anti-EGFR antibody)

[0865] Examples c: TPP-6013 (Anti-CD123 AK)

[0866] TPP-8987 (Anti-CD123 AK)

[0867] TPP-8988 (Anti-CD123 AK)

[0868] TPP-9476 (Anti-CD123 antibody) Examples h: TPP-8382 (Anti-B7H3 antibody)

[0869] Examples: TPP-1015 (Anti-Her2 antibody)

[0870] Examples k: TPP-7006 (Anti-TWEAKR AK)

[0871] TPP-7007 (Anti-TWEAKR AK)

[0872] Examples x: TPP-9574 (Anti-CXCR5 AK) TPP-9580 (Anti-CXCR5 AK)

[0873] The coupling reactions were usually carried out under argon.

[0874] To a solution of the corresponding antibody in PBS buffer at a concentration between 1 mg / mL and 20 mg / mL, preferably in the range of approximately 10 mg / mL to 15 mg / mL, between 2 and 5 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dissolved in PBS buffer, were added and stirred for 1 h at room temperature. The solution of the respective antibody used can be at the concentration specified in the exemplary embodiments or, if necessary, diluted with PBS buffer to approximately half the specified starting concentration to achieve the preferred concentration range. Subsequently, depending on the desired effect,

[0875] The reaction is carried out by adding between 2 and 12 equivalents, preferably about 5-10 equivalents, of the maleimide precursor or halide precursor to be coupled as a solution in DMSO. The amount of DMSO should not exceed 10% of the total volume. The mixture was stirred for 60-240 min at room temperature for maleimide precursors and for 8-24 h at room temperature for halide precursors.

[0876] subsequently via PBS-equilibrated PD 10 columns (Sephadex) ® G-25, GE Healthcare) and eluted with PBS buffer. Unless otherwise specified, 5 mg of the corresponding antibody was typically used in PBS buffer for reduction and subsequent coupling. After purification via the PD-10 column, solutions of the corresponding ADC in 3.5 mL of PBS buffer were obtained.

[0877] Subsequently, the sample was concentrated by ultracentrifugation and, if necessary, diluted back with PBS buffer. If required, the concentration was repeated by ultrafiltration after back-dilution with PBS buffer to improve the separation of low-molecular-weight components. For biological testing, concentrations in the final ADC samples were adjusted to the range of 0.5–15 mg / mL by back-dilution, if necessary. The ADC solutions were prepared using the formulas described in the...

[0878] The protein concentration specified in each of the exemplary embodiments was determined. Furthermore, the antibody loading (drug / mAb ratio) was determined according to the parameters described in section B-6.

[0879] methods described were determined.

[0880] The ADCs shown in the examples can vary depending on the linker.

[0881] They may also be present, to a greater or lesser extent, in the form of hydrolyzed open-chain succinic acid amides linked to the antibodies.

[0882] In particular, the KSP-l ADCs, which are linked via the linker substructure

[0883] The antibodies linked to thiol groups can also be selectively produced into the ADCs linked via open-chain succinic acid amides by buffering after coupling and stirring for approximately 20-24 hours at pH 8 according to Scheme 28.

[0884] #1 represents the sulfur bridge to the antibody and #2 the linkage point to the modified KSP inhibitor

[0885] Such ADCs, in which the linker is linked to the antibodies via hydrolyzed open-chain succinic acid amides, can also be specifically produced according to the small-scale and large-scale couplings listed here as examples:

[0886] To a solution of 2-5 mg of the corresponding antibody in PBS buffer in

[0887] In a concentration range between 1 mg / mL and 20 mg / mL, preferably in the range of approximately 5 mg / mL to 15 mg / mL, between 2 and 7 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dissolved in PBS buffer, were added and stirred at room temperature for 30 min to 1 h. Subsequently, depending on the desired loading, between 2 and 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, 15-20 equivalents can also be used. The amount of DMSO should not exceed 10% of the total volume. The mixture was stirred at room temperature for 60-240 min. The eluate was diluted with PBS buffer pH 8 to a concentration of 1-5 mg / mL and then passed through a PD 10 column (Sephadex) equilibrated with PBS buffer pH 8. ®G-25 (GE Healthcare) was added and eluted with PBS buffer pH 8. The eluate was stirred overnight at room temperature under argon. The solution was then processed by

[0888] Ultracentrifugation to concentrate and dilute back to pH 7.2 with PBS buffer.

[0889] Medium scale coupling:

[0890] 20-200 mg of the antibody in question in PBS buffer (c ~ 5-15 mg / mL) were mixed under argon with 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). The mixture was stirred for 30 min at room temperature, and then 2-20, preferably 5-10 equivalents of a maleimide precursor compound dissolved in DMSO were added. To achieve higher DARs, 15-20 equivalents can also be used. After a further 1.5-2 h stirring at room temperature, the mixture was diluted with PBS buffer previously adjusted to pH 8. This solution was then processed through PD-10 columns (Sephadex) equalized with PBS buffer at pH 8. ®G-25 (GE Healthcare) 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 overnight at room temperature under argon. If necessary, the solution was then buffered again to pH 7.2. The ADC solution was concentrated by ultracentrifugation, diluted back to pH 7.2 with PBS buffer, and then, if necessary, again to a concentration of approximately 10 mg / mL.

[0891] concentrated. In the structural formulas shown, AKi can have the meaning

[0892] Examples a: TPP-981 Cetuximab (partially reduced)- S§ 1

[0893] Examples c: TPP-6013 (Anti-CD123 AK) (partially reduced)- S§ 1 TPP-8987 (Anti-CD123 AK) (partially reduced)- S§ 1 TPP-8988 (Anti-CD123 AK) (partially reduced)- S§ 1 TPP-9476 (Anti-CD123 AK) (partially reduced)- S§ 1

[0894] Examples: TPP-1015 (Anti-Her2 antibody) (partially reduced)- S§1

[0895] Examples h: TPP-8382 (Anti-B7H3 AK) (partially reduced)- S§ 1

[0896] Examples k: TPP-7006 (Anti-TWEAKR AK) (partially reduced)- S§ 1 TPP-7007 (Anti-TWEAKR AK) (partially reduced)- S§ 1

[0897] Examples x: TPP-9574 (Anti-CXCR5 AK) (partially reduced)- S§ 1 TPP-9580 (Anti-CXCR5 AK) (partially reduced)- S§ 1 where

[0898] § 1 the linkage with the succinimide group or with any isomeric hydrolyzed open-chain succinic acid amides that may result from it, or the

[0899] Alkylene residue means, and

[0900] S stands for the sulfur atom of a cysteine ​​residue of the partially reduced antibody.

[0901] B-5. General method for coupling to lysine side chains

[0902] The following antibodies were used in the coupling reactions: Examples a: TPP-981 Cetuximab (Anti-EGFR antibody) Examples c: TPP-6013 (Anti-CD123 antibody) TPP-8987 (Anti-CD123 antibody) TPP-8988 (Anti-CD123 antibody) TPP-9476 (Anti-CD123 antibody) Examples e: TPP-1015 (Anti-Her2 antibody) Examples k: TPP-7006 (Anti-TWEAKR antibody) TPP-7007 (Anti-TWEAKR antibody) Examples x: TPP-9574 (Anti-CXCR5 antibody) TPP-9580 (Anti-CXCR5 antibody)

[0903] The coupling reactions were usually carried out under argon.

[0904] To a solution of the corresponding antibody in PBS buffer at a concentration between 1 mg / mL and 20 mg / mL, preferably around 10 mg / mL, between 2 and 8 equivalents of the precursor compound to be coupled were added as a solution in DMSO, depending on the desired loading. After stirring at room temperature for 30 minutes to 6 hours, the same amount of precursor compound in DMSO was added again. The amount of DMSO should not exceed 10% of the total volume. After another 30 minutes to 6 hours of stirring at room temperature, the mixture was passed through PBS-equilibrated PD-10 columns (Sephadex® G-25, GE Healthcare) and eluted with PBS buffer.

[0905] Purification via the PD10 column yielded solutions of the respective ADCs in PBS buffer. Subsequently, concentration was achieved using

[0906] Ultracentrifugation was performed and the sample was diluted with PBS buffer if necessary. If required, the following was performed to improve the separation of low-molecular-weight components:

[0907] Concentration by ultrafiltration was repeated after re-dilution with PBS buffer. For biological testing, concentrations in the final ADC samples were adjusted to the range of 0.5–15 mg / mL by re-dilution, if necessary. The protein concentration specified in the examples was determined for the ADC solutions. Furthermore, the antibody loading (drug / mAb ratio) was determined according to the methods described in section B-6.

[0908] In the structural formulas shown, AK2 has the meaning of examples a: TPP-981 Cetuximab (Anti EGFR AK) - NH§ 2

[0909] Examples c: TPP-6013 (Anti-CD123 AK) - NH§ 2

[0910] TPP-8987 (Anti-CD123 AK) - NH§ 2

[0911] TPP-8988 (Anti-CD123 AK) - NH§ 2

[0912] TPP-9476 (Anti-CD123 AK) - NH§ 2 Examples: TPP-1015 (Anti-Her2 antibody) - NH§ 2

[0913] Examples k: TPP-7006 (Anti-TWEAKR AK) - NH§ 2

[0914] TPP-7007 (Anti-TWEAKR AK) - NH§ 2 Examples x: TPP-9574 (Anti-CXCR5 AK) - NH§ 2

[0915] TPP-9580 (Anti-CXCR5 AK) - NH§ 2

[0916] where

[0917] § 2 the linkage with the carbonyl group means, and

[0918] NH represents the side-chain amino group of a lysine residue of the antibody. Further purification and characterization of the inventive compounds

[0919] After the reaction was completed, in some cases the reaction mixture was concentrated, for example by ultrafiltration, and then desalted and purified by chromatography, for example using a Sephadex® G-25. Elution was performed, for example, with phosphate-buffered saline (PBS). The solution was then sterile-filtered and frozen. Alternatively, the conjugate can be lyophilized.

[0920] B-6. Determination of antibody, toxophore loading and the proportion of open cysteine ​​adducts. For protein identification, in addition to molecular weight determination according to

[0921] Deglycosylation and / or denaturation, a tryptic digestion was performed, which, after denaturation, reduction and derivatization, confirmed the identity of the protein based on the detected tryptic peptides.

[0922] Of the solutions obtained that are described in the exemplary embodiments

[0923] The toxophore loading of conjugates in PBS buffer (referred to in the tables as DAR, drug-to-antibody ratio) was determined as follows:

[0924] The toxophore loading of lysine-linked ADCs was determined by mass spectrometric determination of the molecular weights of the individual

[0925] Conjugate species. The antibody conjugates were first deglycosylated using PNGaseF, the sample was acidified, and then separated / desalted by HPLC.

[0926] The spectra were analyzed mass spectrometrically using ESI-MicroTofo (Bruker Daltonics). All spectra across the signal in the TIC (Total Ion Chromatogram) were summed, and the

[0927] Molecular weight of the different conjugate species based on MaxEnt

[0928] Deconvolution was calculated. After signal integration of the different species, the DAR (drug / antibody ratio) was then calculated. For this, the sum of the toxophore-weighted integration results of all species was divided by the sum of the singly weighted integration results of all species.

[0929] The toxophore loading of cysteine-linked conjugates was determined by reversed-phase chromatography of reduced and denatured ADCs. Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-dithiothreitol (DTT) (500 mM, 3 μM) were added to the ADC solution (1 mg / mL, 50 μM). The mixture was incubated for one hour at 55 °C and analyzed by HPLC.

[0930] HPLC analysis was performed on an Agilent 1260 HPLC system with detection at 220 nm. A Polymer Laboratories PLRP-S Polymerie Reversed Phase column (catalog number PL1912-3802) (2.1 x 150 mm, 8 μηι particle size, 1000 μm) 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. Mobile phase A consisted of 0.05% trifluoroacetic acid (TFA) in water, and mobile phase B consisted of 0.05% trifluoroacetic acid in acetonitrile.

[0931] The detected peaks were assigned to the light chain (L0) and heavy chain (H0) of the unconjugated antibody by comparing their retention times. Peaks detected exclusively in the conjugated sample were assigned to the light chain with one toxophore (L1) and to the heavy chains with one, two, and three toxophores (H1, H2, H3).

[0932] The average toxophore loading of the antibody (called DAR, drug-to-antibody ratio) was determined from the peak areas calculated by integration as twice the sum of the HC loading and the LC loading. The LC loading was calculated by dividing the sum of the toxophore-weighted integration results of all LC peaks by the sum of the singly weighted integration results of all LC peaks, and the HC loading was calculated by dividing the sum of the toxophore-weighted integration results of all HC peaks by the sum of the singly weighted integration results of all HC peaks. In isolated cases, the toxophore loading could not be calculated exactly due to co-elution of some peaks.

[0933] In cases where sufficient HPLC separation of the light and heavy chains was not possible, the toxophore loading of cysteine-linked conjugates was determined by mass spectrometric determination of the

[0934] Molecular weights of the individual conjugate species on the light and heavy chains.

[0935] Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-dithiothreitol (DTT) (500 mM, 3 μM) were added to an ADC solution (1 mg / mL, 50 μM). The mixture was incubated for one hour at 55 °C and analyzed by mass spectrometry after online desalination using ESI-MicroTofo (Bruker Daltonik).

[0936] To determine the drug-to-antibody ratio (DAR), all spectra were summed using the signal in the total ion chromatogram (TIC), and the molecular weight of the different conjugate species on the light and heavy chains was calculated based on MaxEnt deconvolution. The average toxophore loading of the antibody was determined from the peak areas calculated by integration as twice the sum of the heavy chain (HC) loading and the light chain (LC) loading. The LC loading was calculated as the sum of the toxophore-weighted integration results of all LC peaks divided by the sum of the singly weighted integration results of all LC peaks, and the HC loading was calculated as the sum of the toxophore-weighted integration results of all LC peaks.

[0937] Integration results of all HC peaks divided by the sum of the singly weighted integration results of all HC peaks.

[0938] For the open constructs, the molecular weight area ratio of the closed to the open cysteine ​​adduct (molecular weight delta 18 Daltons) of all singly conjugated light and heavy chain variants was determined to ascertain the proportion of the open cysteine ​​adduct. The mean value across all variants yielded the proportion of the open cysteine ​​adduct.

[0939] B-7. Verification of the antigen binding of the ADC

[0940] The binding capacity of the binder to the target molecule was verified after successful coupling. Various methods are known to those skilled in the art for this purpose; for example, the affinity of the conjugate can be determined using ELISA technology or

[0941] Surface plasmon resonance analysis (BIAcore™ measurements) can be used to verify the conjugate concentration. A specialist can measure this using standard methods.

[0942] for example, antibody conjugates can be determined by protein assay (see also Doronina et al.; Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007; 1:102:616-623).

[0943] Examples of metabolites

[0944] Example M1

[0945] N-{(2S)-2-amino-4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamic acid

[0946]

[0947] Intermediate C1 10D were converted into the title compound by 1 hour hydrogenation over 10% palladium on activated carbon in ethanol under normal hydrogen pressure at RT.

[0948] LC-MS (Method 1): R t = 1.78 mins; MS (ESIpos): m / z = 714 [M+H] + .

[0949] The ADCs shown below as examples can release the preferred metabolite M1, which has preferred pharmacological properties.

[0950] Implementation examples of ADCs

[0951] The ADCs depicted in the structural forms of the exemplary embodiments, which were coupled to the cysteine ​​side chains of the antibodies via maleimide residues, exist predominantly in the respective ring-opened or ring-closed forms, depending on the linker and the coupling protocol. However, a small proportion of the other form may be present in the preparation. The coupling reactions were carried out under argon. All larger batches for in vivo experiments were sterile filtered at the end of the preparation.

[0952] Examples 1

[0953]

[0954] Exemplary Regulation A:

[0955] 5 mg of the corresponding antibody in 0.5 ml PBS (c=10 mg / mL) were mixed under argon with a solution of 0.029 mg TCEP in 0.05 ml PBS buffer. The mixture was stirred for 30 min at room temperature, and then 0.26 mg (0.00023 mmol) of intermediate Q1 dissolved in 50 μL DMSO was added. After a further 90 min of stirring at room temperature, the mixture was diluted to a volume of 2.5 mL with PBS buffer previously adjusted to pH 8 and then filtered through a PD-10 column equilibrated to pH 8 with PBS buffer.

[0956] (Sephadex ® G-25 (GE Healthcare) was added and eluted with PBS buffer pH 8. The eluate was stirred overnight at room temperature under argon. Subsequently, by

[0957] Ultracentrifugation to concentrate and re-diluted with PBS buffer (pH 7.2). Example procedure B:

[0958] 30 mg of the corresponding antibody in 3 ml of PBS (c=10 mg / mL) were mixed under argon with a solution of 0.172 mg TCEP in 0.3 ml of PBS buffer. The mixture was stirred for 30 min at room temperature, and then 1.57 mg (0.0014 mmol) of intermediate Q1 dissolved in 300 μL DMSO was added. After a further 90 min of stirring at room temperature, the mixture was diluted to a volume of 5 mL with PBS buffer previously adjusted to pH 8 and then analyzed using a PD-10 column (Sephadex) equilibrated to pH 8 with PBS buffer. ® G-25, GE Healthcare) and eluted with PBS buffer pH 8. The eluate was diluted to a volume of 7.5 mL with PBS buffer pH 8 and stirred overnight at room temperature under argon. This solution was then filtered through a PD-10 column (Sephadex) equilibrated with PBS buffer pH 7.2. ®G-25 (GE Healthcare) and eluted with PBS buffer pH 7.2. Subsequently, the solution was concentrated by ultracentrifugation, diluted back to PBS buffer (pH 7.2), concentrated again, and filtered under sterile conditions.

[0959] The following ADCs were manufactured in analogy to these specifications and characterized as shown in the table:

[0960] Example Target Antibody Prescription C [mg / mL] DAR

[0961] TPP-

[0962] 1 a-981 EGFR 981 A 1.85 2.5

[0963] 1 C-6013 CD123 6013 A 2.0 2.4

[0964] 1 C-9476 CD123 9476 A 1.96 3.1

[0965] 1 e-1015 HER2 1015 A 1.75 3.3

[0966] 1 h-8382 B7H3 8382 B 1 1 .01 3.5

[0967] 1k-7006 TWEAKR 7006 A 1.8 2.9

[0968] 1k-7007 TWEAKR 7007 B 7.84 3.3

[0969] 1x-9574 CXCR5 9574 A 1.26 2.9 Examples 2

[0970]

[0971] Exemplary Regulation A:

[0972] To 5 mg of the antibody in question in 0.5 ml of PBS (c = 10 mg / mL), 0.2 mg of intermediate Q2 dissolved in 50 µL DMSO was added under argon 5 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified using a Sephadex column, concentrated by ultracentrifugation, and re-diluted with PBS (pH 7.2).

[0973] Exemplary Regulation B:

[0974] To 30 mg of the antibody in question in 3 ml of PBS buffer (pH 7.2) (c = 10 mg / mL), 1 mg of intermediate Q2 dissolved in 50 µL DMSO was added under argon 4 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 5 ml with PBS buffer (pH 7.2), purified using a Sephadex column, and then...

[0975] Ultracentrifugation concentrated, diluted back to PBS (pH7.2) and again

[0976] Concentrated and sterile filtered. Exemplary procedure C:

[0977] To 50 mg of the antibody in question in 5 ml of PBS buffer (pH 7.2) (c = 10 mg / mL), 2.5 Eq of intermediate Q2 dissolved in 250 μί DMSO was added under argon 2.5 Eq. After stirring at room temperature for 1 h, the same amount was added again, and the mixture was stirred for another hour at room temperature. The mixture was then diluted to 7.5 ml with PBS buffer (pH 7.2), purified using a Sephadex column, and then concentrated by ultracentrifugation. It was then diluted back to PBS (pH 7.2) and stirred again.

[0978] concentrated and sterile filtered.

[0979] Exemplary Regulation D:

[0980] To 1000 mg of the antibody in question in 150 ml of PBS buffer (pH 7.2) (c = 6.7 mg / mL), 36 mg of intermediate Q2 dissolved in 7.5 ml of DMSO were added under argon 4.5 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then purified by cross-flow filtration, concentrated, and filtered under sterile conditions.

[0981] Example Target Antibody Prescription C [mg / mL] DAR

[0982] TPP-

[0983] 2a-981 EGFR 981 A 2.23 4.5

[0984] 2c-6013 CD123 6013 A 2.32 4.9

[0985] 2c-8987 CD123 8987 B 8.86 5.8

[0986] 2C-8988 CD123 8988 B 9.81 3.6

[0987] 2C-9476B CD123 9476 B 10.27 4.2

[0988] 2C-9476C CD123 9476C 9.22 3.4

[0989] 2C-9476D CD123 9476 D 15.83 6.3

[0990] 2e-1015 HER2 1015 A 2.05 5.4

[0991] 2k-7006 TWEAKR 7006 A 2.09 5.9

[0992] 2k-7007 TWEAKR 7007 B 9.32 3.4

[0993] 2x-9574 CXCR5 9574 B 9.5 4.8

[0994] 2X-9580 CXCR5 9580 B 10.12 4.8

[0995] Examples 3

[0996]

[0997] Exemplary Regulation A:

[0998] To 5 mg of the antibody in question in 0.5 ml of PBS (c = 10 mg / mL), 0.2 mg of intermediate Q3 dissolved in 50 µL DMSO was added under argon 5 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified using a Sephadex column, concentrated by ultracentrifugation, and re-diluted with PBS (pH 7.2).

[0999] Exemplary Regulation B:

[1000] To 30 mg of the antibody in question in 3 ml of PBS buffer (pH 7.2) (c = 10 mg / mL), 1 mg of intermediate Q3 dissolved in 50 μL DMSO was added under argon 4 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified using a Sephadex column, concentrated by ultracentrifugation, re-diluted with PBS (pH 7.2), and again

[1001] concentrated and sterile filtered.

[1002] Example Target Antibody Prescription C [mg / mL] DAR

[1003] TPP-

[1004] 3a-981 EGFR 981 A 1.99 5.0

[1005] 3C-9476 CD123 9476 A 2.09 5.8 Example Target Antibody Prescription C [mg / mL] DAR

[1006] TPP-

[1007] 3e-1015 HER2 1015 A 2.06 6.3

[1008] 3k-7007 TWEAKR 7007 A 2.1 1 5.3

[1009] 3x-9574 CXCR5 9574 A 2.02 4.5

[1010] Examples 4

[1011] Exemplary Regulation A:

[1012] 5 mg of the antibody in question in 0.4 ml of PBS buffer (pH 7.2) (c=12.5 mg / mL) were mixed under argon with a solution of 0.029 mg TCEP in 0.05 ml of PBS buffer. The mixture was stirred for 30 min at room temperature, and then 0.275 mg (0.00023 mmol) of intermediate Q4 dissolved in 50 μL DMSO was added. After a further 90 min of stirring at room temperature, the mixture was diluted with PBS buffer to a total volume of 2.5 mL. This solution was then processed through a PD-10 column equilibrated with PBS buffer (pH 7.2).

[1013] (Sephadex ® G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2).

[1014] The mixture was then concentrated by ultracentrifugation and diluted back to PBS buffer (pH 7.2). Example Target Antibody Prescription C [mg / mL] DAR

[1015] TPP-

[1016] 4c-9476 CD123 9476 A 2.06 3.5

[1017] 4k-7007 TWEAKR 7007 A 1.87 4.0

[1018] 4x-9574 CXCR5 9574 A 1.93 3.6

[1019] Examples 5

[1020] Exemplary Regulation A:

[1021] To 5 mg of the antibody in question in 0.5 mL of PBS (c = 10 mg / mL), 0.2 mg of intermediate Q5 dissolved in 50 µL of DMSO was added under argon 5 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 2.5 mL with PBS buffer (pH 7.2), purified using a Sephadex column, concentrated by ultracentrifugation, and re-diluted with PBS (pH 7.2).

[1022] Example Target Antibody Prescription C [mg / mL] DAR

[1023] TPP-

[1024] 5C-9476 CD123 9476 A 2.37 5.3

[1025] 5e-1015 HER2 1015 A 2.33 5.5

[1026] 5k-7007 TWEAKR 7007 A 2.18 5.6

[1027] 5X-9574 CXCR5 9574 A 1.88 6.8 Examples 6

[1028]

[1029] Exemplary Regulation A:

[1030] To 5 mg of the antibody in question in 0.4 ml PBS (c = 12.5 mg / mL), 0.18 mg of intermediate Q6 dissolved in 50 µL DMSO was added under argon 5 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified using a Sephadex column, and then...

[1031] Ultracentrifugation concentrated and diluted back to PBS (pH7.2).

[1032] Example Target Antibody Prescription C [mg / mL] DAR

[1033] TPP-

[1034] 6a-981 EGFR 981 A 2.39 4.9

[1035] 6C-9476 CD123 9476 A 1.8 5.3

[1036] 6e-1015 HER2 1015 A 2.23 6.2

[1037] 6k-7007 TWEAKR 7007 A 2.57 5.6 Examples 7

[1038]

[1039] Exemplary Regulation A:

[1040] 5 mg of the corresponding antibody in 0.4 ml PBS (c=12.5 mg / mL) were mixed under argon with a solution of 0.029 mg TCEP in 0.05 ml PBS buffer. The mixture was stirred for 30 min at room temperature, and then 0.24 mg (0.00023 mmol) of intermediate Q7 dissolved in 50 μL DMSO was added. After a further 90 min of stirring at room temperature, the mixture was diluted to a volume of 2.5 mL with PBS buffer previously adjusted to pH 8 and then sampled using a PD-10 column (Sephadex) equilibrated to pH 8 with PBS buffer. ® G-25 (GE Healthcare) was added and eluted with PBS buffer pH 8. The eluate was stirred overnight at room temperature under argon. Subsequently, by

[1041] Ultracentrifugation to concentrate and dilute back to pH 7.2 with PBS buffer.

[1042] Example Target Antibody Prescription C [mg / mL] DAR

[1043] TPP-

[1044] 7a-981 EGFR 981 A 2.03 3.4

[1045] 7c-9476 CD123 9476 A 1.53 4.0

[1046] 7e-1015 HER2 1015 A 1.88 3.8

[1047] 7k-7007 TWEAKR 7007 A 1.99 3.6 Examples 8

[1048]

[1049] Exemplary Regulation A:

[1050] To 5 mg of the antibody in question in 0.5 mL of PBS (c = 10 mg / mL), 0.2 mg of intermediate Q8 dissolved in 50 μL DMSO was added under argon 5 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 2.5 mL with PBS buffer (pH 7.2), purified using a Sephadex column, concentrated by ultracentrifugation, and re-diluted with PBS (pH 7.2).

[1051] Example Target Antibody Prescription C [mg / mL] DAR

[1052] TPP-

[1053] 8a-981 EGFR 981 A 2.32 6.5

[1054] 8C-9476 CD123 9476 A 2.37 6.9

[1055] 8e-1015 HER2 1015 A 1.46 6.6

[1056] 8k-7007 TWEAKR 7007 A 2.43 6.7 Examples 9

[1057]

[1058] Exemplary Regulation A:

[1059] To 5 mg of the antibody in question in 0.5 mL of PBS (c = 10 mg / mL), 0.2 mg of intermediate Q9 dissolved in 50 μL DMSO was added under argon 5 Eq. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The mixture was then diluted to 2.5 mL with PBS buffer (pH 7.2), purified using a Sephadex column, concentrated by ultracentrifugation, and re-diluted with PBS (pH 7.2).

[1060] Example Target Antibody Prescription C [mg / mL] DAR

[1061] TPP-

[1062] 9a-981 EGFR 981 A 2.34 4.4

[1063] 9C-9476 CD123 9476 A 2.65 4.2

[1064] 9e-1015 HER2 1015 A 2.22 4.8

[1065] 9k-7007 TWEAKR 7007 A 2.14 3.8 Examples 10

[1066]

[1067] Exemplary Regulation A:

[1068] 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 7.2) (c=10 mg / mL) were mixed under argon with a solution of 0.029 mg TCEP in 0.05 ml of PBS buffer. The mixture was stirred for 30 min at room temperature, and then 0.295 mg (0.00023 mmol) of intermediate Q10 dissolved in 50 μL DMSO was added. After a further 20 h of stirring at room temperature, the mixture was diluted with PBS buffer to a total volume of 2.5 ml. This solution was then analyzed using a PD-10 column equilibrated with PBS buffer (pH 7.2).

[1069] (Sephadex ® G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2).

[1070] The mixture was then concentrated by ultracentrifugation and diluted back to PBS buffer (pH 7.2).

[1071] Exemplary rule C for achieving a higher DAR:

[1072] 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 7.2) (c=10 mg / mL) were mixed under argon with a solution of 0.057 mg TCEP in 0.05 ml of PBS buffer. The mixture was stirred for 30 min at room temperature, and then 0.59 mg (0.00053 mmol) of intermediate Q10 dissolved in 50 μL DMSO was added. After a further 20 h of stirring at room temperature, the mixture was diluted with PBS buffer to a total volume of 2.5 mL. This solution was then processed through a PD-10 column equilibrated with PBS buffer (pH 7.2).

[1073] (Sephadex ® G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2).

[1074] The mixture was then concentrated by ultracentrifugation and diluted back to PBS buffer (pH 7.2). Example Target Antibody Prescription C [mg / mL] DAR

[1075] TPP-

[1076] 10a-981 EGFR 981 A 1.9 3.2

[1077] 10C-9476 CD123 9476 A 1.83 2.7

[1078] 10C-9476 CD123 9476 C 1.97 4.8

[1079] HD

[1080] 10e-1015 HER2 1015 A 1.83 3.4

[1081] 10k-7007 TWEAKR 7007 A 1.9 4.7

[1082] I Ox-9574 CXCR5 9574 A 1.41 3.8

[1083] I Ox-9574 CXCR5 9574 C 0.97 6.3

[1084] HD

[1085] Examples 11

[1086]

[1087] Exemplary Regulation A:

[1088] 5 mg of the antibody in question in 0.4 ml of PBS buffer (pH 7.2) (c=12.5 mg / mL) were mixed under argon with a solution of 0.029 mg TCEP in 0.05 ml of PBS buffer. The mixture was stirred for 30 min at room temperature, and then 0.32 mg (0.00023 mmol) of intermediate Q1 1 dissolved in 50 μL DMSO was added. After a further 20 h of stirring at room temperature, the mixture was diluted with PBS buffer to a total volume of 2.5 mL. This solution was then processed through a PD-10 column (Sephadex) equilibrated with PBS buffer (pH 7.2). ® G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2).

[1089] The mixture was then concentrated by ultracentrifugation and diluted back to PBS buffer (pH 7.2).

[1090]

[1091] The following ADCs were produced for comparison purposes:

[1092] Reference example R1:

[1093]

[1094] Such ADCs were disclosed in WO2015 / 096982 and WO2016 / 096610 with various antibodies, such as cetuximab and trastuzumab.

[1095] For comparison purposes, the precursor intermediate F194 revealed there was also implemented with TPP-6013 (Anti-CD123 AK). The following ADCs were used.

[1096] Used for comparison purposes: Example Target antibody C [mg / mL] DAR

[1097] TPP-

[1098] R1 a EGFR 981 1 .67 1.9

[1099] R1 c CD123 6013 0.42 2.9

[1100] R1 e HER2 1015 1 .39 2.4

[1101] Rix CXCR5 9574 1 .28 2.2

[1102] Reference example R2:

[1103] Such ADCs were described in WO2016 / 096610 with an aglycosylated anti-TWEAKR

[1104] Antibodies revealed. For comparison purposes, the precursor revealed there was

[1105] Intermediate F291 was also implemented with TPP-9574 (anti-CXCR5 antibody), TPP-981 (anti-EGFR), and TPP-1015 (anti-HER2 antibody). The following ADCs were used for comparison purposes:

[1106]

[1107] For reference examples R1, the metabolite formed from them, example 98, was described in WO2015 / 096982. For reference examples R2, the identical metabolite, example M9, was described in WO2016 / 096610 and is listed here as reference example R3M.

[1108] Reference example R3M: N-(3-Aminopropyl)-N-{(1 R)-1 -[1-benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydrox acetamide

[1109]

[1110] The manufacturing process was described in WO2015 / 096982 as Example 98.

[1111] The biological data for these reference compounds, which are in the aforementioned

[1112] Registrations disclosed or collected using the new reference connections are described in Chapter C.

[1113] C: Assessment of biological efficacy

[1114] The biological effect of the compounds according to the invention can be demonstrated by the assays described below: a. C-1a Determination of the cytotoxic effect of the ADCs. The analysis of the cytotoxic effect of the ADCs is carried out on various cell lines:

[1115] NCI-H292: human mucoepidermoid lung carcinoma cells, ATCC-CRL-1848,

[1116] Standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine) + 10% FCS (Sigma #F2442), TWEAKR positive; EGFR positive.

[1117] BxPC3: human pancreatic cancer cells, ATCC-CRL-1687, standard medium: RPMI 1640 (biochrome; #FG1215, stabilized glutamine) + 10% FCS (Sigma #F2442), TWEAKR-positive

[1118] LoVo: human colorectal cancer cells, ATCC No. CCL-229, culture for MTT assay: standard medium: Kaighn's + L-glutamine (Invitrogen 21 127) + 10% heat-inactivated FCS (Gibco, No. 10500-064). Culture for CTG assay: RPMI 1640 (Biochrom;

[1119] #FG1215, rod. Glutamine) + 10% FCS (Sigma #F2442). TWEAKR positive.

[1120] KPL4: human breast cancer cell line, Bayer Pharma AG (identity checked and confirmed on July 19, 2012 at DSMZ), standard medium: RPMI 1640 (Gibco company; #21875-059, stab. L-glutamine) + 10% heat inactivated FCS (Gibco company, No. 10500-064); HER2 positive.

[1121] 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 (Gibco, No. 10500-064); EGFR-positive, TWEAKR-positive

[1122] MOLM-13: human acute monocytic leukemia cells (AML-M5a), DSMZ, No. ACC 554, standard medium: RPMI 1640 (Gibco company; #21875-059, stab. L-glutamine) + 20% heat inactivated FCS (Gibco company, No. 10500-064); CD123 positive.

[1123] MV-4-1 1: Human biphenotypic B myelomonocytic leukemia cells obtained from peripheral blood, ATCC-CRL-9591, standard medium: IMDM (ATCC: 30-2005), + 10% heat-inactivated FCS (Gibco, No. 10500-064); CD123-positive. NB4: Human acute promyelocytic leukemia cells obtained from bone marrow, DSMZ, No. ACC 207, standard medium: RPMI 1640 + GlutaMAX I (Invitrogen 61870) + 10% heat inactivated FCS (Gibco, No. 10500-064) + 2.5 g of glucose (20% glucose solution, Gibco, No.19002) + 10 mM Hepes (Invitrogen 15630) + 1 mM sodium pyruvate (Invitrogen 1 1360); CD123 negative

[1124] Rec-1: human mantle cell lymphoma cells (B cell non-Hodgkin's lymphoma) ATCC CRL-3004, standard medium: RPMI 1640 + GlutaMAX I (Invitrogen 61870) + 10% heat inactivated FCS (Gibco, No. 10500-064) + 10 mM) CXCR5 positive

[1125] U251: human glioblastoma cells, standard medium: RPMI 1640 (Biochrom; #FG1215, Stab. Glutamine) + 10% FCS (Biochrom; #S0415); B7H3 positive

[1126] 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, University of Münster; standard medium: RPMI 1640 (Biochrome; #FG1215, stabilized glutamine) + 10% FCS (Biochrome; #S0415), culture analogous to Rec-1 cells; CXCR5 positive

[1127] The cells are cultivated according to standard methods, as specified by the American Tissue Culture Collection (ATCC) or the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) for the respective cell lines.

[1128] CTG assay

[1129] The cells were cultivated according to the standard method, using the methods described under C-1

[1130] The growth media specified. For the procedure, the cells were detached with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG #L2143), pelleted, resuspended in culture medium, counted and seeded into a 96-hole culture plate with a white bottom (Costar #3610) (in 75µl / µl, the following cell counts per hole: NCI-H292: 2500 cells / hole, BxPC3: 2500 cells / hole, LoVo: 3000 cells / hole) and incubated in an incubator at 37°C and 5% carbon dioxide. The suspension cells were counted and seeded into a 96-hole culture plate with white substrate (Costar #3610) (in 75μI / ϋ3θϊι, the following cell counts per hole: Rec-1 : 3000 cells / hole, HBL-1 : 6000

[1131] Cells / hole). After 24 hours, the antibody-drug conjugates were added to the cells in 25 μL culture medium (quadrivalent concentration), resulting in final antibody-drug conjugate concentrations of 3 x 10 "7 M to 3 x 10 "11M was reached on the cells (triplicates). Subsequently, the cells were incubated at 37°C and 5%

[1132] Carbon dioxide was used for incubation. In a parallel plate, cell viability at the start of the drug treatment (day 0) was determined using the Cell Titer Glow (CTG) Luminescent Cell Viability Assay (Promega #G7573 and #G7571). For this purpose, 10 μL of the substrate was added to each cell culture, the plates were then covered with aluminum foil, shaken for 2 minutes at 180 rpm using a plate shaker, left to stand on the lab bench for 8 minutes, and then measured with a luminometer (Victor X2, Perkin Elmer). The substrate detects the ATP content in the living cells, generating a luminescence signal whose amplitude is directly proportional to cell viability. After 72 h of incubation with the antibody-drug conjugates, the viability of these cells was also determined using the Cell Titer Glow Luminescent Cell Viability Assay as described above.The IC50 of growth inhibition compared to day 0 was calculated from the measured data using the DRC (Dose Response Curve) Analysis Spreadsheet with a 4-parameter fit. The DRC Analysis.

[1133] Spreadsheet is a Biobook Spreadsheet developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook Suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).

[1134] MTT assay

[1135] The cells were cultivated using the standard method described under C-1

[1136] The cells were detached with a solution of Accutase in PBS (Biochrom AG #L2143), pelleted, resuspended in culture medium, counted, and seeded into a 96-well white bottom culture plate (Costar #3610) (NCI H292: 2500 cells / well; SK-HEP-1: 1000 cells / well; KPL-4: 1200 cells / well; in a total volume of 100 μL). The cells were then incubated at 37°C and 5% carbon dioxide. After 48 hours, the medium was changed. The antibody-drug conjugates were then added to 10 μL of culture medium at concentrations of 10 "5 M to 10 "13M was pipetted to the cells (triplicates) before the mixture was incubated in an incubator at 37°C and 5% carbon dioxide. The suspension cells were counted and seeded into a 96-well white bottom culture plate (Costar #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 hours of incubation at 37°C and 5% carbon dioxide, the medium was changed and the

[1137] Antibody, active ingredient, conjugates or metabolites in 10μI culture medium at concentrations of 10 "5 M to 10 "13M was pipetted to the cells (triplicates) in 90 μL incubation. The mixture was incubated at 37°C and 5% carbon dioxide. After 96 h, cell proliferation was detected using the MTT assay (ATCC, Manassas, Virginia, USA, catalog no. 30-101 OK). 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 resulting dye was detected at 570 nm (Infinite M1000 pro, [Company Name]).

[1138] Tecan). From the measured data, the IC50 of growth inhibition was calculated using the DRC (dose-response curve). Proliferation without

[1139] A test substance, but otherwise identically treated cells, is defined as a 100% value.

[1140] In the following Tables 1a and 1b, the ICso values ​​are more representative.

[1141] Examples of implementation from these assays are listed:

[1142] Table 1 a

[1143] NCI-H292 L0V0 SKHep-1 BxPC3 KPL-4

[1144] MOLM 13 MV-4-11

[1145] ICso [M ] ICso [M] ICso [M] ICso [M] ICso [M]

[1146] Beispiel ICso [M] ICso [M]

[1147] MTT CTG MTT CTG [MTT] MTT MTT

[1148] / CTG

[1149] 1 a-981 2.65E-1 1 6.70E-08

[1150] l c-6013 3.15E-10 2.29E-08

[1151] 1 C-9476 2.28E-09 3.19E-09

[1152] 1 e-1015 1 .51 E-09

[1153] 1 k-7006 1.12E-10 5.37E-1 1 3.62E-1 1 1 .04E-10

[1154] 1 k-7007 6.56E-1 1 6.70E-1 1 8.42E-1 1 9.61 E-1 1

[1155] 2a-981 5.50E-12 1.79E-10

[1156] 2C-6013 1 .78E-1 1 4.77E-10

[1157] 2C-8987 4.59E-10 1 .26E-10

[1158] 2C-8988 8.91 E-1 1 4.39E-09

[1159] 2c-

[1160] 9.15E-10 5.08E-10

[1161] 9476B NCI-H292 LoVo SKHep-1 BxPC3 KPL-4

[1162] MOLM 13 MV-4-11

[1163] ICso [M ] ICso [M] ICso [M] ICso [M] ICso [M]

[1164] Beispiel ICso [M] ICso [M]

[1165] MTT CTG MTT CTG [MTT] MTT MTT

[1166] / CTG

[1167] 2c-

[1168] 1.3E-08 2.01 E-09

[1169] 9476C

[1170] 2c-

[1171] 1.10E-09 6.80E-11

[1172] 9476D e-1015 1.43E-10 k-7006 5.86E-11 1.50E-11 2.04E-11 9.92E-11 k-7007 8.82E-11 1.50E-11 7.54E-11

[1173] 3a-981 7.73E-13 1.11 E-10

[1174] C-9476 2.00E-10 1.05E-11 e-1015 3.64E-11 k-7007 5.49E-11 1.50E-11 9.80E-11

[1175] 4a-981 8.40E-10 1.57E-10

[1176] c-9476 8.40E-10 1.57E-10

[1177] k-7007 7.74E-10 1.50E-11 1.18E-10

[1178] C-9476 1.08E-09 4.40E-11 e-1015 4.50E-10 k-7007 1.12E-10 1.50E-11 3,03E-10

[1179] X-9574

[1180] 6a-981 1.68E-12 5.00E-07

[1181] C-9476 4.46E-10 1.24E-10 e-1015 5.54E-10k-7007 6.94E-11 4.93E-11 1.89E-10

[1182] 7a-981 2.34E-10

[1183] C-9476 1.09E-09 6.41 E-10 e-1015 4.51E-10 NCI-H292 LoVo SKHep-1 BxPC3 KPL-4

[1184] MOLM 13 MV-4-11

[1185] ICso [M ] ICso [M] ICso [M] ICso [M] ICso [M]

[1186] Beispiel ICso [M] ICso [M]

[1187] MTT CTG MTT CTG [MTT] MTT MTT

[1188] / CTG

[1189] 7k-7007 1.03E-10 1.43E-09 4.39E-10

[1190] 8a-981 9,87E-12 3,96E-08

[1191] 8C-9476 3,36E-10 3.46E-11

[1192] 8e-1015 4.30E-10

[1193] 8k-7007 1.84E-10 3.66E-11 4.17E-10

[1194] 9a-981 1.00E-11 4.29E-08

[1195] 9C-9476 5.00E-07 1.44E-07

[1196] 9e-1015 1.39E-10

[1197] 9k-7007 1.45E-10 3.17E-11 2.69E-10

[1198] 10a-981 1.00E-12 5.00E-07

[1199] IOc-9476 1.41E-09 8.42E-10

[1200] 10C-9476

[1201] 1.79E-10 5.45E-11

[1202] HD

[1203] 10e- 3.78E-11 1015

[1204] 10k-7007 9.60E-11 1.50E-11 1.29E-10

[1205] 11a-981 1.98E-12 2.59E-08

[1206] 11C-9475 9.61 E-08 6.90E-08

[1207] 11e- 2.37E-10 1015

[1208] 11k-7007 1.74E-10 1.62E-10 Tablelb

[1209]

[1210] Table 1c below lists the ICso values ​​of the reference samples from these assays.

[1211] MOLM 13 Rec-1 ICso NCI-H292 SKHep-1 KPL-4

[1212] Example ICso [M] [M] ICso [M] ICso [M] ICso [M]

[1213] MTT CTG MTT MTT [MTT]

[1214] R1a 6.14E-11 1.85E-10

[1215] R1c 1.24E-07

[1216] R1e 1.55E-08

[1217] Rix 3.00E-07

[1218] R2a 2.10E-10 6.02E-08

[1219] R2e 4.39E-08

[1220] R2x 3.00E-07 The reported efficacy data refer to the embodiments described in this experimental section with the specified drug / mAB ratios. Values ​​may vary with other drug / mAB ratios. The IC50 values ​​are mean values ​​from several independent experiments or single values. The antibody-drug conjugates exhibited selective activity against their respective isotype controls, which contained the corresponding linker and toxophore. For the CD123-directed ADCs, target specificity was additionally demonstrated by testing on a CD123-negative cell. The ADCs according to the invention generally show significantly improved cytotoxic potency compared to the corresponding reference examples.

[1221] C-1 b Determination of the inhibition of the kinesin spindle protein KSP / Eq5 by selected examples

[1222] The motor domain of the human kinesin spindle protein KSP / Eg5 (tebu-bio company /

[1223] Cytoskeleton Inc., No. 027EG01-XL) was incubated at room temperature for 5 min in 15 mM PIPES, pH 6.8 (5 mM MgCl₂ and 10 mM DTT, Sigma) at a concentration of 10 nM with 5 g / ml Taxol (Sigma, No. T7191-5MG). The freshly prepared mixture was aliquoted into a 384 MTP (Corning) tube. The inhibitors under investigation were then added at concentrations ranging from 1.0 x 10⁻⁶ M to 1.0 x 10⁻¹³ M, along with ATP (final concentration 500 μM; Sigma). Incubation continued for 2 h at room temperature. ATPase activity was detected by measuring the formation of inorganic phosphate with malachite green (Biomol). After adding the reagent, the mixture was incubated for 50 minutes at room temperature before absorbance was detected at a wavelength of 620 nm. Monastrol (Sigma, M8515-1 mg) and ispinesib (AdooQ Bioscience A10486) were used as positive controls.The individual data points of the dose-response curve represent eight times the value.

[1224] These determinations represent ICso values. The ICso values ​​are mean values ​​from two independent experiments. The untreated sample served as the 100% control. Table 2 below summarizes the ICso values ​​of representative examples from the described assay and the corresponding cytotoxicity data (MTT assay): Table 2

[1225]

[1226] The stated efficacy data refer to the exemplary embodiments described in this experimental section. C-1 c Enzymatic Assays

[1227] a: Cathepsin B assay

[1228] For each cathepsin B-cleavable prodrug to be investigated, a reaction was prepared in a 0.5 ml microreaction vessel (Eppendorf). The enzyme used here was derived from human liver tissue. 2 μg of cathepsin B (Sigma C8571 25 μg) was added and diluted to a total volume of 20 ml with 50 mM sodium phosphate buffer, pH 6.0, and 2 mM DTT. Then, 50 μL of the substrate solution to be investigated was pipetted in. The reaction was incubated in a thermoblock (Thermo Fisher Scientific) at 40°C with continuous shaking at 300 rpm.

[1229] The enzymatic reaction was kinetically controlled. For this purpose, a 10 μL sample was taken at different time points. The sample was immediately treated with 20 μL of ice-cold methanol to stop the enzymatic reaction and then frozen at -20°C. The chosen sampling times were after 10 minutes, 2 hours, 4 hours, and 24 hours. The samples were analyzed by reverse-phase HPLC (Agilent Technologies 1200 Series). Determining the released toxophore allowed for the determination of the half-life of the enzymatic reaction.

[1230] Reaction b: Legumain Assay

[1231] The legumain assay was performed using recombinant human enzyme. The rhLegumain enzyme solution (Catalog # 2199-CY, R&D Systems) was diluted to the desired concentration in 50 mM sodium acetate buffer / 100 mM NaCl, pH 4.0, and pre-incubated for 2 h at 37°C. rhLegumain was then adjusted to a final concentration of 1 ng / µL in 50 mM MES buffer / 250 mM NaCl, pH 5.0. A separate reaction was performed for each legumain-cleavable prodrug to be tested in a microreaction vessel (0.5 ml, [Company Name]).

[1232] Eppendorf). For this purpose, the substrate solution was adjusted to the desired concentration (2x concentration) using 50 mM MES buffer, 250 mM NaCl, pH 5.0. For the kinetic measurement of the enzymatic reaction, 250 mM of the

[1233] Legumain solution was introduced, and the enzyme reaction was initiated by adding 250 µL of the substrate solution (final concentration: 3 µL). 50 µL samples were taken at various time points. These samples were immediately treated with 100 µL of ice-cold methanol to stop the enzymatic reaction and then frozen at -20°C. The chosen sampling time points were 0.5 h, 1 h, 3 h, and 24 h. The samples were subsequently analyzed by RP-HPLC and LC-MS. Determining the released toxophore allowed for the determination of the half-life (h / 2) of the enzymatic reaction.

[1234] As representative examples to demonstrate legumain-mediated cleavage, model compounds A and B were prepared as substrates in the legumain assay.

[1235] Reference example model connection A

[1236] N-(Pyridin-4-ylacetyl)-L-alanyl-L-alanyl-N 1-[(2S)-4-[{(1 R)-1 -[1-benzyl-4-(2,5-difluorophenyl)- 1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1 -(methylamino)-1-oxobutan-2-yl]-L-aspartamide

[1237] First, trifluoroacetic acid (2S)-2-amino-4-[{(1 R)-1 -[1-benzyl-4-(2,5- difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-methylbutanamide was prepared as described in WO 2015096982 A1. Then, from this

[1238] The title compound was prepared by coupling with intermediate L103 in DMF in the presence of HATU and N,N-diisopropylethylamine.

[1239] LC-MS (Method 1): R t = 0.86 min; MS (ESIpos): m / z = 902 [M+H] + .

[1240] Reference example model connection B

[1241] N-(Pyridin-4-ylacetyl)-L-alanyl-N-methyl-L-alanyl-N 1-[(2S)-4-[{(1 R)-1-[1 -benzyl-4-(2,5-difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1 -(methylamino)-1-oxobutan-2-yl]-L-aspartami

[1242]

[1243] First, trifluoroacetic acid (2S)-2-amino-4-[{(1 R)-1 -[1 -benzyl-4-(2,5- difluorophenyl)-1 H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-methylbutanamide was prepared as described in WO 2015096982 A1. Then, from this

[1244] The title compound was prepared by coupling with intermediate L1 18 in DMF in the presence of HATU and N,N-diisopropylethylamine.

[1245] LC-MS (Method 1): R t = 0.83 min; MS (ESIpos): m / z = 916 [M+H] + .

[1246] Model compound A was cleaved to the target compound by Legumain under the conditions described above with a half-life of 0.4 h.

[1247]

[1248] Model compound B was cleaved to the target compound by Legumain under the conditions described above with a half-life of 0.5 h.

[1249]

[1250] C-2 Internalisation sassav

[1251] Internalization is the key process for enabling the specific and efficient delivery of the cytotoxic payload into antigen-expressing cancer cells via antibody-drug conjugates (ADCs). This process is monitored by fluorescent labeling of specific antibodies and an isotype control antibody. First, the fluorescent dye was conjugated to lysine residues of the antibody. Conjugation was performed with a 2- to 10-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, No. 87768; elution buffer:

[1252] DULBECCO 'S PBS (Sima-Aldrich, No. D8537) was used to eliminate excess dye and adjust the pH. The protein solution was concentrated using VIVASPIN 500 columns (Sartorius stedim biotec). The antibody dye load was determined by spectrophotometric analysis (NanoDrop) followed by calculation (D / P = Ad e Sprotein :(A280-0, 1 6Adye)Sdye). The dye loads of the antibodies tested here and the isotype control were of a comparable order of magnitude. Cell binding assays were performed to demonstrate that the

[1253] The coupling did not result in any change in antibody affinity.

[1254] The labeled antibodies were used in the internalization assay. Before the start of treatment, cells (2x10 4 / well) in 100 µL medium in a 96-MTP (fat, black, clear bottom No. 4308776, Applied Biosystems). After 18 h incubation at 37°C / 5% CO2, the medium was changed and labeled antibodies were added at varying concentrations (10, 5, 2.5, 1, 0.0 g / mL). The same treatment regimen was performed with the labeled isotype control (negative control). The selected

[1255] Incubation times were 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6 h, and 24 h. Fluorescence measurements were performed using the InCellAnalyzer 1000 (GE Healthcare). Kinetic evaluation was conducted by measuring the parameters granule counts / cell and total granule intensity / cell.

[1256] Antibodies were investigated for their internalization capacity after binding to the receptor. Cells with different receptor expression levels were selected for this purpose. Target-mediated specific internalization was observed with the antibodies, whereas the isotype control showed no internalization.

[1257] C-2b internalisation assay with suspension cells

[1258] The coupling of the fluorescent dye was performed as described under C2. The antigen under investigation is expressed by hematopoietic suspension cells; therefore, internalization was investigated using a FACS-based internalization assay.

[1259] Cells with different target expression levels were examined - The cells (5x10 4The cells ( / well) were seeded into 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 samples were incubated at 37°C for varying durations (1 h, 2 h, 6 h, triple determination). The isotype control was treated under identical conditions. A parallel sample was treated and incubated at a constant temperature of 4°C (negative control). FACS analysis was performed using the Guava flow meter.

[1260] Cytometers (Millipore) were used. Kinetic evaluation was performed by measuring fluorescence intensity, and the analysis was carried out using guavaSoft 2.6 software (Millipore). Significant and specific internalization of the targets and target-specific antibodies described here was detected in various cells. The isotype controls showed no internalization.

[1261] C-2c Co-Localization Studies of Anti-CD123 Antibodies: The generation of the active metabolite of the antibody-drug conjugate occurs via lysosomal degradation due to the linker. Accordingly, intracellular trafficking after internalization is of essential importance. Studies investigating the co-localization of the antibody with markers specific to the lysosomal organelle (e.g., surface molecules or small GTPases) enable the selection of antibodies with a desired profile. For this purpose, target-positive cells (5x10) were used. 4 / well) in 100 μL total volume in a 96-MTP (Greiner bio-one, CELLSTAR, 650 180, U-bottom). After addition of the CypHer5E-labeled anti-target antibody (final concentration 20 mg / ml), the samples (duplicates at each 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 selected incubation time, the lysosome-specific marker was added to the samples to be analyzed. The lysosomes were stained with CytoPainter LysoGreen indicator reagent (final concentration 1:2000; abcam, ab176826). After incubation, 200 μL of ice-cold FACS buffer (DULBECCO) was added. ' S PBS (Sigma-Aldrich, No. D8537) + 3% FBS (heat-inactivated FBS, Gibco, No. 10500-064) were added, and the cell suspension was centrifuged at 400 x g, 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 successful

[1262] After centrifugation, the supernatant was discarded, and the cell pellet was reconstituted in 30 μL ice-cold FACS buffer. The samples were then subjected to immediate FACS / image analysis (FlowSight amnis, Millipore). Co-localization was evaluated using specific software (Co-Localization Software IDEAS Application v6.1). Table 3 summarizes the results from this assay using the anti-CD123 antibody as an example.

[1263]

[1264] The humanized antibodies TPP-9476 and TPP-8987 show a significantly improved profile compared to the parental murine antibody.

[1265] C-3 In vitro tests for determining cell permeability

[1266] The cell permeability of a substance can be determined by means of measurements in w ' / roTesting in a flux assay using Caco-2 cells [MD Troutman and DR

[1267] Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. For this purpose, the cells were cultured on 24-hole filter plates for 15-16 days. To determine permeation, the respective test substance was applied to the cells in a HEPES buffer either apically (A) or basally (B) and incubated for 2 h. Samples were taken from the cis and trans compartments after 0 h and after 2 h. The samples were separated by HPLC (Agilent 1200, Böblingen, Germany) using reverse-phase columns. The HPLC system was coupled via a Turbo Ion Spray interface to a triple quadropole mass spectrometer API 4000 (AB SCIEX Deutschland GmbH, Darmstadt, Germany). Permeability was determined using a P ap p-value was assessed, 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 if the ratio of P a (BA) to P app(AB) (efflux ratio) was >2 or <0.5.

[1268] The permeability of B to A [P] is of crucial importance for toxophores that are released intracellularly. a (BA)] and the ratio of P app (BA) to P app (AB) (efflux ratio): The lower this permeability, the slower the active and passive transport processes of the substance through the monolayer of Caco-2 cells, so that the substance remains in the cell longer after intracellular release. This intracellular retention of the metabolite increases the probability of a

[1269] Interaction with the biochemical target (here: kinesin spindle protein, KSP / Eg5), leading to improved cytotoxic activity. Table 4 below lists permeability data for representative examples from this assay:

[1270] Table 4

[1271]

[1272] The metabolite M1, which can be formed from the binder-drug conjugates according to the invention, shows both a significantly reduced transport out of the cell and a reduced efflux ratio compared to the reference metabolite R3M, which can be formed from the binder-drug conjugates of reference examples 2.

[1273] C-4 In vitro tests to determine the substrate properties of P-glucoprotein (P-qp)

[1274] Many tumor cells express transporter proteins for drugs, which is often associated with the development of resistance to cytostatic agents. Substances that are not substrates of such transporter proteins, such as P-glycoprotein (P-gp) or BCRP, could therefore exhibit an improved efficacy profile. The substrate properties of a substance for P-gp (ABCB1) were determined using a flux assay with LLC-PK1 cells that overexpress P-gp (L-MDR1 cells) [AH Schinkel et al., J. Clin. Invest. 96, 1698-1705 (1995)]. For this purpose, the LLC-PK1 or L-MDR1 cells were cultured on 96-hole filter plates for 3-4 days. To determine permeation, the respective test substance, alone or in the presence of an inhibitor (such as ivermectin or verapamil), was applied to the cells either apically (A) or basally (B) in a HEPES buffer and incubated for 2 h. Samples were taken from the cis and trans compartments after 0 h and after 2 h.The samples were separated by HPLC using reverse-phase columns. The HPLC system was coupled to an API 3000 triple quadropole mass spectrometer (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo Ion Spray interface. Permeability was determined using a P. ap The p-value was evaluated 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 if the efflux ratio P app (BA) to P app (AB) >2 was.

[1275] Further criteria for evaluating P-gp substrate properties include comparing efflux ratios in L-MDR1 and LLC-PK1 cells, or the efflux ratio in the presence or absence of an inhibitor. If these values ​​differ by more than a factor of 2, the substance in question is considered a P-gp substrate.

[1276] C-5 Pharmacokinetics

[1277] Following intravenous administration of 5 mg / kg of Example 2c-9476 (DAR 6.3) and Example 2c-9476 (DAR 3.4) into male Wistar rats, the plasma concentrations of the ADCs were measured by ELISA and the pharmacokinetic parameters such as clearance (CL), area under the curve (AUC) and half-life (ti / 2) were calculated.

[1278] Table 5 summarizes the pharmacokinetic parameters of example 2c-9476 with DAR 6.3 and DAR 3.4.

[1279] In this preliminary rat PK study following IV administration, a typical IgG profile was observed for both examples. No significant differences were found between example 2c-9476 with DAR 6.3 and DAR 3.4.

[1280] Analysis to quantify the ADCs used

[1281] The antibody portion of the ADCs was determined as total IgG concentration in plasma samples and tumor lysates using a ligand-binding assay (ELISA) with a sandwich ELISA format. This ELISA was qualified and validated for determination in plasma and tumor samples. The 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 monkey anti-human IgG(H+L) antibody and horseradish peroxidase (HRP). After a further washing step, the HRP substrate OPD was added, and the color development was monitored.

[1282] Absorption was monitored at 490 nm. Standard samples with known IgG concentrations were fitted using a 4-parameter equation. Within the lower (LLOQ) and upper (ULOQ) limits of quantification, the unknown concentrations were determined by interpolation.

[1283] C5a: Identification of ADC metabolites after internalization in vitro

[1284] Method description:

[1285] Internalization studies with immunoconjugates are performed to analyze intracellularly produced metabolites. For this purpose, human

[1286] Lung tumor cells NCI H292 (3x10 5 The ADCs (1 / 3 well) were seeded into 6-well plates and incubated overnight (37 °C, 5% CO2). They were treated with 10 g / mL (66 nM) of the ADC under investigation. Internalization was performed at 37 °C and 5% CO2. Cell samples were taken at various time points (0, 4, 24, 48, 72 h) for further analysis. The supernatants (approx. 5 mL) were harvested and, after centrifugation (2 min, room temperature, 1000 rpm Heraeus Variofuge 3.0R), stored at -80 °C. The cells were washed with PBS, detached with Accutase, and the cell count determined. After rewashing, a defined cell count (2 x 10⁻⁶) was determined. 5) mixed with 100 ml of Lysis Buffer (Mammalian Cell Lysis Kit (Sigma MCL1)) and shaken continuously

[1287] (Thermomixer, 15 min, 4°C, 650 rpm) in Protein LoBind tubes (Eppendorf Cat. No. 0030 108.1 16). After incubation, the lysate is centrifuged (10 min, 4°C, 12000 g, Eppendorf 5415R) and the supernatant is collected. The obtained supernatant is stored at -80°C. All samples are then analyzed as follows.

[1288] To process 50 μί of culture residue / cell lysate, these are treated with 150 μί

[1289] Precipitating reagent (methanol) was added and the mixture was shaken for 10 seconds.

[1290] The precipitation reagent contains an internal standard (ISTD) at a suitable concentration (usually in the range of 20–100 g / L). After centrifugation for 10 minutes at 1881 g, the supernatant is transferred to an autosampler vial, filled with 300 µL of a buffer matched to the mobile phase, shaken again, and centrifuged for 10 minutes at 1881 g.

[1291] The cell lysate and supernatant samples are then measured using the API6500 triple quadrupole mass spectrometer coupled to an HPLC from AB SCIEX Deutschland GmbH.

[1292] For calibration, the empty lysate or supernatant is mixed with appropriate concentrations (0.1 - 1000 g / L). The limit of detection (LLOQ) is approximately 0.2 μg / L.

[1293] Quality controls for validity testing contain 4 and 40 μg / L.

[1294] C5b: Identification of ADC metabolites in vivo

[1295] Following intravenous administration of 3–30 mg / kg of various ADCs, plasma and tumor concentrations of the ADC and any potentially occurring metabolites can be measured, and pharmacokinetic parameters such as clearance (CL), area under the curve (AUC), and half-life (ti / 2) can be calculated. Analysis for the quantification of potentially occurring metabolites is also available.

[1296] The measurement of compounds in plasma, tumor, liver and kidney is carried out after precipitation of the proteins with usually methanol by high-performance liquid chromatography (HPLC) coupled to a triple quadrupole mass spectrometer (MS).

[1297] To process 50 μM of plasma, it is mixed with 150 μM of precipitation reagent (usually methanol) and 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 for 10 minutes at 1881 g, the supernatant is transferred to a

[1298] The autosampler vial is transferred, filled with 300 µL of a buffer matched to the mobile phase, and shaken again. When processing tumor or organ material, the respective material is mixed with 3 to 20 times 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 phenylmethylsulfonylfluoride (Sigma, St. Louis, MO) at a final concentration of 1 mM. Depending on the tissue type (hard: tumor; soft: liver, kidney), the lysis and

[1299] The homogenization program of the Prescellys 24 Lysis and Homogenization Instrument (Bertin Technologies) was selected (www.prescellys.com). The homogenized samples were left to stand overnight at 4°C. 50 µL of the homogenate was transferred to an autosampler vial, filled with 150 µL of methanol including ISTD, shaken for 10 seconds, and then left to stand for 5 minutes. After adding 300 µL of ammonium acetate buffer (pH 6.8) and briefly shaking, the sample was centrifuged for 10 minutes at 1881 g.

[1300] For calibration, plasma samples and corresponding blank matrix samples with concentrations of 0.6–1000 μg / L are added to the plasma sample and tissue sample sample, respectively. The limit of detection (LOQ) ranges from 1 to 20 μg / L, depending on the sample or tissue type. The plasma and matrix samples are then measured using the API4500 triple quadrupole mass spectrometer from AB SCIEX Deutschland GmbH, coupled to an HPLC system.

[1301] Quality controls for validity testing include 4, 40 and 400 μg / L.

[1302] Table 6 shows metabolite concentrations in the MOLM-13 xenograft mouse model measured in tumor, liver, kidney, and plasma 24 h after administration of 5 mg / kg of the ADC from Example 2c-9476 (n=3). The measured metabolite was: Metabolite M1. nc = not calculated; LOQ: limit of quantification. Table 6:

[1303]

[1304] The application of the ADC according to the invention, example 2c-9476, with a legumaine-cleavable linker led to a significantly selective accumulation of the active agent in the target tissue (tumor) compared to other healthy organs / tissues.

[1305] C-6 efficacy test in vivo

[1306] The efficacy of the conjugates according to the invention was tested in vivo, for example, using xenograft models. Those skilled in the art are familiar with methods by which the efficacy of the compounds according to the invention can be tested (see, e.g., WO 2005 / 08171 1; Polson et al., Cancer Res. 2009 Mar 15;69(6):2358-64). For example, rodents (e.g., mice) were inoculated with a tumor cell line expressing the target molecule of the binder. Subsequently, the inoculated animals were administered either a conjugate according to the invention, an isotype antibody control conjugate, a control antibody, or isotonic saline solution. The application was performed once or repeatedly. After an incubation period of several days, the

[1307] Tumor size was determined by comparing conjugate-treated animals with the control group. The conjugate-treated animals showed a smaller tumor size.

[1308] C-6a. Growth inhibition / regression of experimental tumors in mice

[1309] Human tumor cells expressing the antigen for the antibody-drug conjugate are inoculated subcutaneously into the flank of immunosuppressed mice, such as NMR nude or SCID mice. One to ten million cells are detached from the cell culture, centrifuged, and resuspended in medium or medium / Matrigel. The cell suspension is injected subcutaneously into the mouse. Within a few days, a tumor develops. Treatment begins after tumor establishment, at approximately 40 mm in size. 2 To investigate the effect on larger tumors, the treatment can also be started only when the tumor size is 50-100 mm. 2 to begin.

[1310] Treatment with APDCs and ADCs is administered intravenously (IV) into the tail vein of the mouse. The ADC is administered at a volume of 5 ml / kg.

[1311] The treatment regimen depends on the pharmacokinetics of the antibody. With the conjugates according to the invention, the standard treatment is once a week for 2 or 3 weeks. For a more timely assessment, a regimen with a

[1312] A single treatment is suitable. However, the treatment can also be continued, or a second cycle of three may be necessary at a later date.

[1313] Follow up with treatment days.

[1314] Typically, 8 animals are used per treatment group. In addition to the groups receiving the active substances, one group is treated as a control group with only the buffer, following the same protocol.

[1315] During the experiment, the tumor area is regularly measured in two dimensions (length / width) using calipers. The tumor area is then determined by length x width. The comparison of the mean tumor area of ​​the treatment group with that of the control group is expressed as the T / C area.

[1316] If all groups in the experiment are terminated simultaneously after the end of treatment, the tumors can be removed and weighed. The comparison of the mean tumor weights of the treatment group with the control group is reported as T / C weight.

[1317] C-6b. Effectiveness of the ADCs according to the invention in various

[1318] Tumor models

[1319] Tumor cells (e.g., NCI-H292, REC-1, MOLM-13, and MV-4-1) are inoculated subcutaneously into the flank of female NMRI-nude mice (Janvier). At a tumor size of 40 mm 2The patient is treated intravenously with the antibody-drug conjugate. Following treatment, tumor growth is monitored if necessary. Treatment with the ADCs according to the invention leads to a significant 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 from the tumor area on the respective day of the end of the experiment, calculated according to

[1320] Start of treatment.

[1321] Table 7:

[1322] Example Antigen Tumor Model Dose Dose Scheme T / C area

[1323] 2k-7007 TWEAKR NCI-H292 5 mg / kg Q7dx3 0.09 (Day 25)

[1324] (human

[1325] lung cancer)

[1326] 2x-9574 CXCR5 REC-1 (human 10 mg / kg Q7dx3 0.20 (Day 24)

[1327] Mantle cell lymphoma

[1328] 2C-9476D CD123 MOLM-13 5 mg / kg Q7dx2 0.15 (Day 17)

[1329] (human acute

[1330] myelological

[1331] Leukemia)

[1332] 2C-9476D CD123 MV-4-1 1 5 mg / kg Q7dx2 0.16 (Tag 18)

[1333] (human acute

[1334] myelological

[1335] Leukemia)

Claims

Patent claims 1. Binder-active ingredient conjugates of formula (I) in the X3 stands for C; or X3 stands for N; or X3 stands for N; or X3 stands for C; or X3 stands for C. R 1 stands for hydrogen or methyl R 2 stands for Methyl, Ethyl, -CH2-CH(CH3)2, -CH2-C(=0)OH or iso-Propyl, R 3 for Methyl, Ethyl, -CH2-CH(CH3)2 or -CH2-C(=0)-NH2 M for the group #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH, #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH, #-C(=0)-CH(CH3)-NH-C(=0)-CH2-W, #-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH, #-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH, #-C(=0)-CH2-W, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)2-8 -C(=0)-###, #-C(=0)- (CH2)3-C(=0)-###, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)5-W“ #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)-## or #-C(=0)-CH(CH3)-NH-C(=0)-(CH2-CH2-0)i-8-(CH2)2-NH-C(=0)-CH2-## stands, W for the group n represents a number from 1 to 50, represents a binder or a derivative thereof, preferably an antibody or an antigen-binding antibody fragment, represents the binding to the compound, represents the binding to a sulfur atom of a cysteine ​​side chain of the binder, ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

2. Binder-active ingredient conjugates of formula (I), according to claim 1, in the X2 for C X3 stands for N; R 1 stands for hydrogen or methyl R 2stands for Methyl, -CH2-CH(CH3)2, -CH2-C(=0)OH or iso-propyl, R 3 for Methyl, -CH2-CH(CH3)2 or -CH2-C(=0)-NH2 M for the group #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH, #-C(=0)-CH(CH3)-NH-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH, #-C(=0)-CH(CH3)-NH-C(=0)-CH2-W, #-C(=0)-CH2-NH-C(=0)-CH2-CH(##)-COOH, #-C(=0)-CH2-NH-C(=0)-CH(##)-CH2-COOH, #-C(=0)-CH2-W, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-###, #-C(=0)- (CH2)3-C(=0)-###, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)5-W, #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)-## or #-C(=0)-CH(CH3)-NH-C(=0)-(CH2-CH2-0)4-(CH2)2-NH-C(=0)-CH2-## stands, W for the group n represents a number from 1 to 50, AK for a binder or a derivative thereof, preferably for a Antibody or an antigen-binding antibody fragment is present, # stands for the bond at the connection, ## for the bonding to a sulfur atom of a cysteine ​​side chain of the Binders stands, ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

3. Binder-active ingredient conjugates of formula (I), according to claims 1 and 2, in the R 1 stands for hydrogen or methyl R 2 stands for methyl or isopropyl, R 3 stands for Methyl or -CH2-C(=0)-NH2 M for the group #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for, where n is a number from 1 to 50, AK for a binder or a derivative thereof, preferably for a Antibody or an antigen-binding antibody fragment is present, # stands for the bond at the connection, ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

4. Binder-active ingredient conjugates of formula (I), according to claims 1 to 3, in R 1 stands for methyl, R 2 stands for methyl, R 3 for -CH2-C(=0)-NH2 M for the group #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for, where n is a number from 1 to 50, AK for a binder or a derivative thereof, preferably for a Antibody or an antigen-binding antibody fragment is present, # stands for the bond at the connection, ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

5. Binder-active ingredient conjugates of formula (I), according to claims 1 to 4, in R 1 stands for methyl, R 2stands for methyl, R 3 for -CH2-C(=0)-NH2 M for the group #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for, n stands for a number from 1 to 20, AK for a binder or a derivative thereof, preferably for a Antibody or an antigen-binding antibody fragment is present, # stands for the bond at the connection, ### represents the bond to an N atom of a lysine side chain of the binder, as well as its salts, solvates and salts of these solvates.

6. Binder-active ingredient conjugates of formula (I), according to claims 1 to 5, in R 1 stands for methyl, R 2 stands for methyl, R 3 for -CH2-C(=0)-NH2 M for the group #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for n, which is a number from 1 to 20 and AK stands for an anti-CD 123 antibody, an anti-CXCR5 antibody, an anti-B7H3 antibody, an anti-TWEAKR antibody, an anti-Her2 antibody or an anti-EGFR antibody, or for an antigen-binding antibody fragment of these. # stands for the bond at the connection, ### for bonding to a nitrogen atom of a lysine side chain of the Antibodies (AK) or the antigen-binding antibody fragment thereof, as well as their salts, solvates and salts of these solvates.

7. Binder-active ingredient conjugates of formula (I), according to claims 1 to 6, in R 1 stands for methyl, R 2 stands for methyl, R 3 for -CH2-C(=0)-NH2 M for the group #-C(=0)-CH(CH3)-NH-C(=0)-(CH2)3-C(=0)-### stands for n, which is a number from 1 to 20 and Antibody for an anti-CD123 antibody selected from the group consisting of TPP-9476, TPP-8988, TPP-8987 and TPP-6013, for an anti-CXCR5 antibody selected from the group consisting of TPP-9574 and TPP-9580, for an anti-B7H3 antibody TPP-8382, for an anti-TWEAKR antibody selected from the group consisting of TPP-7006 and TPP-7007, for an anti-Her2 antibody TPP-1015, or for an anti-EGFR antibody TPP-981 or for an antigen-binding antibody fragment of these, # stands for the bond at the connection, ### for bonding to a nitrogen atom of a lysine side chain of the Antibodies (AK) or the antigen-binding antibody fragment thereof, as well as their salts, solvates and salts of these solvates.

8. Binder-active ingredient conjugates of formula (I), according to claims 1 to 7, of the structures 170 171 WO 2018 / 114578 WO 2018 / 114578 174 in which AK1 stands for an antibody that has a sulfur atom attached to a cysteine ​​molecule. The side chain is tied. AK2 stands for an antibody that has a nitrogen atom attached to a lysine molecule. The side chain is tied. n 1 to 50 is, as well as their salts, solvates and salts of these solvates.

9. Binder-active ingredient conjugates according to claim 8, wherein n 1 to 20 is, as well as their salts, solvates and salts of these solvates.

10. Binder-active ingredient conjugates according to claims 8 and 9, wherein n 1 to 8 is, as well as their salts, solvates and salts of these solvates.

1. Binder-active ingredient conjugates according to claims 8 to 10, wherein n 4 to 8 is, as well as their salts, solvates and salts of these solvates. Binder-active ingredient conjugates, according to one or more of claims 1 to 1 1 , 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-9580 (anti-CXCR5) and TPP 9574 (anti-CXCR5), or an antigen-binding fragment of these. Binder-active ingredient conjugates according to one or more of claims 1 to 12, wherein AK (AK1. AK2) stands for an anti-CD123 antibody TPP-6013, for an anti-CD123 antibody TPP-8987, for an anti-CD123 antibody TPP-8988 or for an anti-CD123 antibody TPP-9476, or an antigen-binding fragment of these.

14. Binder-active ingredient conjugates according to one or more of claims 1 up to 13, where AK (AK1 , AK2) (i) for an anti-B7H3 antibody comprising a variable heavy chain region (VH) comprising the variable CDR1 heavy chain sequence (H-CDR1) as represented by SEQ ID NO: 52, the variable CDR2 heavy chain sequence (H-CDR2) as represented by SEQ ID NO: 53 and the variable CDR3 heavy chain sequence (H-CDR3) as represented by SEQ ID NO: 54, and a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1) as represented by SEQ ID NO: 56, the variable CDR2 light chain sequence (L-CDR2) as represented by SEQ ID NO: 57 and the variable CDR3 light chain sequence (L-CDR3) as represented by SEQ ID NO: 58, (ii) for an anti-CD123 antibody comprising a variable heavy chain region (VH) comprising the variable CDR1 heavy chain sequence (H-CDR1) as represented by SEQ ID NO: 22, the variable CDR2 heavy chain sequence (H-CDR2) as represented by SEQ ID NO: 23 and the variable CDR3 heavy chain sequence (H-CDR3) as represented by SEQ ID NO: 24, and a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1) as represented by SEQ ID NO: 26, the variable CDR2 light chain sequence (L-CDR2) as represented by SEQ ID NO: 27 and the variable CDR3 light chain sequence (L-CDR3) as represented by SEQ ID NO: 28, (iii) for an anti-CD123 antibody comprising a variable heavy chain region (VH) comprising the variable CDR1 heavy chain sequence (H-CDR1) as represented by SEQ ID NO: 62, the variable CDR2 heavy chain sequence (H-CDR2) as represented by SEQ ID NO: 63 and the variable CDR3 heavy chain sequence (H-CDR3) as represented by SEQ ID NO: 64, and a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1) as represented by SEQ ID NO: 66, the variable CDR2 light chain sequence (L-CDR2) as represented by SEQ ID NO: 67 and the variable CDR3 light chain sequence (L-CDR3) as represented by SEQ ID NO: 68, (iv) for an anti-CD123 antibody comprising a variable heavy chain region (VH) comprising the variable CDR1 heavy chain sequence (H-CDR1) as represented by SEQ ID NO: 72, the variable CDR2 heavy chain sequence (H-CDR2) as represented by SEQ ID NO: 73 and the variable CDR3 heavy chain sequence (H-CDR3) as represented by SEQ ID NO: 74, and a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1) as represented by SEQ ID NO: 76, the variable CDR2 light chain sequence (L-CDR2) as represented by SEQ ID NO: 77 and the variable CDR3 light chain sequence (L-CDR3) as represented by SEQ ID NO: 78, (v) for an anti-CD123 antibody comprising a variable heavy chain region (VH) comprising the variable CDR1 heavy chain sequence (H-CDR1) as represented by SEQ ID NO: 82, the variable CDR2 heavy chain sequence chain (H-CDR2), as represented by SEQ ID NO: 83 and the variable CDR3 sequence of the heavy chain (H-CDR3), as represented by SEQ ID NO: 84, as well as a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as represented by SEQ ID NO: 86, the variable CDR2 sequence of the light chain (L-CDR2), as represented by SEQ ID NO: 87 and the variable CDR3 sequence of the light chain (L-CDR3), as represented by SEQ ID NO: 88, (vi) for an anti-CXCR5 antibody comprising a variable heavy chain region (VH) comprising the variable CDR1 heavy chain sequence (H-CDR1) as represented by SEQ ID NO: 92, the variable CDR2 heavy chain sequence (H-CDR2) as represented by SEQ ID NO: 93 and the variable CDR3 heavy chain sequence (H-CDR3) as represented by SEQ ID NO: 94, and a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1) as represented by SEQ ID NO: 96, the variable CDR2 light chain sequence (L-CDR2) as represented by SEQ ID NO: 97 and the variable CDR3 light chain sequence (L-CDR3) as represented by SEQ ID NO: 98, or (vii) represents an anti-CXCR5 antibody comprising a variable heavy chain region (VH) comprising the variable CDR1 heavy chain sequence (H-CDR1) as represented by SEQ ID NO: 102, the variable CDR2 heavy chain sequence (H-CDR2) as represented by SEQ ID NO: 103 and the variable CDR3 heavy chain sequence (H-CDR3) as represented by SEQ ID NO: 104, and a variable light chain region (VL) comprising the variable CDR1 light chain sequence (L-CDR1) as represented by SEQ ID NO: 106, the variable CDR2 light chain sequence (L-CDR2) as represented by SEQ ID NO: 107 and the variable CDR3 light chain sequence (L-CDR3) as represented by SEQ ID NO: 108, or represents an antigen-binding fragment of these antibodies.

15. Binder-active ingredient conjugates according to one or more of claims 1 up to 14, where AK (AK1 , AK2) (i) for an anti-B7H3 antibody comprising a variable heavy chain region (VH) as represented by SEQ ID NO: 51 and a variable light chain region (VL) as represented by SEQ ID NO: 55, (ii) for an anti-CD123 antibody comprising a variable heavy chain region (VH) as represented by SEQ ID NO: 21 and a variable light chain region (VL) as represented by SEQ ID NO: 25, (iii) for an anti-CD123 antibody comprising a variable heavy chain region (VH) as represented by SEQ ID NO: 61 and a variable light chain region (VL) as represented by SEQ ID NO: 65, (iv) for an anti-CD123 antibody comprising a variable heavy chain region (VH) as represented by SEQ ID NO: 71 and a variable light chain region (VL) as represented by SEQ ID NO: 75, (v) for an anti-CD123 antibody comprising a variable heavy chain region (VH) as represented by SEQ ID NO: 81 and a variable light chain region (VL) as represented by SEQ ID NO: 85, (vi) for an anti-CXCR5 antibody comprising a variable heavy chain region (VH) as represented by SEQ ID NO: 91 and a variable light chain region (VL) as represented by SEQ ID NO: 95, or (vii) represents an anti-CXCR5 antibody comprising a variable heavy chain region (VH) as represented by SEQ ID NO: 101 and a variable light chain region (VL) as represented by SEQ ID NO: 105, or an antigen-binding fragment of these antibodies.

16. Binder-active ingredient conjugates according to one or more of claims 1 up to 15, where AK (AK1 , AK2) (i) for an anti-B7H3 antibody comprising a heavy chain region as represented by SEQ ID NO: 59 and a light chain region as represented by SEQ ID NO: 60, (ii) for an anti-CD123 antibody comprising a heavy chain region as represented by SEQ ID NO: 29 and a light chain region as represented by SEQ ID NO: 30, (iii) for an anti-CD123 antibody comprising a heavy chain region as represented by SEQ ID NO: 69 and a light chain region as represented by SEQ ID NO: 70, (iv) for an anti-CD123 antibody comprising a heavy chain region as represented by SEQ ID NO: 79 and a light chain region as represented by SEQ ID NO: 80, (v) for an anti-CD123 antibody comprising a heavy chain region as represented by SEQ ID NO: 89 and a light chain region as represented by SEQ ID NO: 90, (vi) for an anti-CXCR5 antibody comprising a heavy chain region as represented by SEQ ID NO: 99 and a light chain region as represented by SEQ ID NO: 100, or (vii) represents an anti-CXCR5 antibody comprising a heavy chain region as represented by SEQ ID NO: 109 and a light chain region as represented by SEQ ID NO: 110, or an antigen-binding fragment of these antibodies.

17. Binder-active ingredient conjugates according to any one of claims 1 to 16, wherein the The antibody or the antigen-binding antibody fragment binds to an extracellular target molecule. Binder-drug conjugates according to any one of claims 1 to 17, wherein the antibody or the antigen-binding antibody fragment binds to an extracellular cancer target molecule. Binder-drug conjugates according to any one of claims 1 to 18, wherein the antibody or the antigen-binding antibody fragment, after binding to its The extracellular target molecule is internalized by the target cell through binding.

20. Pharmaceutical composition comprising at least one binder-active ingredient conjugate according to one or more of the preceding claims in Combination with an inert, non-toxic, pharmaceutically suitable excipient.

21. Binder-drug conjugates according to one or more of the preceding Claims for use in a method for the treatment and / or prophylaxis of diseases.

22. Binder-drug conjugates according to one or more of the preceding Claims for use in a method for treating hyperproliferative and / or angiogenic diseases 23. Binder-drug conjugates according to one or more of the preceding Claims for use in a method for treating cancer and tumors.

24. Binder-drug conjugates according to one or more of the preceding Claims for use in a method for treating cancer and tumors in combination with one or more therapeutic approaches to cancer immunotherapy or with one or more active substances directed against a molecular target from cancer immunotherapy.