HK40016986A - Antibody drug conjugates (adcs) having enzymatically cleavable groups - Google Patents

Description

Antibody-drug-conjugates (ADC) with enzymatically cleavable groups

Introduction and prior art

The present invention relates to novel binding agent-active substance-conjugates (ADCs) with improved properties, active metabolites of these ADCs and methods of making the same. The invention also relates to the use of these conjugates for the treatment and/or prevention of diseases and the use of these conjugates for the preparation of a medicament for the treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders, such as cancer. Such treatment may be administered as monotherapy or in addition in combination with other agents or other therapeutic measures. According to the invention, the binding agent is preferably an antibody.

Cancer is a consequence of uncontrolled cell growth of a wide variety of tissues. In many cases, new cells infiltrate into existing tissues (invasive growth) or they migrate into distant organs. Cancer occurs in a wide variety of organs and often has a tissue-specific course. Thus, the term cancer as a generic concept describes a large group of specific disorders of different organs, tissues and cell types.

The tumor may optionally be removed at an early stage by surgery or radiation therapy. Metastatic tumors are usually only palliatively treated with chemotherapeutic drugs. The goal here is to achieve the best combination of improved quality of life and extended life.

Conjugates of a binder protein with one or more active substance molecules are known, in particular 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 linking unit (Verkn ü pfungseinheit), after introduction of the ADC into tumor cells and subsequent lysis of the conjugate, the cytotoxic agent itself or other cytotoxic effective metabolites formed therefrom are then released within the tumor cells and can directly and selectively exhibit their effect therein, in this way damage to normal tissue can be kept within significantly narrower limits than in conventional chemotherapy of cancer [ see, for example, j.m. Lambert,Curr. Opin. Pharmacol. 5543 ℃ 549 (2005), A.M. Wu and P.D. Senter,Nat. Biotechnol. 23, 1137-1146 (2005); P. D. Senter,Curr. Opin. Chem. Biol. 13235-,Bioconjugate Chem. 21, 5-13 (2010)]. Thus, WO2012/171020 describes ADCs in which multiple toxic cluster molecules are linked to an antibody via a polymer linker. As possible poison clusters, WO2012/171020 mentions, inter alia, the substances SB 743921, SB715992 (Ispinesib), MK-0371, AZD8477, AZ3146 and ARRY-520.

The last-mentioned substances are the so-called kinesin spindle protein inhibitors. Kinesin spindle proteins (KSP, also known as Eg5, HsEg5, KNSL1, or KIF11) are kinesin-like motor proteins essential for the functioning of bipolar mitotic spindles. Inhibition of KSP leads to mitotic arrest and longer apoptosis (Tao et al, cancer cell 2005 Jul 8(1), 39-59). After the discovery of the earliest cell penetrating KSP inhibitors Monastrol, KSP inhibitors have been established as a novel class of chemotherapeutic drugs (Mayer et al, Science 286: 971-974, 1999) and are the subject of a series of patent applications (e.g., WO 2006/044825; WO 2006/002236; WO 2005/051922; WO 2006/060737; WO 03/060064; WO03/040979 and WO 03/049527). However, since KSP is active only during the short period of the mitotic phase, KSP inhibitors must be present at sufficiently high concentrations during this phase. WO2014/151030 discloses ADCs comprising certain KSP inhibitors. ADCs with KSP inhibitors, which also comprise an enzymatically cleavable linker, which however do not have an optimal profile of action, are also disclosed in patent applications WO2015/096982 and WO 2016/096610.

Legumain (Legumain) is a tumor-associated asparaginyl endopeptidase (s. Ishii, Methods enzyme 1994, 244, 604; j.m. Chen et al j. biol. chem. 1997, 272, 8090) and has been used to process prodrugs of small cytotoxic molecules such as doxorubicin and etoposide derivatives and the like (w. Wu et al cancer res. 2006, 66, 970; l. Stern et al Bioconjugate chem. 2009, 20, 500; k.m. bajuri et al mceedd chem 2011, 6, 54).

Other lysosomal enzymes are, for example, cathepsins or glycosidases, for example β -glucuronidase, which have also been used to release active substances by enzymatic cleavage of prodrugs. The group which is enzymatically cleavable in vivo is in particular a 2-8-oligopeptide group or a glycoside. Peptide cleavage sites are disclosed inBioconjugate Chem. 2002, 13, 855 amber 869 andBioorganic & Medicinal Chemistry Letters8 (1998) 3341-3346 andBioconjugate Chem. 1998, 9, 618-626. These include, for example, valine-alanine, valine-lysine, valine-citrulline, alanine-lysine and phenylalanine-lysine (optionally with an additional amide group).

Summary of The Invention

The prior art discloses various antibody-active substance-conjugates with enzymatically cleavable linkers, however they do not have an optimal profile of action, e.g. with respect to their broad utility on different cells. It is therefore an object of the present invention to provide more effective compounds which exhibit a durable apoptotic effect after administration at relatively low concentrations and are therefore useful in the treatment of cancer. Here, in one aspect, the profile of metabolites released from within the ADC cells plays an important role. Typically, the metabolites formed by the ADC are substrates for efflux pumps and/or have high permeability across cell membranes. Both phenomena may lead to short retention times and thus to suboptimal apoptotic effects within the tumor cells.

The subject of the present invention is therefore a binder-active substance-conjugate (ADC) with a specific cluster-linker composition which, in combination with an antibody, has a particularly interesting profile of action in terms of intensity and breadth of action. To further improve the tumor selectivity of ADCs and their metabolites, binder conjugates have been provided with peptide linkers that can be released by tumor-associated lysosomal enzymes such as legumain or cathepsin. Thus, tumor selectivity is determined not only by the selection of antibodies, but also by enzymatic cleavage of peptide derivatives, for example by tumor-associated enzymes such as legumain. Furthermore, the metabolites released from the binding agent-active substance-conjugates (ADCs) according to the invention in tumor cells are also characterized by a property profile of particular interest. They exhibit a low efflux from tumor cells and lead to a high active substance exposure in the tumor. Thus, a high effect is achieved in tumor cells, whereas, due to the poor permeability, only low systemic cytotoxic effects are present, which results in lower off-target toxicity.

Kinesin spindle protein inhibitors used according to the invention have an amino group essential for this effect. By modifying the amino group with a peptide derivative, the effect on kinesin spindle protein is blocked and thus the development of cytotoxic effects is also inhibited. These peptide derivatives may also be part of the linker of the antibody. However, if the peptide residue or peptide linker can be cleaved from the active substance by tumor-associated enzymes such as legumain or cathepsin, the effect can be re-established in the tumor tissue in a targeted manner. The specific property profile of the metabolites formed in the tumor is ensured by further modification of the kinesin spindle protein inhibitor at other positions than the amino group in the molecule, however, the further modification does not compromise the high efficacy at the target.

Furthermore, for certain embodiments, the structure of the ADC according to the invention achieves high loadings of antibody (referred to as DAR, drug to antibody ratio) which surprisingly herein has no negative impact on the physicochemical and pharmacokinetic behavior of the ADC.

Surprisingly, we have now found binding agent-active substance-conjugates of formula (I)

Wherein

X₁Represents N and a nitrogen-containing compound which is a nitrogen-containing compound,

X₂represents N, and

X₃represents C;

or

X₁Represents N and a nitrogen-containing compound which is a nitrogen-containing compound,

X₂represents C, and

X₃represents N;

or

X₁Represents a group selected from the group consisting of CH and CF,

X₂represents C, and

X₃represents N;

or

X₁Which represents a radical of NH or a radical of NH,

X₂represents C, and

X₃represents C;

or

X₁Represents a group of a compound represented by the formula CH,

X₂represents N, and

X₃represents a compound represented by the formula (I),

R¹represents hydrogen or a methyl group,

R²represents methyl, ethyl, -CH₂-CH(CH₃)₂、-CH₂-C (= O) OH or isopropyl,

R³represents methyl, ethyl, -CH₂-CH(CH₃)₂or-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-W，

#-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH₂-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)_2-8 -C(=O)-###，

#-C(=O)- (CH₂)₃-C(=O)-###，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₅-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂) - # # or

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂-CH₂-O)_1-8-(CH₂)₂-NH-C(=O)-CH₂-##，

W represents the following group

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or antigen-binding fragment,

# represents a bond to the compound,

# represents the bond to the sulfur atom of the cysteine side chain of the binding agent,

# # # represents the bond to the nitrogen atom of the lysine side chain of the binder,

as well as salts, solvates and salts of these solvates have superior properties compared to known conjugates.

Preference is given to those binder-active substance-conjugates of the formula (I),

wherein

X₁Represents a group of a compound represented by the formula CH,

X₂represents a compound represented by the formula (I),

X₃represents N and a nitrogen-containing compound which is a nitrogen-containing compound,

R¹represents hydrogen or a methyl group,

R²represents methyl, -CH₂-CH(CH₃)₂、-CH₂-C (= O) OH or isopropyl,

R³represents methyl, -CH₂-CH(CH₃)₂or-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-W，

#-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH₂-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

#-C(=O)- (CH₂)₃-C(=O)-###，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₅-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂) - # # or

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂-CH₂-O)₄-(CH₂)₂-NH-C(=O)-CH₂-##，

W represents the following group

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or antigen-binding fragment,

# represents a bond to the compound,

# represents the bond to the sulfur atom of the cysteine side chain of the binding agent,

# # # represents the bond to the nitrogen atom of the lysine side chain of the binder,

and salts, solvates and salts of such solvates.

Especially preferred are those binding agent-active substance-conjugates of formula (I),

wherein

R¹Represents hydrogen or a methyl group,

R²represents a methyl group or an isopropyl group,

R³represents methyl or-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or antigen-binding fragment,

# represents a bond to the compound,

# # # represents the bond to the nitrogen atom of the lysine side chain of the binder,

and salts, solvates and salts of such solvates.

Very particular preference is given to those binding agent-active substance-conjugates of the formula (I), in which

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or antigen-binding fragment,

# represents a bond to the compound,

# # # represents the bond to the nitrogen atom of the lysine side chain of the binder,

and salts, solvates and salts of such solvates.

Particular preference is given to those binder-active substance-conjugates of the formula (I),

wherein

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 20,

AK represents a binding agent or a derivative thereof, preferably an antibody or antigen-binding fragment,

# represents a bond to the compound,

# # # represents the bond to the nitrogen atom of the lysine side chain of the binder,

and salts, solvates and salts of such solvates.

Selected are those binding agent-active substance-conjugates of formula (I),

wherein

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 20, and

AK represents an anti-CD 123 antibody, an anti-CXCR 5 antibody, an anti-B7H 3 antibody, an anti-TWEAKR antibody, an anti-Her 2 antibody or an anti-EGFR antibody or an antibody fragment representing a binding antigen thereof,

# represents a bond to the compound,

# # represents a bond to a nitrogen atom of a lysine side chain of an Antibody (AK) or an antigen-binding antibody fragment,

and salts, solvates and salts of such solvates.

Selected especially those binding agent-active substance-conjugates of formula (I),

wherein

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 20, and

AK represents an anti-CD 123 antibody selected from TPP-9476, TPP-8988, TPP-8987 and TPP-6013, represents an anti-CXCR 5 antibody selected from TPP-9574 and TPP-9580, represents an anti-B7H 3 antibody TPP-8382, represents an anti-TWEAKR antibody selected from TPP-7006 and TPP-7007, represents an anti-Her 2 antibody TPP-1015 or represents an anti-EGFR antibody TPP-981 or represents antigen-binding antibody fragments thereof,

# represents a bond to the compound,

# # represents a bond to a nitrogen atom of a lysine side chain of an Antibody (AK) or an antigen-binding antibody fragment,

and salts, solvates and salts of such solvates.

Preferred are those binding agent-active agent-conjugates of the above formula, wherein AK represents a binding agent that specifically binds to an extracellular cancer target molecule. In a preferred embodiment, the binding agent is internalized by a target cell by binding after binding to its extracellular target molecule on the target cell. Preferably, the binding agent is an antibody or a fragment that binds an antigen.

In a preferred subject 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 CXCR 5; CD123, CXCR5, and B7H3 are particularly preferred.

In a preferred subject of the invention, the binding agent AK is an anti-CD 123 antibody, an anti-CXCR 5 antibody, an anti-B7H 3 antibody, an anti-TWEAKR antibody, an anti-Her 2 antibody or an anti-EGFR antibody or an antibody fragment thereof binding to an antigen.

Particularly preferred are those binder-active agent-conjugates of the formula, wherein AK (AK1, AK2) represents an antibody selected from TPP-8382 (anti-B7H 3), TPP-6013 (anti-CD 123), TPP-8987 (anti-CD 123), TPP-8988 (anti-CD 123), TPP-9476 (anti-CD 123), TPP-9574 (anti-CXCR 5) and TPP-9580 (anti-CXCR 5), or an antigen-binding fragment thereof. Here, antibodies TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD 123) are preferred. The exact structure (sequence) of these antibodies can be found in the table: protein sequence of the antibody, text and sequence listing behind this table.

Particularly preferred are those binder-active agent-conjugates of formula (I) wherein AK represents an antibody selected from TPP-8382 (anti-B7H 3), TPP-6013 (anti-CD 123), TPP-8987 (anti-CD 123), TPP-8988 (anti-CD 123), TPP-9476 (anti-CD 123), TPP-9574 (anti-CXCR 5) and TPP-9580 (anti-CXCR 5). Here, Antibodies (AK) TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD 123) are preferred.

Drawings

FIG. 1: an annotation sequence of a preferred antibody of the binding agent-active substance-conjugate. Shown are the protein sequences of the heavy and light chains of IgG, as well as the VH and VL regions of these antibodies. The important regions (VH and VL regions in IgG, and CDR regions (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3)) are annotated below the sequence.

FIG. 2: sequence listing of preferred antibodies of binder-active substance-conjugate and sequence listing of target protein.

Detailed Description

The present invention provides conjugates of a binding agent or derivative thereof and one or more active agent molecules, wherein the active agent molecule is a kinesin spindle protein inhibitor (KSP inhibitor).

The following describes the binding agents that may be used according to the present invention, the KSP inhibitors that may be used according to the present invention, and the linkers that may be used according to the present invention, which may be used in combination without limitation. In particular, the binding agents indicated as preferred or particularly preferred in each case can be used in combination with the KSP inhibitors indicated as preferred or particularly preferred in each case, optionally in combination with the linkers indicated as preferred or particularly preferred in each case.

Particularly preferred KSP inhibitor-conjugates (binder-active agent-conjugates)

Particularly preferred KSP inhibitor-conjugates according to the invention are those in which AK (AK)₁、AK₂) Represents a binding agent or a derivative thereof (preferably an antibody), and n represents a number from 1 to 50, preferably from 1 to 20, preferably from 1 to 8, particularly preferably from 4 to 8. AK (alkyl ketene dimer)₁Preferably represents an antibody that binds to a KSP inhibitor via a cysteine residue; AK (alkyl ketene dimer)₂Preferably represents an antibody that binds to a KSP inhibitor via a lysine residue. The binding agents or antibodies used herein are preferably those described as preferred in the specification.

Here, the following binder-active substance-conjugates are particularly preferred:

。

preferred are those binder-active agent-conjugates of the formula, wherein AK (AK1, AK2) represents a binder that specifically binds to an extracellular cancer target molecule. In a preferred embodiment, the binding agent is internalized by a target cell by binding after binding to its extracellular target molecule on the target cell.

In a preferred subject 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 B7H 3.

In a preferred subject of the invention, said binder AK (AK)₁、AK₂) Is an anti-CD 123 antibody, an anti-CXCR 5 antibody, an anti-B7H 3 antibody, an anti-TWEAKR antibody, an anti-Her 2 antibody, or an anti-EGFR antibody or antigen-binding antibody fragment thereof.

Particularly preferred are those binder-active agent-conjugates of the formula, wherein AK (AK1, AK2) represents an antibody selected from TPP-8382 (anti-B7H 3), TPP-6013 (anti-CD 123), TPP-8987 (anti-CD 123), TPP-8988 (anti-CD 123), TPP-9476 (anti-CD 123), TPP 9574 (anti-CXCR 5) and TPP-9580 (anti-CXCR 5), or an antigen-binding fragment thereof. Here, antibodies TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD 123) are preferred. The exact structure (sequence) of these antibodies can be found in the table: protein sequence of the antibody, text and sequence listing behind this table.

Preparation of KSP inhibitor-linker-intermediates and conjugates

The conjugates according to the invention are prepared as follows: by first providing a linker for its low molecular weight KSP inhibitor. The intermediate obtained in this way is then reacted with a binding agent, preferably an antibody.

For intermediates conjugated to lysine residues and subsequent conjugation to antibodies, the reaction can be illustrated as follows:

in the above reaction scheme, X₁、X₂、X₃、R¹、R²、R³And AK₂Has the meaning given in formula (I), and where R⁴Represents methyl, and n represents 0 or 1.

The synthesis of building block A has been described in WO 2015/096982. Peptide derivatives B and C were prepared by classical methods of peptide chemistry. Intermediates C and D were coupled using HATU in DMF in the presence of N, N-diisopropylethylamine at room temperature. Subsequently, the benzyloxycarbonyl protecting group and the benzyl ester were cleaved hydrogenolytically on 10% palladium on activated carbon. The fully deprotected intermediate is then reacted with 1,1' - [ (1, 5-dioxopentane-1, 5-diyl) bis (oxy) ] dipyrrolidine-2, 5-dione in the presence of N, N-diisopropylethylamine in DMF at room temperature to give ADC precursor molecule E. The activated ester was then conjugated to the corresponding antibody as described in section B-5.

For intermediates conjugated to cysteine residues and subsequent conjugation to antibodies, the reaction can be illustrated as follows:

in the above reaction scheme, X₁、X₂、X₃、R¹、R²、R³And AK₁Has the meaning given in formula (I), and where R⁴Represents methyl and n represents 1.

Using a similar procedure, compounds in which n represents 0 can also be prepared.

The synthesis of building block A has been described in WO 2015/096982. Peptide derivatives B and C were prepared by classical methods of peptide chemistry. Intermediates C and D were coupled using HATU in DMF in the presence of N, N-diisopropylethylamine at room temperature. Subsequently, the benzyloxycarbonyl protecting group and the benzyl ester were cleaved hydrogenolytically on 10% palladium on activated carbon. The fully deprotected intermediate is then reacted with 1- {6- [ (2, 5-dioxopyrrolidin-1-yl) oxy ] -6-oxohexyl } -1H-pyrrole-2, 5-dione in the presence of N, N-diisopropylethylamine in DMF at room temperature to give ADC precursor molecule E. The maleimide derivative was then conjugated to the corresponding antibody as described in section B-4.

Depending on the linker, the succinimide-linked ADC can be converted to an open-chain succinamide after conjugation, which has a favorable stability profile.

The reaction (ring opening) can be effected at a pH of 7.5 to 9, preferably pH8 at a temperature of 25 ℃ to 37 ℃, for example by stirring. The preferred stirring time is 8 to 30 hours.

For intermediates coupled to cysteine residues and subsequent coupling to antibodies and subsequent ring opening of the succinimide ring, the reaction can be illustrated as follows:

in the above reaction scheme, X₁、X₂、X₃、R¹、R²、R³And AK₁Has the meaning given in formula (I), and where R⁴Represents methyl, and n represents 0 or 1.

The synthesis of building block A has been described in WO 2015/096982. Peptide derivatives B and C were prepared by classical methods of peptide chemistry. Intermediates C and D were coupled using HATU in DMF in the presence of N, N-diisopropylethylamine at room temperature. Subsequently, the benzyloxycarbonyl protecting group and the benzyl ester were cleaved hydrogenolytically on 10% palladium on activated carbon. The fully deprotected intermediate is 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 ADC precursor molecule E. The maleimide derivative is then conjugated to the corresponding antibody at small-scale or medium-scale conjugation as described in section B-4.

Binding agents

In the broadest sense, the term "binding agent" is understood to mean a molecule that binds to a target molecule present on the particular target cell population to which the binding agent-active substance-conjugate is directed. The term binding agent is to be understood in its broadest sense and also includes e.g. lectins, proteins capable of binding to specific sugar chains or phospholipid binding proteins. Such binding agents include, for example, high molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (e.g., aptamers (US5,270,163) (reviewed by Keefe AD. et al, nat. rev. Drug discov. 2010; 9:537-550) or vitamins), and all other cell binding molecules or substances. Binding proteins are, for example, antibodies and antibody fragments or mimetics, such as Affibodies (Affibodies), adnectins, anticollins, darpins, avimers, nanobodies (reviewed by Gebauer m et al, current. Opinion in chem. biol. 2009; 13: 245-. Binding peptides are, for example, ligands of a ligand/receptor pair, e.g., VEGF of a ligand/receptor pair VEGF/KDR, transferrin of a ligand/receptor pair transferrin/transferrin receptor, or a cytokine/cytokine receptor, e.g., TNF α of a ligand/receptor pair TNF α/TNF α receptor.

The binding agent may be a binding protein. Preferred embodiments of the binding agent are an antibody, an antibody fragment that binds an antigen, a multispecific antibody or a mimobody.

The literature also discloses various possibilities for covalent coupling (conjugation) of organic molecules to binding agents and in particular antibodies. It is preferred according to the present invention that the poison cluster is conjugated to the antibody via one or more sulphur atoms of cysteine residues of the antibody and/or via one or more NH groups of lysine residues of the antibody. However, the toxic cluster may also be bound to the antibody via a free carboxyl group of the antibody or via a sugar residue.

"target molecule" is understood in the broadest sense to mean a molecule present in a target cell population and may be a protein (e.g. a receptor for a growth factor) or a non-peptide molecule (e.g. a sugar or a phospholipid). It is preferably a receptor or antigen.

The term "extracellular" target molecule describes a target molecule that is located outside a cell, attached to the cell, or part of a target molecule that is located outside a cell, i.e., a binding agent can bind to its extracellular target molecule on an intact cell. The extracellular target molecule may be anchored in or be a component of the cell membrane. Those skilled in the art are aware of methods for identifying extracellular target molecules. For proteins, this can be done by determination of the transmembrane domain(s) and the orientation of the protein in the membrane. These data are typically stored in protein databases (e.g., SwissProt).

The term "cancer target molecule" describes a target molecule that is present in greater amounts on one or more cancer cell types than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell species as compared to non-cancer cells of the same tissue type, wherein selectivity describes at least a two-fold enrichment on cancer cells as compared to non-cancer cells of the same tissue type ("selective cancer target molecule"). The use of cancer target molecules allows selective treatment of cancer cells by the conjugates according to the invention.

The binding agent may be attached to the linker via a bond. The binding agent may be attached via a heteroatom of the binding agent. Heteroatoms which may be used for attachment of the binding agent according to the invention are sulfur (in one embodiment via a thiol group of the binding agent), oxygen (in accordance with the invention via a carboxyl or hydroxyl group of the binding agent) and nitrogen (in one embodiment via a primary or secondary amino or amide group of the binding agent). These heteroatoms may be present in the natural binder or introduced by chemical or molecular biological methods. According to the present invention, the attachment of the binding agent to the toxic cluster has only a slight effect on the binding activity of the binding agent to the target molecule. In a preferred embodiment, the linkage has no effect on the binding activity of the binding agent on the target molecule.

According to the present invention, the term "antibody" is to be understood in its broadest sense and includes immunoglobulin molecules, such as intact or modified monoclonal, polyclonal or multispecific antibodies (e.g., bispecific antibodies). The immunoglobulin molecule preferably comprises a molecule having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), typically connected by disulfide bridges. Each heavy chain comprises a heavy chain variable domain (abbreviated VH) and a heavy chain constant domain. The heavy chain constant domain may, for example, comprise three domains CH1, CH2 and CH 3. Each light chain comprises a variable domain (abbreviated VL) and a constant domain. The light chain constant domain comprises a domain (abbreviated CL). VH and VL domains can be further subdivided into regions of hypervariability, also known as complementarity determining regions ("complementarity determining regions"), and regions of low sequence variability ("framework regions", abbreviated as FRs). Typically, each VH and VL region is composed of three CDRs and up to four FRs. For example, from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. Antibodies may be obtained from every species suitable for this, e.g. rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin. The antibody may be, for example, human, humanized or chimeric.

The term "monoclonal" antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for naturally occurring mutations (which may occur in minor numbers). Monoclonal antibodies recognize a single antigen binding site with high specificity. The term monoclonal antibody does not relate to a specific method of preparation.

The term "intact" antibody refers to an antibody comprising a domain that binds an antigen and constant domains of a light chain and a heavy chain. The constant domains may be naturally occurring domains or variants thereof that vary in amino acid positions, and may also be aglycosylated.

The term "modified intact" antibody refers to intact antibodies fused via their amino-or carboxy-termini to another polypeptide or protein not derived from the antibody by means of a covalent bond (e.g., a peptide bond). In addition, antibodies can be modified such that a reactive cysteine is introduced at a specific position to facilitate coupling to a toxic cluster (see Junutula et al, NatBiotechnol. 2008 Aug; 26(8): 925-32).

"amino acid modification" or "mutation" herein refers to amino acid substitution, insertion and/or deletion in a polypeptide sequence. Preferred amino acid modifications herein are substitutions. "amino acid substitution" or "substitution" herein refers to the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W describes a variant of the parent polypeptide in which the tyrosine at position 50 is replaced by a tryptophan. A "variant" of a polypeptide describes a polypeptide having substantially the same amino acid sequence as a reference polypeptide, typically a native or "parent" polypeptide. A polypeptide variant may have one or more amino acid substitutions, deletions and/or insertions at specific positions in the native amino acid sequence.

The term "human" antibody refers to an antibody that is obtainable from, or is a synthetic human antibody, "synthetic" human antibody is an antibody that is partially or even fully obtainable from synthetic sequences based on analysis of human antibody sequences via computer modeling.

The term "humanized" or "chimeric" antibody describes an antibody that consists of non-human and human portions of sequence. In these antibodies, a portion of the sequence of a human immunoglobulin (the acceptor) is replaced by a portion of the sequence of a non-human immunoglobulin (the donor). In many cases, the donor is a murine immunoglobulin. In the case of humanized antibodies, the amino acids of the CDRs of the acceptor are replaced by the amino acids of the donor. Sometimes, the amino acids of the framework are also replaced by the corresponding amino acids of the donor. In some cases, the humanized antibody contains amino acids that are not included in either the recipient or the donor and are introduced during optimization of the antibody. In the case of chimeric antibodies, the variable domain of the donor immunoglobulin is fused to the constant region of a human antibody. Such "humanized" and "chimeric" antibodies also include aglycosylated variants prepared by deglycosylation from PNGaseF or by mutation of N297(Kabat numbering) of the heavy chain to any other amino acid.

The term Complementarity Determining Region (CDR) as used herein refers to those amino acids of the variable antibody domain that are required for binding to an antigen. Typically, each variable region has three CDR regions, which are referred to as CDR1, CDR2, and CDR 3. Each CDR region may comprise amino acids according to the Kabat definition and/or amino acids according to the hypervariable loop defined by Chotia. The region comprising, for example, approximate amino acid positions 24-34(CDR1), 50-56(CDR2) and 89-97(CDR3) of the variable light chain/domain (VL) and the regions of approximate amino acid positions 31-35(CDR1), 50-65(CDR2) and 95-102(CDR3) of the variable heavy chain/domain (VH) according to the definition of Kabat (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). According to the definition of Chotia, the region comprising, for example, the approximate amino acid positions 26-32(CDR1), 50-52(CDR2) and 91-96 (CDR3) of the variable light chain (VL) and the approximate amino acid positions 26-32(CDR1), 53-55(CDR2) and 96-101(CDR3) of the variable heavy chain (VH) are included (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some cases, the CDRs may comprise amino acids from CDR regions defined according to Kabat and Chotia.

Antibodies can be classified into different classes according to the amino acid sequence of the heavy chain constant domain. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, several of which can be divided into further subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant domains corresponding to the different species are called [ alpha/α ], [ delta/δ ], [ epsilon/ε ], [ gamma/γ ], and [ my/μ ]. The three-dimensional structure and subunit structure of antibodies are known.

The term "functional fragment" or "antigen-binding antibody fragment" of an antibody/immunoglobulin is defined as an antibody/immunoglobulin fragment that still comprises the antigen-binding domain of the antibody/immunoglobulin (e.g., the variable domain of an IgG). An "antigen-binding domain" of an antibody typically comprises one or more hypervariable regions of the antibody, for example the CDR, CDR2 and/or CDR3 regions. However, the "framework" or "framework" regions of an antibody may also play a role in binding the antibody to an antigen. The framework regions constitute the framework of the CDRs. The antigen-binding domain preferably 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 amino acids 4 to 111 of the variable heavy chain, particularly preferably the complete variable light and heavy chains, i.e. amino acids 1-109 of the VL and 1 to 113 of the VH (numbering according to WO 97/08320).

"functional fragments" or "antigen-binding antibody fragments" of the present invention include, but are not limited to, Fab ', F (ab')₂And Fv fragments, diabodies, single domain antibodies (DAb), linear antibodies, single chain antibodies (single chain Fv, abbreviated as scFv); and multispecific antibodies, such as bi-and trispecific antibodies, formed from Antibody fragments, c.a. K Borrebaeck editors (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford university press; r. Kontermann&S. Duebel, edit (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). Antibodies other than "multispecific" or "multifunctional" other antibodies are those with the same binding site. Multispecific antibodies may be specific for different epitopes of an antigen or may be specific for epitopes of more than one antigen (see, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al, 1991, j. immunol. 147: 6069; U.S. patent No. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al, 1992, j. immunol. 148: 15471553). Can construct F (ab')₂Or a Fab molecule, such that the number of intermolecular disulfide interactions that occur between Ch1 and CL domains can be reduced or completely prevented.

An "epitope" refers to a protein determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitopic determinants are typically composed of chemically active surface groups of molecules such as amino acids or sugar side chains or combinations thereof, and typically have specific three-dimensional structural properties as well as specific charge properties.

A "functional fragment" or "antigen-binding antibody fragment" may be fused via its amino-or carboxy-terminus by means of a covalent bond (e.g. a peptide bond) to another polypeptide or protein not derived from an antibody. In addition, antibodies and antigen-binding fragments can be modified as follows to allow the introduction of reactive cysteines at specific positions to facilitate coupling to the toxic cluster (see Junutula et al, Nat Biotechnol. 2008 Aug; 26(8): 925-32).

Monoclonal antibodies can be prepared by methods known to those of ordinary skill in the art (K ö hler and Milstein, Nature, 256, 495-497, 1975.) human and humanized monoclonal antibodies can be prepared by methods known to those of ordinary skill in the art (Olsson et al, MethEnzymol. 92, 3-16, or Cabilly et al, U.S. Pat. No. 4,816,567, or Boss et al, U.S. Pat. No. 4,816,397).

Those of ordinary skill in the art will recognize various methods for making human antibodies and fragments thereof, for example, by transgenic mice (N Lonberg and D Huszar, Int Rev immunol. 1995; 13(1):65-93) or phage display technology (Clackson et al, Nature 1991, 8/15; 352(6336): 624-8). The antibodies of the invention can be obtained, for example, from a recombinant antibody library based on the amino acid sequences of a variety of antibodies established from a large number of healthy volunteers. Antibodies can also be prepared by known recombinant DNS techniques. The nucleic acid sequence of the antibody may be obtained by conventional sequencing or may be obtained from a publicly accessible database.

The "isolated" antibody or binding agent has been purified to remove other components of the cell. Contaminating components of the cells that may interfere with diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptidic or non-peptidic components of the cells. Preferred are antibodies or binding agents that have been purified to a degree of greater than 95% by weight (e.g., as determined by Lowry method, UV-Vis spectroscopy, or by SDS capillary gel electrophoresis) based on the antibody or binding agent. Furthermore, an antibody which has been purified so that at least 15 amino acids of the amino-terminal or internal amino acid sequence can be determined or which has been purified to homogeneity, wherein homogeneity is determined by SDS-PAGE under reducing or non-reducing conditions (which can be detected by means of Coomassie blue staining or preferably by silver staining). However, antibodies are typically prepared by one or more purification steps.

The term "specifically binds" or "specifically binds" refers to binding to a predetermined targetAn antibody or binding agent to an antigen/target molecule. Specific binding of an antibody or binding agent is generally described as having a binding capacity of at least 10^-7Affinity of M (as Kd value; i.e.preferably having a ratio of 10)^-7M smaller Kd value) of an antibody or binding agent, wherein the antibody or binding agent has at least twice the affinity for a 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 binding agent does not preclude the binding of the antibody or binding agent to multiple antigens/target molecules (e.g., orthologs of different species). The antibody preferably has a mass of at least 10^-7M (as Kd value; i.e. preferably having a ratio of 10)^-7Those with Kd values smaller than M), preferably at least 10^-8M, particularly preferably 10^-9M to 10^-11Affinity of M. The Kd values can be determined, for example, by means of surface plasmon resonance spectroscopy.

The antibody-active substance-conjugates of the invention likewise exhibit affinities within these ranges. The affinity is preferably substantially unaffected by the conjugation of the active substance (in general, the decrease in affinity is less than an order of magnitude, in other words, for example, up to 10^-8M is reduced to 10^-7 M)。

The antibodies used according to the invention are also preferably characterized by high selectivity. High selectivity exists when the antibodies according to the invention exhibit an affinity for the target protein which is at least 2-fold, preferably 5-fold or particularly preferably 10-fold higher than for other antigens which are independent of one another, such as human serum albumin, for example, the affinity can be determined, for example, by means of surface plasmon resonance spectroscopy.

Furthermore, the antibodies used according to the invention are preferably cross-reactive. In order to be able to simplify and better explain preclinical studies, such as toxicological or utility studies (for example in xenografted mice), it is advantageous if the antibodies used according to the invention bind not only human target proteins but also species target proteins in the species used for the study. In one embodiment, the antibodies used according to the invention are cross-reactive to a target protein of at least one other species in addition to the human target protein. For toxicology and utility studies, it is preferred to use species of the rodent, canine and non-human primates families. Preferred rodent species are mouse and rat. Preferred non-human primates are macaques, orangutans and macaques with a long tail.

In one embodiment, the antibody used according to the invention is cross-reactive, in addition to the human target protein, to a target protein of at least one other species selected from the group consisting of mouse, rat and cynomolgus monkey (cynomolgus monkey). Especially preferred are antibodies for use according to the invention which are cross-reactive at least with respect to a mouse target protein in addition to a human target protein. Preferred are cross-reactive antibodies whose affinity for the target protein of other non-human species differs from that of the human target protein by no more than 50-fold, in particular by no more than 10-fold.

Antibodies against cancer target molecules

The target molecule against which the binding agent, e.g. antibody or antigen-binding fragment thereof, is directed is preferably a cancer target molecule. The term "cancer target molecule" describes a target molecule that is present in greater amounts on one or more cancer cell types than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell species as compared to non-cancer cells of the same tissue type, wherein selectivity describes at least a two-fold enrichment on cancer cells as compared to non-cancer cells of the same tissue type ("selective cancer target molecule"). The use of cancer target molecules allows selective treatment of cancer cells by the conjugates according to the invention.

Antibodies specific for antigens, e.g. cancer cell antigens, can be prepared by a person of ordinary skill in the art by means of methods known to him (e.g. recombinant expression) or can be obtained commercially (e.g. from Merck KGaA, germany). Examples of known commercially available antibodies in cancer therapy are Erbitux (cetuximab, Merck KGaA), Avastin (Bevacizumab, Roche) and Herceptin (trastuzumab, Genettech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1 kappa type that binds with high affinity (Kd = 5 nM) to the extracellular domain of the human epidermal growth receptor in a cell-based assay. The antibody was recombinantly produced in CHO cells. All these antibodies can also be prepared as aglycosylated variants of these antibodies by either deglycosylation with PNGase F or mutation to any amino acid by N297(Kabat numbering) of the heavy chain.

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

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

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

Cancer target molecules are known to those skilled in the art. Examples of which are listed below.

Examples of cancer target molecules are:

(1) EGFR (EGF receptor, NCBI reference NP-005219.2, NCBI Gene ID: 1956)

(2) Mesothelin (SwissProt Reference Q13421-3), wherein mesothelin is encoded by amino acids 296-598. Amino acids 37-286 encode megakaryocyte-enhancing factors. Mesothelin is anchored in the cell membrane by a GPI anchor and is localized extracellularly.

(3) Carbonic anhydrase IX (CA9, SwissProt Reference Q16790), NCBI Gene ID: 768)

(4) C4.4a (NCBI reference NP-055215.2; synonyms LYPD3, NCBI Gene ID: 27076)

(5) CD52 (NCBI reference NP-001794.2)

(6) Her2 (ERBB2; NCBI reference sequence NP-004439.2; NCBI Gene ID: 2064)

(7) CD20 (NCBI reference NP-068769.2)

(8) Lymphocyte activation antigen CD30 (SwissProt ID P28908)

(9) Lymphocyte adhesion molecule CD22 (SwissProt ID P20273; NCBI Gene ID: 933)

(10) Bone marrow cell surface antigen CD33 (SwissProt ID P20138; NCBI Gene ID: 945)

(11) Transmembrane glycoprotein NMB (GPNMB, SwissProt ID Q14956, NCBI Gene ID: 10457)

(12) Adhesion molecule CD56 (SwissProt ID P13591)

(13) Surface molecule CD70 (SwissProt ID P32970, NCBI Gene ID: 970)

(14) Surface molecule CD74 (SwissProt ID P04233, NCBI Gene ID: 972)

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

(16) Surface protein mucin-1 (MUC1, SwissProt ID P15941, NCBI Gene ID: 4582)

(17) Surface protein CD138 (SwissProt ID P18827)

(18) Integrin alphaV (NCBI reference sequence: NP-002201.1, NCBI Gene ID: 3685)

(19) Teratocarcinoma-derived growth factor 1 protein TDGF1 (NCBI reference sequence: NP-003203.1, NCBI Gene ID: 6997)

(20) Prostate specific membrane antigen PSMA (Swiss Prot ID: Q04609; NCBI Gene ID: 2346)

(21) Tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317, NCBI Gene ID: 1969)

(22) Surface protein SLC44A4 (NCBI reference sequence: NP-001171515.1, NCBI Gene ID: 80736)

(23) Surface protein BMPR1B (SwissProt: O00238)

(24) Transporter SLC7A5 (SwissProt: Q01650)

(25) Epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8, Gene ID: 26872)

(26) Ovarian cancer antigen MUC16 (SwissProt: Q8WXI7, Gene ID: 94025)

(27) Transporter SLC34A2 (SwissProt: O95436, Gene ID: 10568)

(28) Surface protein SEMA5b (SwissProt: Q9P283)

(29) Surface protein LYPD1 (SwissProt: Q8N2G4)

(30) Endothelin receptor type B EDNRB (SwissProt: P245630, NCBI Gene ID: 1910)

(31) Ring finger protein RNF43 (SwissProt: Q68DV7)

(32) Prostate cancer associated protein STEAP2 (SwissProt: Q8NFT2)

(33) Cation channel TRPM4 (SwissProt: Q8TD43)

(34) Complement receptor CD21 (SwissProt: P20023)

(35) B-cell antigen receptor complex associated protein CD79B (SwissProt: P40259, NCBI Gene ID: 974)

(36) Cell adhesion antigen CEACAM6 (SwissProt: P40199)

(37) Dipeptidase DPEP1 (SwissProt: P16444)

(38) Interleukin receptor IL20R alpha (SwissProt: Q9UHF4, NCBI Gene ID: 3559)

(39) Proteoglycan BCAN (SwissProt: Q96GW7)

(40) EpHB2 as ephrin receptor (SwissProt: P29323)

(41) Prostate stem cell-associated protein PSCA (NCBI reference: NP-005663.2)

(42) Surface protein LHFPL3 (SwissProt: Q86UP9)

(43) Receptor protein TNFRSF13C (SwissProt: Q96RJ3)

(44) B-cell antigen receptor complex associated protein CD79a (SwissProt: P11912)

(45) Receptor protein CXCR 5(CD 185; SwissProt: P32302; NCBI Gene ID 643, NCBI reference sequence: NP-001707.1)

(46) Ion channel P2X5 (SwissProt: Q93086)

(47) Lymphocyte antigen CD180 (SwissProt: Q99467)

(48) Receptor protein FCRL1 (SwissProt: Q96LA6)

(49) Receptor protein FCRL5 (SwissProt: Q96RD9)

(50) MHC class II molecule Ia antigen HLA-DOB (NCBI reference sequence: NP-002111.1)

(51) T-cell protein VTCN1 (SwissProt: Q7Z7D3)

(52) TWEAKR (FN14, TNFRSF12A, NCBI reference sequence: NP-057723.1, NCBI Gene ID: 51330)

(53) Lymphocyte antigen CD37 (Swiss Prot: P11049, NCBI Gene ID: 951)

(54) FGF receptor 2, FGFR2 (NCBI Gene ID: 2263; official notation: FGFR 2). The FGFR2 receptor occurs in different splice variants (α, β, IIIb, IIIc). All splice variants can serve as target molecules.

(55) Transmembrane glycoprotein B7H3 (CD276; NCBI Gene ID: 80381 NCBI reference sequence: NP-001019907.1, Swiss Prot: Q5ZPR3-1)

(56) B cell receptor BAFFR (CD268; NCBI Gene ID:115650)

(57) Receptor protein ROR 1 (NCBI Gene ID: 4919)

(58) Surface receptor CD123 (IL3RA; NCBI Gene ID: 3563; NCBI reference sequence: NP-002174.1; Swiss-Prot: P26951)

(59) Receptor protein syncytial (NCBI Gene ID 30816)

(60) Aspartic acid beta-hydroxylase (ASPH; NCBI Gene ID 444)

(61) Cell surface glycoprotein CD44 (NCBI Gene ID: 960)

(62) CDH15 (cadherin 15, NCBI Gene ID: 1013)

(63) Cell surface glycoprotein CEACAM 5(NCBI Gene ID: 1048)

(64) Cell adhesion molecule L1-like (CHL1, NCBI Gene ID: 10752)

(65) The somatic tyrosine kinase c-Met (NCBI Gene ID: 4233)

(66) notch ligand DLL3 (NCBI Gene ID: 10683)

(67) Ephrin A4 (EFNA4, NCBI Gene ID: 1945)

(68) Isonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3, NCBI Gene ID: 5169)

(69) Blood coagulation factor III (F3, NCBI Gene ID: 2152)

(70) FGF receptor 3 (FGFR3, NCBI Gene ID: 2261)

(71) Folic acid hydrolase FOLH1 (NCBI Gene ID: 2346)

(72) Folate receptor 1 (FOLR1; NCBI Gene ID: 2348)

(73) Guanylate cyclase 2C (GUCY2C, NCBI Gene ID: 2984)

(74) KIT proto-oncogene receptor tyrosine kinase (NCBI Gene ID: 3815)

(75) Lysosomal associated Membrane protein 1 (LAMP1, NCBI Gene ID: 3916)

(76) Lymphocyte antigen 6 complex, locus E (LY6E, NCBI Gene ID: 4061)

(77) Protein NOTCH3 (NCBI Gene ID: 4854)

(78) Protein tyrosine kinase 7 (PTK7, NCBI Gene ID: 5754)

(79) Binder cell adhesion molecule 4 (PVRL4, NECTN 4, NCBI Gene ID: 81607)

(80) Transmembrane protein syndecan 1 (SDC1, NCBI Gene ID: 6382)

(81) SLAM family member 7 (SLAMF7, NCBI Gene ID: 57823)

(82) Transporter SLC39A6 (NCBI Gene ID: 25800)

(83) SLIT-and NTRK-like family member 6 (SLITRK6, NCBI Gene ID: 84189)

(84) Cell surface receptor TACTD 2 (NCBI Gene ID: 4070)

(85) Receptor protein TNFRSF8 (NCBI Gene ID: 943)

(86) The receptor protein TNFSF13B (NCBI Gene ID: 10673)

(87) Glycoprotein TPBG (NCBI Gene ID: 7162)

(88) Cell surface receptor TROP2 (TACTD 2, NCBI Gene ID: 4070)

(89) Galanin-like G protein-coupled receptor KISS1R (GPR54, NCBI Gene ID: 84634)

(90) The transporter SLAMF6 (NCBI Gene ID: 114836).

In a preferred subject 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 B7H 3.

In another particularly preferred subject matter of the invention, the binding agent binds to an extracellular cancer target molecule selected from the group consisting of: the cancer target molecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particular CD123, CXCR5 and B7H 3.

In another particularly preferred subject matter of the invention, the binding agent specifically binds to an extracellular cancer target molecule selected from the group consisting of cancer target molecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particular CD123, CXCR5 and B7H 3. In a preferred embodiment, the binding agent is internalized by the target cell upon binding to its extracellular target molecule on the target cell. This results in the uptake of the binder-active agent-conjugate (which may be an immunoconjugate or ADC) by the target cell. The binding agent is then preferably treated intracellularly, preferably with the aid of lysosomes.

In one embodiment, the binding agent is a binding protein. In a preferred embodiment, the binding agent is an antibody, an antibody fragment that binds an antigen, a multispecific antibody or a mimobody.

Preferred mimetibodies are affibodies, adnectins, anticalins, darpins, avimers or nanobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies.

In a preferred embodiment, the binding agent is an antibody or an antibody fragment that binds an antigen, more preferably an isolated antibody or an isolated antibody fragment that binds an antigen.

Preferred antigen-binding antibody fragments are Fab, Fab ', F (ab')₂And Fv fragments, diabodies, DAb, linear antibodies, and scFv. Especially preferred are Fab, diabody and scFv.

In a particularly preferred embodiment, the binding agent is an antibody. Particularly preferred are monoclonal antibodies or antibody fragments that bind to an antigen. More particularly preferred are antibody fragments of human, humanized or chimeric antibodies or binding antigens thereof.

Antibodies that bind to cancer target molecules or antibody fragments that bind to antigens can be prepared by one of ordinary skill in the art using known methods, such as chemical synthesis or recombinant expression. Binding agents for cancer target molecules are commercially available or can be prepared by one of ordinary skill in the art using known methods, such as chemical synthesis or recombinant expression. Other methods for preparing antibodies or antibody fragments that bind to antigens are described in WO2007/070538 (see page 22 for "antibodies"). The person skilled in the art knows how to build so-called phage display libraries (e.g. Morphosys HuCAL Gold) and use them for the discovery of antibodies or antigen-binding antibody fragments (see WO2007/070538, AK example 1 on page 24 and thereafter and on page 70, AK example 2 on page 72). Other methods for preparing antibodies using DNA libraries from B cells are described, for example, on page 26 (WO 2007/070538). Methods for humanizing antibodies are described on pages 30-32 of WO2007070538 and described in detail in Queen et al, Pros. Natl. Acad. Sci. USA 86:10029-10033, 1989 or WO 90/0786. Furthermore, Methods for recombinant expression of proteins in general, and Antibodies in particular, are known to those skilled in the art (see, e.g., Berger and Kimrnel (Guide to Molecular Cloning technologies, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al, (Molecular Cloning: A Laboratory Manual, (second edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3), Current Protocols in Molecular Biology, (F.M. Austabel et al [ eds. ], Current Protocols, Green Publishing industries, Inc./John Wiley & Man, Man.; Antibodies et al, (monomer in Biology; wild animal: 19881; wild animal, Inc.; wild animal & S.; plant Laboratory A.; plant et al; wild animal & S.; plant et al; wild animal & S.; plant Laboratory A.; plant et al; wild animal & S. 1998), cold Spring Harbor Laboratory Press (1998)). The skilled person is aware of the corresponding vectors, promoters and signal peptides necessary for the expression of the protein/antibody. Common methods are also described in WO2007/070538, pages 41-45. Methods for preparing IgG1 antibodies are described, for example, on page 74 of WO2007/070538 and example 6 thereafter. Methods that can be used to determine the internalization of an antibody upon binding to its antigen are known to those skilled in the art and are described, for example, in WO2007/070538, page 80. The skilled person is able to use the method described in WO2007/070538, which has been used to prepare carbonic anhydrase ix (mn) antibodies similarly to the preparation of antibodies with other target molecule specificities.

Bacterial expression

One skilled in the art knows in which way an antibody, antigen-binding fragment thereof or variant thereof can be made by means of bacterial expression.

Expression vectors suitable for bacterial expression of a desired protein are constructed by inserting a DNA sequence encoding the desired protein in a functional reading frame, together with appropriate translation initiation and termination signals, and with a functional promoter. The vector comprises one or more phenotypically selectable markers and an origin of replication, such thatAllowing the vector to be retained and self-amplified in the host if desired. Prokaryotic cell hosts suitable for transformation include, but are not limited to, E.coli, Bacillus subtilis (B.) (Bacillus subtilis) Salmonella typhimurium (Salmonella typhimurium) And from the genus Pseudomonas (Pseudomonas) Streptomyces (I), (II)Streptomyces) And Staphylococcus genus (Staphylococcus) The various strains of (1). Bacterial vectors may be based, for example, on phage, plasmid or phagemid. These vectors may contain a selectable marker and a bacterial origin of replication, which may be derived from commercially available plasmids. Many commercially available plasmids generally 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.

After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed (de-reprimert)/induced by suitable means, such as by changing the temperature or chemical induction, and the cells are cultured for an additional period of time. The cells are typically harvested by centrifugation, digested by physical or chemical means if necessary (aufgeschlossen), and the resulting crude extract is retained for further purification.

Accordingly, another embodiment of the present invention is an expression vector comprising a nucleic acid encoding the novel antibody of the present invention.

Fragments of the antibodies or binding antigens of the invention include naturally purified products, products derived from chemical synthesis, and products produced by recombinant techniques in prokaryotic hosts such as E.coli, Bacillus subtilis, Salmonella typhimurium, and various species from the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably E.coli.

Mammalian cell expression

One skilled in the art knows how to prepare antibodies, antigen-binding fragments thereof, or variants thereof by expression in mammalian cells.

Preferred regulatory sequences for expression in a mammalian cell host include viral elements that result in high expression in mammalian cells, such as promoters and/or expression amplicons derived from Cytomegalovirus (CMV) (e.g., the CMV promoter/enhancer), simian virus 40(SV40) (e.g., the SV40 promoter/enhancer), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyoma viruses. Expression of the antibody may 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 description of viral regulatory elements and their sequences reference is made to, for example, U.S.5,168,062 by Stinski, U.S.4,510,245 by Bell et al, and U.S.4,968,615 by Schaffner et al. Recombinant expression vectors can likewise include an origin of replication and a selectable marker (see, e.g., U.S.4,399,216, 4,634,665 and U.S.5,179,017). Suitable selectable markers include genes that confer resistance to a substance, such as G418, puromycin, hygromycin, blasticidin, gemithromycin (Zeocin)/Bleomycin (Bleomycin) or methotrexate, or a selectable marker that causes auxotrophy of the host cell when the vector has been introduced into the cell, such as glutamine synthetase (Bebbington et al, Biotechnology (N Y) 1992 Feb;10(2): 169-75).

For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the neo gene confers resistance to G418, from Aspergillus terreus (Zhang)Aspergillus terreus) The bsd gene of (a) confers resistance to blasticidin, puromycin N-acetyl transferase confers resistance to puromycin, the Sh ble gene product confers resistance to germicin, and the E.coli hygromycin resistance gene (hyg or hph) confers resistance to hygromycin. Selectable markers such as DHFR or glutamine synthetase also facilitate amplification techniques that combine MTX and MSX.

Transfection of the expression vector into a host cell can be performed by standard techniques, in particular by electroporation, nuclear transfection, calcium phosphate precipitation, lipofection, polycation-based transfection, such as Polyethyleneimine (PEI) based transfection and DEAE-dextran transfection.

Mammalian host cells suitable for expression of antibodies, antigen-binding fragments thereof, or variants thereof include Chinese hamster ovary (CHO cells), such as CHO-K1, CHO-S, CHO-K1SV [ including DHFR-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-.

Expression of antibodies, antigen-binding fragments thereof, or variants thereof may also be carried out in a transient or semi-stable manner in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO-Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEALT BN 85, CHOS-XE, CHO-3E7, or CAP-T cells (e.g., such as Durocher et al, Nucleic Acids Res. 2002 Jan 15;30(2): E9).

In some embodiments, the expression vector is constructed in such a way that the protein to be expressed is secreted into the cell culture medium in which the host cell is grown. Antibodies, antigen-binding fragments thereof, or variants thereof can also be obtained from cell culture media by protein purification methods known to those skilled in the art.

Purification of

Antibodies, antigen-binding fragments thereof or variants thereof can be obtained and purified from recombinant cell cultures by well-known methods including, for example, ammonium sulfate precipitation or ethanol precipitation, acid extraction, protein a chromatography, protein G chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, Hydrophobic Interaction Chromatography (HIC), affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997 2001), e.g., chapters 1,4, 6, 8,9, 10.

The antibody or antigen-binding fragment thereof or variant thereof of the present invention includes naturally purified products, products of chemical synthesis, and products produced by recombinant techniques in prokaryotic or eukaryotic host cells. Eukaryotic cell hosts include, for example, yeast cells, higher plant cells, insect cells, and mammalian cells. Depending on the host cell chosen for recombinant expression, the protein expressed may be in glycosylated or non-glycosylated form.

In a preferred embodiment, the antibody is purified (1) above 95% by weight (as measured, for example, by the Lowry method, by UV-Vis spectroscopy, or by SDS capillary gel electrophoresis (e.g., using a Caliper LabChip gxi, GX 90, or biorad bioanalyzer instrument)), and in a more preferred embodiment to a degree of greater than 99% by weight, (2) to a degree suitable for determining at least 15 residues of the N-terminal or internal amino acid sequence, or (3) to homogeneity (as determined by SDS-PAGE under reducing or non-reducing conditions by mackose blue staining or, preferably, silver staining).

Typically, the isolated antibody is obtained by means of at least one protein purification step.

anti-CD 123 antibodies

According to the present invention, anti-CD 123 antibodies may be used.

The term "anti-CD 123 antibody" or "antibody that specifically binds to CD 123" refers to an antibody that binds to 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 a specific embodiment, the antibody is bound with a dissociation constant (K.sub.m) ≦ 1 μ M ≦ 100 nM ≦ 10nM ≦ 1nM, ≦ 0.1 nM ≦ 0.01 nM, or ≦ 0.001nM_D) Binds to CD 123.

Sun et al (Sun et al, 1996, Blood 87(1):83-92) describe the generation and properties of monoclonal antibody 7G3 that binds to the N-terminal domain of IL-3R α, CD 123. U.S. Pat. No. 6,177,078 (Lopez) relates to anti-CD 123 antibody 7G 3. Chimeric variants of this antibody (CSL360) are described in WO 2009/070844, and humanized versions (CSL362) are described in WO 2012/021934. The sequence of the 7G3 antibody is disclosed in EP 2426148. This sequence constitutes the starting point for a humanized antibody obtained by CDR grafting ("CDR grafting").

An antibody that is particularly well internalized upon cell surface antigen binding is the anti-CD 123 antibody 12F1 disclosed by Kuo et al (Kuo et al, 2009, bioconjugateg chem. 20(10): 1975-82). Antibody 12F1 binds with higher affinity to CD123 than antibody 7G3 and internalizes significantly faster than 7G3 upon cell surface antigen binding. Bispecific scFv immune fusion proteins based on 12F1 are disclosed in WO 2013/173820. Antibody TPP-6013 is a chimeric variant of 12F 1.

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

Humanized variants of the murine 7G3 antibody and the murine 12F1 antibody were generated based on CDR grafting ("CDR grafting") into a human framework and subsequent optimization, and are preferred examples in the context of the present invention.

anti-CD 123 antibodies TPP-9476, TPP-8988, TPP-8987 and TPP-6013 are particularly preferred in the context of the present invention.

anti-CXCR 5 antibodies

According to the invention, anti-CXCR 5 antibodies may be used.

The term "anti-CXCR 5 antibody" or "antibody that specifically binds to CXCR 5" refers to an antibody that binds to the cancer target molecule CXCR5(NCBI reference sequence: NP _ 001707.1; SEQ ID NO: 112), preferably with sufficient affinity for diagnostic and/or therapeutic applications. In particular embodiments, the antibody exhibits a dissociation constant (K) of < 1 μ M, < 100 nM, < 10nM, < 1nM, < 0.1 nM, < 0.01 nM, or < 0.001nM_D) Binds to CXCR 5.

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 EP 2195023.

Hybridoma cells for rat antibody RF8B2 (ACC2153) were purchased from DSMZ and the sequences of the antibodies were identified by standard methods. This sequence constitutes the starting point for a humanized antibody obtained by CDR grafting ("CDR grafting").

Humanized variants of this antibody were generated based on CDR grafting ("CDR grafting") into germline sequences.

These antibodies and antigen-binding fragments may be used in the context of the present invention.

The anti-CXCR 5 antibodies TPP-9574 and TPP-9580 are particularly preferred in the context of the present invention.

anti-B7H 3 antibodies

According to the present invention, anti-B7H 3 antibodies can be used.

The term "anti-B7H 3 antibody" or "antibody that specifically binds to B7H 3" refers to an antibody that binds to the cancer target molecule B7H3 (NCBI reference sequence: NP-001019907.1 SEQ ID NO:113), preferably with sufficient affinity for diagnostic and/or therapeutic applications. In particular embodiments, the antibody binds to B7H3 with a dissociation constant (KD) of < 1 μ M, < 100 nM, < 10nM, < 1nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM.

Examples of antibodies and antigen-binding fragments that bind to B7H3 are known to those skilled in the art and are described, for example, in WO201109400, EP1773884, and WO 2014061277. EP2121008 describes the anti-B7H 3 antibody 8H9 and its CDR sequences.

These antibodies and antigen-binding fragments may be used in the context of the present invention.

A preferred embodiment of the anti-B7H 3 antibody is obtained by screening an antibody phage display library against cells expressing mouse recombinant B7H3 (mouse CD276; gene ID: 102657) and human B7H3 (human CD276; gene ID: 80381). The obtained antibody was converted into a human IgG1 form. The anti-B7H 3 antibody TPP-8382 is a preferred example.

The anti-B7H 3 antibody TPP-8382 is particularly preferred in the context of the present invention.

anti-TWEAKR antibodies

According to the invention, anti-TWEAKR antibodies may be used.

The term "anti-TWEAKR antibody" or "antibody that specifically binds to TWEAKR" refers to an antibody that binds to the cancer target molecule TWEAKR (NCBI reference sequence: NP _057723.1 SEQ ID NO:114), preferably with sufficient affinity for diagnostic and/or therapeutic applications. In particular embodiments, the antibody binds to TWEAKR with a dissociation constant (KD) of ≦ 1 μ M, ≦ 100 nM, ≦ 10nM, ≦ 1nM, ≦ 0.1 nM, ≦ 0.01 nM, or ≦ 0.001 nM.

Examples of antibodies that bind to TWEAKR are disclosed in, for example, WO2009/020933(a2), WO2009/140177 (a2), WO 2014/198817 (a1), and WO 2015/189143 (a 1). These antibodies and antigen-binding fragments may be used in the context of the present invention.

ITEM-4 is an anti-TWEAKR antibody described by Nakayama et al (Nakayama, et al, 2003, Biochem Biophy Res Comm, 306: 819. sup. 825). Humanized variants of this antibody based on CDR grafting ("CDR grafting") are described in Zhou et al (Zhou et al, 2013, J investDermatol.133(4):1052-62) and WO 2009/020933. These antibodies and antigen-binding fragments may be used in the context of the present invention.

anti-TWEAKR antibodies TPP-7006 and TPP-7007 are particularly preferred in the context of the present invention. These are humanized variants of the antibody ITEM-4. These antibodies and antigen-binding fragments are preferably useful in the context of the present invention.

anti-HER 2 antibody:

according to the invention, anti-HER 2 antibodies may be used.

The term "anti-HER 2 antibody" or "antibody that specifically binds to HER 2" refers to an antibody that binds to the cancer target molecule HER2 (NCBI reference sequence: NP _004439.2 SEQ ID NO:115), preferably with sufficient affinity to be useful for diagnostic and/or therapeutic applications. In particular embodiments, the antibody binds HER2 with a dissociation constant (KD) of < 1 μ M, < 100 nM, < 10nM, < 1nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM.

An example of an antibody that binds to cancer target molecule Her2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody that is particularly useful in the treatment of breast cancer. In a particularly preferred embodiment, the anti-HER 2 antibody is TPP-1015 (trastuzumab analog).

In addition to trastuzumab (INN 7637, CAS number: RN:180288-69-1) and pertuzumab (CAS number: 380610-27-5), other examples of antibodies that bind to HER2 are antibodies as disclosed in WO 2009/123894-A2, WO 200/8140603-A2, or WO 2011/044368-A2. An example of an anti-HER 2 conjugate is trastuzumab-Emtansine (INN No. 9295). These antibodies and antigen-binding fragments may be used in the context of the present invention.

The anti-HER 2 antibody TPP-1015 (similar to trastuzumab) is particularly preferred in the context of the present invention.

anti-EGFR antibodies

According to the invention, anti-EGFR antibodies may be used.

The term "anti-EGFR antibody" or "antibody that specifically binds 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 sufficient affinity for diagnostic and/or therapeutic applications. In particular embodiments, the antibody binds EGFR with a dissociation constant (KD) of < 1 μ M, < 100 nM, < 10nM, < 1nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM.

In a preferred embodiment, the anti-EGFR antibody is selected from TPP-981, cetuximab, panitumumab, nimotuzumab. In a particularly preferred embodiment, the anti-EGFR antibody is TPP-981.

Other embodiments of EGFR antibodies are:

Zaumumab/2F 8/HuMax-EGFr from Genmab A/S (WO 02/100348, WO 2004/056847, INN No. 8605)

Neiximab/11F 8, Imclone/IMC-11F8 from Imclone Systems Inc. [ Eli Lilly & Co ] Inc. (WO 2005/090407 (EP 01735348-A1, US 2007/0264253-A1, US 7,598,350, WO 2005/090407-A1), INN No. 9083)

matuzumab/anti-EGFR MAb, Merck KGaA/anti-EGFR MAb, Takeda/EMD 72000/EMD-6200/EMD-72000 and EMD-55900/MAb 425/monoclonal antibody 425 from Merck KGaA/Takeda (WO 92/15683, INN # 8103 (matuzumab))

RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/RO4858696/RO5083945 from Glycart Biotechnology AG (Roche HoldingAG) Inc. (WO 2010/112413-A1, WO 2010/115554)

GT-MAB 5.2-GEX/CetuGEX from Glycotope GmbH (WO 2008/028686-A2 (EP 01900750-A1, EP 01911766-A1, EP 02073842-A2, US 2010/0028947-A1)

ISU-101 from Isu Abxis Inc (ISU Chemical Co Ltd)/Scancell (WO 2008/004834-A1)

ABT-806/mAb-806/ch-806/anti-EGFR monoclonal antibody 806 from Ludwig Institute for Cancer Research/Abbott/Life Science Pharmaceuticals (WO 02/092771, WO 2005/081854 and WO 2009/023265)

SYM-004 (consisting of two chimeric IgG1 antibodies (992 and 1024)) from Symphogen A/S (WO 2010/022736-A2)

MR 1-1/MR 1-1KDEL from IVAX Corp (Teva Pharmaceutical Industries Ltd) (Duke university), (patent: WO2001/062931-A2)

Antibodies against the deletion mutant EGFRvIII from Amgen/Abgenix (WO 2005/010151, US 7,628,986)

SC-100 from Scancell Ltd (WO 01/088138-A1)

MDX-447/EMD 82633/BAB-447/H447/MAb, EGFR, Metarex/Merck KgaA from Bristol-Myers Squibb (US)/Merck KGaA (DE)/Takeda (JP) Co., WO 91/05871, WO 92/15683)

anti-EGFR-Mab from Xencor (WO 2005/056606)

DXL-1218/anti-EGFR monoclonal antibody (cancer), lnnexus, from lnnexus Biotechnology Inc, Pharmaprojects PH 048638.

Anti-carbonic anhydrase IX antibodies

Examples of antibodies that bind the cancer target molecule carbonic anhydrase IX are described in WO 2007/070538-a2 (e.g. claims 1-16).

Anti-c4.4a antibodies:

examples of c4.4a antibodies and antigen-binding fragments are described in WO 2012/143499 a 2. The sequences of the antibodies are given in table 1 of WO 2012/143499 a2, wherein each row shows the respective CDR amino acid sequences of the variable light or variable heavy chains of the antibodies listed in column 1.

anti-CD 20 antibody:

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's lymphoma. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-CD 52 antibody:

an example of an antibody that binds to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS number: 216503-57-0) is a humanized antibody for the treatment of chronic lymphocytic leukemia. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

Anti-mesothelin antibody:

examples of anti-mesothelin antibodies are described in, for example, WO 2009/068204. All antibodies and antigen-binding fragments disclosed in WO2009/068204 may be used in the context of the invention disclosed herein. Particularly preferably, the antibody disclosed in WO2009/068204 is MF-T.

anti-CD 30 antibodies

Examples of antibodies that bind to the cancer target molecule CD30 and that can be used for the treatment of cancer (e.g. hodgkin's lymphoma) are Brentuximab, itumumab and antibodies as disclosed in WO 2008/092117, WO 2008/036688 or WO 2006/089232. An example of an anti-CD 30 conjugate is Brentuximab Vedotin (INN No. 9144). These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-CD 22 antibodies

Examples of antibodies that bind to the cancer target molecule CD22 and that can be used to treat cancer (e.g., lymphoma) are Inotuzumab and epratuzumab. Examples of anti-CD 22 conjugates are Inotuzumab Ozagamycin (INN No. 8574) or anti-CD 22-MMAE and anti-CD 22-MC-MMAE (CAS nos: 139504-50-0 and 474645-27-7, respectively). These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-CD 33 antibodies

Examples of antibodies that bind to the cancer target molecule CD33 and that can be used to treat cancer (e.g., leukemia) are gemumab and lintuzumab (INN 7580). An example of an anti-CD 33 conjugate is gemumab-ozagramycin. These antibodies and antigen-binding fragments may be used in the context of the present invention.

anti-NMB antibodies

An example of an antibody that binds to cancer target molecule NMB and can be used to treat cancer (e.g., melanoma or breast cancer) is Glembatumumab (INN 9199). An example of an anti-NMB conjugate is Glembatumumab Vedotin (CAS number: 474645-27-7). These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-CD 56 antibodies

An example of an antibody that binds to the cancer target molecule CD56 and that can be used to treat cancer (e.g., multiple myeloma, small cell lung cancer, MCC, or ovarian cancer) is lovamab. An example of an anti-CD 57 conjugate is lovalizumab maytansine (CAS number: 139504-50-0). These antibodies and antigen-binding fragments may be used in the context of the present invention.

anti-CD 70 antibodies

Examples of antibodies that bind to the cancer target molecule CD70 and that can be used to treat cancer (e.g., non-hodgkin's lymphoma or renal cell carcinoma) are disclosed in WO 2007/038637-a2 and WO 2008/070593-a 2. An example of an anti-CD 70 conjugate is SGN-75 (CD70 MMAF). These antibodies and antigen-binding fragments may be used in the context of the present invention.

anti-CD 74 antibodies

An example of an antibody that binds to the cancer target molecule CD74 and that can be used to treat cancer (e.g., multiple myeloma) is melalizumab. An example of an anti-CD 74 conjugate is Mirablizumab-doxorubicin (CAS number: 23214-92-8). These antibodies and antigen-binding fragments may be used in the context of the present invention.

anti-CD 19 antibodies

Examples of antibodies that bind the cancer target molecule CD19 and that can be used to treat cancer (e.g., non-hodgkin's lymphoma) are disclosed in WO 2008/031056-a 2. Examples of other antibodies and anti-CD 19 conjugates (SAR3419) are disclosed in WO 2008/047242-a 2. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

Anti-mucin antibodies

Examples of antibodies that bind to the cancer target molecule mucin-1 and that can be used to treat cancer (e.g., non-hodgkin's lymphoma) are the antibodies disclosed in Clivatuzumab or WO 2003/106495-a2, WO 2008/028686-a 2. Examples of anti-mucin conjugates are disclosed in WO 2005/009369-a 2. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-CD 138 antibodies

Examples of antibodies and conjugates thereof that bind to the cancer target molecule CD138 that can be used to treat cancer (e.g., multiple myeloma) are disclosed in WO 2009/080829-a1, WO 2009/080830-a 1. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

Anti-integrin-alphav antibodies

Examples of antibodies that bind to the cancer target molecule integrin alphaV and that can be used in the treatment of cancer (e.g., melanoma, sarcoma, or carcinoma) are infliximab (CAS No: 725735-28-4), abciximab (CAS No: 143653-53-6), edazumab (CAS No: 892553-42-3), or the antibodies disclosed in US 7,465,449, EP 719859-A1, WO 2002/012501-A1, or WO 2006/770629-A2. Examples of anti-integrin alphav conjugates are Intetumumab-DM4 and other ADCs disclosed in WO 2007/024536-a 2. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-TDGF 1 antibodies

Examples of antibodies that bind to the cancer target molecule TDGF1 and that may be used for the treatment of cancer are the antibodies disclosed in WO 02/077033-a1, US 7,318,924, WO 2003/083041-a2 and WO 2002/088170-a 2. Examples of anti-TDGF 1 conjugates are disclosed in WO 2002/088170-a 2. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-PSMA antibodies

Examples of antibodies that bind to the cancer target molecule PSMA and that may be used in the treatment of cancer (e.g., prostate cancer) are the antibodies disclosed in WO 97/35616-A1, WO 99/47554-A1, WO 01/009192-A1, and WO 2003/034903. Examples of anti-PSMA conjugates are disclosed in WO 2009/026274-a1 and WO 2007/002222. These antibodies and antigen-binding fragments may be used in the context of the present invention.

anti-EPHA 2 antibodies

Examples of antibodies that bind the cancer target molecule EPHA2 and that can be used to prepare conjugates and for the treatment of cancer are disclosed in WO 2004/091375-a 2. These antibodies and antigen-binding fragments may be used in the context of the present invention.

anti-SLC 44A4 antibody

Examples of antibodies that bind the cancer target molecule SLC44a4 and that can be used to prepare conjugates and for the treatment of cancer (e.g. pancreatic or prostate cancer) are disclosed in WO2009/033094-a2 and US2009/0175796-a 1. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-HLA-DOB antibodies

An example of an antibody that binds to the cancer target molecule HLA-DOB is the antibody Lym-1 (CAS number: 301344-99-0), which can be used to treat cancer such as non-Hodgkin's lymphoma. Examples of anti-HLA-DOB conjugates are disclosed in, for example, WO 2005/081711-a 2. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-VTCN 1 antibodies

Examples of antibodies that bind to the cancer target molecule VTCN1 and that can be used to prepare conjugates and for the treatment of cancer (e.g. ovarian, pancreatic, lung or breast cancer) are disclosed in WO 2006/074418-a 2. These antibodies and antigen-binding fragments thereof may be used in the context of the present invention.

anti-FGFR 2 antibodies

Examples of anti-FGFR 2 antibodies and antigen-binding fragments are described in WO 2013076186. The sequences of the antibodies are shown in tables 9 and 10 of WO 2013076186. Antibodies, antigen-binding fragments and variants of antibodies derived from the antibodies designated M048-D01 and M047-D08 are preferred.

Preferred antibodies and antigen-binding antibody fragments for the binding agent-active substance conjugates according to the invention

In the present application, the binding agent-active substance-conjugates refer to the following preferred antibodies as shown in the following table: TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580.

Table: protein sequence of the antibody:

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 CDR sequences (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) of the variable region of the heavy chain (VH) or of the variable region of the light chain (VL) given in the above table. Preferably, the antibody comprises a given heavy chain variable region (VH) and/or light chain variable region (VL). Preferably, the antibody comprises a given heavy chain region (IgG heavy chain) and/or a given light chain region (IgG light chain).

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

TPP-1015 is an anti-HER 2 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO:12, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO:13, and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO:14, and a light chain variable region (VL) comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO:16, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO:17, and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 18.

TPP-6013 is an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO:22 (H-CDR1), the heavy chain variable CDR2 sequence shown in SEQ ID NO:23 (H-CDR2) and the heavy chain variable CDR3 sequence shown in SEQ ID NO:24 (H-CDR3), and a light chain variable region (VL) comprising the light chain variable CDR1 sequence shown in SEQ ID NO:26 (L-CDR1), the light chain variable CDR2 sequence shown in SEQ ID NO:27 (L-CDR2) and the light chain variable CDR3 sequence shown in SEQ ID NO:28 (L-CDR 3).

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

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

TPP-8382 is an anti-B7H 3 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence shown in SEQ ID NO:52 (H-CDR1), a heavy chain variable CDR2 sequence shown in SEQ ID NO:53 (H-CDR2) and a heavy chain variable CDR3 sequence shown in SEQ ID NO:54 (H-CDR3), and a light chain variable region (VL) comprising a light chain variable CDR1 sequence shown in SEQ ID NO:56 (L-CDR1), a light chain variable CDR2 sequence shown in SEQ ID NO:57 (L-CDR2) and a light chain variable CDR3 sequence shown in SEQ ID NO:58 (L-CDR 3).

TPP-8987 is an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence as set forth in SEQ ID NO:62 (H-CDR1), a heavy chain variable CDR2 sequence as set forth in SEQ ID NO:63 (H-CDR2) and a heavy chain variable CDR3 sequence as set forth in SEQ ID NO:64 (H-CDR3), and a light chain variable region (VL) comprising a light chain variable CDR1 sequence as set forth in SEQ ID NO:66 (L-CDR1), a light chain variable CDR2 sequence as set forth in SEQ ID NO:67 (L-CDR2) and a light chain variable CDR3 sequence as set forth in SEQ ID NO:68 (L-CDR 3).

TPP-8988 is an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO:72 (H-CDR1), the heavy chain variable CDR2 sequence shown in SEQ ID NO:73 (H-CDR2) and the heavy chain variable CDR3 sequence shown in SEQ ID NO:74 (H-CDR3), and a light chain variable region (VL) comprising the light chain variable CDR1 sequence shown in SEQ ID NO:76 (L-CDR1), the light chain variable CDR2 sequence shown in SEQ ID NO:77 (L-CDR2) and the light chain variable CDR3 sequence shown in SEQ ID NO:78 (L-CDR 3).

TPP-9476 is an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO:82, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO:83 and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO:84, and a light chain variable region (VL) comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO:86, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO:87 and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 88.

TPP-9574 is an anti-CXCR 5 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence shown in SEQ ID NO:92 (H-CDR1), a heavy chain variable CDR2 sequence shown in SEQ ID NO:93 (H-CDR2) and a heavy chain variable CDR3 sequence shown in SEQ ID NO:94 (H-CDR3), and a light chain variable region (VL) comprising a light chain variable CDR1 sequence shown in SEQ ID NO:96 (L-CDR1), a light chain variable CDR2 sequence shown in SEQ ID NO:97 (L-CDR2) and a light chain variable CDR3 sequence shown in SEQ ID NO:98 (L-CDR 3).

TPP-9580 is an anti-CXCR 5 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO:102, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO:103 and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO:104, and a light chain variable region (VL) comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO:106, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO:107 and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO: 108.

TPP-981 is an anti-EGFR antibody preferably comprising the heavy chain variable region (VH) as shown in SEQ ID NO:1 and the light chain variable region (VL) as shown in SEQ ID NO: 5.

TPP-1015 is an anti-HER 2 antibody preferably comprising the heavy chain variable region (VH) shown in SEQ ID NO:11 and the light chain variable region (VL) shown in SEQ ID NO: 15.

TPP-6013 is an anti-CD 123 antibody preferably comprising the heavy chain variable region (VH) as shown in SEQ ID NO:21 and the light chain variable region (VL) as shown in SEQ ID NO: 25.

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

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

TPP-8382 is an anti-B7H 3 antibody preferably comprising the heavy chain variable region (VH) as shown in SEQ ID NO:51 and the light chain variable region (VL) as shown in SEQ ID NO: 55.

TPP-8987 is an anti-CD 123 antibody preferably comprising the heavy chain variable region (VH) as set forth in SEQ ID NO:61 and the light chain variable region (VL) as set forth in SEQ ID NO: 65.

TPP-8988 is an anti-CD 123 antibody preferably comprising the heavy chain variable region (VH) as set forth in SEQ ID NO:71 and the light chain variable region (VL) as set forth in SEQ ID NO: 75.

TPP-9476 is an anti-CD 123 antibody preferably comprising the heavy chain variable region (VH) as set forth in SEQ ID NO:81 and the light chain variable region (VL) as set forth in SEQ ID NO: 85.

TPP-9574 is an anti-CXCR 5 antibody preferably comprising the heavy chain variable region (VH) as shown in SEQ ID NO:91 and the light chain variable region (VL) as shown in SEQ ID NO: 95.

TPP-9580 is an anti-CXCR 5 antibody preferably comprising the heavy chain variable region (VH) as shown in SEQ ID NO:101 and the light chain variable region (VL) as shown in SEQ ID NO: 105.

TPP-981 is an anti-CD 123 antibody preferably comprising a heavy chain region as shown in SEQ ID NO. 9 and a light chain region as shown in SEQ ID NO. 10.

TPP-1015 is an anti-HER 2 antibody preferably comprising the heavy chain region shown in SEQ ID NO:19 and the light chain region shown in SEQ ID NO: 20.

TPP-6013 is an anti-CD 123 antibody preferably comprising a heavy chain region as shown in SEQ ID NO:29 and a light chain region as shown in SEQ ID NO: 30.

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

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

TPP-8382 is an anti-B7H 3 antibody preferably comprising a heavy chain region as shown in SEQ ID NO:59 and a light chain region as shown in SEQ ID NO: 60.

TPP-8987 is an anti-CD 123 antibody preferably comprising a heavy chain region as shown in SEQ ID NO:69 and a light chain region as shown in SEQ ID NO: 70.

TPP-8988 is an anti-CD 123 antibody preferably comprising a heavy chain region as shown in SEQ ID NO:79 and a light chain region as shown in SEQ ID NO: 80.

TPP-9476 is an anti-CD 123 antibody preferably comprising a heavy chain region as shown in SEQ ID NO. 89 and a light chain region as shown in SEQ ID NO. 90.

TPP-9574 is an anti-CXCR 5 antibody preferably comprising a heavy chain region as shown in SEQ ID NO:99 and a light chain region as shown in SEQ ID NO: 100.

TPP-9580 is an anti-CXCR 5 antibody preferably comprising a heavy chain region as shown in SEQ ID NO:109 and a light chain region as shown in SEQ ID NO: 110.

Isotopes, salts, solvates, isotopic variations

The invention also encompasses all suitable isotopic variations of the compounds of the invention. Isotopic variations of the compounds of the present invention are understood herein to mean where at least one atom within the compounds of the present invention has been exchanged for another atom of the same atomic number, but having an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be incorporated into the compounds of the invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as²H (deuterium),³H (tritium),¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、³²P、³³P、³³S、³⁴S、³⁵S、³⁶S、¹⁸F、³⁶Cl、⁸²Br、¹²³I、¹²⁴I、¹²⁹I and¹³¹I. particular isotopic variations of the compounds of the present invention, particularly those into which one or more radioactive isotopes are incorporated, may be advantageous, for example, for examining the mechanism of action or the distribution of the active species in the body; due to the relatively easy manufacturability and detectability, in particular³H or¹⁴C-isotopically labelled compounds are suitable for this purpose. Furthermore, due to the higher metabolic stability of the compounds, the incorporation of isotopes (e.g., deuterium) can result in specific therapeutic benefits, such as increased in vivo half-life or reduced active dose requirements; such modifications of the compounds of the invention may therefore optionally also constitute preferred embodiments of the invention. Isotopic variations of the compounds of the present invention can be prepared by methods known to those skilled in the art, for example by methods described further below and by the procedures described in the examples, by using the respective reagents and/or the corresponding isotopic variations of the starting compounds.

Preferred in the context of the present inventionSalt (salt)Are physiologically acceptable salts of the compounds of the invention. Also contemplated are compositions not suitable per se for pharmaceutical applications but useful forFor example, isolating or purifying a salt of a compound of the invention.

Physiologically acceptable salts of the compounds of the invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, for example the following acid salts: 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.

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

In the context of the present inventionSolvatesAre described as forms of the compounds of the invention which form complexes by coordination in the solid or liquid state with solvent molecules. Hydrates are a particular form of solvates in which coordination is made with water. Preferred solvates in the context of the present invention are hydrates.

Therapeutic uses

Hyperproliferative disorders which the compounds of the invention are useful for treating include in particular the class of cancer and tumor disorders. In the context of the present invention, these are understood to mean in particular the following conditions, but not limited to them: breast cancer and breast tumors (breast cancer includes ductal and lobular forms, and in situ: (in situ)) Tumors of the respiratory tract (small and non-small cell cancers, bronchial carcinomas), tumors of the brain (e.g. tumors of the brain stem and hypothalamus, astrocytomas, ependymomas, glioblastomas, gliomas, medulloblastomas, meningiomas and neuroectodermal and pineal tumors), tumors of the digestive organs (cancers of the esophagus, stomach, gall bladder, small intestine, large intestine, rectum and anus), tumors of the liver (in particular hepatocellular carcinomas, cholangiocellular carcinomas and carcinomas of the colon)Mixed hepatocyte cholangiocellular carcinoma), tumors of the head and neck region (laryngeal, hypopharynx, nasopharynx, oropharynx, lips and oral cavity cancers, oral melanoma), skin tumors (basal cell tumors, spinoliome, squamous cell carcinoma, kaposi's sarcoma, malignant melanoma, non-melanoma skin cancers, merkel cell skin cancers, mast cell tumors), tumors of the supporting and connective tissue (especially soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, chondrosarcomas, fibrosarcomas, angiosarcomas, leiomyosarcomas, liposarcomas, lymphosarcomas and rhabdomyosarcomas), tumors of the eye (especially intraocular melanoma and retinoblastoma), tumors of the endocrine and exocrine glands (such as thyroid and parathyroid, pancreatic and salivary gland carcinomas, adenocarcinomas), tumors of the urethra (tumors of the bladder, penis, renal pelvis, kidney and ureter), and tumors of the reproductive organs (endometrial carcinoma in women), Cervical, ovarian, vaginal, vulvar and uterine cancers, prostate and testicular cancer in males). These also include proliferative disorders of the blood, lymphatic system and spinal cord, both in solid form and as circulating cells, such as leukemias, lymphomas and myeloproliferative disorders, for example acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia and AIDS-related lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt's lymphoma and lymphomas in the central nervous system.

These well characterized diseases in humans may also occur with comparable etiologies in other mammals and are equally treatable herein with the compounds of the present invention.

The binding agent-or antibody-active substance-conjugates (ADCs) described herein directed against CD123 may preferably be used for the treatment of CD123 expression disorders, such as CD123 expressing cancers. Typically, such cancer cells have a measurable amount of CD123 at the protein (e.g., using an immunoassay) or RNA level. Some of these cancer tissues show elevated levels of CD123, preferably measured in the same patient, compared to non-cancer tissues of the same type. Optionally, CD123 content is measured before the start of the treatment of cancer with the antibody-active substance-conjugate (ADC) according to the invention (patient stratification). Binding agent-active agent-conjugates (ADCs) directed against CD123 may preferably be used for the treatment of CD123 expressing disorders, such as CD123 expressing cancers, e.g. tumours of hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies. Examples of cancers associated with CD123 expression include myeloid disorders, such as Acute Myeloid Leukemia (AML) and myelodysplastic syndrome (MDS). Other types of cancer include B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, blast cell plasmocytic dendritic cell tumor (BPDCN), Hodgkin's lymphoma, immature T-cell acute lymphocytic leukemia (immature T-ALL), Burkitt's lymphoma, follicular lymphoma, Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL). The methods of the invention include treating a patient having a CD 123-expressing cancer, wherein the methods comprise administering an antibody-active agent-conjugate (ADC) according to the invention.

Treatment of the above cancers, including treatment of solid tumors and their metastatic or circulatory forms, with the compounds of the present invention.

In the context of the present invention, the terms "treatment" or "treating" are used in the conventional sense and refer to the care, care and care of a patient for the purpose of combating, alleviating, attenuating or alleviating a disease or health abnormality and improving the survival status impaired by the disease, for example in the case of cancer.

A further subject of the present invention is therefore the use of the compounds of the invention for the treatment and/or prophylaxis of disorders, in particular of the disorders mentioned above.

A further subject of the present invention is the use of the compounds of the invention for the preparation of a medicament for the treatment and/or prophylaxis of disorders, in particular of the disorders mentioned above.

Further subject matter of the present invention is the use of the compounds of the invention in a method for the treatment and/or prophylaxis of disorders, in particular of the disorders mentioned above.

Further subject matter of the present invention are methods for the treatment and/or prophylaxis of disorders, in particular of the disorders mentioned above, using an effective amount of at least one compound according to the invention.

The compounds of the present invention may be used alone or, if desired, in combination with one or more other pharmacologically active substances, provided that such combination does not cause undesirable and unacceptable side effects. A further subject of the present invention is therefore a medicament containing at least one compound according to the invention and one or more further active substances, in particular for the treatment and/or prophylaxis of the abovementioned disorders.

For example, the compounds of the present invention may be combined with known anti-hyperproliferative, cytostatic, cytotoxic or immunotherapeutic substances used to treat cancer. As suitable combined active substances, mention may be made, by way of example, of:

131I-chTNT, Abarelix (Abarelix), Abiraterone, aclarubicin, Adamantimab, Ado-Trastuzumab Emtansin, Afatinib, Abeprister, Addison, Alemtuzumab (Alemtuzumab), alemtric acid, Aliretin A acid (Alitretinin), altretamine, amifostine, aminoglutethimide, hexyl-5-aminoacetylpropionate, amrubicin, amsacrine, anastrozole, Ancistin, anethothion, Anetumab ravtansine, angiotensin II, antithrombin III, aprepitant, Acitumomab, Arglabin, diarsene, asparaginase, Abuzumab, Abelumab, acitinib, azacitidine, Belotecan, bendamustine, Levelizumab, Bevavacizumab (Bevavacizumab), Bevacizumab, Begonin, Begonimus, and Begonin, Buserelin, bosutinib, Brentuximab vedotin, busulfan, Cabazitaxel (Cabazitaxel), cabozantinib, calcitonin, calcium folinate, calcium levofolinate, capecitabine, carolomab, carbamazepine, carboplatin, Carboquuon, carboplatin, carminomide, carmofluor, carmustine, cetuximab (Catumaxomab), celecoxib, simethionin, ceritinib, cetuximab, chlorambucil, chlorthalidomide, mechlorethamine, cidofovir, cinacalcet, cisplatin, cladribine, clofababine (Clofarabin), cobitinib, Copaliside, clinatapase (Crisastapase), crizotinib, cyproterone, cytarabine, dacarbazine, dalargininil, danielinib, Difutazone, Difletiritin, daunomycin (Deisantiotubrin), daunorubicin, daunomycin, doxamide, doxepin, etc, Deslorelin, dexrazoxane, dibromospiroammonium chloride, didehydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine, doxorubicin + estrone, dronabinol, Durvalumb, eculizumab, etiloamine, endostatin, enocitabine, enzalutamide, Epacadostat, epirubicin, epithiandrol, epoetin alpha, epoetin beta, epoetin zeta, eptaplatin, Eribulin (Eribulin), erlotinib, esomeprazole, estramustine, etoposide, ethinylestradiol, everolimus, exemestane, fadrozole, fentanyl, tolmetin, floxuridine, fludarabine, fluorouracil, flutamide, foscarnet, fotametant, fosaprepin, fotemepin, fotemustine, fulvestrant, valbuterol, gavalbuterol, gazedride, gazedrine, Gd, dimehypolatrine (PA-B), DTE-D-, Gallium nitrate, ganirelix, gefitinib, gemcitabine, Gemtuzumab (Gemtuzumab), carboxypeptidase, glutathione (Glutoxim), goserelin, granisetron, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), histamine dihydrochloride, Histrelin (Histrelin), hydroxyurea, I-125 particles, lansoprazole, ibandronic-Ibritumomab (Ibritumomab-Tiuxetan), ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, Improsulfan (Improsulfan), indostron, incadronic acid, ingenol mebutate, interferon alpha, interferon beta, interferon gamma, iobitol, iobenguan (123I), iomeprol, iomepril (Ipimelimumab), irinotecan, itraconazole, ixabepilone (Izodicam), and Ixabepimelan (Ixapril), and the like, Lanreotide, lansoprazole, Lansoprazol, Lapatinib (Lapatinib), Lasochloline, lenalidomide (Lenalidomide), lenvatinib, lengesitane, lentinan, letrozole, leuprorelin, levotetramisole, levonorgestrel, levothyroxine sodium, Lipegfilgrastim, lisuride, leplatin, cyclonitrosourea, lonidamine, maxol, medroxyprogesterone, megestrol, melarsol, melphalan, mertasane, mercaptopurine, mesna, methadone, methotrexate, methofuramectin, methylaminoketovalerate, methylprednisolone, methyltestosterone, Metirosin, mivamuropeptide (Mifamurtid), Mitefurapine, miboplatin, dibromomannitol, mitoxantrone, Molmitron, Molminticin, Molimimazamoxiflorin, Moxib, Movadaminolide, Nadamine hydrochloride, Napaleon, Naltrexone, nateglinide, netilmimab, nedaplatin, nelarabine (Nelarabin), neridronic acid, netupitant/palonosetron, nivolumab pentotriptide, Nilotinib (Nilotinib), nilutamide, nimorazole, Nimotuzumab, pyrimidineurea, nidanib, nitracridine (nitacrin), nivolumab, atropium, octreotide, Ofatumumab (Ofatumumab), olaparib, olarataumab, hypertriglyceridemic base, omeprazole, ondansetron, gululotin, oritinib, oxaliplatin, oxycodone, oxymetalone, ozagrogamicin, p 53-gene therapy, paclitaxel, palbociclib, paliperidone, Paperuvimine-103 particles, phosphonic acid, furazapine, pegamum (Palmanib, 2-Pazopanib), pegaptamine, Pazopanib, Papanicolazine, Papanicolamib, and other, Pemetrexed, pellucin, perfluorobutane, phosphoramide, pertuzumab, streptolysin (Picibanil), pilocarpine, pirarubicin, pixelstron, Plerixafor, plicamycin, Poliglusam, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, panatin, porphinium sodium, pralatrexate (Pralatrexat), mellophenamine, prednisone, procarbazine, propiconazole, propranolol, quinagolide (Quinagolid), rabeprazole, Racotumomab, chloride 223, ladostinib, ranoxicinol, Raltitrexed (Ratiextred), ramosetron, ramucirumab, ranimustine (Ranimisin), ramucinase, reimine, reinib (Rituximab), Rituximab 186, Rituximab), Rituximab, Regoxib, Regomisin, Rituximab, Regoxib, Reptinib (Rituximab), Reptinib, Retussin, Refate, Reptinib, Renals, Refate, Refat, Rogaratinib, Lapatitan, Romidepsin (Romidepsin), Romotide, Ribosiclib, Lysimazine (153Sm), Sartuzumab, secretin, Stitumomab, Sipuleucel-T, Sizopyran, Sobrozosin, sodium glycinedizole (Natriumglycidazol), Sonedgil, Sorafenib (Sorafenib), Conlidone, streptozotocin, sunitinib, Talaporfin (Talaporfin), Talimogen Laherparpvec, Tamibarotene (Tamibaroton), tamoxifen, Tapentaerythrito, tasonectin (Tasonemerin), Thoteracil (Thoreluukin), Tec [99mTc ] mercaptomomab, 99mTc-HYNIC- [ 3] -Ottor, Gatifloxacin, Fluocidine (Thiaclonimine + Thiaclcilazarin), Thioteracin (Thiaclomutilin), Thiaclopril (Thiaclacin), Thiaclopril (Thiaclonidine), Thiaclonidine (Thiaclonidine), Thiacloniloxacin (Thiaclonicin), Thiaclonidine (Thiaclonicin), Thiaclonicin, Thiaclonidine (Thiaclonicin), Thiaclonidine, Thiaclonicin, Thiacloni, Thioguanine (Tioguanin), Tositumomab (Tocilizumab), topotecan, toremifene, Tositumomab (Tositumomab), Trabectedin (Trabectedin), trametinib, tramadol, trastuzumab, troosulfan (Treosulfan), tretinoin, trifluridine + Tipiracil, trametinib, trilostane, triptorelin, chloroacetohfamide, thrombopoietin, ubenix, Valrubicin (Valrubicin), Vandetanib (vandesib), vapreotide, vartanalanib, verofenib (Vemurafenib), vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodernid, gininostat (Vorinostat), yttrium-90-glass microspheres, neocarzinostat, sethoxylate, zolmitrazol, zolmitriptan.

Furthermore, the compounds of the invention may, for example, be combined with binding agents (e.g. antibodies) that may, for example, bind to the following targets: OX-40, CD137/4-1BB, DR3, IDO1/IDO2, LAG-3 and CD40.

Since the non-cell permeable poison cluster metabolites of the binding agent-active substance-conjugates (ADCs) should not have a damaging effect on the cells of the adaptive immune system, the combination of the binding agent-active substance-conjugates (ADCs) according to the invention with cancer immunotherapy for the treatment of cancer or tumors is another subject of the invention. The intrinsic mechanism of action of the cytotoxic binding agent-active substance-conjugate includes the direct triggering of cell death of tumor cells and the consequent release of tumor antigens that can stimulate an immune response. Furthermore, there are indications that the KSP inhibitor viral cluster class induces a marker called immunocyte death (ICD) in vitro. Thus, the combination of the antibody-active substance-conjugates (ADCs) of the invention with one or more therapeutic agents for cancer immunotherapy or with one or more active substances, preferably antibodies directed against molecular targets for cancer immunotherapy, represents a preferred method for the treatment of cancer or tumors. i) Examples of therapeutic formulations for cancer immunotherapy include immunomodulatory monoclonal antibodies and low molecular weight substances directed against targets for cancer immunotherapy, vaccines, CAR T cells, bispecific T-cell-recruiting antibodies, oncolytic viruses, cell-based vaccination formulations. ii) examples of selected cancer immunotherapy targets suitable for immunomodulating monoclonal antibodies include CTLA-4, PD-1/PDL-1, OX-40, CD137, DR3, IDO1, IDO2, TDO2, LAG-3, TIM-3 CD40, ICOS/ICOSL, TIGIT; GITR/GITRL, VISTA, CD70, CD27, HVEM/BTLA, CEACAM1, CEACAM6, ILDR2, CD73, CD47, B7H3, TLR's. Thus, the combination of a binding agent-active substance-conjugate (ADC) according to the invention with cancer treatment may in one aspect render tumors with weak immunogenicity more immunogenic and enhance the efficacy of cancer immunotherapy and further exert a long lasting therapeutic effect.

Furthermore, the compounds according to the invention may also be used in combination with radiotherapy and/or surgery.

In general, the following objectives are pursued with the combination of the compounds of the present invention with other cytostatic, cytotoxic or immunotherapeutic agents:

improved efficacy in slowing tumor growth, reducing its size or even eliminating it altogether, compared to treatment with a single active substance;

the possibility of using the chemotherapeutic drug used at a lower dose than in the case of monotherapy;

the possibility of a more tolerable treatment with fewer side effects than administration alone;

the possibility of treating a broader spectrum of neoplastic disorders;

achieving higher treatment response rates;

longer patient survival compared to current standard therapy.

In addition, the compounds of the present invention may also be used in combination with radiation therapy and/or surgery.

Further subjects of the invention are pharmaceutical agents comprising at least one compound according to the invention, usually together with one or more inert, non-toxic, pharmaceutically suitable auxiliaries, and their use for the purposes mentioned above.

The compounds of the invention may act systemically and/or locally. For this purpose, they can be administered by suitable means, for example parenterally, possibly by inhalation or as implants or stents.

The compounds of the invention may be administered in a form of administration suitable for these routes of administration.

Parenteral administration may be carried out bypassing a resorption step (e.g. intravenous, intra-arterial, intracardiac, intraspinal or intralumbar) or involving resorption (e.g. intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal). Administration forms suitable for parenteral administration include, in particular, injection and infusion preparations in the form of solutions, suspensions, emulsions or lyophilisates. Parenteral administration, especially intravenous administration, is preferred.

It has generally been found that in the case of parenteral administration it is advantageous to administer an amount of about 0.1 to 20 mg/kg, preferably about 0.3 to 7 mg/kg of body weight to achieve effective results.

Nevertheless, it may optionally be necessary to deviate from the amounts indicated, depending, inter alia, on the body weight, the route of administration, the individual response to the active substance, the type of formulation and the point or time of administration or the time interval. Thus, in some cases less than the minimum amount mentioned above may be sufficient, while in other cases the upper limit mentioned must be exceeded. In the case of larger amounts to be administered, it may be advantageous to divide them into several individual doses within one day.

Examples

The following examples illustrate the invention. The present invention is not limited to these examples.

Unless otherwise indicated, the percentage data in the following tests and examples are percentages by weight; the parts are parts by weight. The solvent ratio, dilution ratio and concentration data for the liquid/liquid solution are in each case based on volume.

The synthesis route is as follows:

for the examples, an exemplary synthetic route to the examples is shown exemplarily in the following scheme:

scheme 1:synthesis of lysine-linked ADCs with legumain cleavable linkers

。

In the above reaction scheme, X₁、X₂、X₃N and AK₂Have the meanings given in formula (I).

a) The method comprises the following steps HATU, DMF, N-diisopropylethylamine, room temperature; b) h₂10% Pd-C, methanol for 1.5 hours, room temperature; c) 1,1' - [ (1, 5-dioxopentane-1, 5-diyl) bis (oxy)]Dipyrrolidine-2, 5-dione, N, N-diisopropylethylamine and DMF, stirring at room temperature overnight; d) AK2 in PBS, 3-5 equivalents of active ester dissolved in DMSO was added under argon, and stirred at room temperature under argon for 60 minutes, and 3-5 equivalents of active ester dissolved in DMSO was added, and stirred at room temperature under argon for 60 minutes, and then passed through PD10 column (Sephadex) equilibrated with PBS buffer (pH7.2)^®G-25, GE Healthcare), and then concentrated by ultracentrifugation and set to the desired concentration with PBS buffer (pH 7.2). Optionally, sterile filtration is also subsequently performed in the case of in vivo batches.

Scheme 2:synthesis of cysteine-linked ADCs

In the above reaction scheme, X₁、X₂、X₃N and AK₁Have the meanings given in formula (I).

a) The method comprises the following steps HATU, DMF, N-diisopropylethylamine, room temperature; b) zinc chloride, trifluoroethanol, 50 ℃, EDTA c): HATU, DMF, N-diisopropylethylamine, room temperature; d) h₂10% Pd-C, methanol for 1.5 hours, room temperature; e) 1- {6- [ (2, 5-dioxopyrrolidin-1-yl) oxy]-6-oxohexyl } -1H-pyrrole-2, 5-dione, N-diisopropylethylamine, DMF, stirring at room temperature; f) AK1 was dissolved in PBS, 3-4 equivalents of TCEP in PBS buffer were added under argon and stirred at room temperature for about 30 minutes, then 5-10 equivalents of Compound E dissolved in DMSO were added, stirred at room temperature for about 90 minutes, then purified by PD10 columns (Sephadex G-25, GE Healthcare) equilibrated with PBS buffer (pH7.2) and then concentrated by ultracentrifugation and set to the desired concentration with PBS buffer (pH7.2)]. Optionally, sterile filtration is also subsequently performed in the case of in vivo batches.

Scheme 3:synthesis of cysteine-linked ADC Via Ring-opened succinimide

In the above reaction scheme, X₁、X₂、X₃N and AK₁Have the meanings given in formula (I).

[ a): HATU, DMF, N-diisopropylethylamine, room temperature; b) zinc chloride, trifluoroethanol, 50 ℃, EDTA c): HATU, DMF, N-diisopropylethylamine, room temperature; d) h₂10% Pd-C, methanol for 1.5 hours, room temperature; e) 1- {2- [ (2, 5-dioxopyrrolidin-1-yl) oxy]-2-oxoethyl } -1H-pyrrole-2, 5-dione, N-diisopropylethylamine, DMF, stirring at room temperature; f) AK1 dissolved in PBS, under argon adding PBS buffer in 3-4 equivalent TCEP and at room temperature stirring for about 30min, then adding dissolved in DMSO in 5-10 equivalent compound E, at room temperature stirring for about 90 min, then by PBS buffer (pH 8) balance PD10 column (Sephadex G-25, GE Healthcare) and buffer to pH8, then at room temperature stirring overnight, but then at room temperature stirringThereafter optionally purified by PD10 columns (Sephadex G-25, GE Healthcare) equilibrated with PBS buffer (pH7.2) and subsequently concentrated by ultracentrifugation and set to the desired concentration with PBS buffer (pH7.2)]. Optionally, sterile filtration is also subsequently performed in the case of in vivo batches.

A. Examples

Abbreviations and acronyms:

ABCB1 ATP-binding cassette subfamily B member 1 (synonyms for P-gp and MDR 1)

abs

Ac acetyl group

ACN acetonitrile

aq. aqueous, water solution

ATP adenosine triphosphate

BCRP breast cancer drug-resistant protein, efflux transporter

BEP 2-bromo-1-ethylpyridinium tetrafluoroborate

Boc tert-butoxycarbonyl

br. Wide (in NMR)

Bsp. example

bxPC3 human tumor cell line

C concentration

ca.About, about

CI chemical ionization (in MS)

DAR drug to antibody ratio

D doublet peak (in NMR)

D days

DC thin layer chromatography

DCI direct chemical ionization (in MS)

DCM dichloromethane

Dd doublet (in NMR)

DMAP 4-N,N-dimethylaminopyridine

DME 1, 2-dimethoxyethane

DMEM Dulbecco's modified Eagle Medium (standardized nutrient Medium for cell culture)

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

D/P dye (fluorescent dye)/protein ratio

Salt solution of DPBS, D-PBS Dulbecco's phosphate buffer

DSMZ German Collection of microorganisms and cell cultures

PBS = DPBS = D-PBS, pH 7.4, from Sigma, number D8537

Consists of the following components:

0.2 g KCl

0.2 g KH₂PO₄(Anhydrous)

8.0 g NaCl

1.15 g Na₂HPO₄(Anhydrous)

By H₂O make-up to 1 liter

Dt doublet triplet (in NMR)

DTT DL-dithiothreitol

d. Th. theoretical (in chemical yield)

EDC N'- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride

EGFR epidemal growth factor receptor = Epidermal growth factor receptor

EI Electron impact ionization (in MS)

ELISA enzyme-linked immunosorbent assay

eq. equivalent

ESI electrospray ionization (in MS)

ESI-MicroTofq ESI-MicroTofq (mass spectrometer name, Tof = fly)Line time sum q = quadrupole)

FCS fetal calf serum

Fmoc (9H-fluoren-9-ylmethoxy) carbonyl

ges. saturation

GTP 5' -guanosine triphosphate

H hours

HATU O- (7-azabenzotriazol-1-yl) -N,N,N',N'-tetramethyluronium hexafluorophosphate

HEPES 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid

HOAc acetic acid

HOAt 1-hydroxy-7-azabenzotriazole

HOBt 1-hydroxy-1H-benzotriazole hydrate

HOSu N-hydroxysuccinimide

HPLC high pressure high performance liquid chromatography

IC₅₀Half maximal inhibitory concentration

i.m. intramuscularly, into muscle

i.v. vein, into vein

Concentration of konz

KPL-4 human tumor cell line

KU-19-19 human tumor cell line

LC-MS liquid chromatography-mass spectrometry combination

LLC-PK1-Zellen Lewis lung cancer porcine kidney cell line

LLC-PK1 cells transfected by L-MDR human MDR1

LoVo human tumor cell line

M multiplet (in NMR)

Me methyl group

MDR1 multidrug resistance protein 1

MeCN acetonitrile

Min minute

MOLM-13 human tumor cell line

MS mass spectrometry

MTT bromination of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl-2HTetrazolium salts

MV-4-11 human tumor cell line

NB4 human tumor cell line

NCI-H292 human tumor cell line

NMM N-methylmorpholine

NMP N-methyl-2-pyrrolidone

NMR nuclear magnetic resonance spectroscopy

NMRI mouse strain from the Naval Medical Research Institute (NMRI)

Nude mouse Nude M ä use (Experimental animal)

NSCLC Non small cell lung cancer

PBS phosphate buffered saline solution

Pd/C activated carbon supported palladium

P-gp P-glycoprotein, transport protein

Enzymes of PNGaseF for sugar cleavage

quant. quantitative (in yield)

Quart quartet (in NMR)

Quint quintet (in NMR)

Rec-1 human tumor cell line

R_fRetention index (in DC)

RT Room temperature

R_tRetention time (in HPLC)

S Single Peak (in NMR)

s.c. subcutaneous, administration under the skin

SCID mice test mice with severe combined immunodeficiency (severe combined immunodeficiency)

SK-HEP-1 human tumor cell line

t triplet (in NMR)

TBAF tetra-n-butylammonium fluoride

TCEP tris (2-carboxyethyl) phosphine

TEMPO (2,2,6, 6-tetramethylpiperidin-1-yl) oxy

Teoc trimethylsilyl ethoxycarbonyl

tertTert

TFA trifluoroacetic acid

THF tetrahydrofuran

T3P^®2,4, 6-tripropyl-1, 3,5,2,4, 6-trioxatriphosphane-2, 4, 6-trioxide

U251 human tumor cell line

UV ultraviolet spectroscopy

volume ratio v/v (of solution)

Z benzyloxycarbonyl.

HPLC and LC-MS methods:

method 1 (LC-MS):

the instrument comprises the following steps: waters ACQUITY SQD UPLC system; column: waters Acquity UPLC HSS T31.8 μ 50 × 1 mm; eluent A: 1l of water +0.25ml of 99% formic acid, eluent B: 1l acetonitrile +0.25ml 99% formic acid; gradient: 0.0min 90% A → 1.2min 5% A → 2.0min 5% A; oven: 50 ℃; flow rate: 0.40 mL/min; and (4) UV detection: 208-400 nm.

Method 2 (LC-MS):

MS instrument type: waters synapset G2S; UPLC instrument type: waters Acquity I-CLASS; column: waters, BEH300, 2.1X 150mm, C181.7 μm; eluent A: 1l of water +0.01% formic acid; eluent B: 1l acetonitrile +0.01% formic acid; gradient: 0.0min 2% B → 1.5min 2% B → 8.5min 95% B → 10.0min 95% B; oven: 50 ℃; flow rate: 0.50 mL/min; and (4) UV detection: 220 nm.

Method 3 (LC-MS):

MS instrument: waters (micromass) QM; HPLC apparatus: agilent 1100 series; column: agilent ZORBAX extended-C183.0X 50mm 3.5 micron; eluent A: 1l of water +0.01mol of ammonium carbonate, eluent B: 1l of acetonitrile; gradient: 0.0min98% A → 0.2min 98% A → 3.0min 5% A → 4.5min 5% A; oven: 40 ℃; flow rate: 1.75 mL/min; and (4) UV detection: 210 nm.

Method 4 (LC-MS):

MS instrument type: waters synapset G2S; UPLC instrument type: waters Acquity I-CLASS; column: waters, HSST3, 2.1X 50mm, C181.8 μm; eluent A: 1l of water +0.01% formic acid; eluent B: 1l acetonitrile +0.01% formic acid; gradient: 0.0min 10% B → 0.3min 10% B → 1.7min 95% B → 2.5min 95% B; oven: 50 ℃; flow rate: 1.20 mL/min; and (4) UV detection: 210 nm.

Method 5 (LC-MS):

the instrument comprises the following steps: waters ACQUITY SQD UPLC system; column: waters Acquity UPLC HSS T31.8 μ 50 × 1 mm; eluent A: 1l of water +0.25ml of 99% formic acid; eluent B: 1l acetonitrile +0.25ml 99% formic acid; gradient: 0.0min 95% a → 6.0min 5% a → 7.5min 5% a oven: 50 ℃; flow rate: 0.35 mL/min; and (4) UV detection: 210-400 nm.

Method 6 (LC-MS):

the instrument comprises the following steps: micromass Quattro Premier and Waters UPLC Acquity; column: thermo Hypersil GOLD 1.9 μ 50 × 1 mm; eluent A: 1l of water +0.5ml of 50% formic acid, eluent B: 1l acetonitrile +0.5ml 50% formic acid; gradient: 0.0min 97% a → 0.5min 97% a → 3.2min 5% a → 4.0min 5% a oven: 50 ℃; flow rate: 0.3 mL/min; and (4) UV detection: 210 nm.

Method 7 (LC-MS):

the instrument comprises the following steps: agilent MS Quad 6150; HPLC: agilent 1290; column: waters Acquity UPLC HSS T31.8 μ 50 × 2.1 mm; eluent A: 1l of water +0.25ml of 99% formic acid; eluent B: 1l acetonitrile +0.25ml 99% formic acid; gradient: 0.0min 90% a → 0.3min 90% a → 1.7min 5% a → 3.0min 5% a oven: 50 ℃; flow rate: 1.20 mL/min; and (4) UV detection: 205-305 nm.

Method 8 (LC-MS):

MS instrument type: waters synapset G2S; UPLC instrument type: waters Acquity I-CLASS; column: waters, HSST3, 2.1X 50mm, C181.8 μm; eluent A: 1l of water +0.01% formic acid; eluent B: 1l acetonitrile +0.01% formic acid; gradient: 0.0min 2% B → 2.0min 2% B → 13.0min 90% B → 15.0min 90% B; oven: 50 ℃; flow rate: 1.20 ml/min; and (4) UV detection: 210 nm.

Method 9: (LC-MS-preparative purification method)

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

Or:

MS instrument: waters, HPLC instrument: waters (column Phenomenex Luna 5. mu.C 18(2) 100A, AXIA Tech.50X 21.2mm, eluent A: water +0.05% formic acid, eluent B: acetonitrile (ULC) with gradient, flow rate: 40 ml/min; UV detection: DAD; 210- & 400 nm).

Method 10: (LC-MS analysis method)

MS instrument: waters SQD; HPLC apparatus: waters UPLC; column: zorbax SB-Aq (Agilent), 50 mm. times.2.1 mm, 1.8 μm; eluent A: water +0.025% formic acid, eluent B: acetonitrile (ULC) +0.025% formic acid; gradient: 0.0min98% A-0.9min 25% A-1.0min 5% A-1.4min 5% A-1.41min 98% A-1.5min 98% A; oven: 40 ℃; flow rate: 0.600 ml/min; and (4) UV detection: DAD; 210 nm.

Method 11 (HPLC):

the instrument comprises the following steps: HP1100 series

Column: merck Chromolith speedROD RP-18e, 50-4.6mm, catalog number 1.51450.0001, Pre-column Chromolith Guard Cartidge Kit, RP-18e, 5-4.6mm, catalog number 1.51470.0001;

gradient: the flow rate is 5 mL/Min;

injection volume 5 μ l;

solvent A: in waterHClO of₄(70%)(4mL/l)，

Solvent B: acetonitrile

Initial 20% B

0.50Min 20% B

3.00Min 90% B

3.50Min 90% B

3.51Min 20% B

4.00Min 20% B

Column temperature: at a temperature of 40 c,

wavelength: 210 nm.

Method 12 (LC-MS):

MS instrument type: thermo Scientific FT-MS; UHPLC + instrument type: thermo Scientific UltiMate 3000; column: waters, HSST3, 2.1X 75mm, C181.8 μm; eluent A: 1l of water +0.01% formic acid; eluent B: 1l acetonitrile +0.01% formic acid; gradient: 0.0min 10% B → 2.5min 95% B → 3.5min 95% B; oven: 50 ℃; flow rate: 0.90 ml/min; and (4) UV detection: 210 nm/optimal integration path 210- & lt300 nm.

Method 13：(LC-MS)：

MS instrument: waters (Micromass) Quattro Micro; instrument Waters UPLC Acquity; column: waters BEH C181.7. mu.50X 2.1 mm; eluent A: 1l of water +0.01mol of ammonium formate, eluent B: 1l of acetonitrile; gradient: 0.0min 95% A → 0.1min 95% A → 2.0min 15% A → 2.5min 15% A → 2.51min 10% A → 3.0min 10% A; oven: 40 ℃; flow rate: 0.5 ml/min; and (4) UV detection: 210 nm.

Method 14：(LC-MS) (MCW-LTQ-POROSHELL-TFA98-10min)

MS instrument type: ThermoFisher scientific LTQ-Orbitrap-XL; HPLC instrument type: agilent 1200 SL; column: agilent, POROSHELL 120, 3X 150mm, SB-C182.7 μm; eluent A: 1l of water +0.1% trifluoroacetic acid; eluent B: 1l acetonitrile +0.1% trifluoroacetic acid; gradient: 0.0min 2% B → 0.3min 2% B → 5.0min 95% B → 10.0min 95% B; oven: 40 ℃; flow rate: 0.75 ml/min; and (4) UV detection: 210 nm.

All reactants or reagents whose preparation is not explicitly described below are purchased from generally available sources. For all other reactants or reagents whose preparation is likewise not described below and which are not commercially available or obtained from sources not generally available, reference is made to the publications describing their preparation.

Starting compounds and intermediates:

intermediate C52

(1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropan-1-amine

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

The combined two batches were purified by preparative RP-HPLC (column: Daiso 300X 100; 10. mu. flow rate: 250mL/min, MeCN/water). The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gives 125.15g (87% of theory) of the compound methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate.

LC-MS (method 1) R_t = 1.18 min; MS (ESIpos): m/z = 295 [M+H]⁺。

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

LC-MS (method 7) R_t = 1.59 min; MS (ESIpos): m/z = 328 [M+H]⁺。

3.60g (11.00mmol) of methyl 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrole-2-carboxylate are initially taken in 90.0mL of THF and 1.04g (27.50mmol) of lithium aluminum hydride (2.4M in THF) are added at 0 ℃. The reaction mixture was stirred at 0 ℃ for 30 minutes. At 0 ℃, saturated sodium potassium tartrate solution was added and ethyl acetate was added to the reaction mixture. The organic phase was extracted three times with saturated sodium potassium tartrate solution. The organic phase was washed once with saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated under vacuum and the residue was dissolved in 30.0mL dichloromethane. 3.38g (32.99mmol) of manganese (IV) oxide were added and stirred at room temperature for 48 hours. An additional 2.20g (21.47mmol) of manganese (IV) oxide was added and stirred at room temperature overnight. The reaction mixture was filtered through celite and the filter cake was washed with dichloromethane. The solvent was evaporated under vacuum and the residue 2.80g (1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrole-2-carbaldehyde) was used in the next synthesis step without further purification.

LC-MS (method 7) R_t = 1.48 min; MS (ESIpos): m/z = 298 [M+H]⁺。

28.21g (94.88mmol) of 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrole-2-carbaldehyde are initially taken with 23.00g (189.77mmol) of (R) -2-methylpropane-2-sulfinamide in 403.0mL of anhydrous THF, and 67.42g (237.21mmol) of titanium (IV) isopropoxide are added and stirred at room temperature overnight. 500mL of a saturated NaCl solution and 1000.0mL of ethyl acetate were added, and the mixture was stirred at room temperature for 1 hour. Filter through celite and wash the filtrate twice with saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under vacuum and the residue was purified using a Biotage Isolera (silica gel, column 1500+340g SNAP, flow rate 200mL/min, ethyl acetate/cyclohexane 1: 10).

LC-MS (method 7) R_t = 1.63 min; MS (ESIpos): m/z = 401 [M+H]⁺。

25.00g (62.42mmol) of (R) -N- { (E/Z) - [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] methylene } -2-methylpropane-2-sulfinamide are initially taken in anhydrous THF under argon and cooled to-78 ℃. 12.00g (187.27mmol) of tert-butyllithium (1.7M solution in pentane) were then added at-78 ℃ and stirred at this temperature for 3 hours. At-78 deg.C, 71.4mL of methanol and 214.3mL of saturated ammonium chloride solution were then added successively, and the reaction mixture was allowed to warm to room temperature and stirred at room temperature for 1 hour. Dilute with ethyl acetate and wash with water. The organic phase is dried over magnesium sulfate and the solvent is evaporated under vacuum. The residue (R) -N- { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } -2-methylpropane-2-sulfinamide was used in the next synthetic step without further purification.

LC-MS (method 6) R_t = 2.97 min; MS (ESIpos): m/z = 459 [M+H]⁺。

28.00g (61.05mmol) (R) -N- { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } -2-methylpropane-2-sulfinamide were placed in advance in 186.7mL 1, 4-dioxane, and then 45.8mL HCl (4.0M) as a solution in 1, 4-dioxane were added. The reaction mixture was stirred at room temperature for 2 hours and the solvent was evaporated under vacuum. The residue was purified by preparative HPLC (column: Kinetix 100X 30; flow rate: 60mL/min, MeCN/water). Acetonitrile was evaporated under vacuum and dichloromethane was added to the aqueous residue. The organic phase is washed with sodium bicarbonate solution and dried over magnesium sulfate. The solvent was evaporated under vacuum and the residue was dried under high vacuum. This gives 16.2g (75% of theory) of the title compound.

LC-MS (method 6) R_t = 2.10 min; MS (ESIpos): m/z = 338 [M-NH₂]⁺, 709 [2M+H]⁺。

¹H-NMR (400 MHz, DMSO-d₆): δ[ppm] = 0.87 (s, 9H), 1.53 (s, 2H), 3.59 (s, 1H), 5.24 (d, 2H), 6.56 (s, 1H), 6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m, 1H),7.26 (m, 2H), 7.34 (m, 2H), 7.46 (m, 1H)。

Intermediate C58

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

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

LC-MS (method 12) R_t = 1.97 min; MS (ESIpos): m/z = 614 (M+H)⁺。

2.06g (3.36mmol) of this intermediate are initially taken in 76 mL of DCM and acylated with 0.81mL (7.17mmol) of 2-chloro-2-oxoethyl acetate in the presence of 2.1 mL of triethylamine. After stirring at room temperature for 20h, 0.36mL of 2-chloro-2-oxoethyl acetate and 0.94 mL of triethylamine are added and the batch is stirred at room temperature for a further 15 min. It is then diluted with 500mL of ethyl acetate and extracted twice with 300 mL of 5% citric acid, twice with 300 mL of saturated sodium bicarbonate solution and once with 100mL of saturated sodium chloride solution, successively, and then dried over magnesium sulfate and concentrated. Drying under high vacuum results in 2.17g (79% of theory) of protected intermediate.

LC-MS (method 1) R_t = 1.48 min; MS (ESIpos): m/z = 714 (M+H)⁺。

2.17 mg (2.64mmol) of this intermediate are dissolved in 54 mL of THF and 27 mL of water, and 26 mL of a2 molar lithium hydroxide solution are added. The batch was stirred at room temperature for 30min, then the pH was adjusted to 3 to 4 using 1.4mL TFA. The batch was concentrated under vacuum. After substantial distillation to remove THF, the aqueous solution was extracted twice with DCM and then concentrated to dryness under vacuum. The residue was purified by preparative HPLC (column: Chromatorex C18). After combining the respective fractions, the solvent was evaporated under vacuum and the residue was lyophilized from acetonitrile/water. This gives 1.1 g (63% of theory) of the title compound.

LC-MS (method 1) R_t = 1.34 min; MS (ESIpos): m/z = 656 (M-H)^-。

¹H-NMR (400 MHz, DMSO-d₆): δ[ppm]0.03 (s, 9H), 0.58 (m, 1H), 0.74-0.92 (m, 11H), 1.40 (m, 1H), 3.3 (m, 2H), 3.7 (m, 1H), 3.8-4.0 (m, 2H), 4.15 (q,2H), 4.9 and 5.2 (2d, 2H), 5.61 (s, 1H), 6.94 (m, 2H), 7.13-7.38 (m, 7H), 7.48(s, 1H), 7.60 (m, 1H), 12.35 (s, 1H).

Intermediate C61

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

The title compound was prepared by coupling 60 mg (0.091 mmol) of intermediate C58 with beta-alanine methyl ester and subsequent ester cleavage with 2M lithium hydroxide solution. This gives 67 mg (61% of theory) of the title compound in 2 steps.

LC-MS (method 1) R_t = 1.29 min; MS (ESIpos): m/z = 729 (M+H)⁺。

Intermediate C102

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

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

LC-MS (method 1) R_t = 1.31 min; MS (ESIpos): m/z = 646 (M-H)^-。

Intermediate C110(D)

N- { (2S) -2-amino-4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } (glycolyl) amino ] butyryl } -beta-alanyl-D-glutamic acid dibenzyl ester

Prepared by the title compound as follows: the dibenzyl D-glutamate, previously liberated from its p-toluenesulfonate by partitioning between ethyl acetate and 5% sodium bicarbonate solution, was coupled with intermediate C61 in the presence of HATU and N, N-diisopropylethylamine, and the Teoc protecting group was subsequently cleaved off by zinc chloride in trifluoroethanol.

LC-MS (method 1) R_t = 1.08 min; MS (ESIpos): m/z = 894 [M+H]⁺。

Intermediate C111

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

First, the dipeptide derivative, di-tert-butyl β -alanyl-D-glutamate, is prepared by conventional peptide chemistry by coupling commercially available N- [ (benzyloxy) carbonyl ] - β -alanine and D-glutamic acid di-tert-butyl ester hydrochloride (1:1) in the presence of HATU and subsequent hydrogenolysis to cleave off the Z protecting group. The title compound was then prepared by: this intermediate was coupled with intermediate C102 in the presence of HATU and N, N-diisopropylethylamine and the Z protecting group was subsequently cleaved by hydrogenation at room temperature under standard hydrogen pressure on 10% palladium on charcoal in DCM/methanol 1:1 for 1 hour.

LC-MS (method 1) R_t = 1.06 min; MS (ESIpos): m/z = 826 [M+H]⁺。

Intermediate C117

N- { (2S) -2- (L-asparaginylamino) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } (glycolyl) amino ] butyryl } -beta-alanyl-D-glutamic acid dibenzyl ester trifluoroacetate

Intermediate C110D and N²- (tert-butoxycarbonyl) -L-asparagine 2, 5-dioxopyrrolidin-1-yl ester (251 mg, 764 μmol) was dissolved in 21 ml DMF and N, N-diisopropylethylamine (363 μ L, 2.01 mmol) was added. The reaction was stirred at room temperature and then purified directly by preparative RP-HPLC (column: Chromatorex C18-10). The solvent was evaporated under vacuum and the residue was lyophilized. The obtained intermediate (578 mg, 52 μmol) was dissolved in 20.0 ml trifluoroethanol. Zinc chloride (426 mg, 3.13 mmol) was added to the reaction mixture and stirred at 50 ℃ for a further 40 minutes. ethylenediamine-N, N, N ', N' -tetraacetic acid (914 mg, 3.13 mmol) was added to the batch and diluted with 20ml of water, TFA (200 μ l) was added and stirring was continued briefly. The batch was filtered and purified by preparative RP-HPLC (column: Chromatorex C18-5; 125X40, flow rate: 100ml/min, MeCN/water, 0.1% TFA gradient). After lyophilization the title compound was obtained.

Intermediate L57

(2S) -4-oxo-2- ({ [2- (trimethylsilyl) ethoxy ] carbonyl } amino) butanoic acid methyl ester

500.0mg (2.72mmol) of L-aspartic acid methyl ester hydrochloride and 706.3mg (2.72mmol) of 2- (trimethylsilyl) ethyl 2, 5-dioxopyrrolidine-1-carboxylate were placed in 5.0mL of 1, 4-dioxane in advance and 826.8mg (8.17mmol) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative RP-HPLC (column: Reprosil 250X 40; 10. mu. flow: 50mL/min, MeCN/water, 0.1% TFA). The solvent is then evaporated under vacuum and the residue is dried under high vacuum. This gives 583.9mg (74% of theory) of the compound (3S) -4-methoxy-4-oxo-3- ({ [2- (trimethylsilyl) ethoxy ] carbonyl } amino) butanoic acid.

LC-MS (method 1) R_t = 0.89 min; MS (ESIneg): m/z = 290 (M-H)^-。

592.9mg of (3S) -4-methoxy-4-oxo-3- ({ [2- (trimethylsilyl) ethoxy ] carbonyl } amino) butanoic acid are initially taken in 10.0mL of 1, 2-dimethoxyethane, cooled to-15 ℃ and 205.8mg (2.04mmol) of 4-methylmorpholine and 277.9mg (2.04mmol) of isobutyl chloroformate are added. After 15 minutes, the precipitate is filtered off with suction and washed twice with 10.0mL of 1, 2-dimethoxyethane each time. The filtrate was cooled to-10 ℃ and 115.5mg (3.05mmol) of sodium borohydride dissolved in 10mL of water were added with vigorous stirring. The phases were separated and the organic phase was washed once each with saturated sodium bicarbonate solution and saturated NaCl solution. The organic phase is dried over magnesium sulfate, the solvent is evaporated under vacuum and the residue is dried under high vacuum. This gives 515.9mg (91% of theory) of the compound N- { [2- (trimethylsilyl) ethoxy ] carbonyl } -L-homoserine methyl ester.

LC-MS (method 1) R_t = 0.87 min; MS (ESIpos): m/z = 278 (M+H)⁺。

554.9mg (2.00mmol) of N- { [2- (trimethylsilyl) ethoxy]Carbonyl } -L-homoserine methyl ester was previously placed in 30.0mL of dichloromethane, and 1.27g (3.0mmol) of dess-martin oxidant and 474.7mg (6.00mmol) of pyridine were added. Stir at room temperature overnight. After 4 hours, the batch is diluted with dichloromethane and the organic phase is taken up in 10% Na₂S₂O₃The solution, 10% citric acid solution and saturated sodium bicarbonate solution were washed three times each. The organic phase is dried over magnesium sulfate and the solvent is evaporated under vacuum. This gives 565.7mg (97% of theory) of the title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm] = 0.03 (s, 9H), 0.91 (m, 2H), 2.70-2.79 (m, 1H), 2.88 (dd, 1H), 3.63 (s, 3H), 4.04 (m, 2H), 4.55 (m, 1H), 7.54(d, 1H), 9.60 (t, 1H)。

Intermediate L95

N- [ (benzyloxy) carbonyl ] -L-valyl-L-alanine

This intermediate is prepared by conventional methods of peptide chemistry starting from N- [ (benzyloxy) carbonyl ] -L-valine and L-alanine tert-butyl ester hydrochloride.

LC-MS (method 12) R_t = 1.34 min; MS (ESIpos): m/z = 323.16 (M+H)⁺。

Intermediate L103

N- (pyridin-4-ylacetyl) -L-alanyl-L-asparagine trifluoroacetate salt

The title compound was prepared by the following method: by conventional peptide chemistry, 4-pyridylacetic acid is first coupled with commercial L-alanyl-L-alanine tert-butyl ester in the presence of HATU and N, N-diisopropylethylamine, followed by deprotection with trifluoroacetic acid, coupling with L-asparagine tert-butyl ester, and subsequent deprotection of the carboxyl group with trifluoroacetic acid.

LC-MS (method 1) R_t = 0.15 min; MS (ESIpos): m/z = 394 (M+H)⁺。

Intermediate L116

N- [ (benzyloxy) carbonyl ] -L-alanyl-N-methyl-L-alanine

The title compound was prepared by classical peptide chemistry starting from commercially available N- [ (benzyloxy) carbonyl ] -L-alanine via coupling with N-methyl-L-alanine tert-butyl ester hydrochloride 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]⁺。

Intermediate L117

N- [ (benzyloxy) carbonyl ] -L-alanyl-N-methyl-L-alanyl-L-asparagine trifluoroacetate salt

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

LC-MS (method 1) R_t = 0.57 min; MS (ESIneg): m/z = 421 [M-H]^- 。

Intermediate L118

N- [ (benzyloxy) carbonyl ] -L-alanyl-N-methyl-L-alanyl-L-alanine

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

LC-MS (method 12) R_t = 1.25 min; MS (ESIneg): m/z = 378 [M-H]^- 。

Intermediate L121

N- [ (benzyloxy) carbonyl ] -L-alanyl-N-methyl-L-alanyl-L-leucine

The title compound was prepared by classical peptide chemistry starting from commercially available L-leucine tert-butyl ester hydrochloride, via coupling with N- [ (benzyloxy) carbonyl ] -L-alanyl-N-methyl-L-alanine (intermediate L116) in the presence of HATU, and finally by cleavage of the tert-butyl ester protecting group with TFA.

LC-MS (method 12) R_t = 0.83 min; MS (ESIneg): m/z = 420 [M-H]^- 。

Intermediate L122

(5S,8S,11S) -11- (2-amino-2-oxoethyl) -8- [2- (benzyloxy) -2-oxoethyl ] -5-methyl-3, 6, 9-trioxo-1-phenyl-2-oxa-4, 7, 10-triazadecane-12-oic acid

The title compound was prepared by classical peptide chemistry starting from commercially available 1-tert-butyl-L-aspartate 4-benzyl ester hydrochloride (1:1) via first coupling with N- (benzyloxy) carbonyl ] -L-alanine 2, 5-dioxopyrrolidin-1-yl ester, followed by cleavage of the tert-butyl ester protecting group with TFA, followed by subsequent coupling with L-asparagine 4-tert-butyl ester in the presence of HATU, and finally another cleavage of the tert-butyl ester TFA protecting group with TFA.

LC-MS (method 1) R_t = 0.76 min; MS (ESIpos): m/z = 543 [M+H]⁺ 。

Intermediate L138

1-bromo-2-oxo-6, 9,12, 15-tetraoxa-3-azaoctadecan-18-oic acid

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

LC-MS (method 5) R_t= 1.05 min, MS (ESIpos) M/z = 386 and 388 (M + H)⁺。

Intermediate Q1

N- [ (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) acetyl]-L-alanyl-N-methyl-L-alanyl-N¹- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-asparagine

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

LC-MS (method 12) R_t = 1.66 min; MS (ESIneg): m/z = 1119 [M-H]^-。

Intermediate Q2

N- {5- [ (2, 5-dioxopyrrolidin-1-yl) oxy]-5-oxovaleryl } -L-alanyl-N-methyl-L-alanyl-N¹- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-asparagine

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

LC-MS (method 1) R_t = 0.93 min; MS (ESIpos): m/z = 1195 [M+H]⁺。

Intermediate Q3

N- {5- [ (2, 5-dioxopyrrolidin-1-yl) oxy]-5-oxovaleryl } -L-alanyl-N¹- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-asparagine

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

LC-MS (method 1) R_t = 0.90 min; MS (ESIneg): m/z = 1181 [M-H]^-。

Intermediate Q4

N- [6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl]-L-alanyl-N-methyl-L-alanyl-N¹- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-asparagine

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

LC-MS (method 1) R_t = 3.2 min; MS (ESIpos): m/z = 1177 [M+H]⁺。

Intermediate Q5

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

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

LC-MS (method 1) R_t = 0.96 min; MS (ESIpos): m/z = 1152 [M+H]⁺。

Intermediate Q6

N- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } (glycolyl) amino ] -2- [ (N- {5- [ (2, 5-dioxopyrrolidin-1-yl) oxy ] -5-oxopentanoyl } -L-valyl-L-alanyl) amino ] butyryl } -beta-alanyl-D-glutamic acid

Starting from compound C110D, the title compound was first prepared 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 charcoal in DCM-methanol 1:1 at room temperature under standard hydrogen pressure for 1 hour, and the deprotected intermediate was then converted to the title compound by reaction with 1,1' - [ (1, 5-dioxopentane-1, 5-diyl) bis (oxy) ] dipyrrolidine-2, 5-dione in the presence of N, N-diisopropylethylamine.

LC-MS (method 1) R_t = 0.98 min; MS (ESIpos): m/z = 1095 [M+H]⁺。

Intermediate Q7

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

Starting from compound C110D, the title compound was first prepared 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 charcoal in DCM-methanol 1:1 at room temperature under standard hydrogen pressure for 1 hour, and the deprotected intermediate was then converted to the title compound by reaction with 1- {2- [ (2, 5-dioxopyrrolidin-1-yl) oxy ] -2-oxoethyl } -1H-pyrrole-2, 5-dione in the presence of N, N-diisopropylethylamine.

LC-MS (method 1) R_t = 0.98 min; MS (ESIpos): m/z = 1021 [M+H]⁺。

Intermediate Q8

N- {5- [ (2, 5-dioxopyrrolidin-1-yl) oxy]-5-oxovaleryl } -L-alanyl-N-methyl-L-alanyl-N¹- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-leucinamide

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

LC-MS (method 1) R_t = 1.02 min; MS (ESIpos): m/z = 1194 [M+H]⁺。

Intermediate Q9

N- {5- [ (2, 5-dioxopyrrolidin-1-yl) oxy]-5-oxovaleryl } -L-alanyl-N-methyl-L-alpha-aspartyl-N¹-{(2S)-4-[{(1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-asparagine

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

LC-MS (method 1) R_t = 0.89 min; MS (ESIpos): m/z = 1225 [M+H]⁺。

Intermediate Q10

N- (bromoacetyl) -L-alanyl-N-methyl-L-alanyl-N¹- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-asparagine

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

LC-MS (method 1) R_t= 0.95 min, MS (ESIpos) M/z = 1104 and 1106 [ M + H ]]⁺。

Intermediate Q11

N- (18-bromo-17-oxo-4, 7,10, 13-tetraoxa-16-azaoctadecane-1-yl) -L-alanyl-N-methyl-L-alanyl-N¹- { (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- [ (3- { [ (1R) -1, 3-dicarboxypropyl]Amino } -3-oxopropyl) amino]-1-oxobut-2-yl } -L-asparagine

The synthesis of the title compound was first carried out by coupling intermediate C111 with intermediate L117 in DMF in the presence of 1.5 equivalents of HATU and 3 equivalents of N, N-diisopropylethylamine. Subsequently, the Z protecting group was removed by hydrogenation on 10% palladium on activated carbon in ethanol at room temperature for 2 hours under hydrogen standard pressure. The deprotected intermediate is then reacted with intermediate L138 in DMF in the presence of 1.5 equivalents HATU and 3 equivalents of N, N-diisopropylethylamine. In the final step, the tert-butyl ester group was cleaved by stirring with 8 equivalents of zinc chloride in trifluoroethanol for 2 hours at 50 ℃ to give the title compound.

LC-MS (method 8) R_t = 4.06 min; MS (ESI-pos): m/z = 1353 [M+H]⁺。

B: preparation of antibody-active substance-conjugates (ADC)

General methods for generating antibodies

The protein sequences (amino acid sequences) 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, are converted into DNA sequences encoding the corresponding proteins by Methods known to the skilled worker and inserted into Expression vectors suitable for transient mammalian cell culture (as described in MichelL R. Dyson and Yves Durocher, Scion blue Ltd, 2007).

General methods for expressing antibodies in mammalian cells

Antibodies such as TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580 are produced in transient mammalian cell culture, such as from Tom et al, Methods Express, Chapter 12: expression Systems, edited by Micheal r, dyson and yvesderocher, as described in Scion Publishing Ltd, 2007.

B-3.General procedure for purification of antibodies from cell supernatants

Antibodies such as TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574, and TPP-9580 were obtained from the cell culture supernatants. The cell supernatant was clarified by cell centrifugation. The cell supernatant was then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. To this end, the column was equilibrated in DPBS pH 7.4(Sigma/Aldrich), cell supernatant was applied, and the column was washed with about 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride. The antibody was eluted in 50mM sodium acetate pH 3.5 + 500 mM sodium chloride and then further purified by gel permeation chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

Commercial antibodies were purified from commercial products by standard chromatography (protein a chromatography, preparative gel filtration chromatography (SEC-size exclusion chromatography)).

B-4. general procedure for coupling to cysteine side chain

The following antibodies were used in this conjugation reaction:

example a TPP-981 Cetuximab (anti-EGFR AK)

Example c TPP-6013 (anti-CD 123 AK)

TPP-8987 (anti-CD 123 AK)

TPP-8988 (anti-CD 123 AK)

TPP-9476 (anti-CD 123 AK)

Example H TPP-8382 (anti-B7H 3 AK)

Example e TPP-1015 (anti-Her 2 AK)

Example k TPP-7006 (anti-TWEAKR AK)

TPP-7007 (anti-TWEAKR AK)

Example x TPP-9574 (anti-CXCR 5 AK)

TPP-9580 (anti-CXCR 5 AK)

The coupling reaction is generally carried out under argon.

2 to 5 equivalents of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) dissolved in PBS buffer are added to a solution of the corresponding antibody in PBS buffer in a concentration range of 1 mg/ml to 20 mg/ml, preferably about 10mg/ml to 15 mg/ml, and stirred at room temperature for 1 hour. For this purpose, the solutions of the respective antibodies used can be used at the concentrations described in the examples, or can optionally also be diluted with PBS buffer to about half the starting concentration to achieve the preferred concentration range. Subsequently, 2 to 12 equivalents, preferably about 5 to 10 equivalents, of the maleimide precursor compound or halide precursor compound to be coupled are added as a solution in DMSO, depending on the desired loading. The amount of DMSO should here not exceed 10% of the total volume. The batch was stirred at room temperature for 60-240 minutes in the case of maleimide precursor and 8-24 hours in the case of halide precursor and then placed on a PD10 column (Sephadex) equilibrated in PBS^®G-25, GE Healthcare) and eluted with PBS buffer. Typically, 5mg of the corresponding antibody in PBS buffer is used for reduction and subsequent conjugation unless otherwise indicated. After purification on a PD10 column, a solution of the corresponding ADC in 3.5 ml of PBS buffer was thus obtained in each case. Then concentrated by ultracentrifugation and the sample is optionally diluted back with PBS buffer. If necessary, for better separation of low molecular weight components, concentration by ultrafiltration was repeated after dilution back with PBS buffer. For biological testing, the concentration in the final ADC sample is adjusted to 0.5-15 mg/ml, optionally by dilution back, if necessary. For the ADC solutions, the protein concentrations described in each of the examples were determined. Further, the same effects as described in B-6 were obtainedThe method of (1) determines the antibody loading (drug/mAb ratio).

Depending on the linker, the ADCs shown in the examples may optionally also be present in more or less extent in the form of hydrolyzed open chain succinamides linked to the antibody.

In particular, KSP-I-ADCs linked to the sulfhydryl group of an antibody via the following linker structure can also be prepared, optionally by rebuffering after conjugation and purposely stirring at pH8 according to scheme 28 into ADC linked via open-chain succinamide for about 20-24 hours

#1 represents the sulfur bridge to the antibody and #2 represents the point of attachment to the modified KSP inhibitor.

Such ADCs in which the linker is present linked to the antibody via a hydrolysed open chain succinamide may also optionally be prepared by small and large scale conjugation as shown herein, for example:

2 to 7 equivalents of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) dissolved in PBS buffer are added to a solution of 2 to 5mg of the corresponding antibody in PBS buffer in a concentration range of 1 mg/ml to 20 mg/ml, preferably about 5 mg/ml to 15 mg/ml, and stirred at room temperature for 30 minutes to 1 hour. Subsequently, 2 to 20 equivalents, preferably about 5-10 equivalents, of the maleimide precursor compound to be coupled are added as a solution in DMSO, depending on the desired loading. To achieve higher DAR, 15-20 equivalents may also be used. The amount of DMSO should here not exceed 10% of the total volume. The batch is stirred at room temperature for 60 to 240 minutes. The eluate was diluted to a concentration of 1-5 mg/ml with PBS buffer pH8 and then placed on a PD-10 column (Sephadex) equilibrated with PBS buffer pH8^®G-25, GE Healthcare) and eluted with PBS buffer pH 8. The eluate was stirred at room temperature under argon overnight. Subsequently, the solution was concentrated by ultracentrifugation and diluted back with PBS buffer (pH 7.2).

Medium scale coupling:

under argon, 2 to 7 equivalents, preferably 3 equivalents, of T are addedA solution of CEP in PBS buffer (c ~ 0.2.2-0.8 mg/ml, preferably 0.5 mg/ml) is added to 20-200 mg of the antibody of interest in PBS buffer (c ~ 5-15 mg/ml. the batch is stirred at room temperature for 30 minutes, then 2-20, preferably 5-10 equivalents of the maleimide precursor compound dissolved in DMSO is added in order to achieve a higher DAR, 15-20 equivalents may also be used, after stirring at room temperature for a further 1.5-2 hours, the batch is diluted with PBS buffer that has been previously adjusted to pH 8. the solution is then placed on a PD10 column (Sephadex) equilibrated with PBS buffer pH8^®G-25, GE Healthcare) and eluted with PBS buffer pH 8. The eluate was diluted to a concentration of 2-7mg/mL with PBS buffer pH 8. The solution was stirred at room temperature under argon overnight. The solution was then re-buffered to pH7.2 if necessary. The ADC solution was concentrated by ultracentrifugation, diluted back with PBS buffer (pH7.2), and then optionally concentrated again to a concentration of about 10 mg/mL.

In the formulae shown, AK1 can have the following meanings here:

example a TPP-981 Cetuximab (partially reduced) -S §¹

Example c TPP-6013 (anti-CD 123 AK) (partially reduced) -S §¹

TPP-8987 (anti-CD 123 AK) (partially reduced) -S §¹

TPP-8988 (anti-CD 123 AK) (partially reduced) -S §¹

TPP-9476 (anti-CD 123 AK) (partially reduced) -S §¹

Example e TPP-1015 (anti-Her 2 AK) (partially reduced) -S §¹

Example H TPP-8382 (anti-B7H 3 AK) (partially reduced) -S §¹

Example k TPP-7006 (anti-TWEAKR) (partially reduced) -S §¹

TPP-7007 (anti-TWEAKR) (partially reduced) -S §¹

Example x TPP-9574 (anti-CXCR 5 AK) (partially reduced) -S §¹

TPP-9580 (anti-CXCR 5 AK) (partially reduced) -S §¹

Wherein

§¹Represents a bond to a succinimide group or to an isomeric hydrolytically cleaved chain succinamide or alkylene residue optionally resulting therefrom,

and is

S represents the sulfur atom of the cysteine residue of the partially reduced antibody.

B-5 general procedure for coupling to lysine side chains

The following antibodies were used in the coupling reaction:

example a TPP-981 Cetuximab (anti-EGFR AK)

Example c TPP-6013 (anti-CD 123 AK)

TPP-8987 (anti-CD 123 AK)

TPP-8988 (anti-CD 123 AK)

TPP-9476 (anti-CD 123 AK)

Example e TPP-1015 (anti-Her 2 AK)

Example k TPP-7006 (anti-TWEAKR AK)

TPP-7007 (anti-TWEAKR AK)

Example x TPP-9574 (anti-CXCR 5 AK)

TPP-9580 (anti-CXCR 5 AK)

The coupling reaction is generally carried out under argon.

Depending on the desired loading, 2 to 8 equivalents of the precursor compound to be coupled are added as a solution in DMSO to a solution of the corresponding antibody in PBS buffer in a concentration range of 1 mg/ml to 20 mg/ml, preferably about 10 mg/ml. After stirring at room temperature for 30 minutes to 6 hours, the same amount of precursor compound in DMSO is added again. The amount of DMSO should here not exceed 10% of the total volume. After stirring for an additional 30 minutes to 6 hours at room temperature, the batch was placed on PD10 columns (Sephadex G-25, GE Healthcare) equilibrated in PBS and eluted with PBS buffer. After purification on a PD10 column, a solution of the corresponding ADC in PBS buffer was thus obtained in each case. Then concentrated by ultracentrifugation and the sample is optionally diluted back with PBS buffer. If necessary, for better separation of low molecular weight components, concentration by ultrafiltration was repeated after dilution back with PBS buffer. For biological testing, the concentration of the final ADC sample is adjusted to 0.5-15 mg/ml, optionally by dilution back, if necessary.

For the ADC solutions, the protein concentrations described in each of the examples were determined. In addition, antibody loading (drug/mAb ratio) was determined using the method described in B-6.

In the formula shown, AK₂The following meanings are given herein

Example a TPP-981 Cetuximab (anti-EGFR AK) -NH §²

Example c TPP-6013 (anti-CD 123 AK) -NH §²

TPP-8987 (anti-CD 123 AK) -NH §²

TPP-8988 (anti-CD 123 AK) -NH §²

TPP-9476 (anti-CD 123 AK) -NH §²

Example e TPP-1015 (anti-Her 2 AK) -NH §²

Example k TPP-7006 (anti-TWEAKR AK) -NH §²

TPP-7007 (anti-TWEAKR AK) -NH §²

Example x TPP-9574 (anti-CXCR 5 AK) -NH §²

TPP-9580 (anti-CXCR 5 AK) -NH §²

Wherein

§²Represents a bond to a carbonyl group

And is

NH represents the side chain amino group of a lysine residue of the antibody.

Further purification and characterization of the conjugates according to the invention

After the reaction is carried out, in some cases, the reaction mixture is concentrated, for example by ultrafiltration, and then desalted and purified by chromatography, for example using Sephadex G-25 columns. Elution is carried out, for example, with Phosphate Buffered Saline (PBS). The solution was then sterile filtered and frozen. Alternatively, the conjugate may be lyophilized.

B-6 determination of antibody, Cluster load and open cysteine adduct content

To identify proteins, in addition to molecular weight determination, deglycosylation and/or denaturation is followed by trypsinization, which confirms the identity of the protein after denaturation, reduction and derivatization by the trypsin peptide found.

The resulting solutions of the conjugates described in the examples in PBS buffer were tested for the loading of the toxic clusters (expressed as DAR in the table, drug/antibody ratio) as follows:

the determination of the loading of the lysine-linked ADCs into the toxic cluster was performed by mass spectrometry of the molecular weight of each conjugate species. Here, the antibody conjugate was deglycosylated with PNGaseF in advance, and the sample was acidified and ESI-MicroTof was used after HPLC separation/desalting_Q(Bruker Daltonik) was analyzed by mass spectrometry. All spectra on the signal in TIC (total ion chromatogram) were added and the molecular weights of the different conjugate species were calculated based on MaxEnt deconvolution. After integration of the signals of the different species, DAR (= drug/antibody ratio) is then calculated. To do this, the sum of the integrated results of all species weighted by the number of poison clusters is divided by the sum of the integrated results of all species simply weighted.

Determination of the toxic cluster loading of cysteine-linked conjugates was determined by reverse phase chromatography of reduced and denatured ADC. Guanidine hydrochloride (GuHCl) (28.6 mg) and DL-Dithiothreitol (DTT) solution (500 mM, 3 μ l) were added to the ADC solution (1 mg/mL, 50 μ l). The mixture was incubated at 55 ℃ for 1 hour and analyzed by HPLC.

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

The peaks detected were assigned by comparing the retention times of the light chain (L0) and heavy chain (H0) of the unconjugated antibody. Peaks detected only in the conjugated samples were assigned to light chains with one toxoproteger (L1) and heavy chains with one, two and three toxoprotegers (H1, H2, H3).

The average loading of antibodies with poison clusters (so-called DAR, drug/antibody ratio) is determined from the peak area determined by integration as twice the sum of HC loading and LC loading, wherein the LC loading is calculated from the sum of the integrated results of all LC peaks weighted by the number of poison clusters divided by the sum of the integrated results of all LC peaks simply weighted, and wherein the HC loading is calculated from the sum of the integrated results of all HC peaks weighted by the number of poison clusters divided by the sum of the integrated results of all LC peaks simply weighted. In individual cases, the viral cluster load may not be accurately determined due to co-elution of certain peaks.

In the case where the light and heavy chains cannot be sufficiently separated by HPLC, the determination of the toxic cluster loading of the cysteine-linked conjugates was performed by mass spectrometric determination of the molecular weight of each conjugate species at the light and heavy chains.

For this, guanidine hydrochloride (GuHCl) (28.6 mg) and DL-Dithiothreitol (DTT) solution (500 mM, 3 μ l) were added to the ADC solution (1 mg/ml, 50 μ l). The mixture was incubated at 55 ℃ for 1 hour and using ESI-MicroTof_Q(Bruker Daltonik) was analyzed by mass spectrometry after on-line desalting.

For DAR (drug: antibody ratio) determination, all spectra on the signal in TIC (total ion chromatogram) were added and the molecular weights of the different conjugate species at the light and heavy chains were calculated based on MaxEnt deconvolution. The average loading of antibody with toxic clusters was determined from the peak area determined by integration as twice the sum of HC loading and LC loading. Here, the LC capacity is calculated from the sum of the integrated results of all LC peaks weighted by the number of toxic clusters divided by the sum of the integrated results of all LC peaks simply weighted, and the HC capacity is calculated from the sum of the integrated results of all HC peaks weighted by the number of toxic clusters divided by the sum of the integrated results of all HC peaks simply weighted.

In the case of the open construct, to determine the content of open cysteine adducts, the molecular weight area ratio of closed to open cysteine adducts (molecular weight Δ 18 daltons) of all mono-conjugated light and heavy chain variants was determined. The average of all variants gives the content of open cysteine adducts.

B-7 verification of antigen-binding of ADC

The ability of the binding agent to bind to the target molecule is examined after the coupling has been performed. Various methods are known to those skilled in the art for this purpose; for example, the affinity of the conjugate can be checked using ELISA techniques or surface plasmon resonance analysis (BIAcore ™ measurement). The concentration of the conjugate can be measured by the skilled person using common methods, for example by protein assays for antibody conjugates (see also Doronina et al; Nature Biotechnol. 2003; 21: 778-.

Examples of metabolites

Example M1

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

Intermediate C110D was converted to the title compound by hydrogenation at room temperature under hydrogen standard pressure on 10% palladium on activated carbon in ethanol for 1 hour.

LC-MS (method 1) R_t = 1.78 min; MS (ESIpos): m/z = 714 [M+H]⁺。

The ADCs exemplarily shown below are capable of releasing the preferred metabolite M1, which has preferred pharmacological properties.

Example ADC

The ADCs shown in the structural formulae of the examples, which are coupled to the cysteine side chains of the antibodies via maleimide residues, are predominantly present in the open-or closed-loop form shown in each case, depending on the linker and coupling procedure. However, the formulations may contain small amounts of the respective other forms. The coupling reaction was carried out under argon. All larger batches used for in vivo testing were sterile filtered at the end of the preparation.

Example 1

Exemplary procedure a:

a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5mg of the corresponding antibody in 0.5mL PBS (c =10mg/mL) under argon. The batch was stirred at room temperature for 30min, and then 0.26 mg (0.00023 mmol) of intermediate Q1 dissolved in 50 μ l DMSO was added. After stirring for a further 90 min at room temperature, the batch was diluted to a volume of 2.5mL with PBS buffer previously adjusted to pH8 and then placed on a PD10 column (Sephadex) equilibrated with PBS buffer pH8^®G-25, GE Healthcare) and eluted with PBS buffer pH 8. The eluate was stirred at room temperature under argon overnight. Subsequently concentrated by ultracentrifugation and diluted back with PBS buffer (pH 7.2).

Exemplary procedure B:

a solution of 0.172 mg TCEP in 0.3mL PBS buffer was added to 30 mg of the corresponding antibody in 3mL PBS under argon (c =10 mg/mL). The batch was stirred at room temperature for 30min, and then 1.57 mg (0.0014 mmol) of intermediate Q1 dissolved in 300 μ l DMSO was added. After stirring for a further 90 min at room temperature, the batch was diluted to a volume of 5mL with PBS buffer previously adjusted to pH8 and then placed on a PD10 column (Sephadex) equilibrated with PBS buffer pH8^®G-25, GE Healthcare) and eluted with PBS buffer pH 8. The eluate was diluted to a volume of 7.5ml with PBS buffer pH8 and stirred at room temperature under argon overnight. The solution was then placed on a PD10 column (Sephadex) equilibrated with PBS buffer pH7.2^®G-25, GE Healthcare) and eluted with PBS buffer pH 7.2. Then concentrated by ultracentrifugation, diluted back with PBS buffer (pH7.2), and again concentrated and sterile filtered.

The following ADCs were prepared similar to these protocols and characterized as shown in the table below:

examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						1a-981	EGFR	981	A	1.85	2.5
1c-6013	CD123	6013	A	2.0	2.4
						1c-9476	CD123	9476	A	1.96	3.1
1e-1015	HER2	1015	A	1.75	3.3
						1h-8382	B7H3	8382	B	11.01	3.5
1k-7006	TWEAKR	7006	A	1.8	2.9
						1k-7007	TWEAKR	7007	B	7.84	3.3
1x-9574	CXCR5	9574	A	1.26	2.9

Example 2

Exemplary procedure a:

5 equivalents (0.2 mg) of intermediate Q2 dissolved in 50 μ l DMSO were added to 5mg of the antibody of interest (c =10mg/ml) in 0.5ml PBS under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 2.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and diluted back with PBS (pH 7.2).

Exemplary procedure B:

4 equivalents (1 mg) of intermediate Q2 dissolved in 50 μ l DMSO were added to 30 mg of the antibody of interest (c =10mg/ml) in 3ml PBS buffer (pH7.2) under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation, diluted back with PBS (pH7.2) and concentrated again and sterile filtered.

Exemplary procedure C:

2.5 equivalents (1 mg) of intermediate Q2 dissolved in 250 μ l DMSO was added to 50 mg of the antibody of interest (c =10mg/ml) in 5ml PBS buffer (pH7.2) under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 7.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation, diluted back with PBS (pH7.2) and concentrated again and sterile filtered.

Exemplary procedure D:

4.5 equivalents (36 mg) of intermediate Q2 dissolved in 7.5ml DMSO were added to 1000 mg of the antibody of interest (c = 6.7 mg/ml) in 150 ml PBS buffer (pH7.2) under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then purified by cross-flow filtration, concentrated and sterile filtered.

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						2a-981	EGFR	981	A	2.23	4.5
2c-6013	CD123	6013	A	2.32	4.9
						2c-8987	CD123	8987	B	8.86	5.8
2c-8988	CD123	8988	B	9.81	3.6
						2c-9476B	CD123	9476	B	10.27	4.2
2c-9476C	CD123	9476	C	9.22	3.4
						2c-9476D	CD123	9476	D	15.83	6.3
2e-1015	HER2	1015	A	2.05	5.4
						2k-7006	TWEAKR	7006	A	2.09	5.9
2k-7007	TWEAKR	7007	B	9.32	3.4
						2x-9574	CXCR5	9574	B	9.5	4.8
2x-9580	CXCR5	9580	B	10.12	4.8

Example 3

Exemplary procedure a:

5 equivalents (0.2 mg) of intermediate Q3 dissolved in 50 μ l DMSO were added to 5mg of the antibody of interest (c =10mg/ml) in 0.5ml PBS under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 2.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and diluted back with PBS (pH 7.2).

Exemplary procedure B:

4 equivalents (1 mg) of intermediate Q3 dissolved in 50 μ l DMSO were added to 30 mg of the antibody of interest (c =10mg/ml) in 3ml PBS buffer (pH7.2) under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then subsequently diluted to 2.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation, diluted back with PBS (pH7.2) and concentrated again and sterile filtered.

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						3a-981	EGFR	981	A	1.99	5.0
3c-9476	CD123	9476	A	2.09	5.8
						3e-1015	HER2	1015	A	2.06	6.3
3k-7007	TWEAKR	7007	A	2.11	5.3
						3x-9574	CXCR5	9574	A	2.02	4.5

Example 4

Exemplary procedure a:

a solution of 0.029 mg TCEP in 0.05 ml PBS buffer was added to 5mg of the antibody of interest (c =12.5 mg/ml) in 0.4 ml PBS buffer (pH7.2) under argon. The batch was stirred at room temperature for 30min, and then 0.275 mg (0.00023 mmol) of intermediate Q4 dissolved in 50 μ l DMSO was added. After stirring for a further 90 minutes at room temperature, the batch was diluted to a total volume of 2.5ml with PBS buffer. The solution was then placed in PD10 equilibrated with PBS buffer (pH7.2)Column (Sephadex)^®G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2). Subsequently concentrated by ultracentrifugation and diluted back with PBS buffer (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						4c-9476	CD123	9476	A	2.06	3.5
4k-7007	TWEAKR	7007	A	1.87	4.0
						4x-9574	CXCR5	9574	A	1.93	3.6

Example 5

Exemplary procedure a:

5 equivalents (0.2 mg) of intermediate Q5 dissolved in 50 μ l DMSO were added to 5mg of the antibody of interest (c =10mg/ml) in 0.5ml PBS under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 2.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and diluted back with PBS (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						5c-9476	CD123	9476	A	2.37	5.3
5e-1015	HER2	1015	A	2.33	5.5
						5k-7007	TWEAKR	7007	A	2.18	5.6
5x-9574	CXCR5	9574	A	1.88	6.8

Example 6

Exemplary procedure a:

5 equivalents (0.18 mg) of intermediate Q6 dissolved in 50 μ l DMSO were added to 5mg of the antibody of interest (c =12.5 mg/ml) in 0.4 ml PBS under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 2.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and diluted back with PBS (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						6a-981	EGFR	981	A	2.39	4.9
6c-9476	CD123	9476	A	1.8	5.3
						6e-1015	HER2	1015	A	2.23	6.2
6k-7007	TWEAKR	7007	A	2.57	5.6

Example 7

Exemplary procedure a:

a solution of 0.029 mg TCEP in 0.05 ml PBS buffer was added to 5mg of the antibody of interest in 0.4 ml PBS under argon (c =12.5 mg/ml). The batch was stirred at room temperature for 30min, and then 0.24 mg (0.00023 mmol) of intermediate Q7 dissolved in 50 μ l DMSO was added. After stirring for a further 90 min at room temperature, the batch was diluted to a volume of 2.5ml with PBS buffer previously adjusted to pH8 and then placed on a PD10 column (Sephadex) equilibrated with PBS buffer pH8^® G-25, GE Healthcare) and eluted with PBS buffer pH 8. The eluate was stirred at room temperature under argon overnight. This was then concentrated by ultracentrifugation and diluted back with PBS buffer (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						7a-981	EGFR	981	A	2.03	3.4
7c-9476	CD123	9476	A	1.53	4.0
						7e-1015	HER2	1015	A	1.88	3.8
7k-7007	TWEAKR	7007	A	1.99	3.6

Example 8

Exemplary procedure a:

5 equivalents (0.2 mg) of intermediate Q8 dissolved in 50 μ l DMSO were added to 5mg of the antibody of interest (c =10mg/ml) in 0.5ml PBS under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 2.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and diluted back with PBS (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						8a-981	EGFR	981	A	2.32	6.5
8c-9476	CD123	9476	A	2.37	6.9
						8e-1015	HER2	1015	A	1.46	6.6
8k-7007	TWEAKR	7007	A	2.43	6.7

Example 9

Exemplary procedure a:

5 equivalents (0.2 mg) of intermediate Q9 dissolved in 50 μ l DMSO were added to 5mg of the antibody of interest (c =10mg/ml) in 0.5ml PBS under argon. After stirring at room temperature for 1 hour, the same amount was added again and the batch was stirred at room temperature for a further 1 hour. The batch was then diluted to 2.5ml with PBS buffer (pH7.2), purified on a Sephadex column, then concentrated by ultracentrifugation and diluted back with PBS (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						9a-981	EGFR	981	A	2.34	4.4
9c-9476	CD123	9476	A	2.65	4.2
						9e-1015	HER2	1015	A	2.22	4.8
9k-7007	TWEAKR	7007	A	2.14	3.8

Example 10

Exemplary procedure a:

under argon, 0.029 mg of TCEPA solution in 0.05 ml of PBS buffer was added to 5mg of the antibody of interest (c =10mg/ml) in 0.5ml of PBS buffer (pH 7.2). The batch was stirred at room temperature for 30min, and then 0.295 mg (0.00023 mmol) of intermediate Q10 dissolved in 50 μ l DMSO was added. After stirring for a further 20h at room temperature, the batch was diluted to a total volume of 2.5ml with PBS buffer. The solution was then placed on a PD10 column (Sephadex) equilibrated with PBS buffer (pH7.2)^® G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2). Subsequently concentrated by ultracentrifugation and diluted back with PBS buffer (pH 7.2).

Exemplary procedure C for obtaining higher DAR:

a solution of 0.057 mg TCEP in 0.05 ml PBS buffer was added to 5mg of the antibody of interest (c =10mg/ml) in 0.5ml PBS buffer (pH7.2) under argon. The batch was stirred at room temperature for 30min, and then 0.59 mg (0.00053 mmol) of intermediate Q10 dissolved in 50 μ l DMSO was added. After stirring for a further 20h at room temperature, the batch was diluted to a total volume of 2.5ml with PBS buffer. The solution was then placed on a PD10 column (Sephadex) equilibrated with PBS buffer (pH7.2)^® G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2). Subsequently concentrated by ultracentrifugation and diluted back with PBS buffer (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						10a-981	EGFR	981	A	1.9	3.2
10c-9476	CD123	9476	A	1.83	2.7
						10c-9476 hD	CD123	9476	C	1.97	4.8
10e-1015	HER2	1015	A	1.83	3.4
						10k-7007	TWEAKR	7007	A	1.9	4.7
10x-9574	CXCR5	9574	A	1.41	3.8
						10x-9574 hD	CXCR5	9574	C	0.97	6.3

Example 11

Exemplary procedure a:

a solution of 0.029 mg TCEP in 0.05 ml PBS buffer was added to 5mg of the antibody of interest (c =12.5 mg/ml) in 0.4 ml PBS buffer (pH7.2) under argon. The batch was stirred at room temperature for 30min, and then 0.32 mg (0.00023 mmol) of intermediate Q11 dissolved in 50 μ l DMSO was added. After stirring for a further 20h at room temperature, the batch was diluted to a total volume of 2.5ml with PBS buffer. The solution was then placed on a PD10 column (Sephadex) equilibrated with PBS buffer (pH7.2)^® G-25, GE Healthcare) and eluted with PBS buffer (pH 7.2). Subsequently concentrated by ultracentrifugation and diluted back with PBS buffer (pH 7.2).

Examples	Target	Antibody TPP-	Protocol	C [mg/ml]	DAR
						11a-981	EGFR	981	A	1.88	1.9
11c-9476	CD123	9476	A	1.89	1.6
						11e-1015	HER2	1015	A	1.69	2.3
11k-7007	TWEAKR	7007	A	1.17	1.9

For comparison purposes, the following ADCs were prepared:

reference example R1:

such ADCs are disclosed in WO2015/096982 and WO2016/096610 with various antibodies such as, for example, cetuximab and trastuzumab. Precursor intermediate F194 disclosed therein was also reacted with TPP-6013 (anti-CD 123 AK) for comparison purposes. The following ADCs were used for comparison purposes:

examples	Target	Antibody TPP-	C [mg/ml]	DAR
					R1a	EGFR	981	1.67	1.9
R1c	CD123	6013	0.42	2.9
					R1e	HER2	1015	1.39	2.4
R1x	CXCR5	9574	1.28	2.2

Reference example R2:

such ADCs are disclosed in WO2016/096610, with non-glycosylated anti-TWEAKR antibodies. For comparison purposes, precursor intermediate F291 disclosed therein was also reacted with TPP-9574 (anti-CXCR 5 AK), TPP-981 (anti-EGFR), and TPP-1015 (anti-HER 2 AK). The following ADCs were used for comparison purposes:

examples	Target	Antibody TPP-	C [mg/ml]	DAR
					R2a	EGFR	981	1.46	3.4
R2e	HER2	1015	1.42	3.5
					R2x	CXCR5	9574	1.41	3.6

For ginsengRatio ofExample R1, WO2015/096982 describe metabolites formed therefrom example 98. For ginsengRatio ofExample R2, WO2016/096610 describe the same metabolite example M9, which is incorporated herein by referenceRatio ofExample R3M.

Reference example R3M:

n- (3-aminopropyl) -N- { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } -2-hydroxyacetamide

The preparation is described in WO2015/096982 as example 98.

Biological data for these reference compounds disclosed in the application or obtained for the novel reference compounds are described in section C.

C：Assessment of biological effectiveness

The biological effect of the compounds according to the invention can be shown by the following assays:

a.C-1adetermination of the cytotoxic Effect of ADC

Analysis of the cytotoxic effects of ADC was performed with various cell lines:

NCI-H292: human mucus epidermoid lung cancer cells, ATCC-CRL-1848, standard medium: RPMI 1640 (Biochrom; # FG1215, stable glutamine) +10% FCS (Sigma; # F2442), TWEAKR-positive; EGFR-positivity.

bxPC 3: human pancreatic cancer cells, ATCC-CRL-1687, standard medium: RPMI 1640 (Biochrom; # FG1215, stabilized glutamine) +10% FCS (Sigma; # F2442), TWEAKR positive.

LoVo: human colorectal cancer cells, ATCC accession number CCL-229, culture for MTT assay: standard medium: kaighn's + L-glutamine (Invitrogen 21127) +10% heat-inactivated FCS (from Gibco, Inc., No. 10500-. Culture for CTG assay: RPMI 1640 (Biochrom; # FG1215, stabilized glutamine) +10% FCS (Sigma # F2442). TWEAKR-positive.

KPL 4: human breast cancer cell line, Bayer Pharma AG (DSMZ identity checked and confirmed at 7/19/2012), standard medium: RPMI 1640 (from Gibco,; # 21875-; HER 2-positive.

SK-HEP-1: human hepatoma cell line, ATCC No. HTB-52, standard medium: MEM containing Earle's salt + Glutamax I (Invitrogen 41090) +10% heat inactivated FCS (from Gibco, accession No. 10500-064); EGFR-positivity, TWEAKR-positivity

MOLM-13: human acute monocytic leukemia cells (AML-M5a), DSMZ, No. ACC 554, standard medium: RPMI 1640 (from Gibco,; # 21875-; CD 123-positive.

MV-4-11: human bi-phenotypic B myelomonocytic leukemia cells obtained from peripheral blood, ATCC-CRL-9591, standard medium: IMDM (ATCC: 30-2005) +10% heat-inactivated FCS (Gibco, code 10500-064); CD 123-positive.

NB 4: human acute promyelocytic leukemia cells obtained from bone marrow, DSMZ, accession ACC 207, standard medium: RPMI 1640+ GlutaMAX I (Invitrogen 61870) +10% heat-inactivated FCS (Gibco, No. 10500-064) +2.5g of glucose (20% glucose solution, Gibco, No. 19002) +10mM Hepes (Invitrogen15630) +1mM sodium pyruvate (Invitrogen 11360); CD 123-negative

Rec-1: 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, accession No. 10500-064) +10mM) CXCR 5-positive

U251: human glioblastoma cells, standard medium: RPMI 1640 (Biochrom; # FG1215, stable glutamine) +10% FCS (Biochrom; # S0415), positive for B7H 3.

HBL-1 human B-cell lymphoma cells (diffuse large B-cell lymphoma) ATT CRL-RRID (advocated resource recognition): CVCL _4213, first described in Abe et al Cancer 61:483-490(1988), professor Lenz, obtained from M ü nster university, standard medium RPMI 1640 (Biochrom;. FG # 1215, stabilized glutamine) +10% FCS (Biochrom;. S0415), similar to the culture of Rec-1 cells; CXCR5 positive

The cells are cultured by standard methods as described by the American Tissue Culture Collection (ATCC) or the Leibniz institute DSMZ-German micro-organism and cell Culture Collection (DSMZ) for the respective cell line.

CTG assay

Cells were cultured by standard methods, with the growth medium shown under C-1. The method is implemented as follows: 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 96-well culture plates with white bottom (Costar #3610) (at 75. mu.l/well with the following cell numbers per well NCI-H292: 2500 cells/well, BxPC 32500 cells/well, LoVo 3000 cells/well) and incubated at 37 ℃ and 5% carbon dioxide in an incubator. The suspension cells were counted and seeded into 96-well culture plates with white bottom (Costar #3610) (75. mu.l/well with the following cell numbers per well: Rec-1: 3000 cells/well, HBL-1: 6000 cells/well). After 24 hours, the antibody-active substance conjugate was placed on the cells in 25. mu.l of medium (4-fold concentration) to reach 3X1 on the cells0^-7 M to 3x10^-11 Final concentration of antibody active-conjugate of M (in triplicate). The cells were then incubated in an incubator at 37 ℃ and 5% carbon dioxide. Cell viability was measured at the beginning of active treatment (day 0) on parallel plates using the Cell Titer Glow (CTG) luminescent Cell viability assay (Promega # G7573 and # G7571). For this, 100 μ l of substrate was added to each cell batch, then the plate was covered with aluminum foil, shaken at 180 rpm for 2 minutes using a plate shaker, left on the laboratory bench for 8 minutes, and then measured using a luminometer (Victor X2, PerkinElmer). The substrate detects the ATP content in living cells, wherein a luminescent signal is generated, the intensity of which is directly proportional to the viability of the cells. After 72 hours incubation with antibody active-conjugate, the viability of these cells was also now determined as described above using the Cell Titer Glow Cell viability assay. Using DRC (dose response Curve) analysis spreadsheet and with 4-parameter fit, the IC of growth inhibition was calculated from the measured data compared to day 0₅₀. DRC analysis spreadsheets are biography spreadsheets developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook Suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).

MTT assay

Cells were cultured by standard methods using the growth medium shown under C-1. The method is implemented as follows: cells were separated with a solution of Accutase in PBS (Biochrom AG # L2143), pelleted, resuspended in culture medium, counted and plated into 96-well culture plates with white bottom (Costar #3610) (NCI H292: 2500 cells/well, SK-HEP-1: 1000 cells/well; KPL-4: 1200 cells/well; in a total volume of 100. mu.l). The cells were then incubated in an incubator at 37 ℃ and 5% carbon dioxide. After 48 hours, the medium was changed. Then the concentration is 10^-5M to 10^-13M of the antibody-active substance-conjugate in 10 μ l of medium was pipetted to the cells (in triplicate) and the batch was then incubated in an incubator at 37 ℃ and 5% carbon dioxide. The suspension cells were counted and seeded into 96-well culture plates (Costar Corp. #3610) (#3610) (MOLM-13: 2000 cells/well; NB 4: 7000 cells) with white bottomA hole; MV-4-11: 5000 cells/well in a total volume of 100. mu.l). After 6 hours of incubation at 37 ℃ and 5% carbon dioxide, the medium was changed and the concentration was 10^-5M to 10^-13M antibody-active substance-conjugates or metabolites in 10. mu.l of culture medium were pipetted into 90. mu.l of cells (in triplicate). The batch was incubated at 37 ℃ and 5% carbon dioxide in an incubator. After 96 hours, cell proliferation was detected by means of the MTT assay (ATCC, Manassas, Virginia, USA; catalog No. 30-1010K). To do this, the MTT reagent was incubated with the cells for 4 hours, and then the cells were lysed overnight by adding a detergent. The dye formed was detected at 570nm (Infinite M1000 pro, Tecan). IC of growth inhibition was calculated from measured data using DRC (dose response Curve)₅₀The value is obtained. The proliferation of cells not treated with the test substance, but otherwise treated identically, is defined as a value of 100%.

Tables 1a and 1b below list the ICs from representative examples of these assays₅₀The value:

table 1c below lists the IC's from the reference examples of these assays₅₀The value is obtained.

The activity data shown are based on the examples described in this experimental section with the indicated drug/mAB ratios. The value may be no longerOptionally at the same drug/mAB ratio. IC (integrated circuit)₅₀Values are the average or individual values of several independent experiments. The effect of the antibody-active substance-conjugate was selective for each isotype control containing the respective linker and poison cluster. For ADCs against CD123, target specificity was additionally demonstrated by testing on CD123 negative cells.

In general, the ADCs according to the invention show a significantly improved cytotoxic efficacy compared to the corresponding reference examples.

C-1b selected examples determination of inhibition of the kinesin spindle protein KSP/Eg5

The motor domain of the human kinesin spindle protein KSP/Eg5 (tebu-bio/Cytoskeleton Inc., No. 027EG01-XL) was combined at 10nM with microtubules (bovine or porcine, Tebu-bio/Cytoskeleton Inc) stabilized with 50. mu.g/ml taxol (Sigma Co., No. T7191-5MG) at 15mM PIPES, pH 6.8 (5mM MgCl. RTM.) at room temperature₂And 10mM DTT, Sigma Co.) for 5 minutes. Freshly prepared mixtures were aliquoted into 384 MTP (from Corning). Then added at a concentration of 1.0 x10^-6M to 1.0 x10^-13M, and ATP (final concentration 500. mu.M, Sigma). Incubate at room temperature for 2 hours. ATPase activity was detected by detecting the inorganic phosphate formed using malachite green (Biomol). After addition of the reagents, incubation was carried out at room temperature for 50 minutes, and then the absorption was measured at a wavelength of 620 nm. The positive controls used were monascin (Monastrol) (Sigma, M8515-1mg) and Ispiesib (AdooQ Bioscience A10486). Each data of the dose-response curves was an eight-fold measurement. IC (integrated circuit)₅₀Values are the average of two independent experiments. The sample that served as the 100% control was not treated with inhibitor.

Table 2 below summarizes the IC's from representative examples of the assays and corresponding cytotoxicity data (MTT assay)₅₀The value is obtained.

TABLE 2

The activity data shown are based on the examples described in this experimental section.

Enzymatic assay for C-1C

a: cathepsin B assay

For each cathepsin B-cleavable prodrug to be detected, batches were formulated in a microreaction container (0.5ml, Eppendorf). The enzyme used here was obtained from human liver tissue. Mu.g of cathepsin B (Sigma C857125. mu.g) was placed in advance and made up to a total volume of 200. mu.l with 50mM sodium phosphate buffer, pH 6.0, 2mM DTT. Then 50. mu.l of the substrate solution to be detected was transferred. The batch was incubated in a heating block (Thermo Fisher Scientific Co.) at 40 ℃ with constant stirring at 300 rpm. Kinetics control the enzymatic reaction. For this, 10. mu.l samples were taken at different time points. To the removed sample was immediately added 20. mu.l of ice-cold methanol to terminate the enzymatic reaction, followed by freezing at-20 ℃. The time points selected for sampling were after 10 min, 2h, 4 h and 24 h. The samples were examined by RP-HPLC analysis (reversed phase HPLC, Agilent technologies, Inc. 1200 series). The determination of the released toxic clusters enables the determination of the half-life t of the enzymatic reaction_1/2。

b: legumain assay

Legumain assays were performed with recombinant human enzymes. Mixing rh legumain solution (catalog No. 2199-CY, R)&D Systems) were diluted to the desired concentration in 50mM sodium acetate buffer/100 mM NaCl, pH 4.0 and preincubated for 2h at 37 ℃. The rh legumain was then adjusted to a final concentration of 1 ng/. mu.l in 50mM MES buffer, 250 mM NaCl, pH 5.0. For each legumain cleavable prodrug to be detected, batches were formulated in a microreaction container (0.5ml, Eppendorf). For this purpose, the substrate solution is adjusted to the desired concentration (double concentration) with 50mM MES buffer, 250 mM NaCl, pH 5.0. For kinetic measurements of the enzymatic reaction, 250. mu.l of legumain solution were initially introduced and the enzymatic reaction was started by adding 250. mu.l of substrate solution (final concentration, single concentration; 3. mu.M). At each time point, 50. mu.l of each sample was taken. To the sample was immediately added 100. mu.l of ice-cold methanol to stop the enzymatic reaction, and then the sample was addedFreezing at-20 ℃. The time points selected for sampling were after 0.5 h, 1h, 3 h and 24 h. The samples were then subsequently analyzed by RP-HPLC analysis and LC-MS analysis. The determination of the released toxic clusters enables the determination of the half-life t of the enzymatic reaction_1/2。

As a representative example showing legumain-mediated cleavage, model compounds a and B were prepared as substrates in the legumain assay.

Reference example model Compound A

N- (pyridin-4-ylacetyl) -L-alanyl-N¹- [ (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- (methylamino) -1-oxobutan-2-yl]-L-asparagine

First, trifluoroacetic acid (2S) -2-amino-4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } (glycolyl) amino ] -N-methylbutanamide was prepared as described in WO2015096982a 1. The title compound was then prepared from this intermediate by coupling with intermediate L103 in DMF in the presence of HATU and N, N-diisopropylethylamine.

LC-MS (method 1) R_t = 0.86 min; MS (ESIpos): m/z = 902 [M+H]⁺。

Reference example model Compound B

N- (pyridin-4-ylacetyl) -L-alanyl-N-methyl-L-alanyl-N¹- [ (2S) -4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl]-2, 2-dimethylpropyl } (glycolyl) amino]-1- (methylamino) -1-oxobutan-2-yl]-L-asparagine

First, trifluoroacetic acid (2S) -2-amino-4- [ { (1R) -1- [ 1-benzyl-4- (2, 5-difluorophenyl) -1H-pyrrol-2-yl ] -2, 2-dimethylpropyl } (glycolyl) amino ] -N-methylbutanamide was prepared as described in WO2015096982a 1. The title compound was then prepared from this intermediate by coupling with intermediate L118 in DMF in the presence of HATU and N, N-diisopropylethylamine.

LC-MS (method 1) R_t = 0.83 min; MS (ESIpos): m/z = 916 [M+H]⁺。

Model compound a was cleaved from legumain under the above conditions with a half-life of 0.4 h to yield the target compound.

Model compound B was cleaved from legumain under the above conditions with a half-life of 0.5 h to yield the target compound.

C-2 internalization assay

Internalization is a key process capable of specifically and efficiently providing a cytotoxic payload in antigen-expressing cancer cells via antibody-active-conjugate (ADC). This process is monitored via fluorescent labeling of the specific antibody and isotype control antibody. To this end, a fluorochrome is first conjugated to the lysine of the antibody. Conjugation was performed at pH 8.3 using a two-to 10-fold molar excess of CypHer5E mononhs ester (batch 357392, GE Healthcare). After coupling, the reaction mixture was purified by gel chromatography (Zeba spin desalting column, 40K, Thermo Scientific, No. 87768; elution buffer, DULBECCO's PBS, Sigma-Aldrich, No. D8537) to eliminate excess dye and to adjust the pH. The protein solution was concentrated using a VIVASPIN 500 column (Sartorius stedim Biotec). Dye load of the antibody was analyzed spectrophotometrically (NanoDrop Corp.) and subsequently calculated (D/P = A)_{Dye material}ε_Protein: (A₂₈₀-0.16A_{Dye material})ε_{Dye material}) To be determined.

The dye loading of the antibodies and isotype controls tested herein were of comparable order. Conjugation did not result in a change in antibody affinity as measured in a cell binding assay.

For internalization, the assay uses a labeled antibody. Cells in 100. mu.l of medium (2X 10) were added before treatment was started⁴Per well) was inoculated into 96-MTP (crude, black, clear bottom, No. 4308776, Applied Biosystems). At 37 deg.C/5% CO₂After 18 hours of incubation, the medium was changed and the labeled antibody was added at different concentrations (10, 5, 2.5, 1, 0.1. mu.g/mL). The same treatment protocol was performed for the labeled isotype control (negative control). Selected incubation times were 0 hours, 0.25 hours, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 6 hours, and 24 hours. Fluorescence measurements were carried out with the aid of an InCellAnalyzer 1000 (GE Healthcare). Kinetic evaluation was performed via measuring parameters particle count/cell and total particle intensity/cell.

Upon binding to the receptor, the internalizing ability of the antibody is detected. For this purpose, cells with different receptor expression levels were selected. Target-mediated specific internalization was observed with the antibody, whereas isotype control did not show internalization.

Internalization assay for C-2b with suspension cells

Coupling of the fluorescent dye was performed as described in C-2. The antigen to be detected is expressed by hematopoietic suspension cells; thus, internalization was detected in FACS-based internalization assays.

Cells with different target expression levels are detected. Cells (5X 10)⁴Perwell) was seeded in 96-MTP (Greiner bio-one, CELLSTAR, 650180, U-base) in a total volume of 100. mu.l. After addition of target specific antibody at a final concentration of 10 μ g/ml, the batches were incubated at 37 ℃ for different time periods (1h, 2h, 6h, in triplicate). Isotype controls were treated under the same conditions. Parallel batches were constantly treated and incubated at 4 ℃ (negative control). FACS analysis was performed with the aid of a Guava flow cytometer (Millipore). Kinetic evaluation was performed by measuring fluorescence intensity and was evaluated by means of guavassoft 2.6 software (Millipore). For the targets and target-specific antibodies described herein, significant and specific antibodies were detected in various cellsPerforming heterogeneous internalization; isotype controls did not show internalization.

Co-localization assay for C-2C anti-CD 123 antibodies

Due to the linker, the active metabolites of the antibody-active-substance conjugate are generated by lysosomal degradation. Thus, intracellular trafficking is crucial after internalization. Co-localization assays with antibodies using markers specific for lysosomal organelles (e.g., surface molecules or small gtpases) enable selection of antibodies with a desired profile. For this, a total volume of 100. mu.l of target-positive cells (5X 10)⁴Perwell) into 96-MTP (Greiner bio-one, CELLSTAR, 650180, U-base). After addition of CypHer5E labeled anti-target antibody (final concentration 20. mu.g/ml), batches (in duplicate at each time point) were placed in an incubator (5% CO)₂) At 37 ℃ for 30min, 2h and 6 h. Lysosome-specific markers were added to the batches to be tested 30min before the end of the selected incubation time. The lysozymes were stained with the Cytopainter LysoGreen indicator (final concentration 1: 2000; abcam, ab 176826). After incubation, 200. mu.l of ice-cold FACS buffer (DULBECCO's PBS, Sigma-Aldrich, code D8537+3% FBS heat-inactivated FBS, Gibco, code 10500-. The cell pellet was resuspended in 300. mu.l ice-cold FACS buffer and centrifuged again (4 min, 400 x g, at 4 ℃). After centrifugation, the supernatant was discarded and the cell pellet was placed in 30 μ l of ice cold FACS buffer. The samples were then immediately subjected to FACS/imaging analysis (FlowSightamnis, Millipore). The co-location is evaluated by means of specific software (co-location software IDEAS Application v 6.1). Table 3 summarizes the results from assays such as for anti-CD 123 antibodies.

TABLE 3

Examples	Co-location [% ]]
		TPP-9476	29
TPP-8987	28
		TPP-8988	41
TPP-6013	43
		7G3	10
Isotype controls	0.2

Humanized antibodies TPP-9476 and TPP-8987 exhibited significantly improved profiles compared to the parent murine antibody.

In vitro assay for C-3 for determining cell permeability

The cell permeability of the substance can be studied by means of in vitro tests in a flux assay using Caco-2 cells [ m.d. Troutman and d.r. Thakker,Pharm. Res. 20 (8), 1210-1224 (2003)]for the determination of the permeation, the various test substances are placed in HEPES buffer on the cell tips (A) or the substrates (B) and incubated for 2 hours, after 0 hour and after 2 hours, samples are taken from the cis-and trans-compartments, the samples are separated by HPLC (Agilent 1200, B ö blingen, Germany) using a reverse phase column, the HPLC system is combined via a Turbo ion spray interface with a triple quadrupole mass spectrometer API 4000 (AB SCIEDeutschland GmbH, Darmstadt, Germany), by means of P_appPermeability was evaluated using the formula disclosed by Schwab et al [ d. Schwab et al,J. Med. Chem. 46, 1716-1725 (2003)]. When P is present_app(B-A) and P_appRatio of (A-B) (outflow ratio)>2 or<At 0.5, the substance is classified as actively transported.

Of critical importance for intracellular released toxic clusters is the B to A permeability [ P_app (B-A)]And P_app(B-A) and P_appRatio of (A-B) (outflow ratio): the lower the permeability, the slower the process of active and passive transport of the substance through the Caco-2 cell monolayer, so that the substance remains in the cell for a longer time after its release in the cell. The longer retention of the metabolite in the cell increases the probability of interaction with the biochemical target (here: kinesin spindle protein, KSP/Eg5), which leads to an improved cytotoxic effect.

Table 4 below lists permeability data from representative examples of this assay:

TABLE 4

Examples	Papp (B-A)[nm/s]	Outflow ratio
			M1	2.7	1.6
R3M	213	16

And can be prepared from ginsengRatio ofReference metabolite R3M formed from the binding agent-active substance-conjugate of example 2 in contrast to that which can be formed from the binding agent-active substance-conjugate according to the inventionMetabolite M1 showed significantly reduced delivery from the cells and a reduced efflux ratio.

In vitro test for determining substrate properties of P-glycoprotein (P-gp) C-4

Many tumor cells express transporters for active substances, which are often accompanied by the development of resistance against cytostatics. Substances that are not substrates for such transporters (e.g., P-glycoprotein (P-gp) or BCRP) may therefore exhibit improved profiles of action.

Substrate properties of the substance for P-gp (ABCB1) were determined by means of flux measurements using LLC-PK1 cells (L-MDR1 cells) overexpressing P-gp [ A.H. Schinkel et al,J. Clin. Invest. 96, 1698-1705 (1995)]to be measured. For this purpose, LLC-PK1 cells or L-MDR1 cells were cultured in 96-well filterplates for 3-4 days. To determine the penetration, the various test substances, alone or in the presence of inhibitors such as Ivermectin (Ivermectin) or Verapamil (Verapamil), were placed on the cell tips (a) or the substrate (B) in HEPES buffer and incubated for 2 hours. After 0 hours and after 2 hours, samples were taken from the cis and trans compartments. The samples were separated by HPLC using a reverse phase column. The HPLC system was used with a triple quadrupole mass spectrometer API 3000 (Applied Biosystems Applera, Darmstadt, germany) via a Turbo ion spray interface. By means of P_appPermeability was evaluated using the formula disclosed by Schwab et al [ d. Schwab et al,J. Med. Chem. 46, 1716-1725 (2003)]. When P is present_app(B-A) and P_appOutflow ratio of (A-B)>2, the material is classified as a P-gp substrate.

As an additional criterion for assessing the properties of P-gp substrates, the efflux ratios in L-MDR1 cells and LLC-PK1 cells, or in the presence or absence of inhibitors, can be compared with one another. If these values differ by more than a factor of 2, the substance involved is a P-gp substrate.

C-5 pharmacokinetics

Following intravenous administration of 5mg/kg of example 2c-9476 (DAR 6.3) and example 2c-9476 (DAR 3.4) in male Wistar rats, the plasma concentration of the ADC was measured by ELISA and pharmacokinetic parameters such as Clearance (CL), under-curve, were calculatedArea (AUC) and half-life (t)_1/2)。

Table 5 summarizes the pharmacokinetic parameters of example 2c-9476 with DAR 6.3 and DAR 3.4.

TABLE 5

Examples		2c-9476C	2c-9476D
				DAR		3.4	6.3
Species (II)		Rat	Rat
				Line of		Wistar	Wistar
Sex		Male sex	Male sex
				Administration of		Intravenous single bolus	Intravenous single bolus
Dosage of administration	[mg / kg]	5.0	5.0
				AUCIs normal	[kg * h / l]	4897	4069
AUC	[mg *h / l]	24484	20343
				ClSubstrate	[ml / h / kg]	0.20	0.25
VSS	[l / kg]	0.063	0.069
				MRT	[h]	306	281
t 1/2	[h]	229	219

In this targeted rat PK study after intravenous administration, a typical IgG profile was observed for both examples. No significant difference could be observed between examples 2c-9476 with DAR 6.3 and DAR 3.4.

Quantitative analysis of the ADC used

The antibody portion of the ADC was determined as the total IgG concentration in plasma samples and tumor lysates using a ligand binding assay (ELISA). Here, a sandwich ELISA format was used. The ELISA has been identified and validated for use in assays in plasma and tumor samples. ELISA plates were coated with goat anti-human IgG-Fc antibody. After incubation with the sample, the plate was washed and incubated with a simian anti-human IgG (H + L) antibody and a detector conjugate of horseradish peroxidase (HRP). After a further washing step, HRP substrate was added to OPD and color development was monitored via absorption at 490 nm. Standard samples with known IgG concentrations were fitted using a 4-parameter equation. The unknown concentrations were determined by interpolation within the lower (LLOQ) and Upper (ULOQ) quantitation limits.

C5 a: identification of ADC metabolites following in vitro internalization

Description of the method:

internalization studies with immunoconjugates were performed to analyze the metabolites formed within the cells. For this purpose, human lung tumor cells NCI H292 (3X 10)⁵Perwell) into 6-well plates and incubated overnight (37 ℃, 5% CO)₂). Cells were treated with 10. mu.g/mL (66nM) of the ADC to be detected. Internalization at 37 ℃ and 5% CO₂The process is carried out as follows. Cell samples were taken at various time points (0, 4, 24, 48, 72h) for further analysis. First, the supernatant (about 5mL) was harvested and stored at-80 ℃ after centrifugation (2 min, RT, 1000 rpmhheraeus Variofuge 3.0R).Cells were washed with PBS, split with Accutase, and cell number determined. After washing again, a specific number of cells (2X 10)⁵) 100mL of lysis buffer (mammalian cell lysis kit (Sigma MCL1) was added and incubated in protein Lobind tubes (eppendorf Cat. No. 0030108.116) with continuous shaking (Thermomixer, 15 min, 4 ℃, 650 rpm). After incubation, the lysates were centrifuged (10 min, 4 ℃,12000 g, eppendorf 5415R) and the supernatants were harvested. The supernatant obtained was stored at-80 ℃. All samples were then analyzed as follows.

To work up 50. mu.L of culture supernatant/cell lysate, 150. mu.L of precipitation reagent (methanol) was added thereto and shaken for 10 seconds. The precipitation reagent contains an Internal Standard (ISTD) at a suitable concentration (typically 20-100. mu.g/L). After centrifugation at 1881g for 10 minutes, the supernatant was transferred to an autosampler vial filled with 300 μ l of buffer matched to the eluent, and again shaken and centrifuged at 1881g for 10 minutes.

Finally, cell lysate samples and supernatant samples were measured on a triple quadrupole mass spectrometer API6500, used in conjunction with HPLC, from AB SCIEX Deutschland GmbH.

For calibration, blank lysate or blank supernatant at the corresponding concentration (0.1-1000. mu.g/l) was added. The detection limit (LLOQ) was about 0.2. mu.g/L.

Quality controls for testing effectiveness contained 4 and 40 μ g/L.

C5 b: identification of ADC metabolites in vivo

After intravenous administration of 3-30 mg/kg of different ADCs, the plasma and tumor concentrations of the ADC and possibly of the metabolites can be measured and pharmacokinetic parameters such as Clearance (CL), area under the curve (AUC) and half-life (t) can be calculated_1/2)。

Quantitative analysis of metabolites that may be present

The measurement of compounds in plasma, tumors, liver and kidney is carried out by High Pressure Liquid Chromatography (HPLC) in combination with triple quadrupole Mass Spectrometer (MS), usually after precipitation of the protein with methanol.

To post-treat 50. mu.L of plasma, 150. mu.L of a precipitation reagent (usually methanol) was added thereto and shaken for 10 seconds. The precipitation reagent contains an Internal Standard (ISTD) at a corresponding concentration (typically 20-100. mu.g/L). After centrifugation at 1881g for 10 minutes, the supernatant was transferred to an autosampler vial filled with 300 μ L of buffer matched to the eluent and shaken again.

In the post-treatment of tumor materials or organ materials, 3 to 20 times the amount of an extraction buffer is added to each material. The extraction buffer contained 50mL of tissue protein extraction reagent (Pierce, Rockford, IL), a complete-protease-inhibitor-mixture of two pellets (Roche Diagnostics GmbH, Mannheim, Germany) and phenyl methanesulfonyl fluoride (Sigma, st. louis, MO) at a final concentration of 1 mM. The lysis and homogenization procedure (Bertin Technologies) of the Prescellerys 24 lysis and homogenization apparatus was selected according to tissue type (hard: tumor; soft: liver, kidney) (www.prescellys.com). The homogenized sample was allowed to stand overnight at 4 ℃. Transfer 50 μ L of the homogenate to an autosampler vial and fill with 150 μ L of methanol including ISTD, shake for 10 seconds, then stand for 5 min. After addition of 300. mu.L of ammonium acetate buffer (pH 6.8) and brief shaking, the sample was centrifuged at 1881g for 10 minutes.

For calibration, plasma was added at a concentration of 0.6-1000. mu.g/L for plasma samples and a corresponding blank matrix was added at a concentration of 0.6-1000. mu.g/L for tissue samples. The limit of detection (LOQ) is 1 to 20. mu.g/L depending on the type of sample or tissue type.

Finally, plasma and matrix samples were measured on a triple quadrupole mass spectrometer API4500, from AB SCIEX Deutschland GmbH, in conjunction with HPLC.

Quality controls for testing effectiveness contained 4, 40 and 400 μ g/L.

Table 6 shows the metabolite concentrations measured in tumor, liver, kidney and plasma in MOLM-13 xenograft mouse model 24 h after administration of 5mg/kg ADC from example 2c-9476 (n = 3). The metabolites measured were: metabolite M1. n.c. = not calculated; LOQ: limit of quantitation

Table 6:

		metabolite M1 MW (mu g/l)	Metabolite M1 SD (mu g/l)	LOQ (µg/l)
					Swelling and swelling treating medicineTumor	Example 2c-9476B	59.5	0.3	2.0-20.0
Liver diseaseZang organs	Example 2c-9476B	< LOQ	n.c.	2.5
					Kidney (Kidney)Zang organs	Example 2c-9476B	10.9	6.5	5.0
Blood circulationPulp and its production process	Example 2c-9476B	< LOQ	n.c.	1.0

Administration of ADC examples 2c-9476 according to the invention with legumain cleavable linkers resulted in a significantly selective enrichment of active agents at the target tissue (tumor) compared to other healthy organs/tissues.

C-6 in vivo efficacy testing

The utility of the conjugates according to the invention is tested in vivo, for example using a xenograft model. The person skilled in the art is familiar with methods available in the prior art for testing the effectiveness of the compounds according to the invention (see, for example, WO 2005/081711; Polson et al, Cancer Res. 2009, 3/15 days; 69(6): 2358-64). To this end, a rodent (e.g., mouse) is inoculated with a tumor cell line that expresses a target molecule of a binding agent. The conjugate according to the invention, the isotype antibody control conjugate or the control antibody or the isotonic salt solution is then administered to the vaccinated animals. Administration occurs once or more frequently. After an incubation time of several days, the size of the tumor was determined by comparing the conjugate treated animals to the control group. The conjugate treated animals exhibited smaller tumor sizes.

Growth inhibition/regression of experimental tumors in C-6a. mice

Human tumor cells expressing the antigen of the antibody-active substance-conjugate are subcutaneously inoculated into the flank of an immunosuppressive mouse (e.g., NMRi nude mouse or SCID mouse). 1 to 10 million cells are isolated from the cell culture, centrifuged and resuspended in culture medium or medium/matrigel. The cell suspension was injected under the skin of the mice.

Within days, tumors grew. After establishing the tumor, therapy is started substantially when the tumor size is 40 mm. To detect the effect on larger tumors, therapy may also be started when the tumor size is 50-100 mm.

Treatment with APDC and ADC was performed via the intravenous (i.v.) route into the tail vein of mice. ADC was administered in a volume of 5 mL/kg.

The treatment regimen depends on the pharmacokinetics of the antibody. As a standard, treatment is achieved once a week for 2 or 3 weeks with a conjugate according to the invention. For rapid assessment, a protocol with a single treatment may also be appropriate. However, treatment may continue further, or a second round with 3 treatment days may follow at a later point in time.

As a standard, 8 animals were used per treatment group. The group as control group was treated with only buffer according to the same protocol except the group to which the active substance was administered.

During the experiment, two-dimensional (length/width) tumor areas were measured periodically using calipers. Tumor area was determined by the line width x. The comparison of the mean tumor area of the treated group to the control group is given as the T/C area.

If all experimental groups were stopped at the same time after the end of the treatment, the tumor could be removed and weighed. The comparison of the mean tumor weights of the treated and control groups is given as T/C weight.

C-6b. the utility of ADCs according to the invention in various tumor models

Tumor cells (e.g., NCI-H292, REC-1, MOLM-13, and MV-4-11) were inoculated subcutaneously into the flank of female NMRI-nude mice (Janvier.) intravenous treatment with an antibody-active-conjugate when the tumor size was ~ 40 mm.

Treatment with ADCs according to the invention results in significant and in some cases sustained inhibition of tumor growth compared to control and conjugated isotype control antibodies. Table 7 shows the T/C values determined for the tumor areas on the day of experiment end (calculated from the start of treatment), respectively.

Claims (24)

1. Binder-active substance conjugates of formula (I) and salts, solvates and salts of these solvates

Wherein

X₁Represents N and a nitrogen-containing compound which is a nitrogen-containing compound,

X₂represents N, and

X₃represents C;

or

X₁Represents N and a nitrogen-containing compound which is a nitrogen-containing compound,

X₂represents C, and

X₃represents N;

or

X₁Represents a group selected from the group consisting of CH and CF,

X₂represents C, and

X₃represents N;

or

X₁Which represents a radical of NH or a radical of NH,

X₂represents C, and

X₃represents C;

or

X₁Represents a group of a compound represented by the formula CH,

X₂represents N, and

X₃represents a compound represented by the formula (I),

R¹represents hydrogen or a methyl group,

R²represents methyl, ethyl, -CH₂-CH(CH₃)₂、-CH₂-C (= O) OH or isopropyl,

R³represents methyl, ethyl, -CH₂-CH(CH₃)₂or-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-W，

#-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH₂-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)_2-8 -C(=O)-###，

#-C(=O)- (CH₂)₃-C(=O)-###，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₅-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂) - # # or

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂-CH₂-O)_1-8-(CH₂)₂-NH-C(=O)-CH₂-##，

W represents the following group

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or an antibody fragment binding to an antigen,

# represents a bond to the compound,

# represents the bond to the sulfur atom of the cysteine side chain of the binding agent,

# represents the bond to the nitrogen atom of the lysine side chain of the binder.

2. A binder-active substance conjugate of formula (I) according to claim 1 as well as salts, solvates and salts of these solvates, wherein

X₁Represents a group of a compound represented by the formula CH,

X₂represents a compound represented by the formula (I),

X₃represents N;

R¹represents hydrogen or a methyl group,

R²represents methyl, -CH₂-CH(CH₃)₂、-CH₂-C (= O) OH or isopropyl,

R³represents methyl, -CH₂-CH(CH₃)₂or-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH(CH₃)-NH-C(=O)-CH₂-W，

#-C(=O)-CH₂-NH-C(=O)-CH₂-CH(##)-COOH，

#-C(=O)-CH₂-NH-C(=O)-CH(##)-CH₂-COOH，

#-C(=O)-CH₂-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

#-C(=O)- (CH₂)₃-C(=O)-###，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₅-W，

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂) - # # or

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂-CH₂-O)₄-(CH₂)₂-NH-C(=O)-CH₂-##，

W represents the following group

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or an antibody fragment binding to an antigen,

# represents a bond to the compound,

# represents the bond to the sulfur atom of the cysteine side chain of the binding agent,

# represents the bond to the nitrogen atom of the lysine side chain of the binder.

3. The binding agent-active substance-conjugates of formula (I) according to claims 1 and 2 as well as salts, solvates and salts of these solvates,

wherein

R¹Represents hydrogen or a methyl group,

R²represents a methyl group or an isopropyl group,

R³represents methyl or-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or an antibody fragment binding to an antigen,

# represents a bond to the compound,

# represents the bond to the nitrogen atom of the lysine side chain of the binder.

4. Binding agent-active substance-conjugates of formula (I) according to claims 1 to 3 as well as salts, solvates and salts of these solvates, wherein

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 50,

AK represents a binding agent or a derivative thereof, preferably an antibody or an antibody fragment binding to an antigen,

# represents a bond to the compound,

# represents the bond to the nitrogen atom of the lysine side chain of the binder.

5. Binding agent-active substance-conjugates of formula (I) according to claims 1 to 4 as well as salts, solvates and salts of these solvates, wherein

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 20,

AK represents a binding agent or a derivative thereof, preferably an antibody or an antibody fragment binding to an antigen,

# represents a bond to the compound,

# represents the bond to the nitrogen atom of the lysine side chain of the binder.

6. Binding agent-active substance-conjugates of formula (I) according to claims 1 to 5 as well as salts, solvates and salts of these solvates, wherein

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 20, and

AK represents an anti-CD 123 antibody, an anti-CXCR 5 antibody, an anti-B7H 3 antibody, an anti-TWEAKR antibody, an anti-Her 2 antibody or an anti-EGFR antibody or an antibody fragment representing a binding antigen thereof,

# represents a bond to the compound,

# # represents the bond to the nitrogen atom of the lysine side chain of an Antibody (AK) or antigen-binding antibody fragment.

7. The binder-active substance conjugate of formula (I) according to claims 1 to 6 as well as salts, solvates and salts of these solvates, wherein

R¹Represents a methyl group, and a salt thereof,

R²represents a methyl group, and a salt thereof,

R³represents-CH₂-C(=O)-NH₂，

M represents the group

#-C(=O)-CH(CH₃)-NH-C(=O)-(CH₂)₃-C(=O)-###，

n represents a number of 1 to 20, and

AK represents an anti-CD 123 antibody selected from TPP-9476, TPP-8988, TPP-8987 and TPP-6013, represents an anti-CXCR 5 antibody selected from TPP-9574 and TPP-9580, represents an anti-B7H 3 antibody TPP-8382, represents an anti-TWEAKR antibody selected from TPP-7006 and TPP-7007, represents an anti-Her 2 antibody TPP-1015 or represents an anti-EGFR antibody TPP-981 or represents antigen-binding antibody fragments thereof,

# represents a bond to the compound,

# # represents the bond to the nitrogen atom of the lysine side chain of an Antibody (AK) or antigen-binding antibody fragment.

8. Binding agent-active substance-conjugates of formula (I) according to claims 1 to 7 having the following structure as well as salts, solvates and salts of these solvates

Wherein

AK1 represents an antibody attached via the sulfur atom of the cysteine side chain,

AK2 represents an antibody attached via the nitrogen atom of the lysine side chain, and

n represents 1 to 50.

9. The binding agent-active agent-conjugate according to claim 8, wherein the salts, solvates and salts of these solvates are

n represents 1 to 20.

10. The binding agent-active substance-conjugate according to claims 8 and 9, as well as salts, solvates and salts of these solvates, wherein

n represents 1 to 8.

11. The binding agent-active substance-conjugate according to claims 8 to 10, as well as salts, solvates and salts of these solvates, wherein

n represents 4 to 8.

12. The binding agent-active substance-conjugate according to one or more of claims 1 to 11, wherein

AK (AK1, AK2) represents an antibody selected from TPP-8382 (anti-B7H 3), TPP-6013 (anti-CD 123), TPP-8987 (anti-CD 123), TPP-8988 (anti-CD 123), TPP 9476 (anti-CD 123), TPP-9580 (anti-CXCR 5) and TPP 9574 (anti-CXCR 5), or an antigen-binding fragment thereof.

13. The binding agent-active substance-conjugate according to one or more of claims 1 to 12, wherein

AK (AK1, AK2) represents anti-CD 123 antibody TPP-6013,

represents anti-CD 123 antibody TPP-8987,

represents the anti-CD 123 antibody TPP-8988 or

Represents anti-CD 123 antibody TPP-9476,

or an antigen-binding fragment thereof.

14. The binding agent-active substance-conjugate according to one or more of claims 1 to 13, wherein AK (AK1, AK2)

(i) Representative of an anti-B7H 3 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO:52, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO:53 and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO:54, and a light chain variable region (VL) comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO:56, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO:57 and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO:58,

(ii) representative of an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO:22 (H-CDR1), the heavy chain variable CDR2 sequence shown in SEQ ID NO:23 (H-CDR2) and the heavy chain variable CDR3 sequence shown in SEQ ID NO:24 (H-CDR3), and a light chain variable region (VL) comprising the light chain variable CDR1 sequence shown in SEQ ID NO:26 (L-CDR1), the light chain variable CDR2 sequence shown in SEQ ID NO:27 (L-CDR2) and the light chain variable CDR3 sequence shown in SEQ ID NO:28 (L-CDR3),

(iii) representative of an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO:62 (H-CDR1), the heavy chain variable CDR2 sequence shown in SEQ ID NO:63 (H-CDR2) and the heavy chain variable CDR3 sequence shown in SEQ ID NO:64 (H-CDR3), and a light chain variable region (VL) comprising the light chain variable CDR1 sequence shown in SEQ ID NO:66 (L-CDR1), the light chain variable CDR2 sequence shown in SEQ ID NO:67 (L-CDR2) and the light chain variable CDR3 sequence shown in SEQ ID NO:68 (L-CDR3),

(iv) representative of an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO:72 (H-CDR1), the heavy chain variable CDR2 sequence shown in SEQ ID NO:73 (H-CDR2) and the heavy chain variable CDR3 sequence shown in SEQ ID NO:74 (H-CDR3), and a light chain variable region (VL) comprising the light chain variable CDR1 sequence shown in SEQ ID NO:76 (L-CDR1), the light chain variable CDR2 sequence shown in SEQ ID NO:77 (L-CDR2) and the light chain variable CDR3 sequence shown in SEQ ID NO:78 (L-CDR3),

(v) representative of an anti-CD 123 antibody comprising a heavy chain variable region (VH) comprising the heavy chain variable CDR1 sequence shown in SEQ ID NO:82 (H-CDR1), the heavy chain variable CDR2 sequence shown in SEQ ID NO:83 (H-CDR2) and the heavy chain variable CDR3 sequence shown in SEQ ID NO:84 (H-CDR3), and a light chain variable region (VL) comprising the light chain variable CDR1 sequence shown in SEQ ID NO:86 (L-CDR1), the light chain variable CDR2 sequence shown in SEQ ID NO:87 (L-CDR2) and the light chain variable CDR3 sequence shown in SEQ ID NO:88 (L-CDR3),

(vi) representative of anti-CXCR 5 antibodies comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO:92, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO:93 and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO:94, and a light chain variable region (VL) comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO:96, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO:97 and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO:98, or

(vii) Representative of an anti-CXCR 5 antibody comprising a heavy chain variable region (VH) comprising a heavy chain variable CDR1 sequence (H-CDR1) shown in SEQ ID NO:102, a heavy chain variable CDR2 sequence (H-CDR2) shown in SEQ ID NO:103 and a heavy chain variable CDR3 sequence (H-CDR3) shown in SEQ ID NO:104 and a light chain variable region (VL) comprising a light chain variable CDR1 sequence (L-CDR1) shown in SEQ ID NO:106, a light chain variable CDR2 sequence (L-CDR2) shown in SEQ ID NO:107 and a light chain variable CDR3 sequence (L-CDR3) shown in SEQ ID NO:108,

or antigen-binding fragments representing these antibodies.

15. The binding agent-active substance-conjugate according to one or more of claims 1 to 14, wherein AK (AK1, AK2)

(I) Represents an anti-B7H 3 antibody comprising the heavy chain variable region (VH) shown as SEQ ID NO:51 and the light chain variable region (VL) shown as SEQ ID NO:55,

(ii) represents an anti-CD 123 antibody comprising the heavy chain variable region (VH) shown as SEQ ID NO:21 and the light chain variable region (VL) shown as SEQ ID NO:25,

(iii) represents an anti-CD 123 antibody comprising the heavy chain variable region (VH) shown as SEQ ID NO:61 and the light chain variable region (VL) shown as SEQ ID NO:65,

(iv) represents an anti-CD 123 antibody comprising the heavy chain variable region (VH) shown as SEQ ID NO:71 and the light chain variable region (VL) shown as SEQ ID NO:75,

(v) represents an anti-CD 123 antibody comprising the heavy chain variable region (VH) shown as SEQ ID NO:81 and the light chain variable region (VL) shown as SEQ ID NO:85,

(vi) represents an anti-CXCR 5 antibody comprising the heavy chain variable region (VH) shown as SEQ ID NO:91 and the light chain variable region (VL) shown as SEQ ID NO:95,

(vii) represents an anti-CXCR 5 antibody comprising the heavy chain variable region (VH) shown as SEQ ID NO:101 and the light chain variable region (VL) shown as SEQ ID NO:105,

or antigen-binding fragments representing these antibodies.

16. The binder-active agent-conjugate according to one or more of claims 1 to 15, wherein AK (AK1, AK2)

(i) Represents an anti-B7H 3 antibody comprising the heavy chain region shown as SEQ ID NO:59 and the light chain region shown as SEQ ID NO:60,

(ii) represents an anti-CD 123 antibody comprising a heavy chain region as shown in SEQ ID NO. 29 and a light chain region as shown in SEQ ID NO. 30,

(iii) represents an anti-CD 123 antibody comprising a heavy chain region as shown in SEQ ID NO:69 and a light chain region as shown in SEQ ID NO:70,

(iv) represents an anti-CD 123 antibody comprising a heavy chain region as shown in SEQ ID NO:79 and a light chain region as shown in SEQ ID NO:80,

(v) represents an anti-CD 123 antibody comprising a heavy chain region as shown in SEQ ID NO. 89 and a light chain region as shown in SEQ ID NO. 90,

(vi) represents an anti-CXCR 5 antibody comprising a heavy chain region as shown in SEQ ID NO:99 and a light chain region as shown in SEQ ID NO:100, or

(vii) Represents an anti-CXCR 5 antibody comprising a heavy chain region as shown in SEQ ID NO:109 and a light chain region as shown in SEQ ID NO:110,

or antigen-binding fragments representing these antibodies.

17. The binding agent-active agent-conjugate according to any one of claims 1 to 16, wherein the antibody or antigen-binding antibody fragment binds to an extracellular target molecule.

18. The binding agent-active agent-conjugate according to any one of claims 1 to 17, wherein the antibody or antigen-binding antibody fragment binds to an extracellular cancer target molecule.

19. The binding agent-active agent-conjugate according to any one of claims 1 to 18, wherein the antibody or antigen-binding antibody fragment is internalized by a target cell by binding after binding to its extracellular target molecule on the target cell.

20. Pharmaceutical composition comprising at least one binding agent-active substance-conjugate according to one or more of the preceding claims in combination with inert, non-toxic, pharmaceutically suitable auxiliaries.

21. The binding agent-active substance-conjugate according to one or more of the preceding claims for use in a method of treatment and/or prevention of a disease.

22. The binding agent-active agent-conjugate according to one or more of the preceding claims for use in a method of treating a hyperproliferative and/or angiogenic disorder.

23. The binding agent-active substance-conjugate according to one or more of the preceding claims for use in a method of treating cancer and tumors.

24. The binder-active-substance conjugate according to one or more of the preceding claims for use in a method of treating cancer and tumors in combination with one or more therapeutic agents for cancer immunotherapy, or with one or more active substances directed against a molecular target of cancer immunotherapy.