HK40008565B - Prodrugs of cytotoxic active agents having enzymatically cleavable groups - Google Patents

Description

Prodrugs of cytotoxic agents with enzymatically cleavable groups

Introduction and Existing Technology

This invention relates to novel prodrugs in which cytotoxic drugs (e.g., kinesin spindle protein inhibitors) are conjugated to groups that are selectively cleaved by tumor-associated proteases and thus release the drug, and the use of these prodrugs or conjugates for the treatment and/or prevention of diseases, and the use of these prodrugs in the preparation of drugs for the treatment and/or prevention of diseases, particularly hyperproliferative and/or angiogenic conditions, such as cancer. Such treatment can be administered as a monotherapy or in combination with other drugs or additional therapeutic measures.

Cancer cells often express specific proteases at higher levels than normal cells. This has led to methods that increase the selectivity of cytotoxic drugs for cancer cells, where the drug binds to groups eliminated by these proteases, resulting in the release of the active ingredient.

This tumor-associated protease is legumain. Legumain is an asparagine acyl endopeptidase (S. Ishii, Methods Enzymol. 1994, 244, 604; J.M. Chen et al. J. Biol. Chem. 1997, 272, 8090), and has been used to process prodrugs of small toxic molecules, such as doxorubicin and etoposide derivatives (W. Wu et al. Cancer Res. 2006, 66, 970; L. Stern et al. Bioconjugate Chem. 2009, 20, 500; K.M. Bajjuri et al. ChemMedChem 2011, 6, 54).

US 2015-0343083 A1 describes a podase-cleavable peptide-active ingredient conjugate of the formula R-Y-Z-Asn-linker-D, wherein the linker is p-aminobenzylcarbamoyl or p-aminobenzyl carbonate, R is a group selected from various chemical groups, D is a cytotoxic drug, Asn is the amino acid asparagine, Y is an amino acid selected from Ala, Thr, Ser, Leu, Arg, Pro, Val, Tyr, and Phe, and Z is an amino acid selected from Ala, Thr, Asn, and Pro, wherein these amino acids are always in the native L configuration.

All prodrugs that can be cleaved by podases described to date contain oligopeptide sequences as substrates for podases.

Invention Overview

The object of this invention is to further improve the tumor selectivity of cytotoxic drugs. To achieve this object, the invention provides prodrugs of cytotoxic drug molecules. In this context, the active ingredient molecule is conjugated to a group cleavable by the enzyme lentinan, wherein the active ingredient and the lentinan-cleavable group are directly linked by a covalent bond or by a self-destructing linker. These prodrugs preferably contain a binding moiety that is internalized by tumor cells and processed intracellularly (preferably via lysosomes) after binding to a receptor on tumor cells. The binding moiety may optionally bind to the active ingredient molecule modified with the lentinan-cleavable group via a linker, such that the two groups (the lentinan-cleavable group and the binding moiety) must be processed independently of each other to form an active metabolite, or the binding moiety may optionally bind to the lentinan-cleavable group via a linker (such that after cleavage of the lentinan-cleavable group, the active ingredient and the binding moiety or its derivatives exist separately). Preferred active ingredient molecules are kinesin spindle protein inhibitors (KSP inhibitors). Preferred binding moieties that are internalized and processed intracellularly (preferably via lysosomes) after binding to a receptor on tumor cells are antibodies. Particularly preferred are antibody-active ingredient conjugates (ADCs), wherein the antibody and the active ingredient are linked to each other via a linker having a group cleavable by podase-based enzymes. Furthermore, prodrug-antibody conjugates (APDCs) are preferred, wherein the antibody binds to a prodrug of the antibody via a linker, and the effect of the active ingredient is masked by the podase-based enzyme-based enzyme-based enzyme group. The podase-based enzyme-based enzyme group bound to the active ingredient used according to the invention has a structural motif reduced to the amino acid asparagine. This reduction increases the stability of lysosomes in healthy organs due to the slowed cleavage by podase-based enzymes, without compromising the high antitumor efficacy (see section C-1c below). As shown by a representative comparison with suitable reference examples containing a tripeptide as a podase-based enzyme-based enzyme group (see section C-1a below), the ADCs and APDCs of the present invention having only one amino acid (asparagine) as an enzymatic cleavage site in the linker exhibit high antitumor efficacy that is surprisingly almost no less than—if any—analogs having a conventional tripeptide substrate (three amino acids) in the linker (see reference examples R1 and R6) (see section C-1a). Therefore, the linker sequence that can be cleaved by the protease can be reduced to just one amino acid unit (one amino acid). Even for conjugates closely associated with amino acids such as glutamine or leucine instead of asparagine (see model compound C RM-C below), it is impossible to show any cleavage by podase in an enzyme assay. Accordingly, the ADC associated with Example 1, which has glutamine instead of asparagine residues in the prodrug load, did not exhibit any cytotoxic activity. The same applies to similar ADCs of Example 6, which have leucine residues in the linker instead of asparagine; they also showed almost no cytotoxic activity compared to the ADCs of Example 6. Although oligopeptide linkers can also be cleaved by various proteases, the ADCs of the present invention show a high preference for cleavage by tumor-asparagine-cleaving proteases (e.g., podase).

The prodrug of the present invention has the following general formula (Ia):

in

La is a self-extinguishing connector.

n is 0 or 1;

D is -D ₁ -(L _b ) _o -(LIG) _p ,

_D1 is a cytotoxic agent.

LIG is the binding site, which, after binding to receptors on tumor cells, is preferably internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lb is the connector.

o and p are each independently 0 or 1.

R is LIG-(L _c ) _e -,

LIG is the binding site, which, after binding to receptors on tumor cells, is preferably internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lc is the connector, and

e is 0 or 1.

or

R is Z ₁ -(C＝O)q-,

q is 0 or 1.

Z ₁ represents a _C1-10 alkyl, C5-10 aryl, _C6-10 aryl- _C1-6 alkyl, _C3-10 heteroalkyl, _C1-10 alkyl-OC6-10 aryl, C5-10 heterocycloalkyl, heteroaryl, heteroarylalkyl, _C5-10 heteroarylalkoxy, _C1-10 alkoxy, _C6-10 aryloxy, _C6-10 aryl-C1-6 alkoxy, _C5-10 heteroalkoxy, _C1-10 alkyl- _OC6-10 aryloxy-, or _{C5-10 heterocycloalkoxy} group, which may be monosubstituted or polysubstituted by the following groups _, either identically or differently: _-NH2 , _-NH - _alkyl , -N(alkyl) ₂ -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2- NH2} , -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or –OH,

It may be _a -H or -( _CH₂ ) _₀- 1 _-Oₓ- ( _CH₂CH₂O ) _v - ^R₁ or _-Oₓ- ( _CH₂CH₂O ) _v ^-R₁ _group .

x is 0 or 1.

v is a number from 1 to 20, and

^R1 can be –H, –alkyl, _-CH2- COOH, _-CH2 - _CH2- COOH, or _-CH2 - _CH2 - _NH2 .

In this context, ^R1 is preferably _C1-12 -alkyl when it is defined as alkyl.

Preferably, the binding portion (LIG) is an antibody or an antigen-binding fragment thereof. The antibody is preferably a human, humanized, or chimeric monoclonal antibody or an antigen-binding fragment thereof, particularly anti-TWEAKR antibodies, anti-EGFR antibodies, anti-B7H3 antibodies, or anti-HER2 antibodies or antigen-binding fragments thereof. Particularly preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007, and TPP-10337; anti-B7H3 antibodies TPP-8382 and TPP-8567; anti-EGFR antibody cetuximab (TPP-981); and anti-HER2 antibodies trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

The prodrug of the present invention preferably contains a binding portion that can bind to tumor cell receptors, and after binding to the receptors, it is usually internalized by tumor cells and processed intracellularly (preferably via lysosomes).

Here, there are preferably two possible implementation schemes.

The binding moiety can optionally be bonded to a cleavable group of the enzyme lentinan via a linker, such that after the cleavable group of lentinan is cleaved, the active ingredient remains separate from the binding moiety or its derivative. Therefore, the compound of embodiment A preferably has the following general formula III':

Where n, e, LIG, La, Lc and _D1 have the definitions given in general formula (Ia).

In this case, -D in general formula (Ia) represents -D1, and -R in general formula (Ia) represents LIG-(L _c ) _e - (implementation A).

Therefore, the present invention preferably relates to compounds of general formula (Ia), wherein

La is a self-extinguishing connector.

n is 0 or 1;

D is -D ₁ -(Lb)o-(LIG) _p ,

o and p are 0

_D1 is a cytotoxic agent.

LIG is the binding site, which, after binding to receptors on tumor cells, is preferably internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lb is the connector.

R is LIG-(Lc) _e- ,

LIG is the binding site, which, after binding to receptors on tumor cells, is preferably internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lc is the connector, and

e is 0 or 1.

Alternatively , the binding moiety may optionally be bonded to the drug molecule via a linker, such that after the cleavage of the podase-cleavable group, the drug is present together with the binding moiety or its derivative.

In this case, -D in general formula (Ia) represents –D ₁ -(Lb)o-LIG, and R- in general formula (Ia) represents Z ₁ -(C(＝O))q-(Scheme B).

Therefore, the compound of embodiment B preferably has the following general formula (IV'):

Where n, o, LIG, La, Lb, and _D1 have the definitions given in general formula (Ia) and R is _Z1 -(C=O)q-, where q is 0 or 1 and _Z1 is _C1-10 -alkyl-, _C5-10 -aryl-, _C6-10 -aryl- _C1-6 -alkyl-, _C3-10 -heteroalkyl-, _C1-10 -alkyl- _OC6-10 -aryl-, _C5-10 -heterocyclic alkyl-, heteroaryl-, _C5-10 -heteroaryl- _C1-6 -alkyl-, _C5-10 -heteroaryl-alkoxy-, _C1-10 -alkoxy-, _C6-10 -aryl-, _C6-10 -aryl- _C1-6 -alkoxy-, _C5-10 -heteroalkoxy-, _C1-10 -alkyl- _OC6-10 -Aryloxy-, _C5-10 -heterocyclic alkoxy, which can be monosubstituted or polysubstituted by the following groups, either identically or differently: _-NH2 , -NH-alkyl, -N(alkyl) ₂ , -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2- NH2} , -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or –OH,

It may be _a -H or -( _CH₂ ) _₀-1 ^-Ox- ( _CH₂CH₂O ) ^vR₁ or -Ox-( _CH₂CH₂O ) _vR₁ group.

x is 0 or 1

v is a number from 1 to 20, and

^R1 can be –H, –alkyl, _-CH2- COOH, _-CH2 - _CH2- COOH, or _-CH2 - _CH2 - _NH2 .

Therefore, the present invention preferably relates to compounds of general formula (Ia), wherein

La is a self-extinguishing connector.

n is 0 or 1;

D is -D ₁ -(Lb)o-(LIG) _p ,

o 0 or 1

p is 1,

_D1 is a cytotoxic agent.

LIG is the binding site, which, after binding to receptors on tumor cells, is preferably internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lb is the connector.

R is Z ₁ -(C＝O)q-,

q is 0 or 1.

Z ₁ represents a _C1-10 alkyl, C5-10 aryl, _C6-10 aryl- _C1-6 alkyl, _C3-10 heteroalkyl, _C1-10 alkyl-OC6-10 aryl, _C5-10 heterocyclic alkyl, heteroaryl, _C5-10 heteroaryl- _C1-6 alkyl, C5-10 heteroarylalkoxy, _C1-10 alkoxy, _C6-10 aryloxy, _C6-10 aryl- _C1-6 alkoxy, _C5-10 heteroalkoxy, _{C1-10 alkyl} - _OC6-10 aryloxy, _{or C5-10 heterocyclic} alkoxy, which may be monosubstituted or polysubstituted by the following groups, either the same or different: _-NH2 , _-NH -alkyl, -N(alkyl) ₂ -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2- NH2} , -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or -OH,

It may be _a -H or -( _CH₂ ) _₀-1 ^-Ox- ( _CH₂CH₂O ) ^vR₁ or -Ox-( _CH₂CH₂O ) _vR₁ group.

x is 0 or 1.

v is a number from 1 to 20, and

^R1 can be –H, –alkyl, _-CH2- COOH, _-CH2 - _CH2- COOH, or _-CH2 - _CH2 - _NH2 .

Attached Figure Description

Figure 1 illustrates the strategy of transglutaminase-catalyzed conjugation site-specific functionalization of non-glycosylated antibodies.

Figures 2A-2Q show the annotated sequences of preferred antibodies for binding partial-drug conjugates. The protein sequences of the heavy and light chains of IgG are shown, along with the VH and VL regions of these antibodies. Below the sequences, important regions are annotated (VH and VL regions in IgG, and CDR regions (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3)).

Figures 3A-3M show sequence listings of preferred antibody sequences and target protein sequences for binding partial-drug conjugates.

Invention Details

First, the following describes the cleavable group of the podase protease and the cytotoxic drug D, which may be used according to the invention, optionally linked to each other by a self-destructive linker. Next, a preferred binding portion LIG according to the invention is described, which, upon binding to a receptor on tumor cells, is internalized by the tumor cells and processed intracellularly (preferably via lysosomes). Various elements of the compounds of the invention may be used in any desired combination without limitation. Specifically, the drug D, described in each case as preferred or particularly preferred, may be used in combination with the binding portion LIG, described in each case as preferred or particularly preferred, and optionally in combination with the linker, described in each case as preferred or particularly preferred.

Groups that can be cleaved by podase

The compounds of general formula (Ia) of the present invention have a group that is cleavable by podases of formula (Ia'):

in

La is a self-extinguishing connector.

n is 0 or 1,

#1 represents the bond that connects to cytotoxic drugs (cytotoxic agents).

R is LIG-(L _c ) _e -,

LIG is the binding site, which, after binding to receptors on tumor cells, is preferably internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lc is the connector, and

e is 0 or 1.

or

R is Z ₁ -(C＝O)q-,

q is 0 or 1.

Z ₁ represents a _C1-10 alkyl, C5-10 aryl, _C6-10 aryl- _C1-6 alkyl, _C3-10 heteroalkyl, _C1-10 alkyl-OC6-10 aryl, _C5-10 heterocyclic alkyl, heteroaryl, _C5-10 heteroaryl- _C1-6 alkyl, C5-10 heteroarylalkoxy, _C1-10 alkoxy, _C6-10 aryloxy, _C6-10 aryl- _C1-6 alkoxy, _C5-10 heteroalkoxy, _{C1-10 alkyl} - _OC6-10 aryloxy, _{or C5-10 heterocyclic} alkoxy, which may be monosubstituted or polysubstituted by the following groups, either the same or different: _-NH2 , _-NH -alkyl, -N(alkyl) ₂ -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2- NH2} , -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _-NH2 , -C(=O)-N(alkyl) ₂ or –OH, or -H or - _{( CH2} ) _0-1 - _Ox- ( _CH2CH2O ) _v - ^R1 or _{-Ox- ( CH2CH2O} ) _v - ^R1 group,

x is 0 or 1.

v is a number from 1 to 20, and

^R1 can be –H, –alkyl, _-CH2- COOH, _-CH2 - _CH2- COOH, or _-CH2 - _CH2 - _NH2 .

Here, ^R1 is preferably _C1-12 -alkyl when it is defined as alkyl.

When R is LIG-(L _c ) _e- , the podase-cleavable group of formula Ia' is also called the podase-cleavable linker (implementation A).

When R is Z ₁ -(C(＝O))q-, the cleavable group of the podase in formula Ia' is also called the cleavable protecting group of the podase (implementation B).

When the cleavable group of the podase in formula Ia' refers to the cleavable protecting group of the podase, q is preferably 1.

_Z1 preferably represents _C1-10 -alkyl-, _C6-10 -aryl- _C1-6 -alkyl-, _C5-10 -heteroaryl- _C1-6 -alkyl-, _C6-10 -aryl- _C1-6 -alkoxy- or _C5-10 -heteroarylalkoxy, which may be monosubstituted or polysubstituted by the following groups, either the same or different: -NH2, -NH-alkyl, -N(alkyl) ₂ , -NH-C(=O) _-alkyl , -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) ₂ -NH2, -S(=O) _{2 -} N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or –OH.

_Z1 is more preferably _C1-3 -alkyl-, _C6-7 -aryl- _C1-6 -alkyl-, _C5-6 -heteroaryl- _C1-3 -alkyl- or _C6-7 -aryl- _C1-6 -alkoxy, which may be monosubstituted or polysubstituted by the following groups, either the same or different: -COOH, -C(＝O)- or –OH.

Self-extinguishing connector L _a

To ensure the effective release of free drug, a so-called self-destructing linker element ( _La ) may optionally be introduced between the enzymatic cleavage site and the drug (Anticancer Agents in Medicinal Chemistry, 2008, 8, 618-637). Drugs can be released through various mechanisms, such as subsequent elimination via an electronic cascade following the initial enzymatic release of the nucleophilic group (Bioorg. Med. Chem., 1999, 7, 1597; J. Med. Chem., 2002, 45, 937; Bioorg. Med. Chem., 2002, 10, 71), or via cyclization of the corresponding linker element (Bioorg. Med. Chem., 2003, 11, 2277; Bioorg. Med. Chem., 2007, 15, 4973; Bioorg. Med. Chem. Lett., 2007, 17, 2241), or a combination of both (Angew. Chem. Inter. Ed., 2005, 44, 4378). An example of such a linker element is shown in the figure below:

The self-destructing linker mentioned here under La in general formulas (Ia) and (Ia') is, for example, one of the following groups:

Where # ₁ represents the bond connecting the carbonyl group, and # ₂ represents the bond connecting the hydroxyl or amino group of D1.

Cytotoxic drugs

In the compound of formula Ia of the present invention, D is a _-D1- (Lb)o-(LIG) _p group, wherein

_D1 is a cytotoxic drug (cytotoxic agent).

LIG stands for binding moiety, which, after binding to receptors on tumor cells, is preferably internalized by the tumor cells and processed within the cells (preferably via lysosomes).

L _b represents the connector, and

o and p are independently 0 or 1.

The preferred cytotoxic agents used are mitomycin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-aminocamptothecin, n8-acetylspermine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, tamethasone, cytarabine, etoposide, camptothecin, paclitaxel, esperamycin, podophyllotoxin, serpentine, vincristine, morpholine-doxorubicin, n-(5,5-diacetoxypentyl)doxorubicin, pyroxine, aurestatin, monomethylaurestatin, dolalastatin, tubulin inhibitors, maytansine, candidacloprid, amatoxins, pyrrolobenzodiazepine derivatives, dihydroindololobenzodiazepine, calcitriol, daunorubicin, and camptophecin. DX8951 (ethanotecan) or kinesin spindle protein inhibitor (KSP inhibitor), wherein the drug is bonded by its hydroxyl or amino group to a _La (when n=1) or carbonyl group according to general formula (Ia) (when n=0). The corresponding derivatization of these drugs can be carried out based on known methods (see, for example, Synthesis, 1999, 1505 on pyroxine; Nat. Struct. Biol., 2002, 9, 337 on camptothecin; Journal of Med. Chem., 2010, 53(3), 1043 on aureatine; ChemMedChem, 2011, 6(1), 54 on doxorubicin; Mol. Cancer. Ther., 2005, 4, 751 on doxorubicin; and J. Biol. Chem, 2008, 283, 9318 on pyrrolobenzodiazepine derivatives (PBD derivatives); see also J. Med. Chem 2013, 56, 7564, and other references in the introduction, J. Med. Chem. 2001, 44, 1341, Oncology). Reports 2011, 26, 629.

Specifically, drug types established as ADC payloads, as outlined in the review by A. Beck et al. (Nature Rev. Drug Discovery; 2017, 16, 315), can also be linked to antibodies via the linker chemistry described herein. Preferably, the cleavable linker in the ADC can be replaced by the linker described herein.

Cytotoxic drugs possessing a free hydroxyl or amino group essential to their efficacy are particularly preferred, especially those possessing a free amino group essential to their efficacy. The efficacy of the cytotoxic drug can be masked by coupling such a group with a cleavable group by a podase inhibitor. This group of drugs includes, for example, doxorubicin having the following formula:

The efficacy of doxorubicin can be masked by conjugating the cleavable group of podase with the free amino group of doxorubicin.

A particularly preferred cytotoxic agent is a kinesin spindle protein inhibitor, such as that disclosed, for example, in International Patent Application WO2015/096982.

This article recommends cytotoxic drugs, especially those kinesin spindle protein inhibitors of formula (II), and their salts, solvates, and salts of solvates:

in

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is N,

X ₂ is C, and

X ₃ is N,

or

_X1 is CH or CF.

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is CH,

X ₂ is N, and

X ₃ is C.

A is -C(=O)-, -S(=O)-, -S(=O) ₂ - or -S(=O) ₂ -NH-,

^R1 is -H, –L-#1, –MOD, or -( _CH2 ) _0-3Z .

Z can be -H, ^-NHY3 , -OY3, ^-SY3 , ^halogen , -C(=O) ^{-NY1Y2 ,} or -C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , -( _CH2CH2O ) _{O-3- ( CH2} ) _O-3Z ' or -CH( _CH2W )Z'.

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' is -H, -NH ₂ , -S(=O) ₃ H, -COOH, -NH-C(=O)-CH ₂ -CH ₂ -CH(NH ₂ )COOH or -(C(=O)-NH-CHY ⁴ ) _1-3 COOH,

W is either -H or -OH.

^Y4 is a linear or branched _C1-6 alkyl-, optionally substituted with -NH-C(=O) _-NH2 , or an aryl or benzyl, optionally substituted with _–NH2 .

^R2 is -H, –L-#1, -MOD, -C(=O) ^-CHY4 - ^NHY5 , or -( _CH2 ) _0-3Z .

Z can be -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C(=O) ^{-NY1Y2 ,} or -C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(=O) _3H , _-NH2 , or -COOH.

^Y4 is a linear or branched _C1-6 alkyl-, optionally substituted with -NHC(=O) _-NH2 , or an aryl or benzyl, optionally substituted with _–NH2 .

Y ⁵ is -H or –C(=O)-CHY ⁶ -NH ₂ ,

^Y6 is a linear or branched _C1-6 -alkyl group.

R ³ is -MOD, –L-#1, or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl groups, which may be substituted with 1 to 3 OH groups, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl groups, 1 to 3 –O-alkyl groups, 1 to 3 –SH groups, 1 to 3 –S-alkyl groups, 1 to 3 –OC(=O)-alkyl groups, 1 to 3 –OC(=O)-NH-alkyl groups, 1 to 3 –NH-C(=O)-alkyl groups, 1 to 3 –NH-C(=O) _-NH -alkyl groups, 1 to 3 –S(=O) ₂ -NH-alkyl groups, 1 to 3 –NH-alkyl groups, 1 to 3 –N(alkyl) ₂ , ₁ to 3 NH2, or 1 to 3 –( _CH2 ) _0-3Z groups.

n is 0, 1, or 2.

Z can be -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C(=O) ^{-NY1Y2 ,} or –C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NHC(＝OCH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z',

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R5 represents -H, _-NH2 , _-NO2 , halogen, -CN, _-CF3 , _-OCF3 , -

CH ₂ F, -CH ₂ F, -SH or -(CH ₂ ) _0-3 Z,

Z is -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C(=O)-NY ¹ Y ² ,

Or –C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z '.

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl, fluoro- _C2-10 -ynyl, hydroxyl, -

_NO₂ , _-NH₂ , -COOH, or halogens,

^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, or _C2-10 -alkenyl group.

Fluoro-C _2-10 -alkenyl, C _2-10 -ynyl, or fluoro-C _2-10 -ynyl, or C _4-10 -cycloalkyl, fluoro-C _4-10 -cycloalkyl, or -(CH ₂ ) _O-2 -(H Z ² ), where H Z ² is a 4- to 7-membered heterocycle having up to two heteroatoms selected from N, O, and S, which may be substituted with –OH, –COOH, -NH ₂ , or –L-#1.

R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ .

–L-#1 is -(Lb) _o -(LIG) _p ,

LIG is the binding site; after binding to receptors on tumor cells, it is internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lb is the connector.

o and p are independently 0 or 1.

–MOD stands for -(NR ¹⁰ )n-(G1)o-G2-G3,

^R10 is -H or _C1 - _C3 -alkyl.

G1 is –NH-C(=O)-, -C(=O)-NH-, or

n is 0 or 1;

o is 0 or 1, and

G2 has a straight-chain or branched hydrocarbon chain with 1-100 carbon atoms, which may be alternated once or more by a group selected from the following: -O-, -S-, -S(＝O)-, -S(＝O) _2- , -NRy-, -NRyC(＝O)-, -C(＝O)NRy-, -NRyNRy-, -S(＝O) _2- NRyNRy-, -C(＝O)-NRyNRy-, -C(＝O)-, ^-CRx ＝NO-, and the straight-chain or branched hydrocarbon chain may be substituted by –NH-C(＝O)-NH2, -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

R_^y is -H, phenyl, C1-C10-alkyl, C2-C10-alkenyl, or C2-C10-alkynyl, each of which can be substituted with –NH-C(=O)-NH₂, -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid.

Rx is -H, C1-C3-alkyl, or phenyl.

G3 is either -H or –COOH, and

-MOD has at least one -COOH group, preferably two -COOH groups, and one amino group in -MOD can be acylated by a podase-cleavable group of formula (Ia’).

This article selects the preferred compounds, among which...

R ³ is –L-#1, or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocyclic alkyl, which may be 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated or trihalogenated alkyl atoms, 1 to 3 –O-alkyl atoms, 1 to 3 –SH atoms, 1 to 3 –S-alkyl atoms, 1 to 3 –OC(=O)-alkyl atoms, 1 to 3 –OC(=O)-NH-alkyl atoms, 1 to 3 –NH-C(=O)-alkyl atoms, 1 to 3 –NH-C(=O)-NH-alkyl atoms, 1 to 3 –S(=O) _n -alkyl atoms, 1 to 3 –S(=O) _2- alkyl atoms. -NH-alkyl, 1 to 3 -NH-alkyl, 1 to 3 -N(alkyl) ₂ , 1 to 3 _NH2 or 1 to 3 -( _CH2 ) _0-3Z groups substituted,

n is 0, 1, or 2.

Z can be -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C(=O) ^{-NY1Y2 ,} or –C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NHC(=O)CH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z', or -(CH ₂ ) _0-3 Z', and

Z' can be -H, -S(＝O) _3H , _-NH2 or –COOH.

Compounds of general formula (II) are preferred, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N.

The efficacy can be masked by conjugating the cleavable group of podase with the free amino group of the compound of formula II.

The kinin spindle protein inhibitors used according to the present invention have amino groups essential for efficacy. By modifying this amino group with peptide derivatives or aspartic acid derivatives, the efficacy of kinin spindle protein is inhibited, thus also inhibiting cytotoxic efficacy. However, if the peptide residues can be released by tumor-associated enzymes such as podocyte protease, the efficacy can be reconstituted in a controlled manner in tumor tissue.

definition

The term "substituted" means that one or more hydrogen atoms on a specified atom or group have been selectively replaced from the specifically indicated group, provided that, in this case, the normal valence state of the specified atom is not exceeded. Combinations of substituents and/or variants are permitted.

The term "optionally substituted" means that the number of substituents may be equal to or not equal to zero. Unless otherwise stated, an optionally substituted group may be replaced by so many optional substituents that it is as many as that can be set by replacing hydrogen atoms with non-hydrogen substituents on any available carbon, nitrogen, or sulfur atom. Typically, the number of optional substituents (if present) may be 1, 2, 3, 4, or 5, especially 1, 2, or 3.

As used herein, for example in the definition of substituents in the general formula compounds of the present invention, the expression "single- or multiple-" means "1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, particularly preferably 1, 2 or 3, and most preferably 1 or 2".

Unless otherwise stated, if a group in the compounds of this invention is substituted, the group may be monosubstituted or polysubstituted. Within the scope of this invention, the definitions of all groups appearing more than once are independent of each other. It is preferred that they be substituted with one, two, or three identical or different substituents. Substitution with one substituent is particularly preferred.

alkyl

Alkyl groups are linear or branched saturated monovalent hydrocarbon groups having 1-10 carbon atoms ( _C1 - _C10 -alkyl), typically 1-6 carbon atoms ( _C1 - _C6 -alkyl), preferably 1-4 carbon atoms ( _C1 - _C4 -alkyl), and particularly preferably 1-3 carbon atoms ( _C1 - _C3 -alkyl).

Preferred examples include:

Methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl and 1,2-dimethylbutyl.

Methyl, ethyl, propyl, isopropyl, and tert-butyl are particularly preferred.

Heteroalkyl

Heteroalkyl groups are straight-chain and/or branched hydrocarbon chains having 1-10 carbon atoms and may be spaced once or more by one or more of the following groups: -O-, -S-, -C(＝O)-, -S(＝O)-, -S(＝O) _2- , -NRy-, -NRyC(＝O)-, -C(＝O)-NRy-, -NRyNRy-, -S(＝O) _2- NRyNRy-, -C(＝O)-NRyNRy-, ^-CRx ＝NO-, and wherein the hydrocarbon chain, including side chains (if present), may be substituted by –NH-C(＝O) _-NH2 , -COOH, -OH, _-NH2 , -NH-C(＝ _NNH2 )-, sulfonamide, sulfone, sulfoxide, or sulfonic acid.

In this context, R y  is -H, phenyl, C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl or C 2 -C 10 -alkynyl in each case, and in each case it can be substituted with -NH-C(=O)-NH2 , -COOH, -OH, -NH 2 , -NH-C(=NNH2 )-, sulfonamide, sulfone, sulfoxide or sulfonic acid.

In this context, ^Rx is -H, _C1 - _C3 -alkyl, or phenyl.

alkenyl

Alkenes are straight-chain or branched monovalent hydrocarbon chains having one or two double bonds and 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms ( _C2 - _C10 -alkenes), especially 2 or 3 carbon atoms ( _C2 - _C3 -alkenes). It is evident that when an alkene contains more than one double bond, the double bonds can be separable or conjugated with each other. For example, the alkenyl group can be ethenyl (or vinyl), propen-2-en-1-yl (or "allyl"), propen-1-en-1-yl, but-3-enyl, but-2-enyl, but-1-enyl, pent-4-enyl, pent-3-enyl, pent-2-enyl, pent-1-enyl, hex-5-enyl, hex-4-enyl, hex-3-enyl, hex-2-enyl, hex-1-enyl, propen-1-en-2-yl (or "isopropenyl"), 2-methylpropen-2-enyl, 1-methylpropen-2-enyl, 2-methylpropen-1-enyl, 1-methyl 1-methylpropenyl, 3-methylbutenyl, 2-methylbutenyl, 1-methylbutenyl, 3-methylbutenyl, 2-methylbutenyl, 1-methylbutenyl, 3-methylbutenyl, 2-methylbutenyl, 1-methylbutenyl, 3-methylbutenyl, 2-methylbutenyl, 1-methylbutenyl, 1,1-dimethylpropenyl, 1-ethylpropenyl, 1-propylvinyl, 1-isopropylvinyl, 4-methylpentanyl, 3-methylpentanyl, 2-methylpentanyl, 1-methylpentanyl 4-Methylpent-3-enyl, 3-methylpent-3-enyl, 2-methylpent-3-enyl, 1-methylpent-3-enyl, 4-methylpent-2-enyl, 3-methylpent-2-enyl, 2-methylpent-2-enyl, 1-methylpent-2-enyl, 4-methylpent-1-enyl, 3-methylpent-1-enyl, 2-methylpent-1-enyl, 1-methylpent-1-enyl, 3-ethylbut-3-enyl, 2-ethylbut-3-enyl, 1-ethylbut-3-enyl, 3-ethylbut-2-enyl, 2-ethylbut-2-enyl, 1-ethyl But-2-enyl, 3-ethylbut-1-enyl, 2-ethylbut-1-enyl, 1-ethylbut-1-enyl, 2-propylprop-2-enyl, 1-propylprop-2-enyl, 2-isopropylprop-2-enyl, 1-isopropylprop-2-enyl, 2-propylprop-1-enyl, 1-propylprop-1-enyl, 2-isopropylprop-1-enyl, 1-isopropylprop-1-enyl, 3,3-dimethylprop-1-enyl, 1-(1,1-dimethylethyl)vinyl, but-1,3-dienyl, pent-1,4-dienyl, or hex-1,5-dienyl. More particularly, the group is vinyl or allyl.

acetylin

An alkynyl group is a straight-chain or branched monovalent hydrocarbon chain with a triple bond and 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms ( _C2 - _C10 -alkynyl), especially 2 or 3 carbon atoms ( _C2 - _C3 -alkynyl). For example, _C2 - _C6... -The alkynyl group can be ethynyl, prop-1-alkynyl, prop-2-alkynyl (or propynyl), but-1-alkynyl, but-2-alkynyl, but-3-alkynyl, pent-1-alkynyl, pent-2-alkynyl, pent-3-alkynyl, pent-4-alkynyl, hex-1-alkynyl, hex-2-alkynyl, hex-3-alkynyl, hex-4-alkynyl, hex-5-alkynyl, 1-methylprop-2-alkynyl, 2-methylbut-3-alkynyl, 1-methylbut-3-alkynyl, 1-methylbut-2-alkynyl, 3-methylbut-1-alkynyl, 1-ethylprop-2-alkynyl, 3-methylpent-4-alkynyl, 2-methylpentyl -4-ynyl, 1-methylpentan-4-ynyl, 2-methylpentan-3-ynyl, 1-methylpentan-3-ynyl, 4-methylpentan-2-ynyl, 1-methylpentan-2-ynyl, 4-methylpentan-1-ynyl, 3-methylpentan-1-ynyl, 2-ethylbutan-3-ynyl, 1-ethylbutan-3-ynyl, 1-ethylbutan-2-ynyl, 1-propylpropan-2-ynyl, 1-isopropylpropan-2-ynyl, 2,2-dimethylbutan-3-ynyl, 1,1-dimethylbutan-3-ynyl, 1,1-dimethylbutan-2-ynyl, or 3,3-dimethylbutan-1-ynyl. More particularly, the alkyl group is ethynyl, propan-1-ynyl, or propan-2-ynyl.

cycloalkyl

Cycloalkyl groups are saturated monovalent monocyclic or bicyclic hydrocarbon groups with 3-12 carbon atoms ( _C3 - _C12 -cycloalkyl).

In this context, a monocyclic hydrocarbon group is a monovalent hydrocarbon group that typically has 3-10 ( _C3 - _C10 -cycloalkyl), preferably 3-8 ( _C3 - _C8 -cycloalkyl), and particularly preferably 3-7 ( _C3 - _C7 -cycloalkyl) carbon atoms.

Preferred examples of monocyclic hydrocarbon groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

Cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl are particularly preferred.

In this context, a bicyclic hydrocarbon group is a hydrocarbon group that typically has 3-12 carbon atoms ( _C3 - _C12 -cycloalkyl), where it should be understood here to represent two fused saturated ring systems that share two directly adjacent atoms. Preferred examples of bicyclic hydrocarbon groups include: bicyclo[2.2.0]hexyl, bicyclo[3.3.0]octyl, bicyclo[4.4.0]decyl, bicyclo[5.4.0]undecyl, bicyclo[3.2.0]heptyl, bicyclo[4.2.0]octyl, bicyclo[5.2.0]nonyl, bicyclo[6.2.0]decyl, bicyclo[4.3.0]nonyl, bicyclo[5.3.0]decyl, bicyclo[6.3.0]undecyl, and bicyclo[5.4.0]undecyl.

Heterocyclic alkyl

Heterocyclic alkyl groups are non-aromatic monocyclic or bicyclic cyclic systems having one, two, three, or four heteroatoms that may be the same or different. The heteroatoms can be nitrogen, oxygen, or sulfur atoms.

The monocyclic ring system according to the invention may have 3-8, preferably 4-7, and particularly preferably 5 or 6 ring atoms.

Preferred examples of heterocyclic alkyl groups having three ring atoms include:

Azacyclopropane.

Preferred examples of heterocyclic alkyl groups having four ring atoms include:

Nitro-heterocyclic butyl and oxo-heterocyclic butyl.

Preferred examples of heterocyclic alkyl groups having 5 ring atoms include:

Pyrrolidinyl, imidazoalkyl, pyrazolealkyl, pyrrolinyl, dioxacyclopentyl, and tetrahydrofuranyl.

Preferred examples of heterocyclic alkyl groups having six ring atoms include:

Piperidinyl, piperazineyl, morpholinyl, dioxanehexyl, tetrahydropyranyl, and thiomorpholinyl.

Preferred examples of heterocyclic alkyl groups having seven ring atoms include:

Azaheptanyl, oxaheptanyl, 1,3-diazaheptanyl, 1,4-diazaheptanyl.

Preferred examples of heterocyclic alkyl groups having eight ring atoms include:

Oxycyclic octyl and nitrogen-containing octyl compounds.

Among monocyclic heterocyclic alkyl groups, 4- to 7-membered saturated heterocyclic groups having up to two heteroatoms selected from O, N, and S are preferred. Morpholinyl, piperidinyl, pyrrolidinyl, and tetrahydrofuranyl are particularly preferred.

According to the present invention, a bicyclic ring system having one, two, three or four heteroatoms that may be the same or different may have 6-12, preferably 6-10, ring atoms, wherein one, two, three or four carbon atoms may be replaced by the same or different heteroatoms from the group of O, N and S.

Preferred examples include: azabicyclo[3.3.0]octyl, azabicyclo[4.3.0]nonyl, diazabicyclo[4.3.0]nonyl, oxazabicyclo[4.3.0]nonyl, thioazabicyclo[4.3.0]nonyl or azabicyclo[4.4.0]decyl and groups derived from other possible combinations according to this definition.

Particularly preferred are perhydrocyclopentadien[c]pyrrolithyl, perhydrofuran[3,2-c]pyridyl, perhydropyrrolo[1,2-a]pyrazinyl, perhydropyrrolo[3,4-c]pyrrolithyl and 3,4-methylenedioxyphenyl.

Heterocyclic alkoxy

Heterocyclic alkoxy groups are heterocyclic alkyl groups that are bonded to the remainder of the molecule via an -O- group.

Alkoxy

Alkoxy groups are linear or branched saturated alkyl ether groups of the formula -O-alkyl, which typically have 1-6 ( _C1 - _C6 -alkoxy) carbon atoms, preferably 1-4 ( _C1 - _C4 -alkoxy) carbon atoms, and particularly preferably 1-3 ( _C1 - _C3 -alkoxy) carbon atoms.

Preferred examples include:

Methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentyloxy, and n-hexyloxy.

Aryl

An aryl group is a monovalent monocyclic or bicyclic aromatic ring system composed of carbon atoms. Examples include naphthyl and phenyl; phenyl or phenyl groups are preferred.

_C6 - _C10 -aralkyl or aralkyl

_C6 - _C10 -aralkyl or aralkyl should be understood to mean a linear or branched, saturated, monovalent hydrocarbon group ( _C1 - _C10 -alkyl) having 1 to 10 carbon atoms bonded to an aryl group as defined above. These should be understood to include, for example, _C6-10 -aryl- _C1-6 -alkyl or benzyl.

heteroaryl

Heteroaryl groups are monovalent monocyclic, bicyclic, or tricyclic aromatic ring systems having 5, 6, 8, 9, 10, 11, 12, 13, or 14 ring atoms ("5- to 14-membered heteroaryl"), particularly 5, 6, 9, or 10 ring atoms, and containing at least one cyclic heteroatom and optionally one, two, or three other cyclic heteroatoms from the group consisting of N, O, and S, and bonded by cyclic carbon atoms or optionally (where valence allows) by cyclic nitrogen atoms.

The heteroaryl group can be a 5-membered heteroaryl group, such as thienyl, furanyl, pyrroleyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiazolyl, or tetrazolyl; or a 6-membered heteroaryl group, such as pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, or triazinyl; or a tricyclic heteroaryl group, such as carbazolyl, acridineyl, or phenazinyl; or a 9-membered heteroaryl group, such as benzofuranyl, benzothienyl, benzooxazolyl, benzoisooxazolyl, benzoimidazolyl, benzothiazolyl, benzotriazolyl, indazole, indolyl, isindolyl, inazinyl, or purine; or a 10-membered heteroaryl group, such as quinolinyl, quinazolinyl, isoquinolinyl, terolinyl, phthalazinyl, quinoxolinyl, or pteridineyl.

Generally, and unless otherwise specified, heteroaryl includes all possible isomer forms, such as tautomers and positional isomers with respect to the connection sites with the remainder of the molecule. Thus, as an explanatory, non-exclusive example, the term pyridyl includes pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl; or the term thiophenyl includes thiophenin-2-yl and thiophenin-3-yl.

_C5 - _C10 -heteroaryl

In the context of this invention, _C5-10 -heteroaryl is a monocyclic or bicyclic aromatic ring system having one, two, three, or four heteroatoms that may be the same or different. Possible heteroatoms include N, O, S, S(=O), and/or S(=O) ₂ . Bond valence can be at any aromatic carbon atom or nitrogen atom.

The monocyclic heteroaryl group according to the present invention has 5 or 6 ring atoms.

Those heteroaryl groups having one or two heteroatoms are preferred. One or two nitrogen atoms are particularly preferred here.

Heteroaryl groups having 5 ring atoms include, for example, the following rings:

Thiophene, thiazolyl, furanyl, pyrrolyl, oxazolyl, imidazole, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, and thiadiazolyl.

Heteroaryl groups having six ring atoms include, for example, the following rings:

Pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, and triazinyl.

The bicyclic heteroaryl group according to the present invention has 9 or 10 ring atoms.

Heteroaryl groups having nine ring atoms include, for example, the following rings:

Phthalide, thiophthalide, indole, isoindole, indazole, benzothiazolyl, benzofuran, benzothiophene, benzoimidazolyl, benzooxazolyl, acrylonitrile, indazinyl, purine, dihydroindole.

Heteroaryl groups having 10 ring atoms include, for example, the following rings:

Isoquinolinyl, quinolinyl, quinazinyl, quinazolinyl, quinoxalinyl, cyclolinyl, phthalazinyl, 1,7- and 1,8-naphthidyl, pteridyl, chromanyl.

heteroaryl

Heteroaryl groups should be understood as linear or branched, saturated monovalent hydrocarbon groups ( _C1 - _C10 -alkyl) having 1 to 10 carbon atoms bonded to a heteroaryl group as defined above. This should be understood to include, for example, _C5-10 -heteroaryl- _C1-6 -alkyl groups.

Aralkoxy or arylalkoxy

Arylalkoxy or arylalkoxy group should be understood to mean a linear or branched, saturated monovalent hydrocarbon group ( _C1 - _C10 alkyl) having 1 to 10 carbon atoms bonded to an aryl group as defined above. This should be understood to include, for example, _C6-10 -aryl- _C1-6 -alkoxy groups.

Aryloxy

Aryloxy group is an aryl group of the formula aryl-O-.

Preferred examples include: phenoxy and naphthoxy.

Hexaalkoxy

An alkoxy group is a straight-chain and/or branched hydrocarbon chain having 1-10 carbon atoms and bonded to the remainder of the molecule by -O-, and may be further substituted by one or more of the following groups: -O-, -S-, -C(＝O)-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , -NR ^y C(＝O)-, -C(＝O)-NR ^y- , -NR ^y NR ^y- , -S(＝O) _2- NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -CR ^x ＝NO-, and wherein the hydrocarbon chain, including the side chain (branched hydrocarbon chain), may be substituted, if present, by -NH-C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , -NH-C(＝NNH ₂ )-, sulfonamide, sulfone, sulfoxide or sulfonic acid.

In this context, R y  is -H, phenyl, C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl or C 2 -C 10 -alkynyl in each case, and in each case it can be substituted with -NH-C(=O)-NH2 , -COOH, -OH, -NH 2 , -NH-C(=NNH2 )-, sulfonamide, sulfone, sulfoxide or sulfonic acid.

In this context, ^Rx is -H, _C1 - _C3 -alkyl, or phenyl.

In the context of this invention, the halogen or halogen atom is fluorine (-F), chlorine (-Cl), bromine (-Br) or iodine (-I).

Fluorine (-F), chlorine (-Cl), and bromine (-Br) are preferred.

The kinin spindle protein inhibitor (cytotoxic agent) according to the present invention preferably has the following formula (IIa), and its salt, solvate and salt of solvate:

in

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is N,

X ₂ is C, and

X ₃ is N,

or

_X1 is CH or CF.

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is CH,

X ₂ is N, and

X ₃ is C.

A is -C(=O)-, -S(=O)-, -S(=O) ₂ - or -S(=O) ₂ -NH-,

^R1 is -H, –L-#1, –MOD, or -( _CH2 ) _0-3Z , where Z is -H, ^-NHY3 , ^-OY3 , ^-SY3 , halogen, -C( ⁼ O) ^-NY1Y2 , or –

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2- ( _CH2CH2O ) _O-3- ( _CH2 )O _{- 3Z} ' or -

CH(CH ₂ W)Z',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' is -H, -NH ₂ , -S(=O) ₃ H, -COOH, -NH-C(=O)-CH ₂ -CH ₂ -

CH( _NH₂ )COOH or -(C(＝O)-NH- ^CHY₄ ) _¹- ³COOH,

W is either -H or -OH.

^Y4 is a linear or branched _C1-6 alkyl group, optionally substituted with -NH-C(=O) _-NH2 .

It may be aryl or benzyl, optionally substituted with -NH ₂ .

^R2 is -H, –L-#1, -MOD, -C(=O) ^-CHY4 - ^NHY5 , or -( _CH2 ) _0-3Z .

Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C( ⁼ O) ^-NY1Y2 , or –

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^Y4 is a linear or branched _C1-6 alkyl group, optionally substituted with -NH-C(=O) _-NH2 .

It may be aryl or benzyl, optionally substituted with -NH ₂ .

Y ⁵ is -H or –C(=O)-CHY ⁶ -NH ₂ ,

^Y6 is a linear or branched _C1-6 -alkyl group.

R ³ is -MOD, –L-#1, or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl, which may be 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl atoms, 1 to 3 –O-alkyl atoms, 1 to 3 –SH atoms, 1 to 3 –S-alkyl atoms, 1 to 3 –OC(=O)-alkyl atoms, 1 to 3 –OC(=O)-NH-alkyl atoms, or 1 to 3 –NH-C(=O)-alkyl atoms.

1 to 3 -NH-C(=O)-NH-alkyl, 1 to 3 -S(=O) _n -alkyl, 1

Up to 3 –S(＝O) _₂ -NH-alkyl, 1 to 3 –NH-alkyl, 1 to 3 –N(alkyl) _₂ , ₁ to 3 NH₂, or 1 to 3 –( _CH₂ ) _₀-3 Z-substituted, where n is 0, 1, or 2.

Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C( ⁼ O) ^-NY1Y2 , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NHC(＝O)CH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z'

Or -(CH ₂ ) _0-3 Z',

Z' can be -H, -S(=O) _3H , _-NH2 , or –C(=O)-OH.

R ⁴ is a group that can be cleaved by the podase Ia'.

^R5 represents -H, _-NH2 , _-NO2 , halogen, -CN, _CF3 , _-OCF3 , _-CH2F , -

_CH₂F , -SH, or -( _CH₂ ) _₀- 3Z

Z represents -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C(=O)-NY ¹ Y ² , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl, fluoro- _C2-10 -ynyl, hydroxyl, _-NO2 , _-NH2 , -COOH, or halogen.

^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl or fluoro-C2-10-ynyl, or _C4-10 -cycloalkyl, fluoro _{-C4-10 -} cycloalkyl or -( _CH2 ) _0-2- ( ^HZ2 ).

HZ ² is a 4- to 7-membered heterocycle having at most two heteroatoms selected from N, O, and S, which can be substituted with -OH, –COOH, -NH ₂ , or –L-#1.

R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ .

–L-#1 is -(Lb)o-(LIG) _p ,

LIG is the binding site; after binding to receptors on tumor cells, it is internalized by the tumor cells and processed within the cells (preferably via lysosomes).

_Lb is the connector.

o and p are independently 0 or 1.

–MOD stands for –(NR ¹⁰ )n-(G1)o-G2-G3,

R ¹⁰ is -H or _C1 - _C3 -alkyl.

G1 is –NH-C(=O)-, -C(=O)-NH-, or

n is 0 or 1;

o is 0 or 1, and

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be alternated once or more by a group selected from the following: -O-, -S-, -S(＝O)-, -S(＝O) _2- , -NRy-, -NRyC(＝O)-, -C(＝O)NRy-, -NRyNRy-, -S(＝O) _2- NRyNRy-, -C(＝O)-NRyNRy-, -C(＝O)-, ^-CRx ＝NO, and the straight-chain or branched hydrocarbon chain may be substituted by –NH-C(＝O)-NH2, -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

R_^y is -H, phenyl, C <sub>_1- C_₁₀-alkyl, C <sub>₂ -C_₁₀-alkenyl, or C <sub>₂ -C_₁₀-alkynyl, each of which can be substituted with –NH-C(=O)-NH_₂ , -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid.

Rx is -H, _C1 - _C3 -alkyl, or phenyl.

G3 is –H or –COOH, and

-MOD has at least one -COOH group, preferably two -COOH groups, and one amino group in -MOD can be acylated by a podase-cleavable group of formula (Ia’).

The preferred compounds here are those, among which

R ³ is –L-#1 or C _1-10 -alkyl, C _6-10 -aryl or C _6-10 -aralkyl, C _5-10 -heteroalkyl, C _1-10 -alkyl-OC _6-10 -aryl or C _5-10 -heterocyclic alkyl, which may be 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated or trihalogenated alkyl atoms, 1 to 3 –O-alkyl, 1 to 3 –SH, 1 to 3 –S-alkyl, 1 to 3 –OC(=O)-alkyl, 1 to 3 –OC(=O)-NH-alkyl, 1 to 3 –NH-C(=O)-alkyl, 1 to 3 –NH-C(=O)-NH-alkyl, 1 to 3 –S(=O) _n -alkyl, 1 to 3 –S(=O) ₂ -NH-alkyl, 1 to 3 –NH-alkyl, 1 to 3 –N(alkyl) ₂ , 1 to 3 NH ₂ or 1 to 3 -(CH ₂ ) _O-3 Z substitutions, where n and Z have the definitions given above.

The -(C(＝O)-NH-CHY ⁴ ) _1-3 -COOH group refers to the presence of one -C(＝O)-NH-CHY ⁴ -COOH group, or two -(C(＝O)-NH-CHY ⁴ ) groups that can be connected to each other, according to -C(＝O)-NH-CHY ⁴ -C(＝O)-NH-CHY ⁴ -COOH, or three groups that can be connected to each other, according to -C(＝O)-NH-CHY ⁴ -C(＝O)-NH-CHY ⁴ -C(＝O)-NH-CHY ⁴ -COOH.

Compounds of general formula (IIa) are particularly preferred, wherein

^X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is CH or CF.

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is H,

X ₂ is N, and

X ₃ is C.

Compounds of general formula (IIa) are particularly preferred, wherein

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is CH,

X ₂ is C, and

X ₃ is N.

Compounds of general formula (IIa) are particularly preferred, among which

_X1 is CH,

X ₂ is C, and

X ₃ is N.

Compounds of general formula (IIa) are preferred, wherein A is –C(＝O)-.

Furthermore, compounds of general formula (IIa) are also preferred, among which

^R1 is –L-#1, -MOD, -H, -COOH, -C( _{＝O)-NH-NH2, -(CH2)1-3NH2, -C(＝O)-NZ”CH2)1-3NH2 and –C ( ＝ O ) -NZ} ” _CH2COOH , and

Z” is either -H or -NH ₂ .

In general formula (IIa), if R4 is –L-#1, ^R1 is preferably –MOD.

More preferably, in general formula (IIa), R4 is –L-#1, ^R1 is –MOD, and ^R3 is not –MOD.

In general formula (IIa), ^R2 is preferably -H.

In general formula (IIa), ^R3 is preferably –L-#1 or -MOD, or _C1-10 -alkyl, which may optionally be substituted with the following groups: –OH, -O-alkyl, -SH, -S-alkyl, -OC(=O)-alkyl, -OC(=O)-NH-alkyl, -NH-C(=O)-alkyl, -NH-C(=O)-NH-alkyl, -S(=O) _n -alkyl, -S(=O) _2- NH-alkyl, -NH-alkyl, -N(alkyl) ₂ or _-NH2 .

Preferably, the alkyl group is a _C1-3 alkyl-.

In general formula (IIa), R ⁵ is preferably -H or -F.

In general formula (IIa), ^R6 and ^R7 are preferably independently -H, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl, fluoro- _C2-10 -ynyl, hydroxyl or halogen.

In general formula (IIa), ^R8 is preferably a branched _C1-5 -alkyl group, especially –C( _CH3 ) _2- ( _CH2 ) _{0-2 –Ry} group, wherein _Ry is –H, –OH, –C(=O) _2H or _–NH2 .

More preferably, ^R8 is a –C( _CH3 ) _2- ( _CH2 ) _–Ry group, wherein _Ry is –H.

In general formula (IIa), R ⁹ is preferably -H or -F.

In general formula (IIa), –MOD is preferably the group QOC-(CHX)x-AM- _CH2 - _CH2 -NH-C(＝O)-.

in

x is a number from 2 to 6.

Q is either -OH or _-NH2

W is independently -H, _-NH2 , COOH or _-CONH2 in the -(CHX) _x- group, and -C(=O)-NH- or -NH-C(=O)- in the AM group.

In general formula (IIa), –MOD is more preferably a group:

QOC-CH ₂ -CH ₂ -CH(COQ)-NH-C(=O)-CH ₂ -CH ₂ -NH-C(=O)-,

HOOC-CH(NH ₂ )-CH ₂ -CH ₂ -C(=O)-NH-CH ₂ -CH ₂ -NH-C(=O)-and

HOOC-CH(NH ₂ )-(CH ₂ ) ₄ -NH-C(=O)-CH ₂ -CH ₂ -NH-C(=O)-,

Where Q is -OH or _-NH2 .

Preferred are compounds of general formula (IIa), their salts, solvates, and salts of solvates, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N,

A is –C(＝O)-,

R ¹ is –L-#1, -H, or –MOD

–L-#1 is -(Lb)o-(LIG) _p ,

LIG is the binding site; after binding to receptors on tumor cells, it is internalized by the tumor cells and processed intracellularly (preferably via lysosomes). Lb is the linker.

o and p are independently 0 or 1.

–MOD is the group –(NR ¹⁰ )n-(G1)o-G2-G3,

R ¹⁰ is -H or _C1 - _C3 -alkyl-.

n is 0 or 1,

G1 is –NH-C-(＝O)- or -C(＝O)-NH-.

o is 0 or 1, and

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) _2- , -

NR ^y C(=O)-, -C(=O)NR ^y -, -NR ^y NR ^y -, -S(=O) ₂ -

NR ^y NR ^y -, -C(=O)-NR ^y NR ^y -, -C(=O)-, -CR ^x =NO-,

Furthermore, straight-chain or branched hydrocarbon chains can be monosubstituted or polysubstituted by the following groups, either identically or differently: –NH-C(＝O) _-NH₂ , –C(＝O) _-NH₂

-COOH, -OH, _-NH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid, where ^Ry is -H, phenyl, C1-C10-alkyl, C2-C10-alkenyl, or C2-C10-alkynyl, each of which can be replaced by –NH-C(=O)-NH₂, -COOH

Substitution with -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid, where Rx is -H, C1-C3-alkyl-, or phenyl-.

G3 is –H or –COOH

^R2 is -H,

R ³ is -MOD, –L-#1, or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl, which may be 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl groups, 1 to 3 –O-alkyl groups, 1 to 3 –SH- groups, or 1 to 3 –S-alkyl groups.

1 to 3 -O-C(=O)-alkyl, 1 to 3 -O-C(=O)-NH-alkyl

1 to 3 -NH-C(=O)-alkyl groups, 1 to 3 -NH-C(=O)-NH-

Alkyl, 1 to 3 -S (=O) _n -alkyl, 1 to 3 -S (=O) ₂ -NH-alkyl, 1 to 3 -NH-alkyl, 1 to 3 -N(alkyl) ₂ , 1 to 3

NH ₂ or 1 to 3 -(CH ₂ ) _O-3 Z groups are substituted.

n is 0, 1, or 2.

Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C( ⁼ O) ^-NY1Y2 , or –

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NHC(=O)CH ₃ )Z',

-(CH ₂ ) _0-3 -CH(NH ₂ )Z' or -(CH ₂ ) _0-3 Z',

Z' can be -H, -S(=O) _3H , _-NH2 , or –C(=O)-OH.

^R4 is a group that can be cleaved by the podase of formula (Ia').

R ⁵ is –H,

^R6 and ^R7 are fluorine.

^R8 is tert-butyl.

R ⁹ is -H,

–L-#1 is -(Lb)o-(LIG) _p ,

LIG is the binding site; after binding to receptors on tumor cells, it is internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lb is the connector.

o and p are independently 0 or 1.

_C1-3 -alkyl is particularly preferred here.

Further preferred are compounds of general formula (IIa), their salts, solvates, and salts of solvates, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N,

A is –C(＝O)-,

R ¹ is –L-#1, -H, or –MOD

–L-#1 is -(Lb)o-(LIG) _p ,

LIG is the binding site; after binding to receptors on tumor cells, it is internalized by the tumor cells and processed intracellularly (preferably via lysosomes). Lb is the linker.

o and p are independently 0 or 1.

–MOD is the group –(NR ¹⁰ )n-(G1)o-G2-G3,

R ¹⁰ is -H or _C1 - _C3 -alkyl-.

n 0,

G1 is -C(＝O)-NH-,

o is 1, and

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following or separated more than once by the same or different groups selected from the following: -O-, -S-, -NRy- or -C(=O)-, and the straight-chain or branched hydrocarbon chain may be mono- or poly-substituted by -C(=O)-NH2 or -COOH.

R_^y is –H or C1-C10-alkyl, which can be converted by –NH-C(＝O)-NH₂, -

COOH, -OH, -NH ₂ substitution,

G3 is –COOH

^R2 is -H,

R ³ is –L-#1,

^R4 is a group that can be cleaved by the podase of formula (Ia').

R ⁵ is –H,

^R6 and ^R7 are fluorine.

^R8 is tert-butyl.

R ⁹ is -H,

–L-#1 is -(Lb)o-(LIG) _p ,

LIG is the binding site; after binding to receptors on tumor cells, it is internalized by the tumor cells and processed intracellularly (preferably via lysosomes). Lb is the linker.

o and p are independently 0 or 1.

Also preferred are compounds of general formula (IIa’), their salts, solvates and salts of solvates.

in

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is N,

X ₂ is C, and

X ₃ is N,

or

_X1 is CH or CF.

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is CH,

X ₂ is N, and

X ₃ is C.

A is a group *-C(＝O)-(CH ₂ ) _x -S-CH ₂ -CH(COOH)-NH-**,

x is 1 or 2.

* indicates a bond that connects drug molecules.

**The bond is a linker (Ia’) to a group that can be cleaved by legume proteases.

R ¹ is –MOD,

–MOD is the group –(NR ¹⁰ )n-(G1)o-G2-G3,

R ¹⁰ is -H or _C1 - _C3 -alkyl-.

When n is 0,

G1 is -C(＝O)-NH-,

o is 1,

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) _2- , -

NR ^y C(=O)-, -C(=O)NR ^y -, -NR ^y NR ^y -, -S(=O) ₂ -NR ^y NR ^y -

-C(＝O)-NR ^y NR ^y -, -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain can be monosubstituted or polysubstituted by the following groups, either the same or different: –NH-C(＝O)-NH ₂ , -C(＝O)-NH ₂ , -COOH, -

OH, _-NH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid,

R_^y is -H, phenyl-, C1-C10-alkyl-, C2-C10-alkenyl-, or C2-C10-ynyl-, each of which can be converted by –NH-C(＝O)-NH₂, -COOH, etc.

Substitution with -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

Rx is -H, C1-C3-alkyl- or phenyl-.

G3 is –H or –COOH

^R2 is –H,

^R3 is a group that can be cleaved by the podase of formula (Ia').

R ⁴ is –H,

^R5 represents -H, _-NH2 , _-NO2 , halogen, -CN, _CF3 , _-OCF3 , -

_CH₂F , _-CH₂F , -SH ^, or -( _CH₂ ) _₀- ₃Z, where Z is -H, ^-OY₃ , ^-SY₃ , halogen, ^-NHY₃ , -C(=O) ^-NY₁Y₂ , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-, fluoro- _C2-10 -alkenyl-, _C2-10 -ynyl-, fluoro- _C2-10 -ynyl-, hydroxyl-

_-NO₂ , _-NH₂ , -COOH or halogens,

^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-,

Fluorine- _C2-10 -alkenyl-, _C2-10 -ynyl-, or fluorine- _C2-10 -ynyl-, or _C4-10-

Cycloalkyl- or fluorine-C _4-10 -cycloalkyl-

R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ .

Of particular preference are those compounds of general formula (IIa’), and their salts, solvates, and salts of solvates, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N,

A is a group *-C(＝O)-(CH ₂ ) _x -S-CH ₂ -CH(COOH)-NH-**,

x is 1 or 2.

* indicates a bond that connects drug molecules.

**The bond is a linker (Ia’) to a group that can be cleaved by legume proteases.

R ¹ is –MOD,

–MOD is the group –(NR ¹⁰ )n-(G1)o-G2-G3,

R ¹⁰ is -H or _C1 - _C3 -alkyl-.

When n is 0,

G1 is -C(＝O)-NH-,

o is 1,

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) _2- , -

NR ^y C(=O)-, -C(=O)NR ^y -, -NR ^y NR ^y -, -S(=O) ₂ -

NR ^y NR ^y -, -C(=O)-NR ^y NR ^y -, -C(=O)-, -CR ^x =NO-, and

Straight-chain or branched hydrocarbon chains can be monosubstituted or polysubstituted by the following groups, either identically or differently: –NH-C(=O) _-NH₂ , -C(=O) _-NH₂ , -

COOH, -OH, _-NH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid,

R_^y is -H, phenyl-, C1-C10-alkyl-, C2-C10-alkenyl-, or C2-C10-alkynyl-, each of which can be substituted with –NH-C(=O)-NH₂, -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid.

Rx is -H, C1-C3-alkyl- or phenyl-.

G3 is –H or –COOH

^R2 is –H,

^R3 is a group that can be cleaved by the podase of formula (Ia).

R ⁴ is –H,

R ⁵ is –H,

^R6 and ^R7 are fluorine.

^R8 is tert-butyl, and

R ⁹ is -H.

Further preferred are compounds of general formula (IIa”), their salts, solvates, and salts of solvates.

in

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is N,

X ₂ is C, and

X ₃ is N,

or

_X1 is CH or CF.

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is CH,

X ₂ is N, and

X ₃ is C.

A is -C(=O)-, -S(=O), -S(=O) ₂ - or -S(=O) ₂ -NH-,

^R1 is a group *-(G1) _o -G2-NH-**,

* indicates the binding site with drug molecules (cytotoxic agents).

** is a bond that the podase Ia'-linked proteinase can cleave.

G1 is -C(＝O)-NH-,

o is 1,

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) _2- , -

NR ^y C(=O)-, -C(=O)NR ^y -, -NR ^y NR ^y -, -S(=O) ₂ -

NR ^y NR ^y -, -C(=O)-NR ^y NR ^y -, -C(=O)-, -CR ^x =NO-, and

Straight-chain or branched hydrocarbon chains can be monosubstituted or polysubstituted by the following groups, either identically or differently: –NH-C(=O) _-NH₂ , -C(=O) _-NH₂ , -

COOH, -OH, _-NH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid,

R_^y is -H, phenyl-, C1-C10-alkyl-, C2-C10-alkenyl-, or C2-C10-ynyl-, each of which can be converted by –NH-C(＝O)-NH₂, -COOH, etc.

Substitution with -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid, where Rx is -H, C1-C3-alkyl-, or phenyl-.

^R2 is –H,

R ³ is an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl group, which may be substituted with 1 to 3 OH groups, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl groups, or 1 to 3 –O-alkyl groups.

1 to 3 -SH-, 1 to 3 -S-alkyl, 1 to 3 -O-C(=O)-alkyl-, 1 to 3 -O-C(=O)-NH-alkyl, 1 to 3 -NH-

C(=O)-alkyl, 1 to 3 -NH-C(=O)-NH-alkyl, 1 to 3 -

S(＝O) _n -alkyl, 1 to 3 –S(＝O) ₂ -NH-alkyl, 1 to 3 -NH-alkyl, 1 to 3 -N(alkyl) ₂ , 1 to 3 NH ₂ or 1 to 3 -

( _CH₂ ) _₀- 3Z group substitution,

n is 0, 1, or 2.

Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C( ⁼ O) ^-NY1Y2 , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NHC(=O)CH ₃ )Z',

-( _CH₂ ) _₀-3 -CH( _NH₂ )Z' or -( _CH₂ ) _₀- 3Z', where Z' is -H, -S(=O) _₃H , _-NH₂ , or –COOH.

R ⁴ is –H,

^R5 represents -H, _-NH2 , _-NO2 , halogen, -CN, _CF3 , _-OCF3 , -

_CH₂F , _-CH₂F , -SH ^, or -( _CH₂ ) _₀- ₃Z, where Z is -H, ^-OY₃ , ^-SY₃ , halogen, ^-NHY₃ , -C(=O) ^-NY₁Y₂ , or –

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-, fluoro- _C2-10 -alkenyl-, _C2-10 -ynyl-, fluoro- _C2-10 -ynyl-, hydroxyl-

_-NO₂ , _-NH₂ , -COOH or halogens,

^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-,

Fluoro- _C2-10 -alkenyl-, _C2-10 -ynyl-, or fluoro- _C2-10 -ynyl-, or _C4-10 -cycloalkyl- or fluoro- _C4-10 -cycloalkyl-,

R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ .

Of particular preference are those compounds of general formula (II”), and their salts, solvates, and salts of solvates, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N,

A is –C(＝O)-,

^R1 is a group *-(G1) _o -G2-NH-**,

* indicates the binding site with drug molecules (cytotoxic agents).

**The bond is a linker (Ia’) to a group that can be cleaved by legume proteases.

G1 is -C(＝O)-NH-,

o is 1,

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following groups or separated more than once by the same or different groups selected from the following groups: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y C(＝O)-, -C(＝O)NR ^y- , -NR ^y NR ^y- , -S(＝O) _2- NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain may be monosubstituted by the following groups or multisubstituted by the same or different groups of the following groups: –NH-C(＝O)-NH ₂ , -C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid,

R_^y is -H, phenyl-, C1-C10-alkyl-, C2-C10-alkenyl-, or C2-C10-alkynyl-, each of which can be substituted with –NH-C(=O)-NH₂, -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid.

Rx is -H, C1-C3-alkyl- or phenyl-.

^R2 is –H,

^R3 is –CH2 _- OH,

R ⁴ is –H,

R ⁵ is –H,

^R6 and ^R7 are fluorine.

^R8 is tert-butyl, and

R ⁹ is –H.

Another preferred option is a compound of general formula (IIa), wherein

^R1 represents –H; –L-#1; –COOH;

QOC-CH ₂ -CH ₂ -CH(COQ)-NH-C(=O)-CH ₂ -CH ₂ -NH-C(=O)-;

HOOC-CH(NH ₂ )-CH ₂ -CH ₂ -C(=O)-NH-CH ₂ -CH ₂ -NH-C(=O)-or

HOOC-CH(NH ₂ )-(CH ₂ ) ₄ -NH-C(=O)-CH ₂ -CH ₂ -NH-C(=O)-,

^R2 is -H,

A is –C(＝O)-,

Q is either -OH or _-NH2

R ³ is -(CH ₂ )OH; -CH(CH ₃ )OH; -CH ₂ -S-CH ₂ CH-(COOH)-NH-C(＝O)-CH ₃ ; -CH(CH ₃ )OCH ₃ ; a phenyl group that can be substituted with 1 to 3 halogen atoms, 1 to 3 amino groups, 1 to 3 alkyl groups or 1 to 3 haloalkyl groups;

HOOC-CH2-CH2-CH(COOH)-NH-C(=O)-CH2-CH2-NH-C(=O)-;

HOOC-CH(NH2)-CH2-CH2-C(=O)-NH-CH2-CH2-NH-C(=O)-;

HOOC-CH(NH2)-(CH2)4-NH-C(=O)-CH2-CH2-NH-C(=O)-or

–CH ₂ -S _x -(CH ₂ ) _0-4 -CHY ⁵ -COOH,

x is 0 or 1.

Y ⁵ is either -H or -NHY ⁶ .

Y ⁶ is –H, –C(＝O)-CH ₃ or –L-#1,

R ⁵ is -H,

^R6 and ^R7 are independently -H, _C1-3 -alkyl, or halogen.

^R8 is a _C1-4 -alkyl group, and

R ⁹ is –H.

The compounds that are particularly preferred here are those in particular, among which

^R6 and ^R7 are independently hydrogen or fluorine, and

^R8 is tert-butyl.

Furthermore, those compounds are preferred, among which...

^R1 is –H, -COOH,

QOC-CH ₂ -CH ₂ -CH(COQ)-NH-C(=O)-CH ₂ -CH ₂ -NH-C(=O)-,

HOOC-CH(NH ₂ )-CH ₂ -CH ₂ -C(=O)-NH-CH ₂ -CH ₂ -NH-C(=O)-or

HOOC-CH(NH ₂ )-(CH ₂ ) ₄ -NH-C(=O)-CH ₂ -CH ₂ -NH-C(=O)-,

^R2 is -H,

A is –C(＝O)-,

^R3 is -( _CH2 )OH, -CH( _CH3 )OH, _-CH2 -S- _CH2CH (COOH)NH-C(＝O) _-CH3 , -CH( _CH3 ) _OCH3 , HOOC-CH2-CH2-CH(COOH)-NH-C(＝O)-CH2-CH2-NH-C(＝O)-, HOOC-CH(NH2)-CH2-CH2-C(＝O)-NH-CH2-CH2-NH-C(＝O)-, HOOC-CH(NH2)-(CH2)4-NH-C(＝O)-CH2-CH2-NH-C(＝O)-, _–CH2 - _Sx- ( _CH2 ) _0-4- ^CHY5 -COOH, or a phenyl group that can be substituted with 1-3 halogen atoms, 1 to 3 amino groups, 1 to 3 alkyl groups, or 1 to 3 haloalkyl groups.

x is 0 or 1.

Y ⁵ is either -H or -NHY ⁶ .

Y ⁶ is –H, –C(＝O)-CH ₃ or –L-#1,

R ⁵ is -H,

^R6 and ^R7 are independently -H, _C1-3 -alkyl, or halogen.

^R8 is a _C1-4 -alkyl group, and

R ⁹ is -H,

One of the substituents ^R1 and ^R3 is –L-#1.

The compounds that are particularly preferred here are those in particular, among which

^R6 and ^R7 are -F, and

^R8 is tert-butyl.

Compounds of general formula (IIb) are also preferred.

in

_X1 , _X2 , _X3 , ^R1 ,

^R2 , ^R4 , ^R5 , ^R6

^R7 , ^R8 , and ^R9 have the definitions given in general formula (IIa), and

A is –C(＝O)-,

B is a single bond, –O- _CH2- or –CH2 _- O-.

^R20 is _–NH2 , -F, _-CF3 , or _-CH3 , and

n is 0, 1, or 2.

Here, compounds of general formula (IIb) are preferred, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N.

Compounds of general formula (IIc) are also preferred.

in

_X1 , _X2 , _X3 , A, ^R1 , ^R3 , ^R4 , ^R6 , ^R7 , ^R8 and ^R9 have the definitions given in general formula (IIa).

Here, compounds of general formula (IIc) are preferred, wherein

_X1 is CH,

X ₂ is C,

X ₃ is N,

A is –C(＝O)-, and

R ³ is -CH ₂ OH, -CH2OCH ₃ , -CH(CH ₃ )OH or -CH(CH ₃ )OCH ₃ .

Furthermore, compounds with the general formula (IId) are also preferred.

in

_X1 , _X2 , _X3 , A, ^R3 , ^R4 , ^R6 , ^R7 , ^R8 and ^R9 have the definitions given in general formula (IIa).

Here, compounds of general formula (IId) are preferred, wherein

_X1 is CH,

X ₂ is C,

X ₃ is N,

A is –C(＝O)-,

^R3 is _–CH2 - _Sx- ( _CH2 ) _0-4 - ^CHY5 -COOH.

x is 0 or 1.

Y ⁵ is -H or -NHY ⁶ , and

Y ⁶ is -H or –C(=O)CH ₃ .

Furthermore, compounds of general formulas (IIa), (IIb), (IIc), and (IId) are also preferred, among which

Z is either -Cl or -Br;

^R1 is -( _CH2 ) _0-3Z ,

Z is –C(＝O)-NY ¹ Y ² ,

Y ¹ is -H, -NH ₂ or -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z';

Y ² is -(CH ₂ CH ₂ O) _0-3 -(CH ₂ ) _0-3 Z', and

Z' is –C(＝O)-OH;

^Y1 is -H,

Y ² is -(CH ₂ CH ₂ O) ₃ -CH ₂ CH ₂ Z', and

Z' is –C(＝O)-OH;

^Y1 is -H,

^Y2 is _{-CH2CH2Z '} ,

Z' is -(C(＝O)NHCHY ⁴ ) ₂ -COOH, and

Y ⁴ has the definition given in general formula (IIa);

·Y ¹ is -H,

^Y2 is _{-CH2CH2Z '} ,

Z' is -(C(＝O)-NHCHY ⁴ ) ₂ -COOH, and

^Y4 is isopropyl or –( _CH2 ) ₃ -NH-C(=O) _-NH2 ;

·Y ¹ is -H,

^Y2 is _{-CH2CH2Z '} ,

Z' is -(C(＝O)-NHCHY ⁴ ) ₂ -COOH, and

^Y4 is _–CH3 or –( _CH2 ) ₃ -NH-C(＝O) _-NH2 ;

• ^Y4 is a linear or branched _C1-6 alkyl group, which may optionally be substituted with –NH-C(＝O) _-NH2 ;

• ^Y4 is isopropyl or _–CH3 ;

·Y ¹ is -H,

^Y2 is _{-CH2CH2Z '} ,

Z' is –C(＝O)-NHCHY ⁴ -COOH, and

^Y4 is optionally _–NH2 -substituted aryl or benzyl;

^Y4 is an aminobenzyl group;

^R2 is –( _CH2 ) _0-3Z ,

Z is –SY ³ , and

^Y3 has the definition given above;

·R ⁴ is –C(＝O)-CHY ⁴ -NHY ⁵ ,

Y4 has the definition given above, and

Y ⁵ is -H;

·R ⁴ is –C(＝O)-CHY ⁴ -NHY ⁵ ,

^Y5 is -C(=O) ^-CHY6 - _NH2 , and

^Y4 and ^Y6 have the definitions given above;

^Y4 is a linear or branched _C1-6 alkyl group, which may optionally be substituted with –NH-C(＝O) _-NH2 .

Furthermore, compounds of general formula (IIa) are preferred, wherein ^R1 , ^R2 or ^R3 is -MOD.

Particularly preferred are those compounds in which ^R3 is -MOD and ^R1 is –L-#1.

in

–MOD is -(NR ¹⁰ )n-(G1)o-G2-G3,

R ¹⁰ is -H or _C1 - _C3 -alkyl;

G1 is –NH-C(=O)-, -C(=O)-NH-, or

n is 0 or 1,

o is 0 or 1.

G2 has a straight-chain or branched hydrocarbon chain with 1-20 carbon atoms, which may be separated once by a group selected from the following or separated more than once by the same or different groups selected from the following: -O-, -S-, -S(＝O)-, -S(＝O) ₂ , ^-NRy- , ^-NRyC (＝O)-, -C(＝O) ^-NRy , ^{-NRy NRy-} , -S(＝O) _2- ^{NRy NRy-} , -C(＝O)-NRy ^NRy- , ^-C (＝O)-, ^-CRx ＝NO-, wherein the straight-chain or branched hydrocarbon chain may be substituted with –NH-C(＝O) _-NH2 , -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

R_^y is -H, phenyl, C1-C10-alkyl, C2-C10-alkenyl, or C2-C10-alkynyl, each of which can be substituted with –NH-C(=O)-NH₂, -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid.

Rx is -H, C1-C3-alkyl, or phenyl.

G3 is either -H or -COOH, and

The –MOD group preferably has at least one –COOH group or preferably two –COOH groups, and one amino group of the –MOD group can be acylated by a podase-cleavable group of formula (Ia’).

More preferably, the –MOD group has at least one -COOH group, for example, in a trimethylammonium ethoxylate group.

Preferably, the -COOH group is at the terminal position.

More preferably, the –MOD group is the group _–CH2 - _Sx- ( _CH2 ) _0-4 - ^CHY5 -COOH.

in

x is 0 or 1.

Y ⁵ is -H or -NHY ⁶ , and

Y6 is -H or –C(=O)CH ₃ .

Furthermore, compounds of general formulas (IIa), (IIb), (IIc), and (IId), their salts, solvates, and salts of solvates are also preferred, wherein...

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is N,

X ₂ is C, and

X ₃ is N,

or

_X1 is CH or CF.

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is CH or CF.

X ₂ is N, and

X ₃ is C,

^R1 is -H, –L-#1, –MOD, or -( _CH2 ) _0-3Z .

Z can be -H, ^-NHY3 , -OY3, ^-SY3 , ^halogen , -C(=O) ^{-NY1Y2 ,} or -C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , -( _CH2CH2O ) _{O-3- ( CH2} ) _O-3Z ' or -CH( _CH2W )Z'.

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' is -H, -NH ₂ , -S(=O) ₃ H, -COOH, -NH-C(=O)-CH ₂ -CH ₂ -CH(NH ₂ )-COOH or -(C(=O)-NH-CHY ⁴ ) _1-3 COOH,

W is either -H or -OH.

^Y4 is a linear or branched _C1-6 alkyl group, optionally substituted with -NHC(=O) _-NH2 , or an aryl or benzyl group, optionally substituted with _–NH2 .

^R2 is -H, -C(=O) ^-CHY4 - ^NHY5 , or -( _CH2 ) _0-3Z .

Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C( ⁼ O) ^-NY1Y2 , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH;

^Y4 is a linear or branched _C1-6 alkyl group, optionally substituted with -NHC(=O) _-NH2 , or an aryl or benzyl group, optionally substituted with _–NH2 .

Y ⁵ is -H or –C(=O)-CHY ⁶ -NH ₂ ,

^Y6 is a linear or branched _C1-6 -alkyl group.

A is -C(=O)-, -S(=O)-, -S(=O) ₂ - or -S(=O) ₂ -NH-,

R ³ represents –L-#1, -MOD, or alkyl, cycloalkyl, aryl, heteroaryl, or heteroalkyl.

Heterocyclic alkyl groups, optionally surrounded by 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl groups, 1 to 3 –O-alkyl groups, 1 to 3 –SH groups, 1 to 3 –S-alkyl groups, 1 to 3 –O-C(=O)-alkyl groups, 1 to 3 –O-

C(＝O)-NH-alkyl, 1 to 3 -NH-C(＝O)-alkyl, 1 to 3 -NH-

C(＝O)-NH-alkyl, 1 to 3 -S(＝O) _n -alkyl, 1 to 3 –S(＝O) ₂ -NH-alkyl, 1 to 3 -NH-alkyl, 1 to 3 -N(alkyl) ₂ , 1 to 3 -

NH(( _CH₂CH₂O ) _{₁- 20H} )-, substituted with 1 to 3 _NH₂ groups or 1 to 3 -( _CH₂ ) _₀- 3Z- groups,

Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C( ⁼ O) ^-NY1Y2 , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NH-C(＝O)CH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z'

Or -(CH ₂ ) _0-3 Z',

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R5 represents -H, -MOD, _-NH2 , _-NO2 , halogen, -CN, _-CF3 , _-OCF3 , and -

CH ₂ F, -CH ₂ F, -SH or -(CH ₂ ) _0-3 Z,

Z represents -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C(=O)-NY ¹ Y ² , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is -H or -( _CH2 ) _0-3Z ', and

Z' can be -H, -S(=O) _3H , _-NH2 , or –C(=O)-OH.

^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl, fluoro- _C2-10 -ynyl, hydroxyl, _-NO2 , _-NH2 , -COOH, or halogen.

^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl, or fluoro- _{C2-10-ynyl, or a C4-10 -} cycloalkyl or fluoro- _C4-10 -cycloalkyl.

R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ .

–MOD is -(NR ¹⁰ ) _n -(G1) _o -G2-G3,

R ¹⁰ is -H or _C1 - _C3 -alkyl.

G1 is –NH-C(=O)-, -C(=O)-NH-, or

n is 0 or 1,

o is 0 or 1.

G2 has a straight-chain or branched hydrocarbon chain with 1-10 carbon atoms, which may be alternated once or more by a group selected from the following: -O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , -NR ^y C(＝O)-, -C(＝O)-NR ^y- , -NR ^y NR ^y- , -S(＝O) _2- NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , wherein the straight-chain or branched hydrocarbon chain may be substituted with -NH-C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

R y  is –H, -C(=O)-, -CRx =NO-, or optionally substituted with NH-C(=O)-NH 2 -, -COOH-, -OH-, -NH2 -, sulfonamide, sulfone, sulfoxide, or sulfonic acid; or a phenyl group, C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, or C 2 -C 10 -ynyl.

^Rx is -H, _C1 - _C3 -alkyl, or phenyl.

G3 is either -H or -COOH, and

The –MOD group preferably has at least one –COOH group, and

Where ^R1 and ^R3 are not both –L-#1.

Compounds of general formulas (IIa), (IIb), (IIc), and (IId) are particularly preferred here, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N.

Compounds of general formulas (IIa), (IIb), (IIc), and (IId) are also particularly preferred here, wherein

R3 is a _C1-10 -alkyl, _C6-10 -aryl, _C6-10 -aralkyl, _C5-10 -heteroalkyl, _C1-10 -alkyl- _OC6-10 -aryl, or C5-10-heterocyclic alkyl, optionally surrounded by 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl atoms, 1 to 3 –O-alkyl atoms, 1 to 3 –SH atoms, 1 to 3 –S-alkyl atoms, 1 to 3 –OC(=O)-alkyl atoms, 1 to 3 –OC(=O)-NH-alkyl atoms, 1 to 3 –NH-C(=O)-alkyl atoms, 1 to 3 –NH-C(=O)-NH-alkyl atoms, 1 to 3 –S(=O) _n -alkyl atoms, 1 to 3 –S(=O) _2- NH-alkyl atoms, 1 to 3 –NH-alkyl atoms, 1 to 3 –N(alkyl) ₂ Substitution with 1 to 3 -NH (( _CH₂CH₂O ) _{¹- 2OH} ), 1 to ₃ NH₂, or 1 to 3 -( _CH₂ ) _₀- 3Z groups, and

Z can be -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C(=O) ^{-NY1Y2 ,} or -C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NH-C(=O)CH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z', or -(CH ₂ ) _0-3 Z', and

Z' can be -H, -S(＝O) _3H , _-NH2 or –COOH.

Other preferred compounds of general formulas (IIa), (IIb), (IIc), and (IId) are those, among which

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is N,

X ₂ is C, and

X ₃ is N,

or

_X1 is CH or CF.

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is CH or CF.

X ₂ is N, and

X ₃ is C,

^R1 is -H, –L-#1, –MOD, or -( _CH2 ) _0-3Z .

Z can be -H, ^-NHY3 , -OY3, ^-SY3 , ^halogen , -C(=O) ^{-NY1Y2 ,} or -C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , -( _CH2CH2O ) _{O-3- ( CH2} ) _O-3Z ' or -CH( _CH2W )Z'.

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' is -H, -NH ₂ , -S(=O) ₃ H, -COOH, -NH-C(=O)-CH ₂ -CH ₂ -CH(NH ₂ )-COOH or -(C(=O)-NH-CHY ⁴ ) _1-3 COOH,

W is either -H or -OH.

^Y4 is a linear or branched _C1-6 alkyl group optionally substituted with –NH-C(=O) _-NH2- or optionally substituted with _–NH2- aryl or benzyl groups.

^R2 is -H, -C(=O) ^-CHY4 - ^NHY5 , or -( _CH2 ) _0-3Z .

Z can be -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C(=O) ^{-NY1Y2 ,} or -C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^Y4 is a linear or branched _C1-6 alkyl group optionally substituted with –NH-C(=O) _-NH2- or optionally substituted with _–NH2- , or a benzyl group.

Y ⁵ is -H or –C(=O)-CHY ⁶ -NH ₂ ,

^Y6 is a linear or branched _C1-6 -alkyl group.

A is -C(=O)-, -S(=O)-, -S(=O) ₂ - or -S(=O) ₂ -NH-,

R ³ is –L-#1, -MOD, or alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl, optionally surrounded by 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogen, dihalogen, or trihalogen alkyl groups, 1 to 3 –O-alkyl groups, 1 to 3 –

SH, 1 to 3 -S-alkyl, 1 to 3 -O-C(=O)-alkyl, 1 to 3 -O-

C(＝O)-NH-alkyl, 1 to 3 -NH-C(＝O)-alkyl, 1 to 3 -NH-

C(＝O)-NH-alkyl, 1 to 3 -S(＝O) _n -alkyl, 1 to 3 –S(＝O) ₂ -NH-alkyl, 1 to 3 -NH-alkyl, 1 to 3 -N(alkyl) ₂ , 1 to 3 -

NH(( _CH₂CH₂O ) _{₁- 20H} )-, substituted with 1 to 3 _NH₂ groups or 1 to 3 -( _CH₂ ) _₀- 3Z- groups,

Z represents -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C( ⁼ O) ^-NY1Y2 , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NH-C(＝O)CH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z'

Or -(CH ₂ ) _0-3 Z',

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R5 represents -H, -MOD, _-NH2 , _-NO2 , halogen, -CN, _-CF3 , _-OCF3 , and -

CH ₂ F, -CH ₂ F, SH or -(CH ₂ ) _0-3 Z,

Z represents -H, -OY ³ , -SY ³ , halogen, -NHY ³ , -C(=O)-NY ¹ Y ² , or -

C(＝O)-OY ³ ,

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z ',

^Y3 is either -H or -( _CH2 ) _0-3Z '.

Z' can be -H, -S(＝O) _3H , _-NH2 , or –COOH.

^R6 and ^R7 are independently -H or halogens.

^R8 is a straight-chain or branched _C1-10 -alkyl or fluoro- _C1-10 -alkyl.

R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ .

–L-#1 is -(Lb) _o -(LIG) _p ,

LIG is the binding site; after binding to receptors on tumor cells, it is internalized by the tumor cells and processed within the cells (preferably via lysosomes).

Lb is the connector.

o and p are each independently 0 or 1.

–MOD is –CH ₂ -S _x -(CH ₂ ) _O-4 -CHY ⁵ -COOH,

x is 0 or 1.

Y ⁵ is either -H or -NHY ⁶ .

^Y6 is -H or –C(=O) _CH3 , and

Where ^R1 and ^R3 are not both –L-#1,

And its salts, solvates and salts of solvates.

Here, compounds of general formulas (IIa), (IIb), (IIc), and (IId) are preferred, wherein

_X1 is CH,

X ₂ is C, and

X ₃ is N.

Compounds of general formulas (IIa), (IIb), (IIc), and (IId) are also preferred here, wherein

R ³ is a _C1-10 -alkyl, _C6-10 -aryl or _C6-10 -aralkyl, C5-10 -heteroalkyl, _C1-10 -alkyl- _OC6-10 -aryl or _C5-10 -heterocyclic alkyl, which may be 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated or _{trihalogenated} alkyl atoms, 1 to 3 -O-alkyl atoms, 1 to 3 -SH atoms, 1 to 3 -S-alkyl atoms, 1 to 3 -OC(=O)-alkyl atoms, 1 to 3 -OC(=O)-NH-alkyl atoms, 1 to 3 -NH-C(=O)-alkyl atoms, 1 to 3 -NH-C(=O)-NH-alkyl atoms, 1 to 3 -S(=O) _n -alkyl atoms, 1 to 3 -S(=O) _2- NH-alkyl atoms, 1-3-NH-alkyl atoms, 1 to 3 -N(alkyl) _{2 atoms} Substitution with 1 to 3 _NH2 or 1 to 3 -( _CH2 ) _O- 3Z groups,

Z can be -H, halogen, ^-OY3 , ^-SY3 , ^-NHY3 , -C(=O) ^{-NY1Y2 ,} or -C(=O) ^-OY3 .

^Y1 and ^Y2 are independently -H, _-NH2 , or -( _CH2 ) _0-3Z '.

Y ³ is -H, -(CH ₂ ) _0-3 -CH(NH-C(=O)CH ₃ )Z', -(CH ₂ ) _0-3 -CH(NH ₂ )Z', or -(CH ₂ ) _0-3 Z', and

Z' can be -H, -S(＝O) _3H , _-NH2 or –COOH.

Unless otherwise defined, the term "alkyl" is preferably _C1 - _C10 -alkyl.

Unless otherwise defined, halogens are fluorine (-F), chlorine (-Cl), and bromine (-Br).

Compounds of the following general formulas (V), (VI) and (VII) are particularly preferred, wherein ^R1 , ^R2 , ^R3 , ^R4 and ^R5 have the definitions given in general formula (IIa):

Compounds of general formulas (V), (VI) and (VII) are particularly preferred, wherein

^R1 , ^R2 and ^R5 are -H, and ^R4 has the definition given in general formula (IIa).

Compounds of general formula (VI) are particularly preferred here.

Binding portion that binds to tumor cell receptors

In its broadest sense, the term "binding moiety" should be understood as a molecule that binds to a target molecule present in a specific target cell population to be treated by a binding moiety-drug conjugate. The term "binding moiety" should be understood in its broadest sense and also includes, for example, lectins, proteins capable of binding specific glycans, or phospholipid-binding proteins. Such binding moieties include, for example, high molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (e.g., aptamers (US5,270,163, review by Keefe AD. et al., Nat. Rev. Drug Discov. 2010; 9:537-550 or vitamins), and all other cell-binding molecules or substances. Binding proteins are, for example, antibodies and antibody fragments or antibody mimics, such as affibodies, adnectin, anticalins, DARPins, avimers, and nanobodies (Gebauer M. et al. review, Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr. Opinion in Pharmacology 2008; 8:608-617). Binding peptides are, for example, ligands of ligand/receptor pairs, such as VEGF of ligand/receptor pairs for VEGF/KDR, or transferrin of ligand/receptor pairs for transferrin/transferrin receptors, or cytokines/cytokine receptors, such as TNFα of ligand/receptor pairs for TNFα/TNFα receptors.

The prodrug according to the invention preferably contains a binding moiety that can bind to a receptor on tumor cells and is typically internalized by the tumor cells after binding to the receptor, preferably processed via lysosomes. In one embodiment, the binding moiety can use a group cleavable by the enzyme lentinan, optionally linked via a linker, such that after the lentinan-cleavable group is cleaved, the active ingredient exists separately from the binding moiety or its derivative. In this case, -D in formula (Ia) represents -D1 and -R in formula (Ia) represents ( _Lc ) _r -LIG (Embodiment A). Alternatively, the binding moiety can be linked to a drug molecule, optionally via a linker, such that after the lentinan-cleavable group is cleaved, the active ingredient exists together with the binding moiety or its derivative. In this case, -D in formula (Ia) represents -D1-(Lb)o-LIG and R in formula (Ia) represents _Z1- (C(=O))q- (Embodiment B).

The compound of embodiment A preferably has the following general formula (III'):

Where n, r, LIG, La, Lc and _D1 have the definitions given in general formula (Ia).

The compound of embodiment B preferably has the following general formula (IV'):

Where n, o, R, LIG, La, Lb and _D1 have the definitions given in general formula (Ia).

The binding portion of LIG is usually a peptide, protein, or a derivative thereof. The corresponding peptides are known from the literature (review by D. and A. Beck-Sickinger, J. Pept. Sci. 2015-21.186; see also B. Forner et al., Specialty Chemicals Magazine, May 2012; V. Ahrens et al., Future Med. Chem. 2012, 4, 1567; W. Tai et al., Mol. Pharmaceutics 2011, 8, 901; R. Soudy et al., J. Med. Chem. 2013, 56, 7564 and other references in the introductions of R. Soudy et al., M. Langer et al., J. Med. Chem. 2001, 44, 1341; C. Gruendker et al., Oncology Reports 2011, 26, 629). The peptide or its derivatives are preferably selected from octreotide, GnRH-III, [D-Tyr ⁶ , β-Ala ¹¹ , Phe ¹³ , Nle ¹⁴ ]BN(6-14), NT(8-13), c(RGDfK), HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH ₂ (SEQ ID NO:161), NAP amide, [Phe ⁷ , Pro ³⁴ ]NPY, HER2-targeting peptide, ATEPRKQYATPRVFWTDAPG (SEQ ID NO:162) or LQWRRDDNVHNFGVWARYRL (SEQ ID NO:163) [the peptide sequence is shown here with standard 1-letter codes for amino acids]. Other peptide sequences can be determined by screening methods such as those described in Umlauf et al., Bioconj. Chem. 2014, Oct. 15; 25(10):1829-37.

In embodiment A, the peptide can be directly bonded (e.g., via its C-terminus) to the N-terminus of a group cleavable by podase-derived peptide bonds. The peptide can also be bonded to the N-terminus of a podase-derived peptide group via a linker _Lc , in which case the linker is preferably bonded to the C or N-terminus of the peptide or to a lysine or cysteine side chain of the peptide.

In embodiment B, the peptide can be directly bonded to the drug molecule. However, the peptide is preferably bonded to the drug molecule via a linker Lb, in which case the linker is preferably bonded to the C or N terminus of the peptide or to the lysine or cysteine side chain of the peptide. Binding to the Lb or peptide typically occurs through the substitution of hydrogen atoms in the drug molecule.

For example, in the case of compounds of general formulas (IIa), (IIb), (IIc), (IId), (V), (VI), or (VII), conjugates can be obtained by substituting the hydrogen atoms in ^R1 , ^R2 , ^R3 , ^R5 , or ^R8 in a manner known to those skilled in the art, wherein one of the substituents ^R1 , ^R2 , ^R3 , ^R5 , or ^R8 is -(Lb)o-LIG. Particularly preferred binding moiety LIG is an antibody or its antigen-binding fragment or derivative that binds to extracellular target molecules of tumor cells. Particularly preferred is an antibody or fragment thereof that binds to one or more cytotoxic drug molecules. Therefore, in the case of embodiment A, the compound of the present invention is an antibody-drug conjugate (ADC) of the following general formula (IIIa'):

Where n, r, La, Lc and _D1 have the definitions given by general formula (Ia), AB represents antibody, and s represents a number from 1 to 20, preferably from 2 to 8, more preferably from 2 to 6, for example 4.

In this context, _D1 is preferably a compound of general formula (IIa), (IIb), (IIc), (IId), (V), (VI) or (VII), wherein the substituents selected from ^R1 , ^R2 , ^R3 , ^R4 , ^R8 do not have the definitions given above under general formulas (IIa), (IIb), (IIc), (IId), (V), (VI) and (VII), but represent a bond connecting La (i.e., a self-destructing linker) or a bond connecting a carbonyl group.

In embodiment B, the compound of the present invention is an antibody-prodrug conjugate (APDC) of the following general formula (IVa'):

Wherein n, o, R, La, and Lb have the definitions given in general formula (Ia), AB represents antibody, and s represents a number from 1 to 20, preferably 2 to 8, particularly preferably 2 to 6, for example 4. In this context, D1 is preferably a compound of general formula (IIa), (IIb), (IIc), (IId), (V), (VI), or (VII), wherein one of the substituents R ⁴ does not have the definitions given above under general formula (IIa), (IIb), (IIc), (IId), (V), (VI), or (VII), but represents a bond connecting _La or a carbonyl group.

The antibodies (e.g., AB in formulas (IIIa) and (IVa) above) are preferably human, humanized, or chimeric monoclonal antibodies or antigen-binding fragments thereof, especially anti-TWEAKR antibodies, anti-EGFR antibodies, anti-B7H3 antibodies, or anti-HER2 antibodies or antigen-binding fragments thereof. Particularly preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007, and TPP-10337; anti-B7H3 antibodies TPP-8382 and TPP-8567; anti-EGFR antibody cetuximab (TPP-981); and anti-HER2 antibodies trastuzumab and TPP-1015, or antigen-binding fragments thereof.

The literature also discloses various options for the covalent coupling (conjugation) of organic molecules to antibodies. According to the present invention, conjugation to antibodies is preferably achieved through one or more sulfur atoms of cysteine residues and/or through one or more NH groups of lysine residues. However, organic molecules can also be bound to antibodies through free carboxyl groups or through sugar residues of the antibody.

Antibodies bind to extracellular target molecules of tumor cells. In the broadest sense, "target molecule" should be understood as a molecule present in the target cell population, which can be a protein (e.g., a receptor for growth factors) or a non-peptide molecule (e.g., a sugar or phospholipid). It is preferably a receptor or antigen.

The term "extracellular" target molecule describes a target molecule located outside the cell that binds to the cell, or a portion of a target molecule located outside the cell, meaning that an antibody can bind to its extracellular target molecule within an intact cell. Extracellular target molecules can be anchored in the cell membrane or are components of the cell membrane. Methods for identifying extracellular target molecules are known to those skilled in the art. For proteins, this can be achieved by determining one or more transmembrane domains and the orientation of the protein within the membrane. This data is typically stored in protein databases (e.g., SwissProt).

The term "cancer target molecule" describes a target molecule that is more abundant in one or more cancer cell types than in non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present in one or more cancer cell types compared to non-cancer cells of the same tissue type, wherein "selective" describes an enrichment of at least twice that in cancer cells compared to non-cancer cells of the same tissue type ("selective cancer target molecule"). The use of cancer target molecules allows for the selective treatment of cancer cells using conjugates according to the invention.

The term "binding portion" according to the present invention should be understood to refer to a binding peptide, a derivative of a binding peptide, a binding protein, or a derivative of a binding protein. The binding portion is connected to the linker by a bond. The binding portion can be linked by heteroatoms of the binding portion. The heteroatoms of the binding portion that can be used for linking according to the present invention are:

Sulfur, through the binding of some thiol groups,

Oxygen, through the binding of partial carboxyl or hydroxyl groups, and

Nitrogen, via primary or secondary amine groups.

More specifically, according to the present invention, the term "binding portion" should be understood to refer to an antibody.

The heteroatoms listed above can be present in natural antibodies or introduced by chemical or molecular biological methods. According to the present invention, the linking of the antibody to the organic group in formula (I) has only a minor effect on the antibody's binding activity with respect to the target molecule.

In a preferred embodiment, the linker has no effect on the binding activity of the binding moiety with respect to the target molecule.

According to the present invention, the term "antibody" should be understood in its broadest sense and includes immunoglobulin molecules, such as intact or modified monoclonal antibodies, polyclonal antibodies, or multispecific antibodies (e.g., bispecific antibodies). Immunoglobulin molecules preferably comprise molecules having four polypeptide chains, two heavy chains (H chains), and two light chains (L chains), which are typically linked by disulfide bridges. Each heavy chain comprises a variable region (abbreviated as VH) and a constant region. The constant region of the heavy chain may, for example, comprise three regions CH1, CH2, and CH3. Each light chain comprises a variable region (abbreviated as VL) and a constant region. The constant region of the light chain comprises a region (abbreviated as CL). The VH and VL regions can be further subdivided into regions with high variability, also known as complementarity-determining regions (abbreviated as CDRs), and regions with lower sequence variability (frame regions, abbreviated as FRs). Typically, each VH and VL region consists of three CDRs and up to four FRs. For example, from the amino terminus to the carboxyl terminus, the sequence is as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibody can be obtained from any suitable species, such as rabbit, llama, camel, mouse, or rat. In one embodiment, the antibody is of human or mouse origin. The antibody can be, for example, human, humanized, or chimeric.

The term "monoclonal" antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that, apart from a few possible naturally occurring mutations, the individual antibodies in the population are identical. Monoclonal antibodies recognize a single antigen-binding site with high specificity. The term "monoclonal antibody" does not imply a specific method of preparation.

The term "complete" antibody refers to an antibody that contains both an antigen-binding region and constant regions of both the light and heavy chains. The constant regions can be naturally occurring regions or variants thereof with numerous modified amino acid positions.

The term "modified intact" antibody refers to an intact antibody that is fused to another polypeptide or protein not derived from the antibody via a covalent bond (e.g., a peptide bond) at its amino or carboxyl terminus. Furthermore, antibodies can be modified to introduce a reactive cysteine residue at a specific position to facilitate conjugation with the toxic cluster (see Junutula et al., Nat Biotechnol. 2008, 26(8): 925-32).

The term "human" antibody refers to an antibody that can be obtained from humans or is a synthetic human antibody. A "synthetic" human antibody is an antibody that can be obtained partially or completely from a synthetic sequence using a computer, based on analysis of the human antibody sequence. Human antibodies can be encoded, for example, by nucleic acids isolated from a human antibody sequence library. An example of such an antibody can be found in [Authors' Name], Nature Biotech. 2000, 18:853-856.

The terms "humanized" or "chimeric" antibodies describe antibodies composed of non-human and non-human sequence portions. In these antibodies, a sequence portion of a human immunoglobulin (receptor) is replaced by a sequence portion of a non-human immunoglobulin (donor). In many cases, the donor is mouse immunoglobulin. In the case of humanized antibodies, amino acids in the receptor's CDR are replaced by amino acids from the donor. Sometimes, amino acids in the frame are also replaced by the corresponding amino acids from the donor. In some cases, humanized antibodies contain amino acids that are not present in either the receptor or the donor, which are introduced during antibody optimization. In the case of chimeric antibodies, the variable region of the donor immunoglobulin is fused to the constant region of the human antibody.

As used herein, the term complementarity-determining region (CDR) refers to those amino acids in the variable antibody region essential for binding to the antigen. Typically, each variable region has three CDR regions, referred to as CDR1, CDR2, and CDR3. Each CDR region may contain amino acids according to Kabat's definition and/or amino acids of the hypervariable ring according to Chotia's definition. According to Kabat's definition, this includes, for example, regions from approximate amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) of the variable light chain and approximate amino acid positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) of the variable heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). According to Chothia's definition, this includes, for example, regions from approximate amino acid positions 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) of the variable light chain and approximate amino acid positions 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) of the variable heavy chain (Chothia and Lesk; J MolBiol 196 901-917 (1987)). In some cases, the CDR may contain amino acids from the CDR region as defined by Kabat and Chothia.

Antibodies can be classified into different categories based on the amino acid sequence of their heavy chain constant regions. There are five main types of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into other subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different categories are called [α], [δ], [ε], [γ], and [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 a fragment of an antibody/immunoglobulin (e.g., the variable region of IgG) that still contains the antigen-binding region of the antibody/immunoglobulin. The "antigen-binding region" of an antibody typically contains one or more hypervariable regions of the antibody, such as CDR, CDR2, and/or CDR3 regions. However, the "framework" or "backbone" region of the antibody can also play a role in the binding of the antibody to the antigen. The frame region forms the backbone of the CDR. Preferably, the antigen-binding region contains 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, and particularly preferably complete variable light and heavy chains, i.e., amino acids 1-109 of VL and 1 to 113 of VH (according to WO97/08320).

The “functional fragment” or “antigen-binding antibody fragment” of this invention nonexclusively includes Fab, Fab', F(ab')2 and Fv fragments, double-chain antibodies, single-domain antibodies (DAbs), linear antibodies, single-chain antibodies (single-chain Fv, abbreviated as scFv); and multispecific antibodies, such as bispecific and trispecific antibodies, which are formed from antibody fragments. (C.A.K. Borrebaeck, ed. (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, ed. (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag.) Antibodies that are different from “multispecific” or “multifunctional” antibodies are antibodies with the same binding site. Multispecific antibodies can be specific to different epitopes of an antigen, or to more than one epitope of an antigen (see, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; Tutt et al., 1991, J. Immunol. 14760 69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol. 14815471553). F(ab')2 or Fab molecules can be constructed such that the amount of intermolecular disulfide interactions occurring between the Ch1 and CL regions can be reduced or completely prevented.

An epitope is a protein determinant that can specifically bind to immunoglobulins or T-cell receptors. Epitope determinants are typically composed of chemically active surface groups of a molecule, such as amino acids or sugar side chains or combinations thereof, and usually have specific three-dimensional structural properties and specific charge properties.

The “functional fragment” or “antigen-binding antibody fragment” can be fused to another polypeptide or protein not derived from the antibody via a covalent bond (e.g., a peptide bond) at its amino or carboxyl terminus. Furthermore, the antibody and antigen-binding fragment can be modified by introducing a reactive cysteine residue at a specific position to facilitate coupling with the toxic cluster (see Junutula et al., Nat Biotechnol. 2008 Aug; 26(8) 925-32).

Polyclonal antibodies can be prepared by methods known to those skilled in the art. Monoclonal antibodies can be prepared by methods known to those skilled in the art (and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies can be prepared by methods known to those skilled in the art (Olsson et al., MethEnzymol. 92, 3-16 or Cabilly et al. US 4,816,567 or Boss et al. US 4,816,397).

Those skilled in the art are familiar with various methods for preparing human antibodies and their fragments, for example, by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13(1)65-93) or phage display technology (Clackson et al., Nature. 1991 Aug 15; 352(6336)624-8). The antibodies of the present invention can be obtained from a recombinant antibody library, which, for example, consists of the amino acid sequences of various antibodies collected from a large number of healthy volunteers. Antibodies can also be generated using known recombinant DNA techniques. The nucleic acid sequences of the antibodies can be obtained by conventional sequencing or from publicly available databases.

The "isolated" antibody or conjugate has been purified to remove other cellular components. Contaminating cellular components that may interfere with diagnostic or therapeutic use are, for example, cellular enzymes, hormones, or other peptide or non-peptide components. Preferred antibodies or conjugates are those purified to more than 95% by weight relative to the antibody or conjugate (e.g., by the Lowry method, UV-Vis spectroscopy, or by SDS capillary gel electrophoresis). Furthermore, the antibody has been purified to the point where at least 15 amino acids of the N-terminal or internal amino acid sequence can be determined, or has been purified to homogeneity, wherein homogeneity is determined by SDS-PAGE under reducing or non-reducing conditions (detection can be performed by means of Coomassie brilliant blue staining or preferably by silver staining). However, antibodies are typically prepared through one or more purification steps.

The term "specific binding" or "specifically binding" refers to an antibody or binding moiety that binds to a predetermined antigen/target molecule. Specific binding of an antibody or binding moiety typically describes an antibody or binding moiety having an affinity of at least ^10⁻⁷ M ( as a Kd value; i.e., ^preferably those with a Kd value less than ^10⁻⁷ M ), wherein the antibody or binding moiety has an affinity for the predetermined antigen/target molecule at least twice as high as its affinity for nonspecific antigens/target molecules (e.g., bovine serum albumin or casein) (which are not the predetermined antigen/target molecule or closely related antigens/target molecules). Specific binding of an antibody or binding moiety does not mean that the antibody or binding moiety cannot bind to multiple antigens/target molecules (e.g., orthologs from different species). The affinity of the antibody is preferably at least ^10⁻⁷ M (as a Kd value; i.e., preferably those with a Kd value less than ^10⁻⁷ M), preferably at least ^10⁻⁸ M, more preferably ^10⁻⁹ M to ^10⁻¹¹ M. The Kd value can be determined, for example, by means of surface plasmon resonance spectroscopy.

The antibody-drug conjugates of the present invention also possess affinity within these ranges. The affinity is preferably substantially unaffected by the drug conjugation (typically, the affinity reduction is less than an order of magnitude, i.e., at most ^10⁻⁸ M to ^10⁻⁷ M).

The antibodies used according to the present invention are also preferably characterized by high selectivity. High selectivity exists when the antibody of the present invention has an affinity for the target protein that is at least 2 times better, preferably 5 times or more preferably 10 times better than that of other independent antigens such as human serum albumin (the affinity can be determined, for example, by means of surface plasmon resonance spectroscopy).

Furthermore, the antibodies used in this invention are preferably cross-reactive. To facilitate and better interpret preclinical studies, such as toxicology or efficacy studies (e.g., in xenograft mice), it is advantageous if the antibodies used according to this invention bind not only to human target proteins but also to species target proteins in the species used for study. In one embodiment, the antibodies used according to this invention, in addition to human target proteins, are cross-reactive with target proteins of at least one other species. For toxicology and efficacy studies, species of rodents, canines, and non-human primates are preferred. Preferred rodent species are mice and rats. Preferred non-human primates are rhesus monkeys, chimpanzees, and macaques.

In one embodiment, the antibody used according to the invention, in addition to the human target protein, is cross-reactive with the target protein of at least one other species selected from a species comprising mice, rats, and cynomolgus monkeys (Macaca fascicularis). Particularly preferred are the antibodies used according to the invention that are at least cross-reactive with the mouse target protein, in addition to the human target protein. Preferably, the cross-reactive antibody has an affinity for the target protein of the other non-human species that differs from its affinity for the human target protein by no more than 50-fold, and more particularly, no more than 10-fold.

Antibodies targeting cancer molecules

The target molecules targeted by the binding portion, such as an antibody or its antigen-binding fragment, are preferably cancer target molecules. The term "cancer target molecule" describes a target molecule that is more abundant in one or more cancer cell types than in non-cancer cells of the same tissue type. Preferably, cancer target molecules are selectively present in one or more cancer cell types compared to non-cancer cells of the same tissue type, wherein "selective" describes an enrichment of at least twice that in cancer cells compared to non-cancer cells of the same tissue type ("selective cancer target molecule"). The use of cancer target molecules allows for the selective treatment of cancer cells using the conjugates according to the invention.

Antibodies that specifically target antigens such as cancer cell antigens can be prepared by those skilled in the art using methods they are familiar with (e.g., recombinant expression) or are commercially available (e.g., Merck KGaA from Germany). Examples of commercially available antibodies known in cancer therapy include cetuximab (Merck KGaA), bevacizumab (Roche), and...

(Trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1κ type that binds with high affinity (Kd = 5 nM) to the extracellular domain of the human epidermal growth receptor in cell-based assays. The antibody was prepared by recombinant synthesis in CHO cells.

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 these are listed below.

Examples of cancer target molecules are:

(1) EGFR (EGF receptor, NCBI reference sequence NP_005219.2, NCBI gene ID: 1956)

(2) Mesothelin (SwissProt Reference Q13421-3), in which amino acids 296-598 encode mesothelin. Amino acids 37-286 encode megakaryocyte-enhancing factor. Mesothelin is anchored in the cell membrane via GPI and is located extracellularly.

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

(4) C4.4a (NCBI reference sequence NP_055215.2; synonym LYPD3, NCBI gene ID: 27076)

(5) CD52 (NCBI reference sequence NP_001794.2)

(6) HER2 (ERBB2; NCBI reference sequence NP_004439.2; NCBI gene ID: 2064)

(7) CD20 (NCBI reference sequence NP_068769.2)

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

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

(10) Myeloid 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 αV (NCBI reference sequence: NP_002201.1, NCBI gene ID: 3685)

(19) Teratoma-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 protein 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 protein 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: P24530, 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) CD79b, a protein associated with the B-cell antigen receptor complex (SwissProt: P40259, NCBI gene ID: 974)

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

(37) Dipeptidase DPEP1 (SwissProt: P16444)

(38) Interleukin receptor IL20Rα (SwissProt:Q9UHF4,NCBI gene ID:3559)

(39) BCAN (SwissProt:Q96GW7)

(40) Liver glycoprotein receptor EPHB2 (SwissProt: P29323)

(41) Prostate stem cell-related protein PSCA (NCBI reference sequence: NP_005663.2)

(42) Surface protein LHFPL3 (SwissProt:Q86UP9)

(43) Receptor protein TNFRSF13C (SwissProt:Q96RJ3)

(44) CD79a (SwissProt: P11912), a protein associated with the B-cell antigen receptor complex.

(45) Receptor protein CXCR5 (CD185; 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 symbol: FGFR2). The FGFR2 receptor occurs in different splice variants (α, β, IIIb, IIIc). All splice variants can act as target molecules.

(55) Transmembrane glycoprotein B7H3 (CD276; NCBI gene ID: 80381; NCBI reference sequence: NP_001019907.1; Swiss Prot: Q5ZPR3-1)

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

(57) 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 syncytiocytine (NCBI gene ID 30816)

(60) Aspartate β-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 CEACAM5 (NCBI gene ID: 1048)

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

(65) Receptor tyrosine kinase c-Met (NCBI gene ID: 4233)

(66) Notch ligand DLL3 (NCBI gene ID: 10683)

(67) Liver glycoprotein A4 (EFNA4, NCBI gene ID: 1945)

(68) Extracellular nucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3, NCBI gene ID: 5169)

(69) Coagulation factor III (F3, NCBI gene ID: 2152)

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

(71) 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) Lysosome-associated membrane protein 1 (LAMP1, NCBI gene ID: 3916)

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

(77) NOTCH3 protein (NCBI gene ID: 4854)

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

(79) nectin cell adhesion molecule 4 (PVRL4, NECTIN4, NCBI gene ID: 81607)

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

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

(82) Transporter protein SLC39A6 (NCBI gene ID: 25800)

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

(84) Cell surface receptor TACSTD2 (NCBI gene ID: 4070)

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

(86) Receptor protein TNFSF13B (NCBI gene ID: 10673)

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

(88) Cell surface receptor TROP2 (TACSTD2, NCBI gene ID: 4070)

(89) Glycopropyl peptide-like G protein-coupled receptor KISS1R (GPR54, NCBI gene ID: 84634)

(90) Transporter protein SLAMF6 (NCBI gene ID: 114836).

In the preferred subject matter of the invention, the cancer target molecules are selected from cancer target molecules (1)-(90), especially TWEAKR, B7H3, EGFR and HER2.

In another particularly preferred subject of the invention, the binding portion binds extracellular cancer target molecules selected from cancer target molecules (1)-(90), especially TWEAKR, B7H3, EGFR and HER2.

In another particularly preferred aspect of the invention, the binding moiety specifically binds to extracellular cancer target molecules selected from cancer target molecules (1)-(90), particularly TWEAKR, B7H3, EGFR, and HER2. In a preferred embodiment, after binding to its extracellular target molecule on a target cell, the binding moiety is internalized by the target cell through binding. This results in the uptake of the binding moiety-drug conjugate (which may be an immunoconjugate or an ADC) by the target cell. The binding moiety is then preferably processed intracellularly, preferably via lysosomes.

In one embodiment, the binding moiety is a binding protein. In a preferred embodiment, the binding moiety is an antibody, an antigen-binding antibody fragment, a multispecific antibody, or an antibody mimic.

Preferred antibody mimics are affibodies, adnectins, anticalins, DARPins, avimers, or nanobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies.

In a preferred embodiment, the binding portion is an antibody or an antibody fragment bound to an antigen, more preferably an isolated antibody or an isolated antibody fragment bound to an antigen.

Preferred antigen-binding antibody fragments are Fab, Fab', F(ab')2 and Fv fragments, double-chain antibodies, DAb, linear antibodies and scFv. Fab, double-chain antibodies and scFv are particularly preferred.

In a particularly preferred embodiment, the binding portion is an antibody. Particularly preferred are monoclonal antibodies or antibody fragments thereof bound to their antigens. Further particularly preferred are human, humanized, or chimeric antibodies or antibody fragments thereof bound to their antigens.

Antibody fragments that bind to cancer target molecules or antigens can be prepared by those skilled in the art using known methods, such as chemical synthesis or recombinant expression. The binding portion for cancer target molecules is commercially available or can be prepared by those skilled in the art using known methods, such as chemical synthesis or recombinant expression. Other methods for preparing antibody fragments that bind to antibodies or antigens are described in WO 2007/070538 (see “Antibodies” on page 22). Those skilled in the art know how to compile methods such as phage display libraries (e.g., Morphosys HuCAL Gold) for discovering antibody fragments that bind to antibodies or antigens (see WO 2007/070538, pages 24 and thereafter, and AK Example 1 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 antibody humanization are described on pages 30-32 of WO2007070538 and in detail in Queen, et al., Pros. Natl. Acad. Sci. USA 8610029-10033, 1989 or WO 90/0786. Furthermore, methods for general protein recombinant expression and, in particular, antibody recombinant expression are known to those skilled in the art (see, for example, Berger and Kimrnel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook et al. (Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology (F.M. Ausabel et al. [edited], Current Protocols, Green Publishing Associates, Inc./John Wiley & Sons, Inc.); Harlow et al. (Monoclonal Antibodies A Laboratories) y Manual, Cold Spring Harbor Laboratory Press (1988, Paul [edit]); (Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow et al., (Using Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)). Proteins/antibodies known to those skilled in the art are known to the public. The appropriate vector, promoter, and signal peptide necessary for in vivo expression are described. Common methods are also described on pages 41-45 of WO 2007/070538. Methods for preparing IgG1 antibodies are described, for example, in Example 6 of WO 2007/070538, on page 74 and thereafter. Methods for determining antibody internalization after binding to its antigen are known to those skilled in the art and are described, for example, on page 80 of WO 2007/070538. Those skilled in the art can use the methods described in WO 2007/070538 for preparing carbonic anhydrase IX (Mn) antibodies, similar to the preparation of antibodies with different target molecule specificities.

Bacterial expression

Those skilled in the art know how antibodies, their antigen-binding fragments, or variants thereof can be prepared by means of bacterial expression.

A suitable expression vector for bacterial expression of a desired protein is constructed by inserting a DNA sequence encoding the desired protein, along with appropriate translation initiation and termination signals and a functional promoter, within a functional reading frame. The vector contains one or more phenotypic optional markers and origins of replication to allow for vector preservation and, if desired, amplification within the host. Suitable prokaryotic hosts for transformation include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, and various species from the genera *Pseudomonas*, *Streptomyces*, and *Staphylococcus*. Bacterial vectors can be based on, for example, bacteriophages, plasmids, or phage particles. These vectors may contain optional markers and bacterial origins of replication derived from commercially available plasmids. Many commercially available plasmids typically contain elements of the well-known cloning vector pBR322 (ATCC 37017). In bacterial systems, many advantageous expression vectors can be selected based on the intended use of the protein to be expressed.

After transforming a suitable host strain and allowing it to grow to an appropriate cell density, the selected promoter is de-inhibited/induced using appropriate methods (e.g., temperature variation or chemical induction), and the cells are cultured for an additional period. Cells are typically collected by centrifugation and, if necessary, broken down physically or chemically, with the resulting crude extract retained for further purification.

Therefore, another embodiment of the present invention is an expression vector containing nucleic acid encoding the novel antibody of the present invention.

The antibodies or antigen-binding fragments of the present invention comprise naturally purified products, chemically synthesized products, and products produced in a prokaryotic host via recombinant technology, such as *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, and various species from the genera *Pseudomonas*, *Streptomyces*, and *Staphylococcus*, with *Escherichia coli* being preferred.

mammalian cell expression

Those skilled in the art know how antibodies, their antigen-binding fragments, or variants thereof can be prepared by means of expression in mammalian cells.

Preferred regulatory sequences for expression in mammalian cell hosts include viral elements that lead to high expression in mammalian cells, such as promoters and/or expression amplicones derived from cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), promoters and/or expression amplicones derived from simian virus 40 (SV40) (e.g., SV40 promoter/enhancer), promoters and/or expression amplicones derived from adenovirus (e.g., adenovirus major late promoter (AdMLP)), and promoters and/or expression amplicones derived from polyomas. Antibody expression can be constitutive or regulatory (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, see, for example, Stinski’s U.S. 5,168,062, Bell et al.’s U.S. 4,510,245, and Schaffner et al.’s U.S. 4,968,615. Recombinant expression vectors may also include origins of replication and optional markers (see, for example, U.S. 4,399,216, 4,634,665, and U.S. 5,179,017). Suitable optional markers include genes that confer resistance to substances such as G418, puromycin, hygromycin, blastomycin, zeocin/bleomycin, or methotrexate, or optional markers that cause host cell nutrient deficiencies when the vector is introduced into the host cell, such as glutamine synthase (Bebbington et al., Biotechnology (NY). 1992 Feb; 10(2):169-75).

For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the neo gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blast fungicide, puromycin N-acetyltransferase confers resistance to puromycin, the Shble gene product confers resistance to zeocin, and the Escherichia coli hygromycin resistance gene (hyg or hph) confers resistance to hygromycin. Optional markers such as DHFR or glutamine synthase can also facilitate amplification techniques that bind to MTX and MSX.

Expression vectors can be transfected into host cells using standard techniques, particularly electroporation, nuclear transfection, calcium phosphate precipitation, lipid transfection, and polycation-based transfections, such as polyethyleneimine (PEI)-based transfection and DEAE-dextran transfection.

Suitable mammalian host cells for expressing antibodies, their antigen-binding fragments, or variants thereof include Chinese hamster ovaries (CHO cells), such as CHO-K1, CHO-S, and CHO-K1SV [including DHFR-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun; 33(2):405-12], which are similar to DHFR - Optional markers are used together, such as those described in R.J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621, and other knockout cells, such as those described in detail in Fan et al., Biotechnol Bioeng. 2012 Apr; 109(4):1007-15, NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells and SP2 cells.

Antibodies, their antigen-binding fragments, or variants thereof may also be expressed in the expression system in a transient or semi-stable manner, such as in HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7, or CAP-T cells (e.g., Durocher et al., Nucleic Acids Res. 2002 Jan 15; 30(2):E9).

In some embodiments, the expression vector is constructed by secreting the protein to be expressed into a cell culture medium in which host cells are grown. Antibodies, their antigen-binding fragments, or variants thereof can be obtained from the cell culture medium using protein purification methods known to those skilled in the art.

purification

Antibodies, their antigen-binding fragments, or variants thereof, can be obtained and purified from recombinant cell cultures using known methods, including ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography (HIC), affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997–2001), e.g., Chapters 1, 4, 6, 8, 9, and 10.

The antibodies or antigen-binding fragments or variants thereof of the present invention include naturally purified products, products of chemical synthesis methods, and products prepared in prokaryotic or eukaryotic host cells by means of recombinant technology. Eukaryotic hosts include, for example, yeast cells, higher plant cells, insect cells, and mammalian cells. Depending on the host cell selected for recombinant expression, the expressed protein can be in a glycosylated or non-glycosylated form.

In a preferred embodiment, the antibody is purified to (1) more than 95% by weight, which is measured, for example by the Lowry method, UV-vis spectroscopy or by SDS capillary gel electrophoresis (e.g., using a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer instrument), and in a more preferred embodiment to more than 99% by weight, (2) to a degree suitable for determining at least 15 groups of the N-terminal or internal amino acid sequence, or (3) homogeneity determined by SDS-PAGE under reducing or non-reducing conditions by means of Coomassie Brilliant Blue or preferably silver staining.

Typically, isolated antibodies are obtained by means of at least one protein purification step.

Preferably, the antigen-binding fragment of the antibody according to any of the foregoing embodiments or the antigen-binding fragment of the antibody according to any of the foregoing embodiments is scFv, Fab, Fab fragment or F(ab’)2 fragment.

Preferably, the antibody or antigen-binding fragment according to any of the foregoing embodiments is a monoclonal antibody or its antigen-binding fragment.

Preferably, the antibody or antigen-binding fragment according to any of the foregoing embodiments is a human, humanized, or chimeric antibody or antigen-binding fragment.

Anti-TWEAKR antibody

According to the present invention, anti-TWEAKR antibodies can 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: 164), preferably having sufficient affinity for diagnostic and/or therapeutic applications. In specific embodiments, the antibody binds to TWEAKR with a dissociation constant (K_{_D}) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

Examples of antibodies binding to TWEAKR are disclosed, for example, in WO2009/020933(A2), WO2009/140177(A2), WO 2014/198817(A1) and WO 2015/189143(A1). These antibody-antigen binding fragments can be used in the context of this invention.

ITEM-4 is an anti-TWEAKR antibody described by Nakayama et al. (Nakayama et al., 2003, Biochem Biophy Res Comm, 306:819-825). Humanized variants of this antibody based on CDR grafting are described by Zhou et al. (Zhou et al., 2013, J Invest Dermatol. 133(4):1052-62) and WO 2009/020933. Humanized variants of ITEM-4 are TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, and TPP-10337. These antibody-antigen binding fragments are used in the context of this invention.

In the context of this invention, anti-TWEAKR antibodies TPP-2090, TPP-2658, TPP-5442, TPP-8825, TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, and TPP-10337 are preferred. More preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, and TPP-10337. Particularly preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007, and TPP-10337.

Anti-B7H3 antibody

According to the present invention, anti-B7H3 antibody can be used.

The term "anti-B7H3 antibody" or "antibody that specifically binds to B7H3" refers to an antibody that binds to the cancer target molecule B7H3 (NCBI reference sequence: NP_001019907.1, SEQ ID NO: 165), preferably having sufficient affinity for diagnostic and/or therapeutic applications. In specific embodiments, the antibody binds to B7H3 with a dissociation constant (K_{_D} ) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

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

These antibody and antigen-binding fragments can be used in the context of this invention.

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

In the context of this invention, anti-B7H3 antibodies TPP-8382 and TPP-8567 are preferred.

Anti-HER2 antibody:

According to the present invention, anti-HER2 antibodies can be used.

The term "anti-HER2 antibody" or "antibody that specifically binds to HER2" refers to an antibody that binds to the cancer target molecule HER2 (NCBI reference sequence: NP_004439.2, SEQ ID NO: 166), preferably having sufficient affinity for diagnostic and/or therapeutic applications. In specific embodiments, the antibody binds to HER2 with a dissociation constant (K_{_D}) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

An example of an antibody that binds to the cancer target molecule HER2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody, particularly used to treat breast cancer. In a particularly preferred embodiment, the anti-HER2 antibody is TPP-1015 (a trastuzumab analog).

Besides trastuzumab (INN 7637, CAS No.: RN: 180288-69-1) and pertuzumab (CAS No.: 380610-27-5), other examples of antibodies binding to HER2 include those disclosed in WO 2009/123894-A2, WO 200/8140603-A2, or WO 2011/044368-A2. An example of an anti-HER2 conjugate is trastuzumab-emtansine (INN No. 9295). These antibodies and antigen-binding fragments are applicable in the context of this invention.

In the context of this invention, anti-HER2 antibodies trastuzumab and TPP-1015 are particularly preferred.

Anti-EGFR antibody

According to the present invention, anti-EGFR antibodies can 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: 167), preferably having sufficient affinity for diagnostic and/or therapeutic applications. In specific embodiments, the antibody binds to EGFR with a dissociation constant (K_{_D}) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤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, and nimotuzumab. In a particularly preferred embodiment, the anti-EGFR antibody is TPP-981 (cetuximab).

Other implementation schemes for EGFR antibodies are as follows:

• Zalumumab/2F8/HuMax-EGFr, from Genmab A/S (WO 02/100348, WO 2004/056847, INN No. 8605)

• Nexituzumab/11F8, ImClone/IMC-11F8, from ImClone Systems Inc. [EliLilly & Co] (WO 2005/090407(EP 01735348-A1,US 2007/0264253-A1,US 7,598,350,WO2005/090407-A1), INN No. 9083)

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

·RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/RO4858696/RO50839 45, from Glycart Biotechnology AG (Roche Holding AG) (WO 2010/112413-A1,WO 2010/115554)

·GT-MAB 5.2-GEX/CetuGEX, from Glycotope GmbH (WO 2008/028686-A2(EP01900750-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 (composed of two chimeric IgG1 antibodies (992 and 1024)) is derived from Symptomogen A/S (WO2010/022736-A2).

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

• Antibody against deletion mutation, 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/H 447/MAb,EGFR,Medarex/Merck KgaA, from Bristol-Myers Squibb(US)/Merck KGaA(DE)/Takeda(JP), (WO 91/05871,WO 92/15683)

• Anti-EGFR-Mab, from Xencor (WO 2005/056606)

• DXL-1218 / Anti-EGFR Monoclonal Antibody (Cancer), InNexus, from InNexus Biotechnology Inc., Pharmaprojects PH048638.

Anti-carbonic anhydrase IX antibody

Examples of antibodies that bind to the cancer target molecule carbonic anhydrase IX are described in WO 2007/070538-A2 (e.g., claims 1-16).

Anti-CD123 antibody

The term "anti-CD123 antibody" or "antibody that specifically binds to CD123" refers to an antibody that binds to the cancer target molecule CD123 (NCBI reference sequence: NP_002174.1; Swiss-Prot: P26951), preferably having sufficient affinity for diagnostic and/or therapeutic applications. In specific embodiments, the antibody binds to CD123 with a dissociation constant (K_{_D}) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

Sun et al. (Sun et al., 1996, Blood 87(1)83-92) described the generation and properties of the monoclonal antibody 7G3, which binds to the N-terminal region of IL-3Rα and CD123. U.S. Patent No. 6,177,078 (Lopez) relates to the anti-CD123 antibody 7G3. A chimeric variant of this antibody (CSL360) is described in WO 2009/070844, and a humanized form (CSL362) is described in WO2012/021934. The sequence of the 7G3 antibody is disclosed in EP2426148. This sequence forms the starting point for humanized antibodies obtained via CDR transplantation.

The antibody that is particularly well internalized after binding to cell surface antigens is the anti-CD123 antibody 12F1, disclosed by Kuo et al. (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82). Antibody 12F1 binds to CD123 with a higher affinity than antibody 7G3, and its internalization is significantly faster than that of 7G3 after binding to cell surface antigens. A bispecific scFv immunofusion protein based on 12F1 is disclosed in WO 2013/173820. Antibody TPP-6013 is a chimeric variant of 12F1.

Humanized variants of these mouse antibodies were generated based on CDR transplantation and optimization in germline sequences.

Anti-CXCR5 antibody

The term "anti-CXCR5 antibody" or "antibody that specifically binds to CXCR5" refers to an antibody that binds to the cancer target molecule CXCR5 (NCBI reference sequence: NP_001707.1), preferably having sufficient affinity for diagnostic and/or therapeutic applications. In specific embodiments, the antibody binds to CXCR5 with a dissociation constant (K_{_D}) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

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

Hybridoma cells used for the rat antibody RF8B2 (ACC2153) were purchased from DSMZ, and the antibody sequence was identified using standard methods. This sequence constitutes the starting point for the humanized antibody obtained via CDR transplantation.

Humanized variants of the antibody were generated based on CDR transplantation in the germline sequence.

Anti-C4.4a antibody:

Examples of C4.4a antibody-antigen binding fragments are described in WO 2012/143499A2. Antibody sequences are given in Table 1 of WO 2012/143499A2, with each row showing the respective CDR amino acid sequence of the variable light chain or variable heavy chain of the antibody listed in column 1.

Anti-CD20 antibody:

An example of an antibody that binds to the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS No.: 174722-31-7) is a chimeric antibody used to treat non-Hodgkin's lymphoma. These antibodies and their antigen-binding fragments are used in the context of this invention.

Anti-CD52 antibody:

An example of an antibody that binds to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS No.: 216503-57-0) is a humanized antibody used to treat chronic lymphocytic leukemia. These antibodies and their antigen-binding fragments are used in the context of this invention.

Anti-mesothelin antibody:

Examples of anti-mesothelin antibodies are described, for example, in WO 2009/068204. All antibodies and antigen-binding fragments disclosed in WO 2009/068204 can be used in the context of the invention disclosed herein. Particularly preferred are the antibodies disclosed in WO 2009/068204, which are MF-T antibodies.

Anti-CD30 antibody

Examples of antibodies that bind to the cancer target molecule CD30 and can be used to treat cancers such as Hodgkin's lymphoma are bentuximab, itolumab, and antibodies disclosed in WO 2008/092117, WO 2008/036688, or WO 2006/089232. An example of an anti-CD30 conjugate is bentuximab-vitoltin (INN 9144). These antibodies and their antigen-binding fragments are used in the context of this invention.

Anti-CD22 antibody

Examples of antibodies that bind to the cancer target molecule CD22 and can be used to treat cancers such as lymphoma are inotuzumab or epazolizumab. Examples of anti-CD22 conjugates are inotuzumab-ozogamycin (INN 8574) or anti-CD22-MMAE and anti-CD22-MC-MMAE (CAS numbers: 139504-50-0 and 474645-27-7, respectively). These antibodies and their antigen-binding fragments are used in the context of this invention.

Anti-CD33 antibody

Examples of antibodies that bind to the cancer target molecule CD33 and can be used to treat cancers such as leukemia are gemtuzumab and lintuzumab (INN 7580). An example of an anti-CD33 conjugate is gemtuzumab-orzomicin. These antibodies and antigen-binding fragments are used in the context of this invention.

Anti-NMB antibody

An example of an antibody that binds to the cancer target molecule NMB and can be used to treat cancers such as melanoma or breast cancer is glemumab (INN 9199). An example of an anti-NMB conjugate is glemumab-vitoline (CAS No.: 474645-27-7). These antibodies and their antigen-binding fragments are used in the context of this invention.

Anti-CD56 antibody

An example of an antibody that binds to the cancer target molecule CD56 and can be used to treat cancers such as multiple myeloma, small cell lung cancer, MCC, or ovarian cancer is lotuzumab. An example of an anti-CD56 conjugate is lotuzumab-mertansine (CAS No.: 139504-50-0). These antibodies and antigen-binding fragments are used in the context of this invention.

Anti-CD70 antibody

Examples of antibodies that bind to the cancer target molecule CD70 and can be used to treat cancers such as non-Hodgkin's lymphoma or renal cell carcinoma are disclosed in WO 2007/038637-A2 and WO 2008/070593-A2. An example of an anti-CD70 conjugate is SGN-75 (CD70 MMAF). These antibodies and antigen-binding fragments are applicable in the context of this invention.

Anti-CD74 antibody

An example of an antibody that binds to the cancer target molecule CD74 and can be used to treat cancers such as multiple myeloma is mirtuzumab. An example of an anti-CD74 conjugate is mirtuzumab-doxorubicin (CAS No.: 23214-92-8). These antibodies and antigen-binding fragments are used in the context of this invention.

Anti-CD19 antibody

Examples of antibodies that bind to the cancer target molecule CD19 and can be used to treat cancers such as non-Hodgkin's lymphoma are disclosed in WO 2008/031056-A2. Examples of other antibodies and anti-CD19 conjugates (SAR3419) are disclosed in WO 2008/047242-A2. These antibodies and their antigen-binding fragments are applicable in the context of this invention.

Anti-mucin antibodies

Examples of antibodies that bind to the cancer target molecule mucin-1 and can be used to treat cancers such as non-Hodgkin's lymphoma are clevipodumab and the antibodies disclosed in WO 2003/106495-A2 and WO 2008/028686-A2. Examples of anti-mucin conjugates are disclosed in WO 2005/009369-A2. These antibodies and their antigen-binding fragments are useful in the context of this invention.

Anti-CD138 antibody

Examples of antibodies that bind to the cancer target molecule CD138 and its conjugates and can be used to treat cancers such as multiple myeloma are disclosed in WO 2009/080829-A1 and WO 2009/080830-A1. These antibodies and their antigen-binding fragments are applicable in the context of this invention.

Anti-integrin-αV antibody

Examples of antibodies that bind to the cancer target molecule integrin αV and can be used to treat cancers such as melanoma, sarcoma, or carcinoma are integrinumab (CAS No.: 725735-28-4), abciximab (CAS No.: 143653-53-6), edazolizumab (CAS No.: 892553-42-3), and the antibodies disclosed in US 7,465,449, EP 719859-A1, WO 2002/012501-A1, or WO2006/062779-A2. Examples of anti-integrin αV conjugates are integrinumab-DM4 and other ADCs disclosed in WO 2007/024536-A2. These antibodies and their antigen-binding fragments are used in the context of this invention.

Anti-TDGF1 antibody

Examples of antibodies that bind to the cancer target molecule TDGF1 and can be used to treat cancer are those disclosed in WO 02/077033-A1, US7,318,924, WO 2003/083041-A2, and WO 2002/088170-A2. Examples of anti-TDGF1 conjugates are disclosed in WO 2002/088170-A2. These antibodies and their antigen-binding fragments are useful in the context of this invention.

Anti-PSMA antibody

Examples of antibodies that bind to the cancer target molecule PSMA and can be used to treat cancers such as prostate cancer are those disclosed in WO 97/35616-A1, WO 99/47554-A1, WO 01/009192-A1, and WO2003/034903. Examples of anti-PSMA conjugates are disclosed in WO 2009/026274-A1 and WO 2007/002222. These antibodies and their antigen-binding fragments are applicable in the context of this invention.

Anti-EPHA2 antibody

Examples of antibodies that bind to the cancer target molecule EPHA2 and can be used to prepare conjugates for cancer treatment are disclosed in WO2004/091375-A2. These antibodies and their antigen-binding fragments are applicable in the context of this invention.

Anti-SLC44A4 antibody

Examples of antibodies that bind to the cancer target molecule SLC44A4 and can be used to prepare conjugates for the treatment of cancers such as pancreatic cancer or prostate cancer are disclosed in WO2009/033094-A2 and US2009/0175796-A1. These antibodies and their antigen-binding fragments are applicable in the context of this invention.

Anti-HLA-DOB antibody

An example of an antibody that binds to the cancer target molecule HLA-DOB is antibody Lym-1 (CAS No.: 301344-99-0), which can be used to treat cancers such as non-Hodgkin's lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, for example, in WO 2005/081711-A2. These antibodies and their antigen-binding fragments are useful in the context of this invention.

Anti-VTCN1 antibody

Examples of antibodies that bind to the cancer target molecule VTCN1 and can be used to prepare conjugates for the treatment of cancers such as ovarian cancer, prostate cancer, lung cancer, or breast cancer are disclosed in WO 2006/074418-A2. These antibodies and their antigen-binding fragments are applicable in the context of this invention.

Anti-FGFR2 antibody

Examples of anti-FGFR2 antibodies and antigen-binding fragments are described in WO2013076186. The sequences of the antibodies are shown in Tables 9 and 10 of WO2013076186. Preferred antibodies, antigen-binding fragments, and antibody variants derived from antibodies designated M048-D01 and M047-D08 are preferred.

Preferred antibody fragments for binding to antigens in the binding moiety-drug conjugate according to the present invention.

In this application, in the context of binding partial-drug conjugates, reference is made to the following preferred antibodies as shown in the table below: TPP-2090, TPP-2658, TPP-5442, TPP-8825, TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, TPP-10337, TPP-1015, TPP-7510, TPP-7511, TPP-8382, and TPP-8567.

Table: Protein sequences of antibodies:

TPP-2090, TPP-2658, TPP-5442, TPP-8825, TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, TPP-10337, TPP-1015, TPP-7510, TPP-7511, TPP-8382, and TPP-8567 are antibodies containing one or more of the CDR sequences (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) of the heavy chain variable region (VH) or light chain variable region (VL) shown in the table above. Preferably, the antibody contains the variable region (VH) of the heavy chain and/or the variable region (VL) of the light chain shown. Preferably, the antibody comprises the indicated region of the heavy chain (IgG heavy chain) and/or the indicated region of the light chain (IgG light chain).

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

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

TPP-2090 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:22, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:23, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:24. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:26, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:27, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:28.

TPP-2658 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:32, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:33, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:34. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:36, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:37, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:38.

TPP-5442 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:42, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:43, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:44. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:46, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:47, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:48.

TPP-7006 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:52, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:53, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:54. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:56, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:57, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:58.

TPP-7007 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:62, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:63, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:64. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:66, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:67, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:68.

TPP-7510 is an anti-HER2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:72, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:73, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:74. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:76, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:77, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:78.

TPP-7511 is an anti-HER2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:82, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:83, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:84. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:86, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:87, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:88.

TPP-8382 is an anti-B7H3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:92, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:93, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:94. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:96, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:97, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:98.

TPP-8567 is an anti-B7H3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:102, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:103, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:104. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:106, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:107, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:108.

TPP-8825 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:112, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:113, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:114. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:116, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:117, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:118.

TPP-10334 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:122, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:123, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:124. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:126, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:127, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:128.

TPP-10335 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:132, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:133, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:134. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:136, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:137, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:138.

TPP-10336 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:142, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:143, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:144. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:146, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:147, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:148.

TPP-10337 is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) contains a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:152, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:153, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:154. The light chain variable region (VL) contains a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:156, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:157, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:158.

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

TPP-1015 is an anti-HER2 antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:11 and a light chain variable region (VL) as shown in SEQ ID NO:15.

TPP-2090 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:21 and a light chain variable region (VL) as shown in SEQ ID NO:25.

TPP-2658 is an anti-TWEAKR antibody, which preferably contains 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-5442 is an anti-TWEAKR antibody, which preferably contains 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-7006 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:51 and a light chain variable region (VL) as shown in SEQ ID NO:55.

TPP-7007 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:61 and a light chain variable region (VL) as shown in SEQ ID NO:65.

TPP-7510 is an anti-HER2 antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:71 and a light chain variable region (VL) as shown in SEQ ID NO:75.

TPP-7511 is an anti-HER2 antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:81 and a light chain variable region (VL) as shown in SEQ ID NO:85.

TPP-8382 is an anti-B7H3 antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:91 and a light chain variable region (VL) as shown in SEQ ID NO:95.

TPP-8567 is an anti-B7H3 antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:101 and a light chain variable region (VL) as shown in SEQ ID NO:105.

TPP-8825 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:111 and a light chain variable region (VL) as shown in SEQ ID NO:115.

TPP-10334 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:121 and a light chain variable region (VL) as shown in SEQ ID NO:125.

TPP-10335 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:131 and a light chain variable region (VL) as shown in SEQ ID NO:135.

TPP-10336 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:141 and a light chain variable region (VL) as shown in SEQ ID NO:145.

TPP-10337 is an anti-TWEAKR antibody, which preferably contains a heavy chain variable region (VH) as shown in SEQ ID NO:151 and a light chain variable region (VL) as shown in SEQ ID NO:155.

TPP-981 is an anti-EGFR antibody, which preferably contains a heavy chain region as in SEQ ID NO:9 and a light chain region as in SEQ ID NO:10.

TPP-1015 is an anti-HER2 antibody, which preferably contains a heavy chain region as in SEQ ID NO:19 and a light chain region as in SEQ ID NO:20.

TPP-2090 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:29 and a light chain region as in SEQ ID NO:30.

TPP-2658 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:39 and a light chain region as in SEQ ID NO:40.

TPP-5442 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:49 and a light chain region as in SEQ ID NO:50.

TPP-7006 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:59 and a light chain region as in SEQ ID NO:60.

TPP-7007 is an anti-TWEAKR antibody, which preferably contains a heavy chain region such as SEQ ID NO:69 and a light chain region such as SEQ ID NO:70.

TPP-7510 is an anti-HER2 antibody, which preferably contains a heavy chain region as in SEQ ID NO:79 and a light chain region as in SEQ ID NO:80.

TPP-7511 is an anti-HER2 antibody, which preferably contains a heavy chain region as in SEQ ID NO:89 and a light chain region as in SEQ ID NO:90.

TPP-8382 is an anti-B7H3 antibody, which preferably contains a heavy chain region such as SEQ ID NO:99 and a light chain region such as SEQ ID NO:100.

TPP-8567 is an anti-B7H3 antibody, which preferably contains a heavy chain region such as SEQ ID NO:109 and a light chain region such as SEQ ID NO:110.

TPP-8825 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:119 and a light chain region as in SEQ ID NO:120.

TPP-10334 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:129 and a light chain region as in SEQ ID NO:130.

TPP-10335 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:139 and a light chain region as in SEQ ID NO:140.

TPP-10336 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:149 and a light chain region as in SEQ ID NO:150.

TPP-10337 is an anti-TWEAKR antibody, which preferably contains a heavy chain region as in SEQ ID NO:159 and a light chain region as in SEQ ID NO:160.

LIG joint connectors ( _Lb and _Lc )

This document discloses various options for the covalent coupling (conjugation) of organic molecules with peptides or proteins such as antibodies (see, for example, K. Lang and J.W. Chin. Chem. Rev. 2014, 114, 4764-4806, M. Rashidian et al. Bioconjugate Chem. 2013, 24, 1277-1294). According to the invention, organic groups are preferably conjugated to antibodies via one or more sulfur atoms of cysteine residues and/or via one or more NH groups of lysine residues, said sulfur atoms being present as free thiols or generated via reduced disulfide bridges. However, KSP inhibitors or prodrugs can also be bound to antibodies via tyrosine residues, glutamine residues, residues of non-natural amino acids, free carboxyl groups, or sugar residues of antibodies.

According to the present invention, drug molecules can also be conjugated to specific conjugation sites of the binding moiety, which improves product homogeneity. Various methods for site-specific conjugation are described in the literature (Agarwal et al., Bioconjug. Chem. 26 , 176-192 (2015); Cal et al., Angew. Chem. Int. Ed. Engl. 53 , 10585-10587 (2014); Behrens et al., MAbs 6 , 46-53 (2014); Panowski et al., MAbs 6 , 34-45 (2014)). In particular, these methods also include enzymatic conjugation methods using, for example, transglutaminases (TGases), glycosyltransferases, or formylglycine synthases (Sochaj et al., Biotechnology Advances 33 , 775-784, (2015)).

According to the present invention, a conjugate specific binding moiety of kinesin spindle protein inhibitor can be provided, wherein the kinesin spindle protein inhibitor is conjugated to the glutamine side chain of the binding moiety.

When the binding site is an antibody, it contains the receptor glutamine, preferably in the constant region. This receptor glutamine can be introduced into glutamine via mutation at an appropriate position (e.g., the N297Q mutation in the heavy chain, Kabat EU number), or by generating deglycosylated or non-glycosylated antibodies (e.g., by enzymatic deglycosylation using PNGase F or by the N297X mutation in the heavy chain, Kabat EU number (where X can be any amino acid other than N)). In the case of the latter deglycosylated or non-glycosylated antibody, the glutamine group Q295 (Kabat EU number) in the heavy chain becomes the receptor glutamine. Antibodies containing the N297A or N297Q mutation (Kabat EU number) are particularly preferred. Therefore, all antibodies described herein also include non-glycosylated variants of these antibodies, prepared by deglycosylation of PNGase F or by an N297 mutation of the heavy chain (Kabat EU number) (Kabat numbering system for antibodies, see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) to any amino acid other than N. Furthermore, all antibodies described herein also contain variants of the antibodies that, due to engineering, contain one or more receptor glutamine residues for transglutaminase-catalyzed reactions.

One method for this type of conjugation site-specific conjugation is described in the literature, which involves conjugation site-specific conjugation of the binding moiety by means of transglutaminase. Transglutaminases (TGases), including bacterial transglutaminase (BTG) (EC2.3.2.13), are a family of enzymes that catalyze the formation of a covalent bond between the γ-carbonyl-amide group of glutamine and the primary amine group of lysine. Because these transglutaminases also accept substrates other than lysine as amine donors, they have been used to modify proteins, including antibodies, at suitable receptor glutamine (Jeger et al., Angewandte Chemie Int. Ed. Engl 49 , 9995-9997 (2010); Josten et al., J. Immunol. Methods 240 , 47-54 (2000); Mindt et al., Bioconjugate Chem. 19 , 271-278 (2008); Dennler et al., Antibody Drug Conjuagtes (Ducry, L., Ed.), pp 205-215, Humana Press. (2013)). On the one hand, transglutaminase has been used to conjugate drugs to antibodies containing artificial glutamine tags, which are introduced into the antibody via genetic engineering to include receptor glutamine residues (Strop et al., Chem. Biol. 20 , 161-167 (2013)). On the other hand, the conserved glutamine residue Q295 (Kabat EU number) in the constant region of the antibody heavy chain has been described as the sole γ-carbonyl-amide donor for bacterial transglutaminase (EC2.3.2.13) in the non-glycosylated IgG1 molecular backbone, and therefore a receptor glutamine, whereas receptor glutamine is absent in the IgG1 backbone when the antibody is glycosylated at the N297 position (Kabat EU number) of the heavy chain (Jeger et al., Angewandte Chemie Int. Ed. Engl 49 , 9995-9997 (2010)). In summary, bacterial transglutaminase can be used to conjugate amine donor substrates, such as drug-proximity constructs, at the receptor glutamine residues of antibodies. Antibodies can be designed to introduce this receptor glutamine by mutation or by generating non-glycosylated antibodies. Such non-glycosylated antibodies can be introduced by deglycosylation of peptidyl N-glycosidase F (PNGase F) or by mutating the N297 site of the heavy chain glycosylation site (Kabat EU number) to any amino acid other than N. Enzymatic conjugation of such non-glycosylated antibodies using bacterial transglutaminase has been described for use with non-glycosylated antibody variants containing mutant N297D, N297Q (Jeger et al., Angewandte Chemie Int. Ed. Engl 49 , 9995-9997 (2010)) or N297S (see patent applications WO2013092998A1 and WO2013092983A2). Enzymatic conjugation of non-glycosylated antibodies using transglutaminase typically yields an ADC with a DAR of 2, where both heavy chains are specifically functionalized at the Q295 position (Kabat EU number). The mutation N297Q in the heavy chain alone provides an additional conjugation site for each heavy chain. This variant conjugation produces an ADC with a DAR of 4, where both heavy chains are specifically functionalized at the Q295 and Q297 positions. Antibody variants with mutations Q295N and N297Q in the heavy chains have only one receptor glutamine residue at the Q297 position (Kabat number) of each heavy chain (Simone Jeger, Site-specific conjugation of tumor-targeting antibodies using transglutaminase, ETH Zurich dissertation (2009)). Several examples exist in the literature describing site-specific conjugation of non-glycosylated antibodies against bacterial transglutaminase (e.g., Dennler et al., Bioconjugate Chemistry 19 , 569-578 (2014); Lhospice et al., Molecular Pharmaceutics 12 , 1863-1871 (2015)). Strategies for site-specific functionalization of transglutaminase-catalyzed non-glycosylated antibodies are summarized in Figure 1.

Coupling—both in a site-specific and site-nonspecific manner—is accomplished using so-called linkers. Linkers can be classified into a group of linkers that can be cleaved in vivo and a group of linkers that are stable in vivo (see L. Ducry and B. Stump, Bioconjugate Chem. 21 , 5-13 (2010)). Linkers that can be cleaved in vivo have groups that can be cleaved in vivo, which can be further distinguished between groups that are chemically cleavable in vivo and groups that are enzymatically cleavable in vivo. “Chemically cleavable in vivo” and “enzymatically cleavable in vivo” refer to linkers or groups that are stable in circulation and are cleaved at or within target cells by different chemical or enzymatic environments (lower pH; elevated glutathione concentration; presence of lysosomal enzymes such as cathepsins or plasminogen lysins, or glycosidases such as β-glucuronidase), thereby releasing low molecular weight KSP inhibitors or their derivatives. Groups that can be chemically cleaved in vivo are particularly disulfides, hydrazones, acetals, and acetalamines; groups that are enzymatically cleavable in vivo are particularly 2-8-oligopeptide groups, especially dipeptide groups or glycosides. Peptide cleavage sites are disclosed in Bioconjugate Chem. 2002, 13, 855-869, Bioorganic & Medicinal Chemistry Letters 8 (1998) 3341-3346, and Bioconjugate Chem. 1998, 9, 618-626. These include, for example, alanine-alanine-asparagine, valine-alanine, valine-lysine, valine-citrulline, alanine-lysine, and phenylalanine-lysine (optionally having additional amide groups).

To ensure the effective release of free drug, a so-called self-destructing linker element (SIG) may optionally be introduced between the enzymatic cleavage site and the drug (Anticancer Agents in Medicinal Chemistry, 2008, 8, 618-637). Drugs can be released through various mechanisms, such as subsequent elimination via an electron cascade after initial enzymatic release of the nucleophilic group (Bioorg. Med. Chem., 1999, 7, 1597; J. Med. Chem., 2002, 45, 937; Bioorg. Med. Chem., 2002, 10, 71), or through cyclization of the corresponding linker element (Bioorg. Med. Chem., 2003, 11, 2277; Bioorg. Med. Chem., 2007, 15, 4973; Bioorg. Med. Chem. Lett., 2007, 17, 2241), or through a combination of both (Angew. Chem. Inter. Ed., 2005, 44, 4378). Examples of such linker elements are shown in the figure below:

Examples of a series of enzymatic steps for drug release using histone deacetylase and cathepsin L are described in Nat. Commun., 2013, 4, 2735 and illustrated in Figures 2A-2Q.

In vivo stable linkers are characterized by high stability (less than 5% metabolites after 24 hours in plasma) and the absence of the aforementioned chemically or enzymatically cleavable groups in vivo.

The connector _-Lb- or _-Lc- preferably has one of the following basic structures (i) and (ii):

(i)–(C＝O) _m –(L1) _n -(L2) _n -

(ii)–(C＝O) _m –L1-SG-L2-

Where m and n are 0 or 1; SG is a (chemically or enzymatically) cleavable group in vivo (especially disulfides, hydrazones, acetals, and acetalamines; or 2-8-oligopeptide groups cleavable by podases, cathepsins, or plasmins); L1 represents a stable organic group in vivo; and L2 represents a coupling group or single bond to the binding site. Here, coupling is preferably performed with cysteine or lysine residues of the antibody. Alternatively, coupling can be performed with tyrosine residues, glutamine residues, or non-natural amino acids of the antibody. Non-natural amino acids may contain, for example, aldehyde or ketone groups (e.g., formylglycine), or azido or alkynyl groups (see Lan & Chin, Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins, Chem. Rev. 2014, 114, 4764-4806).

According to the present invention, the linker base structure (i) is particularly preferred. Through metabolism, administration of the conjugate of the present invention having the linker base structure (i) according to embodiment B, and coupling of the linker with cysteine or lysine residues of the antibody, produces a cysteine or lysine derivative of the following formula:

In each case, L1 is linked to a cytotoxic drug, such as a low molecular weight KSP inhibitor, for example, a compound of formula (IIa), (IIb), (IIc), (IId), (V), (VI), or (VII).

In embodiment A, the conjugate of the present invention having a connector basic structure (i) is given, and after metabolism, a KSP inhibitor is obtained, which preferably has the structure of a compound of the general formula (IIa), (IIa’), (IIa”), (IIb), (IIc), (IId), (V), (VI) or (VII).

According to the invention, the connector base structure (ii) is also preferred, especially when it is bonded to the R1 site in compounds of formula (IIa), (IIb), (IIc), (IId) or (V), particularly when _L1 has one of the following structures:

(a)–NH-( _CH₂ ) _O₄- ( _CHCH₃ ) _O₄ - ^CHY₅ -C(＝O)-Y₇, where

Y ⁵ is either -H or -NHY ⁶ .

^Y6 is -H or –C(=O) _-CH3 , and

^Y7 is a single bond or –NH-( _CH2 ) _{O- 4} –CHNH2-C(＝O)-.

This allows for the obtaining of the corresponding structure after pyrolysis in implementation scheme B.

–NH-( _CH₂ ) _O₄- ( _CHCH₃ ) _O₄ - ^CHY₅ -COOH or

–NH-(CH ₂ ) _0-4- (CHCH ₃ ) _0-4 -CHY ⁵ -C(=O)-NH-(CH ₂ ) _0-4- CHNH ₂ -COOH.

(b) _–CH₂ - _Sx- ( _CH₂ ) _₀-₄ - ^CHY₅ -C(＝O)-, where

x is 0 or 1.

Y ⁵ is -H or -NHY ⁶ , and

^Y6 is -H or –C(=O) _-CH3 ,

This allows for the obtaining of the corresponding structure after pyrolysis in implementation scheme B.

-CH ₂ -S _x -(CH ₂ ) _0-4 -CHY ⁵ -COOH.

In implementation scheme A, the conjugate of the present invention having a connector basic structure (ii) is given, and after metabolism, a KSP inhibitor having a structure of general formula (II) is obtained.

When the linker is attached to a cysteine side chain or cysteine group, L2 is preferably derived from a group that reacts with the thiol group of cysteine. These include haloacetyl groups, maleimides, aziridines, acryloyl compounds, arylated compounds, vinyl sulfones, pyridyl disulfides, TNB thiols, and disulfide reducing agents. These groups typically react with the thiol bond electrophilically, forming a sulfur bridge (e.g., a thioether) or a disulfide bridge. Stable sulfur bridges are preferred.

L2 preferably has the following structure:

in

# ¹ is the junction site with the sulfur atom of the antibody.

# ² is the connection point with _L1 , and

R ₂₂ is -COOH, -C(=O)-OR, -C(=O)R, -C(=O)-NHR or -

C(＝O)N(R) ₂ , and

R is a _C1-3 -alkyl group.

When R ₂₂ is –COOH, it is preferred here.

Particularly preferred are the compounds of the present invention wherein L2 has the following formulas A3 and A4:

in

# ¹ is the junction site with the sulfur atom of the antibody.

# ² is the binding site with the drug molecule.

x is 1 or 2, and

R ²² is –COOH, -C(＝O)-OR, -C(＝O)-R, -C(＝O)-NR ₂ , -C(＝O)-NHR or -C(＝O)-NH ₂ , and

R is a _C1-3 -alkyl group.

Preferably, in this context, R ²² is –COOH, and in particular, in this context, if x is 1, then R ²² is –COOH.

In the compounds of the present invention, the linker Lb or Lc may be bonded to the cysteine side chain or cysteine residue in the binding moiety, and in this case has the following formula:

(i)–(C＝O) _m –(L1) _n -(L2) _n -

and

(ii)–(C＝O) _m –L1-SG-L2

in

m and n are independently 0 or 1.

SG is a chemically or enzymatically cleavable group in vivo (especially disulfides, hydrazones, acetals, and acetalamines; or 2-8-oligopeptide groups cleavable by podases, cathepsins, or plasmins).

L2 is a single bond or one of the following groups:

in

# ¹ indicates the bonding site with the sulfur atom in the bonded portion.

# ² indicates the bonding point with L1.

L1 is –(NR10) _n- (G1) _o- G2-,

R10 is –H or _C1 - _C3 -alkyl.

G1 is –NH-C(＝O)-, -C(＝O)-NH-, or –(CH ₂ ) _O-3 -C(＝O)-NH-.

n is 0 or 1,

o is 0 or 1.

G2 is a straight or branched hydrocarbon chain with 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(＝O)-, -S(＝O) ₂ , -NH-, -C(＝O)-, -CH(COOH)-, -CH( _CH2 -C(＝O) _-NH2 ), -NMe-, -NHNH-, -S(＝O) _2- NHNH-, -NHC(＝O)-, -C(＝O)-NH-, -C(＝O)-NHNH-, having up to 4 heteroatoms selected from N, O and S, 5- to 10-membered aromatic or non-aromatic heterocycles of -S(＝O)- or -S(＝O) _2- , and wherein the side chains of the branched carbon chain may be separated by –NH-C(＝O) _-NH2 , -COOH, -OH, -NH _2. -NH-CNNH _2. Substituted with sulfonamide, sulfone, sulfoxide, or sulfonic acid, or representing one of the following groups:

in

Rx is -H, C1-C3-alkyl, or phenyl.

Preferably, the present invention relates to compounds wherein the linker Lb or Lc is bonded to the cysteine side chain or cysteine residue of the binding moiety and has the following formula:

§-(C＝O) _m -L1-(L2) _n -§§

in

m is 0 or 1.

n is 0 or 1,

§ is the bond that links the drug molecule or the cleavable group of the podase protease of formula (Ia’), and

§§ represents the key connecting the joined parts.

-L2 is one of the following groups

Or is a key, in which

# ¹ indicates the bonding site with the sulfur atom in the bonded portion, acid.

# ² indicates the bonding point with L1.

L1 is –(NR10) _n- (G1) _o- G2-,

R10 is –H or _C1 - _C3 -alkyl.

G1 is –NH-C(＝O)-, -C(＝O)-NH-, or –(CH ₂ ) _O-3 -C(＝O)-NH-.

n is 0 or 1,

o is 0 or 1.

G2 is a straight or branched hydrocarbon chain with 1-100 carbon atoms, which may be selected from the following groups spaced once or from the same or different groups spaced more than once: –O-, -S-, -S(＝O)-, -S(＝O) ₂ , -NH-, -C(＝O)-, -CH(COOH)-, -CH( _CH2 -C(＝O) _-NH2 ), -NMe-, -NHNH-, -

S(=O) ₂ -NHNH-, -NH-C(=O)-, -C(=O)-NH-, -C(=O)-NHNH-

It has up to four heteroatoms selected from N, O, and S, and 5- to 10-membered aromatic or non-aromatic heterocycles of type -S(=O)- or -S(=O) _2- , wherein the side chains of the branched carbon chain can be substituted with –NH-C(=O) _-NH2 , -COOH, -OH, _-NH2 , -NH-

CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid substitution, or representing one of the following groups:

in

Rx is -H, C1-C3-alkyl, or phenyl.

In the compounds of the present invention, the linker Lb or Lc may also be bonded to the cysteine side chain or cysteine residue of the binding site, and in this case has the universal basic structures (i) and (ii).

(i)–(C＝O) _m –(L1) _n -(L4) _n -(C＝O)-§§

and

(ii)–(C＝O) _m –L1-SG-L4-(C＝O)-§§

in

m and n are independently 0 or 1.

SG is a chemically or enzymatically cleavable group in vivo (especially disulfides, hydrazones, etc.).

Acetals and acetalamines; or 2-8-oligopeptide groups that can be cleaved by podases, cathepsins, or plasminogen lysins.

L1 is –(NR10) _n- (G1) _o- G2-,

R10 is –H or _C1 - _C3 -alkyl.

G1 is –NH-C(＝O)-, -C(＝O)-NH-, or –(CH ₂ ) _O-3 -C(＝O)-NH-.

n is 0 or 1,

o is 0 or 1.

G2 is a bond or a straight or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) ₂ , -

NH-, -C(=O)-, -CH(COOH)-, -CH(CH ₂ -C(=O)-NH ₂ )

, -NMe-, -S(=O) ₂ -NHNH-, -NH-C(=O)-, -C(=O)-NH-

-C(=O)-NHNH-, having up to 4 heteroatoms selected from N, O and S, -S(=O)- or -S(=O) _2- , 5- to 10-membered aromatic or non-aromatic heterocycles, wherein the side chains in the branched carbon chain can be –NH-

C(＝O)-NH ₂ , -COOH, -OH, -NH-CNNH ₂ , sulfonamide,

Sulfone, sulfoxide, or sulfonic acid substitution, or representing one of the following groups:

in

Rx is -H, C1-C3-alkyl, or phenyl, and is an acid.

L4 is a single bond or a group.

–(C＝O) _y -G4-,

y is 0 or 1, and

G4 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) ₂ , -NH-, -

C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -S(=O) ₂ -

NHNH-, -C(＝O)-NHNH-, -CH(COOH)-, and 5- to 10- having up to 4 heteroatoms selected from N, O, and S, S(＝O)-, or S(＝O) ₂ .

Aromatic or non-aromatic heterocycles, wherein the side chains of the branched carbon chains can be oxidized by –NH-C(＝O)-NH ₂ , -COOH, -OH, -NH-CN-NH ₂ , etc.

Sulfonamide, sulfone, sulfoxide, or sulfonic acid substitution,

§§ represents the bonding site with the nitrogen atom of the lysine residue in the binding region.

Preferably, the present invention relates to compounds in which the linker Lb or Lc may also be bonded to the lysine side chain or lysine residue of the binding moiety, and in this case has the following formula:

§-(C＝O) _m -L1-(L4) _n -C(＝O)-§§

in

m is 0 or 1.

n is 0 or 1,

§ is the bond that links the drug molecule or the cleavable group of the podase protease of formula (Ia’), and

§§ represents the bond connecting the nitrogen atoms in the bonding region.

L1 is –(NR10) _n- (G1) _o- G2-,

R10 is –H or _C1 - _C3 -alkyl.

G1 is –NH-C(＝O)-, -C(＝O)-NH-, or –( _CH2 ) _O-3- C(＝O)-NH-, where n is 0 or 1.

o is 0 or 1.

G2 is a bond or a straight or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) ₂ , -

NH-, -C(=O)-, -CH(COOH)-, -CH(CH ₂ -C(=O)-NH ₂ )

, -NMe-, -S(=O) ₂ -NHNH-, -NH-C(=O)-, -C(=O)-NH-

, -C(＝O)-NHNH-, having up to 4 heteroatoms selected from N, O and S, -S(＝O)- or -S(＝O) _2- , 5- to 10-membered aromatic or non-aromatic heterocycles, wherein the side chains in the branched carbon chain can be –NH-

C(＝O)-NH ₂ , -COOH, -OH, -NH-CNNH ₂ , sulfonamide,

Sulfone, sulfoxide, or sulfonic acid substitution, or representing one of the following groups:

in

Rx is -H, C1-C3-alkyl, or phenyl, and is an acid.

L4 is a single bond or a group.

–(C＝O) _y -G4-, where y is 0 or 1, and

G4 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(=O)-, -S(=O) ₂ , -NH-, -

C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -S(=O) ₂ -

NHNH-, -C(＝O)-NHNH-, -CH(COOH)-, and having up to 4 heteroatoms selected from N, O, and S, or 5- to 10- of S(＝O)- or S(＝O) ₂ .

Aromatic or non-aromatic heterocycles, and the side chains in their branched carbon chains, if present, can be substituted with –NH-C(＝O) _-NH₂ , -COOH, -OH, or -NH-CN- _NH₂.

It can be replaced by sulfonamide, sulfone, sulfoxide or sulfonic acid.

More preferably, the connector L2 has one or two of the following:

in

# ¹ is the bonding site with the sulfur atom in the bonding region.

# ² is the bonding site with the L1 group.

R22 is -COOH, and

The bonds with sulfur atoms in the bonding portion reach a level greater than 80% in one of these two formulas (based on the total number of bonds between the joint and the bonding portion).

In the conjugates according to the invention, or in mixtures of conjugates according to the invention, the bonds with cysteine residues of the antibody are preferably greater than 80%, more preferably greater than 90% (in each case based on the total number of bonds between the linker and the antibody), and more preferably present in one of the two structures of formula A3 or A4. Here, the structures of formula A3 or A4 are generally present together, preferably in a ratio of 60:40 to 40:60, based on the number of bonds with the antibody. The remaining bonds are then present in the following structures.

in

# ¹ is the junction site with the sulfur atom of the antibody, and

# ² is the L1 junction with the drug molecule.

Preferably, L4 has the following structure:

# ¹ -C(＝O)-(CH ₂ ) _2-20 -C(＝O)-# ²

The chain can be spaced by 1-4 oxygen atoms.

and

# ¹ is the binding site with the nitrogen atom of lysine in the antibody, and

# ² is the L1 binding site with the drug molecule.

According to the present invention, L1 is preferably represented by the following formula:

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2 _w' -# ²

in

# ¹ is the junction site with the sulfur atom of the antibody, and

# ² is the binding site for drug molecules or the binding site for groups that can be cleaved by podases.

^R10 is -H, _-NH2 , or _C1 - _C3 -alkyl.

n is 0 or 1,

o is 0 or 1.

w' is 0 or 1

G1 is –NH-C(=O)-, -C(=O)-NH-, or...

G2 has a straight-chain or branched hydrocarbon chain of 1-100 carbon atoms, composed of arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be separated once or more by one or more of the following groups: -O-, -S-, -S(＝O)-, -S(＝O) ₂ , -NR ^y- , CH(COOH)-, -CH( _CH2 -C(＝O) _-NH2 , -NR ^y C(＝O)-, -C(NH)NR ^y- , -C(＝O)-NR ^y- , -NR ^y NR ^y- , -S(＝O) _2- NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and/or has up to 4 heteroatoms selected from N, O and S, -S(＝O)- or -S(＝O) ₂ - 3- to 10-membered aromatic or non-aromatic heterocycles, wherein the straight or branched carbon chain may be additionally substituted with –NH-C(＝O)-NH ₂ , -COOH, -OH, sulfonamide, sulfone, sulfoxide or sulfonic acid,

Ry is -H, phenyl, C <sub>1- C 10 -alkyl, C <sub>2 -C 10 -alkenyl, or C <sub>2- C 10 -ynyl, each of which can be substituted with –NH-C(=O)-NH2 , -COOH, -OH, -NH 2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

Rx is -H, _C1 - _C3 -alkyl, or phenyl.

In this context, G1 is preferably -NH-C(=O)- or ^R10 is preferably not _-NH2 .

Preferably, G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, consisting of arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be separated once or more by one or more of the following groups: -O-, -S-, -S(=O)-, -S(=O) ₂ , -NH-, -C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -NHNH-, -S(=O) _2- NHNH-, -C(=O)-NHNH-, and 5- to 10-membered aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S, or -S(=O)-.

More preferably, G1 is a straight-chain or branched hydrocarbon chain that may be additionally substituted with –NH-C(＝O)-NH ₂ .

G2 is also preferably a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, composed of arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be separated once or more by one or more of the following groups: -O-, -S-, -S(=O)-, -S(=O) ₂ , -NH-, -

C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -CH(COOH)-, -CH(CH ₂ -

C(=O)-NH ₂ ), -NMe-, -NHNH--S(=O) ₂ -NHNH-, -C(=O)-NHNH-

-CR ^x = NO-, and/or having up to 4 heteroatoms selected from N, O and S, -

S(＝O)- or –S(＝O) _2- of 3- to 10-membered aromatic or non-aromatic heterocycles, wherein the straight or branched hydrocarbon chain may be additionally substituted with –NH-C(＝O) _-NH2 , -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid, and

Rx is -H, _C1 - _C3 -alkyl, or phenyl.

In the context, G2 is preferred.

Preferably, G2 represents a spacer group having the following structure:

in

Rx is -H, _C1 - _C3 -alkyl, or phenyl.

# ¹ is the bond connecting the KSP inhibitor or prodrug, and

# ² is the bond connecting the antibody to the coupling group (e.g., L2).

Straight-chain or branched hydrocarbon chains derived from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups typically contain α,ω-divalent alkyl groups having their respective indicated carbon numbers. Preferred examples include: methylene, ethane-1,2-diyl (1,2-ethylene), propane-1,3-diyl (1,3-propylene), butane-1,4-diyl (1,4-butylene), pentane-1,5-diyl (1,5-pentane), hexane-1,6-diyl (1,6-hexane), heptane-1,7-diyl (1,7-hexane), octane-1,8-diyl (1,8-octane), nonane-1,9-diyl (1,9-nonane), and decane-1,10-diyl (1,10-decane).

A branched hydrocarbon chain is a straight-chain hydrocarbon chain or a straight-chain alkylene chain in which one or more hydrogen atoms are replaced by _C1-10 alkyl groups, thereby forming a branched hydrocarbon chain or side chain (branched hydrocarbon chain).

The hydrocarbon chain may additionally contain cyclic alkylene groups (cycloalkyldiyl groups), such as 1,4-cyclohexanediyl or 1,3-cyclopentanediyl. These cyclic groups may be unsaturated. Specifically, aromatic groups (aryl groups), such as phenylene, may be present in the hydrocarbon chain. Moreover, one or more hydrogen atoms in the cyclic alkylene groups and aryl groups may optionally be substituted with _C1-10 alkyl groups. In this way, an optionally branched hydrocarbon chain is formed. The hydrocarbon chain has a total of 0-100 carbon atoms, preferably 1-50, and particularly preferably 2-25 carbon atoms.

Branched hydrocarbon chains or side chains (branched hydrocarbon chains) can be replaced by -NH-C(=O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid.

A hydrocarbon chain may be separated by one or more of the following groups, one or more times:

-O-, -S-, -S(＝O)-, -S(＝O) _2- , -NH-, -C(＝O)-, -NH-C(＝O)-, -C(＝O)-NH-, -CH(COOH)-, -CH(CH ₂ -C(＝O)-NH ₂ ), -NMe-, -NHNH-, -S(＝O) ₂ -NHNH-, -C(＝O)-NHNH-, and 5- to 10-membered aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from =N-, -O- and -S-, -S(＝O)- or -S(＝O) _2- .

Here, the preferred component is a group.

Other spacer groups in G2 are preferably

More preferably, and referring to the definition above, L1 corresponds to the following simplified expression:

–NR ¹¹ B-

in

R ¹¹ is -H or -NH ₂ .

B is –[(CH ₂ ) _x -(X ⁴ ) _y ]w-(CH ₂ ) _z -,

w is between 0 and 20.

x is between 0 and 5.

y is 0 or 1.

z is between 0 and 5, and

X ⁴ is –O-, -C(=O)-NH-, –NH-C(=O)- or

Preferably, the connector L has the following formula:

in

#3 is the bond that connects the drug molecule or prodrug.

#4 is a bond that connects and binds parts of a peptide or protein.

R ¹¹ is -H or -NH ₂ .

B is –[(CH ₂ ) _x -(X ⁴ ) _y ]w-(CH ₂ ) _z -,

w is between 0 and 20.

x is between 0 and 5.

y is 0 or 1.

z is 1 to 5, and

X ⁴ is –O-, -C(=O)-NH-, –NH-C(=O)- or

The aforementioned linker is particularly preferred in the conjugate of formula (IIa), wherein the linker is coupled to ^R1 by replacing a hydrogen atom, i.e., ^R1 is -L-#1, where #1 is the bond that links the antibody.

In the conjugates according to the invention or mixtures of conjugates according to the invention, the bonds linking the antibody cysteine residues are present to a degree preferably greater than 80%, particularly preferably greater than 90% (in each case based on the total number of bonds between the linker and the antibody).

Here, the two structures of general formula (A5) and (A6) are particularly preferred.

in

# ¹ is the junction site with the sulfur atom of the antibody.

# ² is the connection point with L1.

R ²² is –COOH, -C(＝O)-OR, -C(＝O)-R, -C(＝O)-NH ₂ , -C(＝O)-NR ₂ or -C(＝O)-NHR, and

R is a _C1-3 -alkyl group.

More preferably, R ²² is –COOH.

Here, the structures of general formula A5 or A6 are usually present together, preferably in a ratio of 60:40 to 40:60, based on the number of bonds with the antibody. The remaining bonds then exist in the following structures:

in

#1 and #2 have the definitions given above.

In the above formula §-(C(＝O))m-(L1) _n -L2-§§, the preferred groups L1 are those listed in the table below, where r is 0 to 20, preferably 0 to 15, and particularly preferably 0 to 10:

More preferably, AK1 is an antibody or its antigen-binding fragment. The antibody is preferably a human, humanized, or chimeric monoclonal antibody or its antigen-binding fragment, especially anti-TWEAKR antibody, anti-EGFR antibody, anti-B7H3 antibody, or anti-HER2 antibody, or its antigen-binding fragment. Particularly preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007, and TPP-10337; anti-B7H3 antibodies TPP-8382 and TPP-8567; anti-EGFR antibody cetuximab (TPP-981); and anti-HER2 antibody trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

Alternatively, the preferred option is the base structure of the connector (ii).

(ii)–(C＝O) _m –(L1) _n -SG-L2,

Wherein SG represents a group that can be cleaved by proteases (e.g., cathepsins), and m, n, SG, L1, and L2 have the definitions given above. Particularly preferred are the following groups:

-Val-Ala-C(＝O)-NH- (leads to the cleavage of the amide bond at the C-terminal amide of alanine)

-NH-Val-Lys-C(＝O)-NH- (Cleavage of the amide bond at the C-terminal amide site of lysine)

-NH-Val-Cit-C(＝O)-NH- (Cleavage of the amide bond at the C-terminal amide site of citrulline)

-NH-Phe-Lys-C(＝O)-NH- (Cleavage of the amide bond at the C-terminal amide of lysine)

-NH-Ala-Lys-C(＝O)-NH- (Cleavage of the amide bond at the C-terminal amide site of lysine)

-NH-Ala-Cit-C(＝O)-NH- (Cleavage of the amide bond at the C-terminal amide site of citrulline)

In the context, SG is preferred to be:

in

X is -H or _C1-10 -alkyl, which may also be -NH-C(=O) _-NH2 , -COOH, -OH

It can be replaced by _-NH2 or sulfonic acid.

In the case of transglutaminase-catalyzed conjugation, various options for covalently coupling (conjugating) organic molecules to binding moieties (e.g., antibodies) in a conjugation site-specific manner have been disclosed in the literature (see, for example, Sochaj et al., Biotechnology Advances, 33 , 775-784, (2015), Panowski et al., MAbs 6 , 34-45 (2014)). According to the invention, it is preferred to use transglutaminase to conjugate KSP inhibitors or prodrugs to antibodies via receptor glutamine residues of the antibody. Such receptor glutamine groups can be generated by designing antibodies or by mutations that produce non-glycosylated antibodies. The number of these receptor glutamines in the antibody is preferably 2 or 4. Suitable linkers are used for conjugation. Suitable linker structures are those with free amine donor functionality, which are suitable substrates for transglutaminase. Linkers can be attached to antibodies in various ways.

Preferably, in the case of transglutaminase-catalyzed conjugation, the linker has one of the basic structures (i) and (ii) mentioned above.

(i)–(C＝O) _m –(L1) _n -L2-

(ii)–(C＝O) _m –(L1) _n -SG-L2

in

L1, SG, SG1, and m have the definitions given above.

L2 preferably represents one of the following groups:

in

Ry is a -H or -NH-C(=O)-alkyl group.

# ¹ is the connection point with ^L1 , and

# ² is the connection point with the glutamine residues of the binding site.

Preferably, in the context, Ry is -H or –NH-C(=O) _-CH3 .

Examples of corresponding conjugates have the following structures, wherein MOD and L1 have the definitions given above, AK ₃ is preferably the binding moiety of the antibody, and n is preferably 2 or 4.

Specially selected KSP inhibitor conjugates

According to the present invention, the following KSP inhibitor conjugates are particularly preferred, wherein

AK (AK ₁ ; AK ₂ ; AK ₃ ) is the binding site, preferably an antibody or antigen-binding fragment, and...

n is a number from 1 to 50, preferably from 1 to 20, more preferably from 2 to 8, and even more preferably from 2 to 6.

AK ₁ is preferably an antibody that is bound to a KSP inhibitor via a cysteine residue.

AK ₂ is preferably an antibody that is bonded to a KSP inhibitor via a lysine residue.

AK ₃ is preferably an antibody that binds to a KSP inhibitor via glutamine residues.

The binding sites or antibodies used herein are preferably those described in the specification as preferred binding sites and antibodies.

Particularly preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007 and TPP-10337, anti-B7H3 antibodies TPP-8382 and TPP-8567, anti-EGFR antibody cetuximab (TPP-981) and anti-HER2-antibody trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

The particularly preferred conjugates are:

in

_AK1 and _AK2 are antibody fragments that bind to antibodies or antigens, and

n is a number from 1 to 50, preferably from 1 to 20, more preferably from 2 to 8, and especially from 2 to 6.

Preparation of KSP inhibitors, linker intermediates or prodrugs, linker intermediates, and conjugates

The conjugate according to the invention is prepared by first providing a linker for a low molecular weight KSP inhibitor or its prodrug. The intermediate obtained in this way is then reacted with the binding moiety (preferably an antibody).

Preferably, for coupling with cysteine residues, one of the following compounds reacts with a cysteine-containing binding moiety, such as an antibody, which is optionally partially reduced for this purpose:

in

_X1 is CH,

X ₂ is C,

X ₃ is N,

R is -H or –COOH

K is a linear or branched _C1 - _C6 alkyl group, optionally substituted with _C1 - _C6 alkoxy- or -OH, and

_SG1 , _L1 , _L2 , _L3

_L4 has the definition given above.

In the above formula, and also in the reaction schemes and structural formulas below, the hydrogen atom at the R ⁴ position of formula IIa (i.e., in the -NH ₂ group) can be replaced by a cleavable group of the podase of formula Ia used according to the present invention.

In each of the compounds above and below, the tert-butyl group can be replaced by a cyclohexyl group.

This compound can be used, for example, in the form of its trifluoroacetate. For reaction with the binding moiety, such as an antibody, it is preferable to use the compound in a 2 to 12 molar excess relative to the binding moiety.

Preferably, in order to couple lysine residues, one of the following compounds reacts with a lysine-containing binding moiety, such as an antibody:

In the formula,

_X1 is CH,

X ₂ is C,

X ₃ is N, and

_L4 has the same definition as _L1 , where _L1 has the same definition as above.

For intermediates coupled with cysteine residues, the reaction can be described as follows:

This can correspondingly cause other intermediates and other antibodies to react.

For intermediates coupled with lysine residues, the reaction can be described as follows:

According to the present invention, the following conjugates are obtained:

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

In the above formula, X1 represents CH, X2 represents C and X3 represents N, SG1 and L1 have the same definition as above, L2, L3 and L4 have the same definition as L1; and R and K have the same definition as above.

AK1 is an antigen-binding fragment of an anti-TWEAKR antibody, anti-EGFR antibody, anti-B7H3 antibody, or anti-HER2 antibody, or such antibodies, conjugated to a cysteine residue. AK2 is an antigen-binding fragment of an anti-TWEAKR antibody, anti-EGFR antibody, anti-B7H3 antibody, or anti-HER2 antibody, or such antibodies, conjugated to a lysine residue. Particularly preferred, AK1 and AK2 are anti-TWEAKR antibodies TPP-7006, TPP-7007, TPP-10336, and TPP-10337; anti-B7H3 antibodies TPP-8382 and TPP-8567; anti-EGFR antibody cetuximab (TPP-981); and anti-HER2 antibodies trastuzumab and TPP-1015, or antigen-binding fragments of such antibodies.

Other definitions

The term "transglutaminase," which can also be used interchangeably with "TGase" or "TG," should be understood as referring to an enzyme capable of linking proteins through an acyl transfer reaction between the γ-formamide group of peptide-bound glutamine and the ε-amino group of lysine or a structurally related primary amine (e.g., aminopentyl) or, for example, peptide-bound lysine, which produces an 8-(γ-glutamyl)-lysine isopeptide bond. TGase includes bacterial transglutaminase (BTG), such as the enzyme (protein-glutamine-γ-glutamyltransferase) with EC reference number 2.3.2.13.

When referring to the amino acid residues of an antibody, the expression "receptor glutamine" refers to a glutamine residue that, under appropriate conditions, is recognized by transglutaminase and can be linked to it via a reaction between this specific glutamine and a lysine or a structurally related primary amine (e.g., aminopentyl) catalyzed by transglutaminase. Optionally, the receptor glutamine may be surface-exposed glutamine.

"Amino acid modification" or "mutation" herein refers to the substitution, insertion, and/or deletion of amino acids in a polypeptide sequence. Substitution is the preferred amino acid modification herein. "Amino acid substitution" or "replacement" herein refers to the exchange of an amino acid at a given position in a protein sequence for another amino acid. For example, substitution Y50W describes a variant of the parent polypeptide in which the tyrosine residue at position 50 has been replaced with tryptophan. A "variant" of a polypeptide describes a polypeptide having a substantially identical amino acid sequence to a reference polypeptide (typically the native or "parental" polypeptide). Polypeptide variants may have one or more amino acid substitutions, deletions, and/or insertions at specific positions in the native amino acid sequence.

The term "conjugate site-specific conjugate" describes a conjugate of a binding moiety (preferably an antibody) and a residue (preferably a linker-drug residue), wherein the binding moiety is functionalized at one or more specific sites, preferably at glutamine residues. Transglutaminases (TGases), including bacterial transglutaminase (BTG) (EC 2.3.2.13), exhibit strong specificity in the recognition of glutamine-protein substrates and can catalyze "conjugate site-specific conjugation".

The terms "homogeneous conjugate" or "homogeneous ADC" describe a mixture of conjugates that are specific to the binding site, wherein at least 60%, 70%, 80%, or 90% of the binding moiety has the same number of conjugate residues per binding moiety. In the case of antibodies, this number should be even, preferably 2 or 4.

Isotopes, salts, solvates, isotope variants

This invention also includes all suitable isotopic variants of the compounds of this invention. Isotopic variants of the compounds of this invention should be understood herein as compounds in which at least one atom of the compound of this invention has been replaced by another atom having the same atomic number but having a different atomic mass than that found in nature. Examples of isotopes that can be incorporated into the compounds of this invention are those of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, such as ^2H (deuterium), ^3H ( ^tritium ), ^13C , ^14C , ^15N , ^17O , ^18O , ^32P , ^33P , ^33S , ^34S , ^35S , 36S, ^18F , ^36Cl , ^82Br , ^123I , ^124I , ^129I , and ^131I . Certain isotopic variants of the compounds of the present invention, particularly those incorporated with one or more radioisotopes, can be advantageous for examining mechanisms of action or in vivo distribution of the active ingredient, for example. This is due to their relatively easy preparability and detectability, especially compounds labeled with ^3H or ^14C isotopes, which are well-suited for this purpose. Furthermore, the incorporation of isotopes such as deuterium can lead to specific therapeutic benefits due to increased metabolic stability of the compound, such as a prolonged in vivo half-life or a reduction in the required active dose; therefore, these modifications to the compounds of the present invention may also optionally constitute preferred embodiments of the invention. Isotopic variants of the compounds of the present invention can be prepared by methods known to those skilled in the art, for example by the methods and procedures described in the examples further described below, which involve the use of respective reagents and/or corresponding isotopic modifications of the starting compound.

In the context of this invention, preferred salts are physiologically acceptable salts of the compounds of this invention. Also included are salts that are not inherently suitable for pharmaceutical applications but can be used, for example, to isolate or purify the compounds of this invention.

Physiologically acceptable salts of the compounds of this invention include acid addition salts of inorganic acids, carboxylic acids, and sulfonic acids, such as salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid, and benzoic acid.

Physiologically acceptable salts of the compounds of the present invention also include salts of conventional bases, such as 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 1 to 16 carbon atoms, such as and preferably ethylamine, diethylamine, triethylamine, ethyl diisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine, and 1,2-ethylenediamine.

In the context of this invention, solvates are described as those forms of the compounds of this invention that form complexes by coordination with solvent molecules in a solid or liquid state. Hydrates are a specific form of solvate in which coordination with water occurs. Hydrates are preferred solvates in the context of this invention.

The present invention also includes prodrugs of the compounds of the present invention. In this context, the term "prodrug" refers to a compound that may be biologically active or inactive on its own, but which reacts (e.g., metabolizes or hydrolyzes) during its residence in the body to yield the compounds of the present invention.

Specific implementation plan

The following implementation schemes are particularly preferred:

Implementation Plan A':

APDCs of formula IVa' or IVa” or IVa”', wherein _D1 in formula IVa' or IVa” or IVa”' is a compound of formula (IIe).

in

_X1 is N,

X ₂ is N, and

X ₃ is C

or

_X1 is CH,

X ₂ is C, and

X ₃ is N

or

_X1 is NH,

X ₂ is C, and

X ₃ is C

or

_X1 is CH,

X ₂ is N, and

X ₃ is C,

A is –C(＝O)-,

^R1 is –L-#1, -H, -COOH, -C(＝O)-NHNH ₂ , -(CH ₂ ) _1-3 NH ₂ , -C(＝O)-NZ""CH ₂ ) _1-3 NH ₂ and –C(＝O)-NZ"CH ₂ -C(＝O)-OH,

Z represents -H or _-NH2 .

^R2 is -H,

R ⁴ is a group of formula (Ia).

^R3 is –L-#1 or _C1-10 -alkyl, which may optionally be substituted with the following groups: –OH, -O-alkyl, -SH, -S-alkyl, -OC(=O)-alkyl, -OC(=O)-NH-alkyl, -NH-C(=O)-alkyl, -NH-C(=O)-NH-alkyl, -S(=O) _n -alkyl, -S(=O) _2- NH-alkyl, -NH-alkyl, -N(alkyl) ₂ or _-NH2 .

R ⁵ is either -H or -F.

^R6 and ^R7 are independently -H, _C1-3 -alkyl, fluoro- _C1-3 -alkyl, _C2-4 -alkenyl, fluoro- _C2-4 -alkenyl, _C2-4 -ynyl, fluoro- _C2-4 -ynyl, hydroxyl, or halogen.

^R8 is a branched _C1-5 -alkyl or cyclohexyl group.

R ⁹ is either -H or -F.

–L-#1 is a linker group

§-(C(＝O)) _m -(L1) _n -L2-§§

in

m and n are independently 0 or 1.

§ represents the bond with KSP inhibitors.

§§ represents the bond with the antibody.

L2 is one of the following groups

# ¹ is the junction site with the sulfur atom of the antibody.

# ² is the connection point with L1.

L1 is a group

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ¹⁰ is -H, -NH ₂ , or C _1-3 -alkyl.

G1 is –NHC(＝O)- or

n is 0 or 1,

o is 0 or 1, and

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, selected from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be selected from the following groups spaced once or from the same or different groups spaced more than once: -O-, -S-, -S(=O)-, -S(=O) _2- , -NH-, -C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -NHNH-, -S(=O) _2- NHNH-, -C(=O)-NHNH-, and a 3- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from N, O, and S, or –S(=O)-, wherein the straight-chain or branched hydrocarbon chain may be –

Substitution with NH-C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

# ¹ is the bond that connects to the KSP inhibitor.

# ² is the L2 bond that links the antibody.

One of the substituents ^R1 and ^R3 is a linker group -L-#1, and its salt, solvate and salt of solvate, and the antibody mentioned in formula IVa' or IVa” or IVa”' is a human, humanized or chimeric monoclonal antibody or its antigen-binding fragment, and n in formula IVa' or IVa” or IVa”' is a number from 1 to 10.

Preferably, the antibody here is an anti-TWEAKR antibody, an anti-EGFR antibody, an anti-B7H3 antibody, or an anti-HER2 antibody, or an antigen-binding fragment of these antibodies.

Especially preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007 and TPP-10337, anti-B7H3 antibodies TPP-8382 and TPP-8567, anti-EGFR antibody cetuximab (TPP-981) and anti-HER2 antibody trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

The preferred compounds here are those of formula (IIe), wherein R ₃ is defined as an alkyl group, preferably a _C1-3 alkyl group.

In this context, G2 is preferred.

Alternatively, the linker -L-#1 can be bonded to a lysine side chain or a lysine residue. In this case, it preferably has the following formula:

-§-(SG) _x -L4-C(＝O)-§§

in

§ represents the bond that links the KSP inhibitor.

§§ represents the bond that links the antibody.

x is 0 or 1.

SG is a cleavable group.

L4 is a single bond or a functional group.

–(C(＝O)) _y -G4-y is 0 or 1, and

G4 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, selected from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: -O-, -S-, -S(=O)-, -S(=O) ₂ , -NH-, -C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -NHNH-, -S(=O) _2- NHNH-, -C(=O)-NHNH-, and a 5- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from N, O, and S, -S(=O)-, or –S(=O) _2- , wherein the straight-chain or branched hydrocarbon chain may be –NH-C(=O) _-NH2 Substitution with -COOH, -OH, sulfonamide, sulfone, sulfoxide or sulfonic acid.

In this context, SG is preferably a 2-8 oligopeptide, more preferably a dipeptide.

Preferably, the straight or branched hydrocarbon chains of G4 can be spaced apart.

Implementation Plan B':

APDCs of formula IVa' or IVa” or IVa”', wherein _D1 in formula IVa' or IVa” or IVa”' is a compound of formula (IIf).

in

_X1 is N,

X ₂ is N, and

X ₃ is C,

or

_X1 is CH,

X ₂ is C, and

X ₃ is N,

or

_X1 is NH,

X ₂ is C, and

X ₃ is C,

or

_X1 is CH,

X ₂ is N, and

X ₃ is C,

A is –C(＝O)-,

^R1 can be –L-#1, -H, -COOH, -C(＝O)-NHNH ₂ , -(CH ₂ ) _1-3 NH ₂ , -C(＝O)-NZ“(CH ₂ ) _1-3 NH ₂ and –C(＝O)-NZ“CH ₂ C(＝O)-OH,

Z represents -H or _-NH2 .

^R2 is -H,

^R4 is a group of formula (Ia).

^R3 is –L-#1 or _C1-10 -alkyl, which may optionally be substituted with the following groups: –OH, -O-alkyl, -SH, -S-alkyl, -OC(=O)-alkyl, -OC(=O)-NH-alkyl, -NH-C(=O)-alkyl, -NH-C(=O)-NH-alkyl, -S(=O) _n -alkyl, -S(=O) _2- NH-alkyl, -NH-alkyl, -N(alkyl) ₂ and _-NH2 .

R ⁵ is either -H or -F.

^R6 and ^R7 are independently -H, _C1-3 -alkyl, fluoro- _C1-3 -alkyl, _C2-4 -alkenyl, fluoro- _C2-4 -alkenyl, _C2-4 -ynyl, fluoro- _C2-4 -ynyl, hydroxyl, or halogen.

^R8 is a branched _C1-5 -alkyl group.

R ⁹ is either -H or -F.

–L-#1 is a group

§-(C(＝O)) _m -(L1) _n -L2-§§

in

m is 0 or 1;

§ represents the bond that links the KSP inhibitor.

§§ represents the bond that links the antibody.

L2 is a group

# ¹ is the junction site with the sulfur atom of the antibody.

# ² is the connection point with L1.

L1 is a group

# ¹ -(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

^R10 is -H, _-NH2 , or _C1-C3 -alkyl.

G1 is –NH-C(＝O)- or

n is 0 or 1,

o is 0 or 1.

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, selected from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be selected from the following groups spaced once or from the same or different groups spaced more than once: -O-, -S-, -S(=O)-, -S(=O) _2- , -NH-, -C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -NHNH-, -S(=O) _2- NHNH-, -C(=O)-NHNH-, and a 3- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from N, O, and S, or –S(=O)-, wherein the straight-chain or branched hydrocarbon chain may be –

Substitution with NH-C(＝O)-NH2, -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid.

#1 is the link to the KSP inhibitor.

#2 is the L2 bond that links the antibody.

One of the substituents ^R1 and ^R3 is a linker group -L-#1, and its salt, solvate and salt of solvate, and the antibody mentioned in formula IVa' or IVa” or IVa”' is a human, humanized or chimeric monoclonal antibody or its antigen-binding fragment, and n in formula IVa' or IVa” or IVa”' is a number from 1 to 10.

Preferably, the antibody here is an anti-TWEAKR antibody, an anti-EGFR antibody, an anti-B7H3 antibody, or an anti-HER2 antibody, or an antigen-binding fragment of these antibodies.

Especially preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007 and TPP-10337, anti-B7H3 antibodies TPP-8382 and TPP-8567, anti-EGFR antibody cetuximab (TPP-981) and anti-HER2 antibody trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

The preferred compounds herein are those of formula (IIf), wherein R ₃ is defined as an alkyl group, preferably a _C1-3 alkyl group.

In this context, G2 is preferred.

Alternatively, the linker -L-#1 can be bonded to a lysine side chain or a lysine residue. In this case, it preferably has the following formula:

-§-(SG) _x -L4-C(＝O)-§§

in

§ represents the bond that links the KSP inhibitor.

§§ represents the bond that links the antibody.

x is 0 or 1.

SG is a cleavable group.

L4 is a single bond or a functional group.

–(C(＝O)) _y -G4-y is 0 or 1, and

G4 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, selected from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, and may be selected from the following groups spaced once or from the following same or different groups spaced more than once: -O-,

-S-, -S(＝O)-, -S(＝O) ₂ , -NH-, -C(＝O)-, -NH-C(＝O)-, -C(＝O)-NH-, -NMe-, -NHNH-, -S(＝O) _2- NHNH-, -C(＝O)-NHNH-, and 5- to 10-membered aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S, -S(＝O)- or –S(＝O) _2- , wherein the straight or branched hydrocarbon chain may be substituted with –NH-C(＝O) _-NH2 , -COOH, -OH, sulfonamide, sulfone, sulfoxide or sulfonic acid.

In this context, SG is preferably a 2-8 oligopeptide, more preferably a dipeptide.

Preferably, the straight or branched hydrocarbon chains of G4 can be spaced apart.

Implementation Plan C':

APDCs of formula IVa' or IVa” or IVa”', wherein _D1 in formula IVa' or IVa” or IVa”' is a compound of formula (IIg).

in

_X1 is N,

X ₂ is N, and

X ₃ is C

or

_X1 is CH,

X ₂ is C, and

X ₃ is N

or

_X1 is NH,

X ₂ is C, and

X ₃ is C

or

_X1 is CH,

X ₂ is N, and

X ₃ is C,

A is –C(＝O)-,

R ¹ is –L-#1,

^R2 is -H,

^R4 is a group of formula (Ia).

^R3 is a _C1-10 alkyl group, which may optionally be substituted with a group selected from the following: –OH, -O-alkyl, -SH, -S-alkyl, -OC(=O)-alkyl, -OC(=O)-NH-alkyl, -NH-C(=O)-alkyl, -NH-C(=O)-NH-alkyl, -S(=O) _n -alkyl, -S(=O) ₂ -NH-alkyl, -NH-alkyl, -N(alkyl) ₂ or _-NH2 , or –MOD.

-MOD is a radical

–(NR ¹⁰ ) _n -(G1) _o -G2-H

R ¹⁰ is -H or _C1 - _C3 -alkyl;

G1 is –NH-C(=O)-, -C(=O)-NH-, or

n is 0 or 1,

o is 0 or 1.

G2 is a straight-chain or branched hydrocarbon chain having 1-10 carbon atoms, and may be separated once by a group selected from the following or separated more than once by the same or different groups selected from the following: -O-, -S-, -SO-, -S(＝O) ₂ , ^-NRy- , ^-NRyC (＝O)-, -C(＝O) ^NRy- , ^{-NRy NRy-} , -S(＝O) _2- NRy ^{NRy- ,} -C(＝O)-NRy ^{NRy -} C(＝O)- or ^-CRx ＝NO-, wherein the straight-chain or branched hydrocarbon chain may be substituted with –NH-C(＝O)-NH2, -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid.

Rx is -H, _C1 - _C3 -alkyl, or phenyl.

R_^y is -H, phenyl, C <sub>_1- C_₁₀-alkyl, C <sub>₂ -C_₁₀-alkenyl, or C <sub>₂ -C_₁₀-alkynyl, each of which can be substituted with –NH-C(=O)-NH_₂ , -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid.

R ⁵ is either -H or -F.

^R6 and ^R7 are independently -H, _C1-3 -alkyl, fluoro- _C1-3 -alkyl, _C2-4 -alkenyl,

Fluoro- _C2-4 -alkenyl, _C2-4 -ynyl, fluoro- _C2-4 -ynyl, hydroxyl, or halogen, ^R8 being a branched _C1-5 -alkyl group.

R ⁹ is either -H or -F.

–L-#1 is a linker group

§-(C(＝O)) _m -(L1) _n -L2-§§

in

m and n are independently 0 or 1.

§ represents the bond that links the KSP inhibitor.

§§ represents the bond that links the antibody.

L2 is one of the following groups

# ¹ is the junction site with the sulfur atom of the antibody.

# ² is the connection point with L1.

L1 is a group

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ¹⁰ is -H, -NH ₂ , or C _1-3 -alkyl.

G1 is –NHC(＝O)- or

n is 0 or 1,

o is 0 or 1, and

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, selected from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be separated once by a group selected from the following or separated more than once by the same or different groups selected from the following: -O-, -S-, -S(=O)-, -S(=O) _2- , -NH-, -C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -NHNH-, -S(=O) _2- NHNH-, -C(=O)-NHNH-, and a 3- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from N, O and S, or –S(=O)-, wherein the straight-chain or branched hydrocarbon chain may be substituted with –NH-C(=O) _-NH2 , -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid.

#1 is the link to the KSP inhibitor.

#2 is the L2 bond that links the antibody.

And its salts, solvates and salts of solvates, wherein the antibody mentioned in formula IVa' or IVa” or IVa”' is a human, humanized or chimeric monoclonal antibody or its antigen-binding fragment, and n in formula IVa' or IVa” or IVa”' is a number from 1 to 10.

Preferably, the antibody here is an anti-TWEAKR antibody, an anti-EGFR antibody, an anti-B7H3 antibody, or an anti-HER2 antibody, or an antigen-binding fragment of these antibodies.

Especially preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007 and TPP-10337, anti-B7H3 antibodies TPP-8382 and TPP-8567, anti-EGFR antibody cetuximab (TPP-981) and anti-HER2 antibody trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

The preferred compounds here are those of formula (IIg), wherein R ³ is defined as an alkyl group, preferably a _C1-3 alkyl group.

In this case, –MOD preferably has at least one COOH- group.

Implementation Plan D':

APDCs of formula IVa' or IVa” or IVa”', wherein _D1 in formula IVa' or IVa” or IVa”' is a compound of formula (IIh).

in

_X1 is N,

X ₂ is N, and

X ₃ is C

or

_X1 is CH,

X ₂ is C, and

X ₃ is N

or

_X1 is NH,

X ₂ is C, and

X ₃ is C

or

_X1 is CH,

X ₂ is N, and

X ₃ is C,

A is –C(＝O)-,

^R1 is -H or –COOH.

^R2 is -H,

^R4 is a group of formula Ia.

R ³ is –L-#1,

R ⁵ is either -H or -F.

^R6 and ^R7 are independently -H, _C1-3 -alkyl, fluoro- _C1-3 -alkyl, _C2-4 -alkenyl,

Fluoro- _C2-4 -alkenyl, _C2-4 -ynyl, fluoro- _C2-4 -ynyl, hydroxyl, or halogen, ^R8 being a branched _C1-5 -alkyl group.

R ⁹ is either -H or -F.

–L-#1 is a linker group

§-(C(＝O)) _m -(L1) _n -L2-§§

in

m and n are independently 0 or 1.

§ represents the bond that links the KSP inhibitor.

§§ represents the bond that links the antibody.

L2 is one of the following groups

# ¹ is the junction site with the sulfur atom of the antibody.

# ² is the connection point with L1.

L1 is a group

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ¹⁰ is -H, -NH ₂ , or C _1-3 -alkyl.

G1 is –NHC(＝O)- or

n is 0 or 1,

o is 0 or 1, and

G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, selected from arylene and/or straight-chain and/or branched and/or cyclic alkylene groups, which may be separated once by a group selected from the following or separated more than once by the same or different groups selected from the following: -O-, -S-, -S(=O)-, -S(=O) _2- , -NH-, -C(=O)-, -NH-C(=O)-, -C(=O)-NH-, -NMe-, -NHNH-, -S(=O) _2- NHNH-, -C(=O)-NHNH-, and a 3- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from N, O and S, or –S(=O)-, wherein the straight-chain or branched hydrocarbon chain may be substituted with –NH-C(=O) _-NH2 , -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid.

#1 is the link to the KSP inhibitor.

#2 is the L2 bond that links the antibody.

And its salts, solvates and salts of solvates, wherein the antibody mentioned in formula IVa' or IVa” or IVa”' is a human, humanized or chimeric monoclonal antibody or its antigen-binding fragment, and n in formula IVa' or IVa” or IVa”' is a number from 1 to 10.

Preferably, the antibody here is an anti-TWEAKR antibody, an anti-EGFR antibody, an anti-B7H3 antibody, or an anti-HER2 antibody, or an antigen-binding fragment of these antibodies.

Especially preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007 and TPP-10337, anti-B7H3 antibodies TPP-8382 and TPP-8567, anti-EGFR antibody cetuximab (TPP-981) and anti-HER2 antibody trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

In this context, G2 is preferred.

Implementation Plan E' :

APDCs of formula IVa' or IVa” or IVa”', wherein _D1 in formula IVa' or IVa” or IVa”' is a compound of formula (IIi).

in

R ¹ is –L-#1,

–L-#1 is a linker group

§-(C(＝O)) _m -(L1) _n -L2-§§

in

m and n are independently 0 or 1.

§ represents the bond linking to the KSP inhibitor, §§ represents the bond linking to the antibody, and L2 is a functional group.

R22 can be –COOH, -C(=O)-OC _1-3 -alkyl, -C(=O)-C _1-3 -alkyl, -C(=O)-NH-C _1-3 -alkyl, or -C(=O)-NH2.

#1 is the junction point with the sulfur atom of the antibody.

#2 is the key to connect to L1.

L1 is a group

# ¹ –(NR ¹⁰ ) _n -(G1) _o -G2-# ²

in

R ¹⁰ is –H,

G1 is –NHC(＝O)- or

n is 0 or 1,

o is 0 or 1.

G2 is a _C1-3 -alkyl group.

#1 is the link to the KSP inhibitor.

#2 is the L2 bond that links the antibody.

^R2 and ^R5 are -H.

^R3 is _-CH2OH , and

^R4 is a group of formula (Ia).

And its salts, solvates and salts of solvates, wherein the antibody mentioned in formula IVa' or IVa” or IVa”' is a human, humanized or chimeric monoclonal antibody or its antigen-binding fragment, and n in formula IVa' or IVa” or IVa”' is a number from 1 to 10.

Preferred compounds are those of formula (IIi), wherein R ²² is –COOH.

In the conjugates according to the invention or in mixtures of conjugates according to the invention, the bonds linking the cysteine residues of the antibody are present to a degree preferably greater than 80%, more preferably greater than 90% (in each case based on the total number of bonds between the linker and the antibody).

The present invention particularly prefers conjugates having the following groups as L2.

R ²² has the definition given above.

Typically, conjugates having two types of L2 groups are present, preferably in a ratio of 60:40 to 40:60, based on the number of bonds with the antibody.

The remaining bonds then exist in the following structure.

#1 and #2 have the definitions given above.

Preferably, the antibody here is an anti-TWEAKR antibody, an anti-EGFR antibody, an anti-B7H3 antibody, or an anti-HER2 antibody, or an antigen-binding fragment of these antibodies.

Especially preferred are anti-TWEAKR antibodies TPP-7006, TPP-7007 and TPP-10337, anti-B7H3 antibodies TPP-8382 and TPP-8567, anti-EGFR antibody cetuximab (TPP-981) and anti-HER2 antibody trastuzumab and TPP-1015, or antigen-binding fragments of these antibodies.

Therapeutic uses

Hyperproliferative diseases that can be treated with the compounds of this invention particularly include the cancer and neoplasm disease group. In the context of this invention, these are understood to specifically refer to, but are not limited to, the following diseases: breast cancer and breast tumors (breast cancer includes ductal and lobular forms, as well as in situ), respiratory tract tumors (small cell and non-small cell carcinoma, bronchial cancer), brain tumors (e.g., brainstem and hypothalamic tumors, astrocytomas, ependymomas, glioblastomas, gliomas, medulloblastomas, meningiomas, and neuroectodermal and pineal gland tumors), digestive organ tumors (esophageal, gastric, gallbladder, small intestine, large intestine, rectal, and anal cancers), liver tumors (especially hepatocellular carcinoma, cholangiocarcinoma, and mixed hepatocellular cholangiocarcinoma), head and neck tumors (larynx, hypopharynx, nasopharynx, oropharynx, lip, and oral cavity cancers, oral melanoma), and skin tumors (basal cell carcinoma, spinal cord tumors, etc.). Tumors include: aliometra, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, non-melanoma skin cancer, Merkel cell skin cancer, mast cell tumor; soft tissue tumors (especially soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, chondrosarcoma, fibrosarcoma, angiosarcoma, leiomyosarcoma, liposarcoma, lymphosarcoma, and rhabdomyosarcoma); eye tumors (especially intraocular melanoma and retinoblastoma); endocrine and exocrine gland tumors (e.g., thyroid and parathyroid tumors, pancreatic and salivary gland cancers, adenocarcinoma); urinary tract tumors (bladder, penis, kidney, renal pelvis, and ureter tumors); and reproductive organ tumors (endometrial, cervical, ovarian, vaginal, vulvar, and uterine cancers in women, and prostate and testicular cancers in men). These also include physical forms of blood, lymphatic system, and spinal cord, as well as proliferative diseases that are circulating cells, such as leukemia, lymphoma, and myeloproliferative disorders, such as acute myeloid, acute lymphoblastic, chronic lymphocytic, chronic myeloid, and hairy cell leukemia, as well as AIDS-related lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt's lymphoma, and central nervous system lymphoma.

These well-characterized diseases in humans can also occur in other mammals with comparable causes and can be treated with the compounds of the present invention in the same way.

Treatment of the above-mentioned cancer diseases with the compounds of the present invention includes treatment of solid tumors and treatment of their metastatic or circulating forms.

In the context of this invention, the term “treatment” is used in a conventional sense and refers to the handling, care and nursing of a patient with the aim of combating, reducing, alleviating or relieving a disease or health abnormality and improving the quality of life impaired by the disease, such as in the case of cancer.

Therefore, the present invention further provides the use of the compounds of the present invention for treating and/or preventing diseases, especially the aforementioned diseases.

The present invention further provides the use of the compounds of the present invention in the preparation of medicaments for treating and/or preventing diseases, especially the aforementioned diseases.

The present invention further provides the use of the compounds of the present invention in methods for treating and/or preventing diseases, especially the aforementioned diseases.

The present invention further provides a method for treating and/or preventing diseases, particularly the aforementioned diseases, using an effective amount of at least one compound of the present invention.

The compounds of the present invention can be used alone or, if desired, in combination with one or more other pharmacologically active substances, provided that the combination does not cause undesirable and unacceptable side effects. Therefore, the present invention also provides a formulation comprising at least one compound of the present invention and one or more other pharmaceutical agents, particularly for the treatment and/or prevention of the aforementioned conditions.

For example, the compounds of the present invention can be combined with known anti-proliferation, cell-inhibiting, cytotoxic, or immunotherapeutic substances for the treatment of cancer. Examples of suitable combination drugs include:

131I-chTNT, abarelix, abiraterone, arubibin, adalimumab, ado trastuzumab, afatinib, aflibercept, interleukin, alemtuzumab, alenbanic acid, alitretinoin, hexamethylmelamine, amifostine, aminoglutethimide, hexyl 5-aminolevulinate, amarupin, acridine, anastrozole, anisolet ravtansin, angiotensin II, antithrombin III, arepinephrine Pyrantel, Acimomab, Arglabin, Arsenic Trioxide, Asparaginase, Atezolizumab, Avelumab, Axitinib, Azacitidine, Belotecone, Bendamustine, Besoxumab, Belistat, Bevacizumab, Bexaroten, Bicalutamide, Bismuth subcitrate, Bleomycin, Boratetumab, Bortezomib, Busereline, Bosutinib, Brentuximab Vedotin, Busulfan, Cabazitaxel l), Cabozantinib, Calcitonin, Leucovorin, Levoleucovorin, Capecitabine, Carolomumab, Carbamipine, Carboplatin, Carboquinone, Carfenomib, Carmoflu, Carmustine, Catumaxomab, Celebrex, Cimointerleukin, Ceritinib, Cetuximab, Chlorpheniramine, Chlormadinone, Nitrogen Mustard, Siddofovir, Cinacalcet, Cisplatin, Cladribine, Clodronate, Clofarabin, Cobimetinib, Copanlisib, Cristatinase, Crizotinib Cyclophosphamide, cyproterone acetate, cytarabine, dacarbazine, actinomycin, daratumumab, dabrafenib, darolutamide, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, diprotopeptide, dilorelin, dextromethorphan, dibromospiramycin, dichlorvos, diclofenac, docetaxel, dolasetron, deoxyfluorouridine, doxorubicin, doxorubicin + estrone, dronabinol, durvalumab, ezetoxam, ezetoxam, ezetoxam Licetamide, Endostatin, Enoxabin, Enzalutamide, Epacadostat, Epirubicin, Cyclothrosine, Epoetin Alpha, Epoetin Beta, Epoetin Zeta, Etaplatin, Eribulin, Ellotinib, Esomeprazole, Estrogenus Estradiol, Etoposide, Etoxicoside, Everolimus, Exemestane, Faldroxil, Fentanyl, Fluorosulfan, Fluorouracil, Fludarabine, Flutamide, Leucovorin, Formaptan, Fosapiram, Formosine, Fulvestrant, Gadoxetine, Gadoxetine Dimethicone, Gadoxetine Dimethicone, Gadoxetine Disodium Garlic nitrate (Gd-EOB-DTPA disodium salt), gallium nitrate, ganiriplastic, gefitinib, gemcitabine, gemtuzumab, carboxypeptidase, glutathione, goserelin, gransilone, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), histamine dihydrochloride, histrelin, hydroxyurea, I-125 particles, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin Bismuth subsalicylate, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incardonic acid, phoryl methylbutenoate, interferon α, interferon β, interferon γ, iodide, iodobenzylguanidine (123I), iodomeprazole, ipilimumab, irinotecan, itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, lasocholine, lenalidomi d) Lenvatinib, Levofloxacin, Mushroom Polysaccharide, Letrozole, Leuprorelin, Levotetramazole, Levonorgestrel, Levothyroxine Sodium, Lipefragrance, Ergotamine, Levoplatin, Cyclohexidine Nitrourea, Clonidamine, Masrophenone, Medroxyprogesterone, Melarsone, Phenylephrine Mustard, Meandromethane, Mercaptopurine, Mesna, Methadone, Methotrexate, Methoxyfusin, Methylaminolevulinate, Methylprednisolone, Methyltestosterone, Methyltyrosine, Mifamurtid, Miterfusin, Miplatin iriplatin), dibromomannitol, propaminazone, dibromoceramide, mitomycin, mitotane, mitoxantrone, mogamulizumab, morasilidine, mopiperazine, morphine hydrochloride, morphine sulfate, nabiximols, nafarelin, naloxone + tebuconazole, naltrexone, natosine, necitumumab, nedaplatin, nelarabin, neridonic acid, netupitant/palonosetron, nivolumab, nivolumab pentetre OTID, Nilotinib, Nilumet, Nimozol, Nimotuzumab, Nitrosine Nitrourea, Nintedanib, Nitracrin, Nivolumab, Atorizumab, Octreotide, Ofatumumab, Olaparib, Olaratumab, Homoharringtonine, Omeprazole, Oxytransilone, Oreolus, Osimertinib, Oxaliplatin, Oxycodone, Oxymethylphenidate, Ozomicin, p53 gene therapy, Paclitaxel, Palbociclib, PA Lifermin, Palladium-103 particles, Palonosetron, Pamidronic acid, Panitumumab, Pabipiridine, Pantoprazole, Pazopanib, Pegappain, Pamumumab, Pegylated interferon alpha-2b, Pembrolizumab, Pemetrexed, Pentostatin, Pelomycin, Perfluorobutane, Pephosphatamide, Pertuzumab, picibanil, Pilocarpine, Pirarubicin, Plexafor, Prucalomycin, Polyglucosamine Lusam), polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, panatinib, porphyrin sodium, pralatrexat, pinesol mustard, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, lardotinib, ranoxifene, raltitrexed, ramusetron, ramucirumab, ranimustin, raburicase, propylthiouracil Refametinib, Regorafenib, Rexadolinate, Rhenium-186 etidronate, Rituximab, Rogalatinib, Rorapipsin, Romotide, Roniciclib, Lecithinam (153Sm), Satumomab, Secretin, Sesuximab, Sipuleucel-T, Cizonan, Sobuzosen, Natrium Glycididazol, Sinide Gem, Sorafenib, Stanozolol, Streptozomycin, Sunitinib, Talamobron, Talimogen, Lagerparepvec, Tamibarbitine, Tamoxifen, Tapentadol, Tasonermin, Teclaeukin, Technetium [99mTc]-Mitomomumab, 99mTc-HYNIC-[Tyr3]-Octreotide, Tegafur, Tegafur + Gimeracil + Ooteracil, Temoporphyrin, Temoporphyrin Mozolomide, sirolimus, epipodophyllotoxin thiophene glycoside, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyroid-stimulating hormone alpha, tioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, treosulfan n), retinoic acid, trifluuridine + tipiracil, trametinib, tramostan, triptorelin, chlorocyclophosphamide, thrombopoietin, ubenmex, valrubicin, vandetanib, valproate, vastatin, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vemodilamide, vorinostat, yttrium-90 glass microspheres, neomycin, fentostatin, zoledronic acid, zorubicin.

Alternatively, antibodies can be selected from MPS1 inhibitors or antibodies targeting OX-40, CD137/4-1BB, DR3, IDO1/IDO2, LAG-3, and CD40.

This invention further provides a combination of the present invention's binding moiety-drug conjugate (ADC) with cancer immunotherapy for the treatment of cancer and tumors. The intrinsic mechanism of action of the cytotoxic binding moiety-drug conjugate includes directly triggering cell death in tumor cells, thereby releasing tumor antigens that can stimulate an immune response. Furthermore, there are indications that KSP inhibitor toxic clusters induce markers of so-called immunogenic cell death (ICD) in vitro. Therefore, the combination of the present invention's binding moiety-drug conjugate (ADC) with one or more therapeutic methods for cancer immunotherapy or with one or more drugs (preferably antibodies) targeting molecular targets derived from cancer immunotherapy constitutes a preferred therapeutic method for cancer and tumors. Examples of therapeutic methods for cancer immunotherapy include immunomodulatory monoclonal antibodies and low molecular weight substances targeting targets derived from cancer immunotherapy, vaccines, CAR T cells, bispecific T cell recruitment antibodies, oncolytic viruses, and cell-based vaccination methods. ii) Examples of selected targets suitable for cancer immunotherapy using immunomodulatory 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, and TLR. Therefore, the combination of the present invention's partial-drug conjugate (ADC) with cancer immunotherapy can initially enhance the immunogenicity of weakly immunogenic tumors and improve the effectiveness of cancer immunotherapy, while also demonstrating durable therapeutic effects.

Furthermore, the compounds of this invention can also be used in combination with radiotherapy and/or surgical interventions.

Typically, the following objectives can be achieved by combining the compounds of the present invention with other cell-inhibiting, cytotoxic, or immunotherapeutic agents:

• Compared to treatment using a single active ingredient, it improves the efficacy of slowing tumor growth, reducing tumor size, or even completely eliminating tumors;

• The possibility of using chemotherapy at a lower dose than a single therapy;

• Compared to single administration, it allows for better-tolerated treatment with fewer side effects;

• The possibility of treating a wider range of cancer conditions;

• Achieve a higher response rate to treatment;

• Patients survive longer compared to current standard treatments.

Furthermore, the compounds of this invention can also be used in combination with radiotherapy and/or surgical interventions.

The present invention also provides a medicine comprising at least one compound of the present invention, generally together with one or more inert, non-toxic, pharmaceutically suitable excipients, and its use for the purposes described above.

The compounds of this invention can act systemically and/or locally. For this purpose, they can be administered in appropriate manner, such as parenteral, possibly by inhalation, or as implants or stents.

The compounds of this invention can act systemically and/or locally. For this purpose, they can be administered in appropriate ways, such as by oral, parenteral, pulmonary, nasal, sublingual, tongue, oral cavity, rectal, vaginal, skin, transdermal, conjunctival, ear routes, or as implants or scaffolds.

The compounds of the present invention can be administered in forms suitable for these routes of administration.

Suitable forms of administration for oral administration are those that work according to the prior art and deliver the compound of the invention rapidly and/or in a modified manner and contain the compound of the invention in crystalline and/or amorphous and/or soluble forms, such as tablets (uncoated or coated tablets, such as those with enteric coatings or insoluble or delayed-dissolving coatings that control the release of the compound of the invention), tablets that disintegrate rapidly in the mouth, or films/sheets, films/lyophilized products, capsules (e.g., hard or soft gelatin capsules), sugar-coated tablets, granules, pills, powders, emulsions, suspensions, aerosols, or solutions.

Parenteral administration can bypass reabsorption steps (e.g., intravenous, intra-arterial, intracardiac, intraspinal, or intralumbar) or involve reabsorption (e.g., intramuscular, subcutaneous, intradermal, percutaneous, or intraperitoneal). Forms suitable for parenteral administration include injectable and infusion formulations in the form of solutions, suspensions, emulsions, lyophilized products, or sterile powders.

Other suitable forms of administration via other routes include, for example, inhaled formulations (including powder inhalers, nebulizers), nasal drops, nasal solutions or sprays; tablets, films/sheets or capsules, suppositories, eye drops, ointments, eye washes, ophthalmic inserts, ear drops, ear sprays, ear powders, ear washes or ear plugs, vaginal capsules, aqueous suspensions (washes, shaken mixtures), lipophilic suspensions, emulsions, microemulsions, ointments, creams, transdermal therapy systems (e.g., patches), lotions, pastes, foams, sprinkles, implants or stents.

Parenteral administration is preferred, especially intravenous administration.

The compounds of the present invention can be converted into the described administration form. This can be accomplished by mixing with pharmaceutically suitable excipients in a manner known per se. These excipients include...

• Fillers and carriers (e.g., cellulose, microcrystalline cellulose such as lactose, mannitol, starch, calcium phosphate, etc.),

• Ointment base (e.g., petrolatum, paraffin, triglycerides, wax, lanolin, lanolin alcohol, hydrophilic ointments, polyethylene glycol),

• Suppository base (e.g., polyethylene glycol, cocoa butter, hardened ester),

Solvents (e.g., water, ethanol, isopropanol, glycerol, propylene glycol, fatty oils of medium-chain triglycerides, liquid polyethylene glycol, paraffin),

Surfactants, emulsifiers, dispersants, or wetting agents (e.g., sodium lauryl sulfate, lecithin, phospholipids, fatty alcohols such as sorbitol fatty acid esters such as polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene fatty acid glycerides such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamer, etc.)

• Buffering substances, as well as acids and bases (e.g., phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, tromethamine, triethanolamine),

• Isotonic agents (e.g., glucose, sodium chloride),

• Adsorbent (e.g., highly dispersed silica),

• Tackifiers, gelling agents, thickeners, or adhesives (e.g., polyvinylpyrrolidone, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, starch, carbomer, polyacrylic acids such as alginate, gelatin),

• Disintegrants (e.g., modified starch, sodium carboxymethyl cellulose, sodium glycolate starch, crospyrrolidone, crospyrrolidone, etc.),

• Flow conditioners, lubricants, flow aids, and release agents (e.g., magnesium stearate, stearic acid, talc, highly dispersed silica, etc.)

• Coating agents (e.g., sugar, shellac), and film-forming agents for films or diffusion films that dissolve rapidly or in modified forms (e.g., polyvinylpyrrolidone such as polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, hydroxypropyl methylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylate, polymethacrylate, etc.).

• Capsule materials (e.g., gelatin, hydroxypropyl methylcellulose),

• Synthetic polymers (e.g., polylactide, polyglycolic acid ester, polymethacrylate, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene oxide, polyethylene glycol and their copolymers and block copolymers),

Plasticizers (e.g., polyethylene glycol, polypropylene glycol, glycerin, triacetin, triethyl citrate, dibutyl phthalate),

• Penetration enhancer

• Stabilizers (e.g., antioxidants such as ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate),

• Preservatives (such as parabens, sorbic acid, thimerosal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate),

• Dyes (e.g., inorganic pigments such as iron oxide and titanium dioxide),

• Fragrances, sweeteners, flavor and/or odor correctors.

The present invention further provides pharmaceutical compositions comprising at least one compound of the present invention, generally together with one or more pharmaceutically suitable excipients, and their use for the purposes described above.

Generally, it has been found that, when administered parenterally, a dosage of about 0.1 to 20 mg/kg, preferably about 0.3 to 7 mg/kg body weight, is advantageous for obtaining effective results.

However, it may be optional to deviate from the stated amounts, more precisely, based on body weight, route of administration, individual response to the active ingredient, the nature of the formulation, and the time or interval of administration. Therefore, in some cases, administering amounts below the minimum stated amount may be sufficient, while in others, the stated upper limit must be exceeded. In cases of administering larger amounts, it may be recommended to divide them into several individual doses throughout the day.

The compounds of the present invention can also be in the form of isotopic variants. Therefore, the present invention includes one or more isotopic variants of the compounds of the present invention, especially deuterium-containing compounds.

The term "isotopic variant" of a compound or reagent is defined as a compound having a non-natural amount of one or more isotopes that constitute such a compound.

The term "isotopic variant of the compound of the present invention" is defined as a compound of the present invention having a non-natural amount of one or more isotopes constituting such a compound.

The expression “non-natural abundance” should be understood as referring to the abundance of this isotope at a frequency higher than its natural frequency. The natural frequencies of isotopes used in this regard can be found in “Isotopic Compositions of the Elements 1997”, Pure Appl. Chem., 70(1), 217-235, 1998.

Examples of such isotopes are stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, such as ^2H (deuterium), ^3H (tritium), ^11C , ^13C , 14C, ^15N , ^17O , ^18O , ^32P , ^{33P , 33S, 34S, 35S, 36S, 18F, 36Cl , 82Br , 123I , 124I , 125I , 129I ,} and ^131I .

Regarding the treatment and/or prevention of the conditions described herein, one or more isotopic variants of the compounds of the invention preferably contain deuterium (“deuterium-containing compounds of the invention”). Isotopic variants of the compounds of the invention incorporating one or more radioactive isotopes such as ^3H or ^14C are beneficial, for example, in studies of drug and/or substrate tissue distribution. These isotopes are particularly preferred due to their ease of incorporation and detectability. Positron-emitting isotopes such as ^18F or ^11C can be incorporated into the compounds of the invention. These isotopic variants of the compounds of the invention are suitable for in vivo imaging applications. The deuterium-containing and ^13C -containing compounds according to the invention can be used in preclinical or clinical studies for mass spectrometry analysis.

Isotopic variants of the compounds of the present invention can typically be prepared by methods known to those skilled in the art, as described herein and/or in the examples, by replacing the reagent with an isotopic variant, preferably a deuterium-containing reagent. Depending on the desired deuteration site, in some cases, deuterium from D₂O can be directly incorporated into the compound or incorporated into a reagent that can be used to synthesize these compounds. The useful reagent for incorporating deuterium into the molecule is deuterium gas. A rapid route for deuteration incorporation is the catalytic deuteration of alkene and alkyne bonds. To directly replace hydrogen with deuterium in functionalized hydrocarbons, metal catalysts (i.e., Pd, Pt, and Rh) can also be used in the presence of deuterium gas. Various deuteration reagents and synthetic units are commercially available from companies such as C/D/NIsotopes, Quebec, Canada; Cambridge Isotope Laboratories Inc., Andover, MA, USA; and CombiPhos Catalysts, Inc., Princeton, NJ, USA.

The term "deuterium-containing compound" is defined as a compound according to the invention, wherein one or more hydrogen atoms are replaced by one or more deuterium atoms, and wherein the frequency of deuterium at each deuterated position in the compound of general formula (I) is higher than about 0.015% of the natural frequency of deuterium. More specifically, in the deuterium-containing compounds of the invention, the frequency of deuterium at each deuterated position in the compound of general formula (I) is higher than 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% at that one or more position, preferably higher than 90%, 95%, 96%, or 97%, and even particularly preferably higher than 98% or 99%. It is evident that the frequency of deuterium at each deuterated position is independent of the frequency of deuterium at other deuterated positions.

By selectively incorporating one or more deuterium atoms into the compounds of the present invention, physicochemical properties (e.g., acidity [C.L. Perrin et al., J. Am. Chem. Soc., 2007, 129, 4490], basicity [C.L. Perrin et al., J. Am. Chem. Soc., 2005, 127, 9641], lipophilicity [B. Testa et al., Int. J. Pharm., 1984, 19(3), 271]) and/or the metabolic characterization of the molecule can be altered, resulting in changes in the ratio of the parent compound to metabolites or the amount of metabolites formed. These changes may lead to specific therapeutic benefits and are therefore preferred in certain circumstances. Decreases in metabolic rate and metabolic conversion rate have been reported where metabolite ratios are altered (A.E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). These changes in exposure to the parent compound and metabolites can have significant consequences for the pharmacodynamics, tolerability, and efficacy of the deuterium-containing compounds of the present invention. In some cases, deuteration reduces or eliminates the formation of unwanted or toxic metabolites and enhances the formation of desired metabolites (e.g., nevirapine: A.M. Sharma et al., Chem. Res. Toxicol., 2013, 26, 410; efavirenz: A.E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). In other cases, the primary effect of deuteration is to reduce systemic clearance. As a result, the biological half-life of the compound increases. Potential clinical benefits include the ability to maintain similar systemic exposure, with reduced peak levels and increased trough levels. This may result in lower side effects and enhanced efficacy depending on the pharmacokinetic/pharmacodynamic relationship of the respective compounds. Examples of this deuterium effect are ML-337 (C.J. Wenthur et al., J. Med. Chem., 2013, 56, 5208) and odantangate (K. Kassahun et al., WO2012/112363). Other cases have also been reported where the reduced metabolic rate leads to increased drug exposure without altering systemic clearance (e.g., rofecoxib: F. Schneider et al., Arzneim. Forsch. Drug. Res., 2006, 56, 295; telaprevir: F. Maltais et al., J. Med. Chem., 2009, 52, 7993). Deuterated drugs exhibiting this effect may have reduced dosing requirements (e.g., lower dose amounts or lower doses to achieve the desired effect) and/or produce a lower metabolite load.

The compounds of this invention may possess multiple potential metabolic attack sites. To optimize the aforementioned effects on physicochemical properties and metabolic characteristics, deuterium-containing compounds of this invention with one or more specific modes of deuterium-hydrogen exchange can be selected. More specifically, the deuterium atoms of (one or more) deuterium-containing compounds of this invention are bonded to carbon atoms and/or located at those sites in the compounds of this invention that are attack sites for metabolic enzymes such as cytochrome P450.

Example

The following examples illustrate the executability of the present invention, but the present invention is not limited to these examples.

Unless otherwise stated, percentages in the following tests and examples are weight percentages; parts are parts by weight. Solvent ratios, dilution ratios, and concentration data for liquid/liquid solutions are based on volumetric measurements in each case.

Synthetic route:

As an example of a working embodiment, the following scheme illustrates an illustrative synthetic route.

In these schemes, according to formula IIa, the ^R4 substituent on amino- ^NHR4 can be replaced by the _Z1- (C＝O)-NH-CH( _CH2C (＝O) _NH2 )-C(＝O) group.

In this context,

_Z1 is a _C1-10 alkyl, _C5-10 aryl or C6-10 aralkyl, _C5-10 heteroalkyl, _C1-10 alkyl- _OC6-10 aryl, _C5-10 heterocycloalkyl, heteroaryl, heteroarylalkyl, C5-10 heteroarylalkoxy, _C1-10 alkoxy, _C6-10 aryloxy, _{C6-10 aryl} -C1-10 alkoxy or _C6-10 aralkyloxy, _C5-10 heteroalkoxy, _C1-10 alkyl- _OC6-10 aryloxy- or _{C5-10 heterocycloalkoxy} , which may be monosubstituted or polysubstituted with the following groups: _-NH2 , -C(＝O)-, _-NH -alkyl, -N( _alkyl ) ₂ -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2- NH2} , -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or –OH,

It may be -H or -( _CH₂ ) _₀-1 -Ox- _{( CH₂CH₂O} ) ^vR₁ ,

x is 0 or 1.

v is a number from 1 to 20, and

^R1 has the definition given in equation (II).

Scheme 1: Synthesis of cysteine-linked ADCs

Option 2: Synthesis of cysteine-linked ADCs

Option 3: Synthesis of lysine-linked ADCs

Option 4: Synthesize cysteine-linked ADCs via hydrolysis of succinate.

This method is specifically designed for ADCs with L1 = # ¹ _-CH2 -# ² to convert these ADCs into an open-chain connection.

Option 5: Synthesis of ADC precursor molecules

[a) HATU, DMF, diisopropylethylamine, RT; b) Zinc chloride, trifluoroethanol, 50°C, EDTA.

Option 6: General methods for synthesizing intermediates and ADC precursors

[a) HATU, DMF, N,N-diisopropylethylamine, RT or EDCl, HOBT, N,N-diisopropylethylamine, DMF, RT; b) _H₂ , 10% Pd-C, MeOH, RT; c) _Z₁ -COOH, EDCl, HOBT, N,N-diisopropylethylamine, DMF, RT or _Z₁- COOH, HATU, N,N-diisopropylethylamine, DMF, RT or ^Z₁- COOSu, N,N-diisopropylethylamine, DMF, RT]

Option 7: Synthesis of ADC precursor molecules with linkers cleavable by podases

[a) HATU, DMF, N,N-diisopropylethylamine, RT or EDCl, HOBT, N,N-diisopropylethylamine, DMF, RT; b) _H₂ , 10% Pd-C, MeOH, RT; c) 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, N,N-diisopropylethylamine, DMF, RT]

Option 8: Synthesis of ADC precursor molecules with linkers cleavable by podases

[a) HATU, DMF, N,N-diisopropylethylamine, RT or EDCl, HOBT, N,N-diisopropylethylamine, DMF, RT; b) _H₂ , 10% Pd-C, MeOH, RT; c) 1,1'-[(1,5-dioxolane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, N,N-diisopropylethylamine, DMF, RT]

In addition, other intermediates according to schemes 6, 7 and 8 can be converted into podase-cleavable ADC and APDC precursors.

As an alternative to the benzyloxycarbonyl group shown in schemes 6-8, other protecting groups established in peptide chemistry can be used, and they can be cleaved using corresponding methods known to those skilled in the art. The choice of protecting group strategy is based on requirements related to compatibility with other structural elements present in the molecule. If any remain, the other protecting groups in the molecule can be removed in a final step.

The synthesis can also optionally be rearranged according to its sequence.

Scheme 9: Synthesis of ADCs with cysteine bonds on a headgroup cleavable by podases

[a) HATU, DMF, N,N-diisopropylethylamine, RT; b) 2-5 TCEP equivalents, PBS pH 7.2, stirred at room temperature for 30 min; c) stirred at room temperature under argon for 90 min, then passed through a PD10 column (G-25, GE Healthcare) and buffered to pH 8, stirred overnight under argon at room temperature, followed by concentration by ultracentrifugation and setting to the desired concentration with PBS at pH 7.2]

Scheme 10: Synthesis of an ADC with a lysine-linked linker cleavable by podase.

[a) HATU, DMF, N,N-diisopropylethylamine, RT; b) _H₂ , 10% Pd-C, methanol 1.5 h, RT; c) 1,1'-[(1,5-dioxolane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, N,N-diisopropylethylamine, DMF, stirred overnight at room temperature; d) AK₂ in PBS, with 5 equivalents of the active ester dissolved in DMSO added, stirred for 60 min at room temperature and under argon, then another 5 equivalents of the active ester dissolved in DMSO added, stirred for 60 min at room temperature and under argon, then purified by PD10 column equilibrated with PBS buffer (pH 7.2) (G-25, GE Healthcare), followed by concentration by ultracentrifugation and setting to the desired concentration with PBS buffer (pH 7.2).]

Scheme 11: Synthesis of ADC precursor with a cleavable apical group of podase.

[a) NaBH(OAc) ₃ , HOAc, dichloromethane, RT; b) Chloroacetyl chloride, _NET3 , DCM, RT; c) L-cysteine, _NaHCO3 , DBU, isopropanol/water, 50°C; d) HATU, DMF, diisopropylethylamine, RT; e) Zinc chloride, trifluoroethanol, 50°C; f) d) HATU, DMF, diisopropylethylamine, RT]

Option 12: Conversion of ADC via transglutaminase coupling

[a: 5 mg of AK3 in DPBS at pH 7.4 (c ~ 10 mg/ml), 6 equivalents of virucidal cluster-adaptor precursor, 50 μl of 12.5 μl (1.25 U) of recombinant bacterial transglutaminase aqueous solution (100 U/ml) and 37.5 μl of DPBS at pH 7.4 were added, and incubated at 37°C for 24 h]

A. Example

Abbreviations and acronyms:

ABCB1 ATP-binding box subfamily B member 1 (synonym for P-gp and MDR1)

abs. (waterless)

Acetyl group

ACN Acetonitrile

aq. containing water, aqueous solution

ATP (adenosine triphosphate)

BCRP is a breast cancer drug resistance protein, an efflux transporter protein.

BEP 2-Bromo-1-ethylpyridinium tetrafluoroborate

Boc tert-butoxycarbonyl

br. Broad peak (in NMR)

Example.

BxPC3 human tumor cell line

ca. approximately, about

CI chemical ionization (in MS)

D doublet (in NMR)

D day

DAR (Drug-Antibody Ratio) (Antibody Loading)

TLC (Thin Layer Chromatography)

Direct chemical ionization of DCI (in MS)

DCM dichloromethane

Dd doublet (in NMR)

DMAP 4-N,N-dimethylaminopyridine

DME 1,2-dimethoxyethane

DMEM (Dürburgring Eagle Medium) is a standardized nutrient medium for cell culture.

DMF N,N-dimethylformamide

DMSO (dimethyl sulfoxide)

DPBS, D-PBS, Duchenne phosphate buffered saline solution

D/P dye (fluorescent dye)/protein ratio

PBS (D-PBS), pH 7.4, from Sigma, No. D8537. Composition: 0.2g KCl, 0.2g _KH₂PO₄ (anhydrous), 8.0g NaCl, 1.15g _Na₂HPO₄ ( _anhydrous ). Add _H₂O to a _final volume of 1L.

Dt double triplet (in NMR)

DTT DL-Dithiothreitol

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

EGFR epidermal growth factor receptor

EI electron bombardment ionization (in MS)

ELISA (Enzyme-Linked Immunosorbent Assay)

eq. equivalent

ESI electrospray ionization (in MS)

ESI -MicroTofq (The name of the mass spectrometer, where Tof = time of flight and q = quadrupole)

FCS Fetal Bovine Serum

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

sat. saturated

GTP guanosine-5'-triphosphate

H hours

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

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

HOAc acetic acid

HOAt 1-hydroxy-7-azabenzotriazole

HOBt 1-hydroxy-1H-benzotriazole hydrate

HOSu N-hydroxysuccinimide

HPLC (High-Performance Liquid Chromatography)

_IC50 half-maximal inhibitory concentration

i.m. via muscle, applied to muscle

i.v. via vein, administered intravenously

conc. thick

KPL-4 human tumor cell line

KU-19-19 Human Tumor Cell Line

LC-MS (Liquid Chromatography-Mass Spectrometry)

LLC-PK1 cells Lewis lung cancer porcine kidney cell line

L-MDR human MDR1 transfected LLC-PK1 cells

LoVo human tumor cell line

M multiplets (in NMR)

MDR1 (Multidrug Resistance Protein 1)

MeCN acetonitrile

Min

MS mass spectrometry

MTT 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl-2H-tetrazonium bromide NCI-H292 human tumor cell line

-Nme- Methyl group bonded to a nitrogen atom

NMM N-methylmorpholine

NMP (N-methyl-2-pyrrolidone)

NMR (Nuclear Magnetic Resonance) Spectroscopy

NMRI mouse strains derived from the Naval Medical Research Institute (NMRI)

Nude mice, laboratory animals

NSCLC (Non-Small Cell Lung Cancer)

PBS phosphate-buffered saline solution

Palladium on Pd/C activated carbon

P-gp P-glycoprotein, a type of transport protein

PNGaseF is an enzyme used to cleave sugars.

quant. (in the context of yield)

Quartet (in NMR)

Quintet (in NMR)

_Rf retention index (in TLC)

RT room temperature

R _{_t} retention time (in HPLC)

S-wave singlet (in NMR)

s.c. Subcutaneous, applied under the skin

SCID mice are experimental mice with severe combined immunodeficiency.

SK-HEP-1 human tumor cell line

t triplet (in NMR)

TBAF tetra-n-butylammonium fluoride

TCEP Tris(2-Carboxyethyl)phosphine

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

Teoc trimethylsilylethoxycarbonyl

Uncle tert

TFA (trifluoroacetic acid)

THF tetrahydrofuran

2,4,6-Tripropyl-1,3,5,2,4,6-Trioxatriphosphacyclohexane 2,4,6-trioxide

U251 human tumor cell line

UV spectrum

v/v volume/volume ratio (for solutions)

Z-benzyloxycarbonyl

amino acid abbreviation

Ala = alanine

Arg = Arginine

Asn = Asparagine

Asp = Aspartic acid

Cys = Cysteine

Glu = Glutamic acid

Gln = glutamine

Gly = Glycine

H is histidine

Ile = Isoleucine

Leu = Leucine

Lys = Lysine

Met = Methionine

Nva = valine

Phe = Phenylalanine

Pro = Proline

Serine

Threonine

Trp = Tryptophan

Tyr = Tyrosine

Valine

HPLC and LC-MS methods:

Method 1 (LC-MS ):

Instrumentation: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8μm 50x1mm; Eluent A: 1L water + 0.25ml 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: 0.40ml/min; UV detection: 208-400nm.

Method 2 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; Column: Waters, BEH300, 2.1x150mm, C18 1.7μm; Eluent A: 1L 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: 0.50ml/min; UV detection: 220nm.

Method 3 (LC-MS):

MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100 series; Column: Agilent ZORBAX Extend-C18 3.0x50mm 3.5-micron; Eluent A: 1L water + 0.01mol ammonium carbonate, Eluent B: 1L acetonitrile; Gradient: 0.0min 98% A → 0.2min 98% A → 3.0min 5% A → 4.5min 5% A; Oven: 40℃; Flow rate: 1.75ml/min; UV detection: 210nm.

Method 4 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; Column: Waters, HSST3, 2.1x50mm, C18 1.8μm; Eluent A: 1L 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: 1.20ml/min; UV detection: 210nm.

Method 5 (LC-MS):

Instruments: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8μm 50x1 mm; Eluent A: 1L water + 0.25ml 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: 0.35ml/min; UV detection: 210-400nm.

Method 6 (LC-MS):

Instruments: Micromass Quattro Premier/Waters UPLC Acquity; Column: ThermoHypersil GOLD 1.9μm 50x1mm; Eluent A: 1L water + 0.5ml 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: 0.3ml/min; UV detection: 210nm.

Method 7 (LC-MS):

Instruments: Agilent MS Quad 6150; HPLC: Agilent 1290; Column: Waters Acquity UPLC HST31.8μm 50x2.1mm; Eluent A: 1L water + 0.25ml 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: 1.20ml/min; UV detection: 205-305nm.

Method 8 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; Column: Waters, HSST3, 2.1x50mm, C18 1.8μm; Eluent A: 1L 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: 1.20ml/min; UV detection: 210nm.

Method 9 : LC-MS-Prep purification method for Examples 181-191 (Method LIND-LC-MS-Prep)

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

or

MS instrument: Waters; HPLC instrument: Waters (Phenomenex Luna 5μC18(2)100A column, AXIA Tech. 50x21.2mm, eluent A: water + 0.05% formic acid, eluent B: acetonitrile with gradient (ULC); flow rate: 40ml/min; UV detection: DAD; 210-400nm).

Method 10 : LC-MS analysis method (LIND_SQD_SB_AQ) used in Examples 181-191

MS instrument: Waters SQD; HPLC instrument: Waters UPLC; Column: Zorbax SB-Aq (Agilent), 50 mm x 2.1 mm, 1.8 μm; Eluent A: Water + 0.025% formic acid, Eluent B: Acetonitrile (ULC) + 0.025% formic acid; Gradient: 0.0 min 98% A - 0.9 min 25% A - 1.0 min 5% A - 1.4 min 5% A - 1.41 min 98% A - 1.5 min 98% A; Oven: 40℃; Flow rate: 0.600 ml/min; UV detection: DAD; 210 nm.

Method 11 (HPLC):

Instrument: HP1100 series

Post: Merck Chromolith SpeedROD RP-18e, 50-4.6mm, catalog number 1.51450.0001; Chromolith Guard Cartridge Kit front post, RP-18e, 5-4.6mm, catalog number 1.51470.0001

Gradient: Flow rate 5 ml/min

Injection volume 5 μl

Solvent A: HClO4 (70%) / Water (4 ml/L)

Solvent B: Acetonitrile

Start with 20% B

0.50 min 20% B

3.00 Min 90% B

3.50 min 90% B

3.51 Min 20% B

4.00 min 20% B

Column temperature: 40℃

Wavelength: 210nm.

Method 12 : (LC-MS):

MS instrument type: Thermo Scientific FT-MS; UHPLC+ instrument type: Thermo Scientific UltiMate 3000; Column: Waters, HSST3, 2.1x75mm, C18 1.8μm; Eluent A: 1L 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: 0.90ml/min; UV detection: 210nm / Optimal integration range: 210-300nm.

Method 13 : (LC-MS):

MS instrument: Waters (Micromass) Quattro Micro; Instrument: Waters UPLC Acquity; Column: Waters BEH C18 1.7μm 50x2.1mm; Eluent A: 1L water + 0.01mol ammonium formate, Eluent B: 1 acetonitrile; Gradient: 0.0min 95% A → 0.1min 95% A → 2.0min 15% A → 2.5min 15% A → 2.5min 10% A → 3.0min 10% A; Oven: 40℃; Flow rate: 0.5ml/min; UV detection: 210nm.

All reactants or reagents whose preparation is not explicitly described below were commercially available from normally available sources. For all other reactants or reagents whose preparation is also not described below and which are not commercially available or from normally unavailable sources, refer to published literature which describes their preparation.

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

MS instrument type: ThermoFisher Scientific LTQ-Orbitrap-XL; HPLC instrument type: Agilent 1200SL; Column: Agilent, POROSHELL 120, 3x150mm, SB-C182.7μm; Eluent A: 1L 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: 0.75ml/min; UV detection: 210nm.

Starting compounds and intermediates:

The preparation of starting compounds and suitable intermediates suitable for the preparation of the compounds of the present invention has been described in WO2015/96982A1 and WO2016/96610 A1.

Intermediates C1 to C73, L1 to L73, F1 to F58 and F82 to F91, F103 to F129, F142 to F156, F163 to F180, F192 to F196, F204 to F207, F209 to F218, F235, F236, F238, F241 to F245, F247, F248 and F254 according to WO2015/96610A1 constitute part of the disclosure of this application. Other intermediates described in WO2016/96610A1 also constitute part of the disclosure of this application. When a compound having a specific number (e.g., intermediate C1, L1 or F1) is mentioned below, it means a compound having those numbers according to WO2015/96982 A1. Other starting compounds and intermediates are described below.

Intermediate C102

(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-{[(benzyloxy)carbonyl]amino}butyric acid

First, similar to intermediate C2, intermediate C52 is reduced and alkylated with benzyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutyrate. Then, the secondary amine group is acylated with ethyl 2-chloro-2-oxoacetate, followed by hydrolysis of the two ester groups with a 2M lithium hydroxide methanol solution.

LC-MS (Method 1): R _{_t} = 1.31 min; MS (ESIpos): m/z = 646 (MH) ^- .

Intermediate C109

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-glutamic acid di-tert-butyl ester

First, the dipeptide derivative β-alanyl-L-glutamic acid di-tert-butyl ester was prepared by conventional peptide chemistry methods, through coupling commercially available N-[(benzyloxy)carbonyl]-β-alanine and L-glutamic acid di-tert-butyl ester hydrochloride (1:1) in the presence of HATU, followed by hydrogenolysis to remove the Z protecting group. The title compound was then prepared by coupling this intermediate with intermediate C102 in the presence of HATU and N,N-diisopropylethylamine, followed by hydrogenation in methanol over 10% palladium/activated carbon at standard hydrogen pressure for 45 min to remove the Z protecting group.

LC-MS (Method 1): R _{_t} = 0.99 min; MS (ESIpos): m/z = 826[M+H] ^⁺ .

Intermediate C111

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-D-glutamic acid di-tert-butyl ester

Similar to intermediate C109, synthesize the title compound.

LC-MS (Method 1): R _{_t} = 1.06 min; MS (ESIpos): m/z = 826[M+H] ⁺ .

Intermediate C116

Trifluoroacetic acid/N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(2-{[-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]amino}ethyl)amino]-1-oxobut-2-yl}-L-asparagine

Trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]amino}ethyl)butyramide (1:1) (81.0 mg, 100 μmol) (intermediate F104) and ^N2- (tert-butoxycarbonyl)-L-asparagine 2,5-dioxopyrrolo-1-yl ester (43.0 mg, 131 μmol) were dissolved in 5.0 ml DMF. The reaction mixture was stirred with N,N-diisopropylethylamine (61 μl, 350 μmol) at RT for another 1 h, and then directly purified by preparative RP-HPLC (column: Chromatorex 125x30; 10 μm, flow rate: 75 mL/min, MeCN/water, 0.1% TFA). The solvent was evaporated under reduced pressure and the residue was lyophilized. This yielded 84 mg (88% of the theoretical value) of the compound [(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]amino}ethyl)amino]-1-oxobut-2-yl}amino)-1,4-dioxobut-2-yl]carbamate tert-butyl. LC-MS (Method 1): R _{_t} = 1.09 min; MS (ESIpos): m/z = 907 [M+H] ^₊

[(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]amino}ethyl)amino]-1-oxobut-2-yl}amino)-1,4-dioxobut-2-yl]tert-butyl carbamate (83.0 mg, 91.5 μmol) was dissolved in 5.0 mL of trifluoroethanol. The reaction mixture was mixed with zinc chloride (74.8 mg, 549 μmol) and stirred at 50 °C for 15 min. The mixture was mixed with ethylenediamine-N,N,N',N'-tetraacetic acid (160 mg, 549 μmol) and diluted with 5.0 mL of acetonitrile/water. TFA (20 μl) was added, and the mixture was stirred for another 10 min. The mixture was filtered through a syringe filter and purified by preparative RP-HPLC (column: Chromatorex 125x30; 10 μm, flow rate: 75 mL/min, MeCN/water, 0.1% TFA). The solvent was evaporated under reduced pressure, and the residue was dried under high vacuum. 50 mg (58% of theoretical value) of the title compound was given.

LC-MS (Method 1): R _{_t} = 0.81 min; MS (ESIpos): m/z = 807 [M+H] ⁺

Intermediate C118

N-[(8S)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]ethyl}-2,2-dimethyl-6,9-dioxo-5-oxa-7,10-diaza-2-silazode-12-yl]-D-α-glutamine tert-butyl ester

The title compound was prepared by conventional peptide chemistry methods, by coupling intermediates L119 and C58 in the presence of HATU and then removing the Z protecting group by hydrogenolysis.

LC-MS (Method 1): R _{_t} = 1.05 min; MS (ESIpos): m/z = 885(M+H) ⁺ .

Intermediate C119

N-[(8S)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]ethyl}-2,2-dimethyl-6,9-dioxo-5-oxa-7,10-diaza-2-silazode-12-yl]-D-α-glutamine glycyl tert-butyl ester

Intermediate C119 was prepared by conventional peptide chemistry methods by coupling N-[(benzyloxy)carbonyl]glycine 2,5-dioxopyrrolidone-1-yl ester and intermediate C118 in the presence of HATU, followed by removal of the Z protecting group and hydrogenation at room temperature in methanol/dichloromethane on 10% palladium/activated carbon under standard hydrogen pressure.

LC-MS (Method 1): R _{_t} = 1.03 min; MS (ESIpos): m/z = 942(M+H) ⁺ .

Intermediate C121

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

The title compound was prepared by coupling D-glutamic acid dibenzyl ester (previously released from its p-toluenesulfonate by partitioning between ethyl acetate and 5% sodium bicarbonate solution) with intermediate C61 in the presence of HATU and N,N-diisopropylethylamine, followed by deprotection of the Teoc protecting group with zinc chloride/trifluoroethanol.

LC-MS (Method 1): R _{_t} = 1.05 min; MS (ESIpos): m/z = 894 [M+H] ⁺ .

Intermediate C122

N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-N ^2- (tert-butoxycarbonyl)-D-α-glutamine tert-butyl ester

The title compound was prepared by coupling N-(2-aminoethyl)-N2-(tert-butoxycarbonyl) ^-α -glutamine trifluoroacetate with intermediate C102 in the presence of HATU and N,N-diisopropylethylamine, followed by hydrogenolysis to remove the Z protecting group.

LC-MS (Method 1): R _{_t} = 1.06 min; MS (ESIpos): m/z = 841[M+H] ⁺ .

Intermediate C123

N-[2-({(2S)-2-(L-asparagine amide)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-N ^2- (tert-butoxycarbonyl)-D-α-glutamine tert-butyl ester

The title compound was prepared by coupling ^N2 -[(benzyloxy)carbonyl]-L-asparagine 4-nitrobenzene ester with intermediate C122 in DMF in the presence of N,N-diisopropylethylamine, followed by hydrogenation at RT under standard hydrogen pressure on 10% palladium/activated carbon in DCM/methanol 1:1.

LC-MS (Method 1): R _{_t} = 0.98 min; MS (ESIpos): m/z = 955 [M+H] ^⁺ .

Intermediate C130

{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate 9H-fluorene-9-ylmethyl ester

First, methyl 9H-fluorene-9-ylcarbamate (2.50 g, 3.94 mmol) of [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate (intermediate C67) and triethylamine (1.6 mL, 12 mmol) were added to dichloromethane (200 mL). Chloroacetyl chloride (2.23 g, 19.7 mmol) was added, and the reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed with 10% citric acid solution, water, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic phase was then dried over magnesium sulfate, filtered, and concentrated. The residue was used without further purification. 1.7 g of the title compound was given.

Intermediate C131

S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propyl)amino]-2-oxoethyl}-L-cysteine tert-butyl ester

Under argon atmosphere, TCEP (303 mg, 1.06 mmol) was added to an initial feed of di-tert-butyl L-cysteine dihydrochloride (135 mg, 317 μmol) in 6.0 mL of water and 7.5 mL of isopropanol. The reaction mixture was stirred at room temperature for 30 min. Subsequently, a solution of 9H-fluorene-9-yl methyl carbamate (300 mg, 422 μmol) and 1,8-diazabicyclo(5.4.0)undec-7-ene (760 μl, 5.1 mmol) in 1.5 mL of isopropanol was added, and the reaction mixture was stirred at 50 °C for 1 h. The mixture was diluted with ethyl acetate, and the organic phase was washed with water and a saturated sodium chloride solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue can be used further without purification. 360 mg of the title compound was obtained.

LC-MS (Method 1): R _{_t} = 1.17 min; MS (ESIpos): m/z = 851 [M+H] ⁺

Intermediate C132

S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propyl)amino]-2-oxoethyl}-N-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentoxa-5-azaeicosano-20-yl)-L-cysteine tert-butyl ester

S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[(9H-fluorene-9-ylmethoxy)carbonyl]amino}propyl)amino]-2-oxoethyl}-L-cysteine tert-butyl ester (361 mg, 424 μmol) was dissolved in anhydrous DMF (3 mL), and 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaeicosano-20-oleic acid (186 mg, 509 μmol) was added. HATU (193 mg, 509 μmol) and N,N-diisopropylethylamine (300 μl, 1.7 mmol) were added to the mixture, and the reaction mixture was stirred at room temperature for 5 minutes. The mixture was quenched with 1 ml of water and 0.1% TFA, and then directly purified by preparative HPLC (elution buffer: ACN/water + 0.1% TFA, gradient). 167 mg of the target compound was obtained.

LC-MS (Method 1): R _{_t} = 1.56 min; MS (ESIpos): m/z = 1198 [M+H] ⁺

Intermediate C133

S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentoxa-5-azaeicosano-20-yl)-L-cysteine tert-butyl ester

Morpholine (160 μl, 1.8 mmol) was added to a DMF (5 mL) solution of S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[(9H-fluorene-9-ylmethoxy)carbonyl]amino}propyl)amino]-2-oxoethyl}-N-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxa-5-azaeicosano-20-yl)-L-cysteine tert-butyl ester (214 mg, 178 μmol), and the mixture was stirred at room temperature for 5 hours. The mixture was quenched with water + 0.1% TFA and directly purified by preparative HPLC (acetonitrile/water + 0.1% TFA gradient) to give 111 mg of the target compound.

LC-MS (Method 1): R _{_t} = 1.03 min; MS (ESIpos): m/z = 976 [M+H] ⁺

Intermediate C134

S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[N2-(pyridin-4-ylacetyl)-L-asparaginyl]amino}propyl)amino]-2-oxoethyl}-N-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentoxa-5-azaeicosano-20-yl)-L-cysteine tert-butyl ester

S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentoxa-5-azaeicosano-20-yl)-L-cysteine tert-butyl ester (27.2 mg, 27.9 μmol) and ^N2- (pyridin-4-ylacetyl)-L-asparagine (8.40 mg, 33.4 μmol, intermediate L136) were dissolved in anhydrous DMF (3 ml), and HATU (12.7 mg, 33.4 μmol) and N,N-diisopropylethylamine (15 μl, 84 μmol) were added. The reaction mixture was stirred at room temperature for 10 minutes. The mixture was quenched with 1 ml of water and 0.1% TFA, and then directly purified by preparative HPLC (elution buffer: ACN/water + 0.1% TFA, gradient). 23 mg of the target compound was obtained.

LC-MS (Method 12): R _{_t} = 2.12 min; MS (ESIpos): m/z = 1209 [M+H] ⁺

Intermediate C135

N-(15-amino-4,7,10,13-tetraoxapentadecan-1-acyl)-S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[N2-(pyridin-4-ylacetyl)-L-asparaginyl]amino}propyl)amino]-2-oxoethyl}-L-cysteine trifluoroacetate

S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[N ^2- (pyridin-4-ylacetyl)-L-asparaginyl]amino}propyl)amino]-2-oxoethyl}-N-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-penta-5-azaeicosano-20-yl)-L-cysteine tert-butyl ester (63.6 mg, 52.6 μmol) was dissolved in trifluoroethanol (3.0 mL, 41 mmol), and zinc chloride (43.0 mg, 316 μmol) was added. The reaction mixture was stirred at 50 °C for 2 hours and 20 minutes. An additional 6 equivalents of _ZnCl₂ were added, and the mixture was stirred at 50 °C for 1 hour. Ethylenediamine-N,N,N',N'-tetraacetic acid (184 mg, 631 μmol) was added to the mixture and allowed to cool to room temperature. Water (+0.1% TFA) was added to the reaction mixture, which was then filtered and purified by preparative RP-HPLC (flow rate: 50 mL/min, MeCN/water, 0.1% TFA). The solvent was evaporated under reduced pressure, and the residue was lyophilized. 40 mg of the title compound was given.

LC-MS (Method 1): R _{_t} = 0.75 min; MS (ESIneg): m/z = 1051 [MH] ^-

Intermediate C136

[2-({(2S)-2-(L-asparagine amide)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]carbamate trifluoroacetate

First, intermediate C58 was coupled with benzyl 2-aminoethylcarbamate hydrochloride (1:1) in the presence of HATU and N,N-diisopropylethylamine. The Teoc protecting group was then removed by stirring in trifluoroethanol containing 8 equivalents of zinc chloride at 50 °C for 2 h. Next, the resulting intermediate was coupled with ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester in the presence of N,N-diisopropylethylamine. In the final step, the Boc protecting group was removed by stirring in trifluoroethanol containing 6 equivalents of zinc chloride at 50 °C for 1 h to obtain the target compound. LC-MS (Method 1): R_{_t} = 0.91 min; MS (ESIpos): m/z = 804 [M+H] ^⁺ .

Intermediate C137

N-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]sulfonyl}ethyl)-N ² -[(benzyloxy)carbonyl]-D-α-glutamine benzyl ester trifluoroacetate

The title compound was prepared from intermediate L81 by coupling to intermediate C58 in the presence of HATU and N,N-diisopropylethylamine. In the next step, the Z protecting group was removed by hydrogenation for 30 min at room temperature on 10% palladium/activated carbon in DCM/methanol 1:1 over 10% hydrogen at standard hydrogen pressure. The deprotected intermediate was then converted to the title compound by coupling with (2R)-5-(benzyloxy)-2-{[(benzyloxy)carbonyl]amino}-5-oxovalerate in the presence of 2 equivalents of HATU and 3 equivalents of N,N-diisopropylethylamine, and finally by deprotection with 6 equivalents of zinc chloride (stirred in trifluoroethanol at 50 °C for 1 h).

LC-MS (Method 12): R _{_t} = 1.89 min; MS (ESIpos): m/z = 1001(M+H) ⁺ .

Intermediate C138

N-[(8S)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]ethyl}-2,2-dimethyl-13,13-dioxo-6,9-dioxo- ⁵ -oxa-13λ6-thia-7,10-diaza-2-silazopentane-15-yl]-D-α-glutamine tert-butyl ester

The title compound was prepared from intermediate C58 by coupling to intermediate L81 in the presence of HATU and N,N-diisopropylethylamine. In the next step, the Z protecting group was removed by hydrogenation for 1 hour at room temperature on 10% palladium/activated carbon in DCM/methanol 1:1 at standard hydrogen pressure. The deprotected intermediate was then converted to the title compound by coupling with (2R)-2-{[(benzyloxy)carbonyl]amino}-5-tert-butoxy-5-oxovalerate in the presence of HATU and N,N-diisopropylethylamine. Finally, the Z protecting group was removed by hydrogenation for 2 hours at room temperature on 10% palladium/activated carbon in DCM/methanol 1:1.

LC-MS (Method 1): R _{_t} = 1.14 min; MS (ESIpos): m/z = 977(M+H) ⁺ .

Intermediate C139

N-{(2S)-2-(L-asparagine amide)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-D-glutamic acid di-tert-butyl ester

The title compound was synthesized by coupling intermediate C111 to 1.5 equivalents of ^N2 -[(benzyloxy)carbonyl]-L-aspartic acid 4-nitrobenzene in DMF in the presence of 3 equivalents of N,N-diisopropylethylamine, followed by removal of the Z protecting group by hydrogenation at standard hydrogen pressure and room temperature in 10% palladium/activated carbon in DCM/methanol 1:1 for 2 h.

LC-MS (Method 1): R _{_t} = 1.02 min; MS (ESIpos): m/z = 940[M+H] ⁺ .

Intermediate C140

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl- ^N1 , ^N5 -dibenzyl-D-glutamine trifluoroacetate

First, ^N1 , ^N5 -dibenzyl-D-glutamine trifluoroacetate was prepared by coupling N-(tert-butoxycarbonyl)-D-glutamic acid with benzylamine in the presence of HATU and N,N-diisopropylethylamine, followed by removal of the Boc protecting group using TFA. Then, this intermediate was coupled with Boc-β-alanine in the presence of HATU and N,N-diisopropylethylamine, and the Boc protecting group was removed using a DCM solution of TFA. The resulting compound was then coupled with intermediate C58 in the presence of HATU and N,N-diisopropylethylamine, and finally, the Teoc protecting group was removed using a zinc chloride-trifluoroethanol solution to prepare the title compound.

LC-MS (Method 1): R _{_t} = 0.95 min; MS (ESIpos): m/z = 892[M+H] ^⁺ .

Intermediate C141

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}-β-alanyl-L-asparagine trifluoroacetate

First, intermediate C61 was coupled with L-aspartic acid tert-butyl ester in the presence of 1.5 equivalents of HATU and 3 equivalents of N,N-diisopropylethylamine. Subsequently, the Teoc protecting group and tert-butyl ester were removed by stirring with a solution of 6 equivalents of zinc chloride in trifluoroethanol at 50 °C, thus yielding the title compound.

LC-MS (Method 1): R _{_t} = 0.95 min; MS (ESIpos): m/z = 892[M+H] ^⁺ .

Intermediate C142

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl ester

To a DMF (10 mL) solution of N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanine (745 mg, 1.02 mmol, intermediate C61), D-glutamic acid di-tert-butyl hydrochloride (363 mg, 1.23 mmol), HATU (505 mg, 1.33 mmol), and N,N-diisopropylethylamine (530 μl, 3.1 mmol) were added, and the reaction mixture was stirred at room temperature for 10 min. The reaction mixture was mixed with ethyl acetate and washed with water and saturated sodium chloride solution. The organic phase was dried over magnesium sulfate and concentrated, and the residue was purified by preparative RP-HPLC (gradient, MeCN/water + 0.1% TFA). The solvent is evaporated under reduced pressure, and the residue is dried under high vacuum.

LC-MS (Method 1): R _{_t} = 1.58 min; MS (ESIpos): m/z = 970 [M+H] ⁺

Intermediate C143

N-[(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}{[(methylsulfonyl)oxy]acetyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl ester

At 0 °C, a solution of N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}-amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl ester (257 mg, 264 μmol of intermediate C142) in dichloromethane (20 mL) was mixed with methanesulfonyl chloride (49 μl, 630 μmol) and triethylamine (92 μl, 660 μmol), and the reaction mixture was stirred at 0 °C for 1 hour. The mixture was then diluted with dichloromethane and washed with saturated sodium bicarbonate solution (3×) and saturated sodium chloride solution. The organic phase was dried over magnesium sulfate and concentrated; the residue was further converted without further purification.

LC-MS (Method 1): R _{_t} = 1.52 min; MS (ESIpos): m/z = 1048 [M+H] ⁺

Intermediate C144

N-[(2S)-4-[({[(2R)-2-amino-3-tert-butoxy-3-oxopropyl]thioalkyl(sulphanyl)}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-({[2-(trimethyl-silyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl trifluoroacetate

Under argon atmosphere, TCEP (687 mg, 2.40 mmol) was added to the initial feed of L-cysteine di-tert-butyl dihydrochloride (306 mg, 719 μmol) in 25 ml of water and 50 ml of isopropanol. The reaction mixture was stirred at room temperature for 30 minutes. Subsequently, a solution of N-[(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}{[(methyl-sulfonyl)oxy]acetyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl ester (1.00 g, 958 μmol of intermediate C143) and 1,8-diazabicyclo(5.4.0)undec-7-ene (1.7 mL, 11 mmol) in 35 mL of isopropanol was added, and the reaction mixture was stirred at 50 °C for 14 hours. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed with water and saturated sodium chloride solution. The organic phase was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by preparative RP-HPLC (gradient, MeCN/water + 0.1% TFA). The solvent was evaporated under reduced pressure, and the residue was dried under high vacuum. 923 mg of the title compound was obtained.

LC-MS (Method 12): R _{_t} = 2.46 min; MS (ESIpos): m/z = 1129 [M+H] ⁺

Intermediate C145

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}({[(2R)-3-tert-butoxy-2-({N ² -[(9H-fluoren-9-ylmethoxy)carbonyl]-L-asparaginyl}amino)-3-oxopropyl]thioalkyl}acetyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl ester

To N-[(2S)-4-[({[(2R)-2-amino-3-tert-butoxy-3-oxopropyl]thioalkyl}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl ester trifluoroacetic acid (60.0 mg, 48.2 μmol intermediate C144) and N ² HATU (22.0 mg, 57.9 μmol) and N,N-diisopropylethylamine (25 μl, 140 μmol) were added to a DMF (3.0 mL) solution of -[(9H-fluorene-9-ylmethoxy)carbonyl]-L-asparagine (20.5 mg, 57.9 μmol), and the reaction mixture was stirred at room temperature for 10 min. 1 mL of water + 0.1% TFA was added to the mixture, and it was directly purified by preparative HPLC (elution: ACN/water + 0.1% TFA, gradient). 71 mg of the target compound was obtained.

LC-MS (Method 12): R _{_t} = 3.15 min; MS (ESIpos): m/z = 1466 [M+H] ⁺

Intermediate C146

N-[(2S)-4-[({[(2R)-2-(L-asparagine)-3-tert-butoxy-3-oxopropyl]thioalkyl}-acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl trifluoroacetate

First, N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}({[(2R)-3-tert-butoxy-2-({ ^N2 -[(9H-fluoren-9-ylmethoxy)carbonyl]-L-asparagineyl}amino)-3-oxopropyl]thioalkyl}acetyl)amino]-2-({[2-(trimethylsilyl)ethoxy]-carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid di-tert-butyl ester (63.5 mg, 43.3 μmol, intermediate C145) was added to DMF (3 ml), followed by morpholine (38 μl, 430 μmol), and the mixture was stirred at room temperature for 18 hours. Add 1 ml of water and 0.1% TFA to the mixture, and purify it directly by preparative HPLC (elution buffer: ACN/water + 0.1% TFA, gradient). 38 mg of the target compound was obtained.

LC-MS (Method 1): R _{_t} = 1.25 min; MS (ESIpos): m/z = 1243 [M+H] ⁺

Intermediate L81

Benzyl trifluoroacetic acid/{2-[(2-aminoethyl)sulfonyl]ethyl}carbamate

In the presence of N,N-diisopropylethylamine, 250 mg (1.11 mmol) of 2,2'-sulfonyl diethylamine was coupled with 92.3 mg (0.37 mmol) of 1-{[(benzyloxy)carbonyl]oxy}pyrrolidine-2,5-dione in DMF. The mixture was then purified by HPLC to give 70 mg (47% of the theoretical value) of the title compound.

C-MS (Method 12): R _{_t} = 0.64 min; MS (ESIpos): m/z = 257.11(M+H) ⁺ .

Intermediate L103

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

The title compound was prepared by conventional peptide chemistry methods by first coupling 4-pyridineacetic acid with commercially available L-alanyl-L-alanine tert-butyl ester in the presence of HATU and N,N-diisopropylethylamine, then deprotecting it with trifluoroacetic acid, coupling it with L-aspartic acid tert-butyl ester, and then deprotecting the carboxyl group with trifluoroacetic acid.

LC-MS (Method 1): R _{_t} = 0.15 min; MS (ESIpos): m/z = 394(M+H) ⁺ .

Intermediate L119

N-(2-aminoethyl) ^-N2 -[(benzyloxy)carbonyl]-D-α-glutamine tert-butyl trifluoroacetate

Intermediate L119 was prepared by conventional peptide chemistry methods, in the presence of HATU, by coupling commercially available (2R)-2-{[(benzyloxy)carbonyl]amino}-5-tert-butoxy-5-oxovaleric acid (1.00 g, 2.96 mmol) with (2-aminoethyl)carbamate tert-butyl ester (560 μl, 3.6 mmol), followed by acidic removal of the Boc protecting group with a dichloromethane solution of TFA.

LC-MS (Method 1): R _{_t} = 0.62 min; MS (ESI-pos): m/z = 380(M+H) ⁺ .

Intermediate L136

N ^2- (pyridin-4-ylacetyl)-L-asparagine

The title compound was prepared by coupling pyridin-4-ylacetic acid hydrochloride with L-aspartic acid tert-butyl ester in the presence of HATU and N,N-diisopropylethylamine, followed by removal of the tert-butyl protecting group by dichloromethane solution of trifluoroacetic acid.

LC-MS (Method 12): R _{_t} = 0.65 min; MS (ESIneg): m/z = 250 [MH ^]

Intermediate L137

N ^2- (pyridin-4-ylacetyl)-L-glutamine

The title compound was prepared by coupling pyridin-4-ylacetic acid hydrochloride (1:1) with L-glutamic acid tert-butyl ester in the presence of HATU and N,N-diisopropylethylamine, followed by removal of the tert-butyl protecting group by dichloromethane solution of trifluoroacetic acid.

LC-MS (Method 12): R _{_t} = 0.59 min; MS (ESIneg): m/z = 264 [MH ^]

Intermediate L138

1-Bromo-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecyl-18-oleic acid

The title compound was prepared by coupling 1-amino-3,6,9,12-tetraoxapentadecan-15-oleic 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 F104

Trifluoroacetic acid (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-amino}ethyl)butyramide

The title compound was prepared starting from intermediate C58. First, 15 mg (0.023 mmol) of intermediate C58 was reacted with 11 mg (0.036 mmol) of intermediate L1 in the presence of 13 mg (0.034 mmol) HATU and 10 μl N,N-diisopropylethylamine. After stirring at room temperature for 60 min, the mixture was concentrated, and the residue was purified by preparative HPLC. 12.3 mg (63% of the theoretical value) of the protected intermediate was obtained.

LC-MS (Method 1): R _{_t} = 1.3 min; MS (EIpos): m/z = 837[M+H] ⁺ .

In the second step, the intermediate was dissolved in 3 mL of 2,2,2-trifluoroethanol. 12 mg (0.088 mmol) of zinc chloride was added, and the mixture was stirred at 50 °C for 2 hours. Subsequently, 26 mg (0.088 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid and 2 mL of 0.1% trifluoroacetic acid aqueous solution were added. The mixture was purified by preparative HPLC. After concentrating the appropriate fraction and freeze-drying the residue from acetonitrile/water, 8.1 mg (68% of the theoretical value) of the title compound was given.

LC-MS (Method 1): R _{_t} = 0.89 min; MS (ESIpos): m/z = 693(M+H) ⁺ .

General methods for synthesizing APDC or ADC precursors (intermediate series S)

The F-series (F1-F305) intermediates previously described in prior disclosures WO2015/96982A1 and WO2016/096610A1 can optionally be converted into APDC precursor S after adapting to a synthetic route or protecting group strategy. As shown by way of example in Schemes 1a and 1b, in the case of releasing the N-terminal amino group of asparagine that is cleavable by podase in the APDC precursor molecule, this can be modified in a final step with substituted acyl or alkyl groups having various structures to improve the performance profile. Protein-reactive groups (e.g., maleimide or active ester) can optionally be introduced into the synthesis at a later time.

An exemplary method is described here:

Dissolve 0.037 mmol of intermediate F1-Fx in 1-20 ml, preferably 5-10 ml, of a suitable solvent, such as DMF, DMSO, DCM, chloroform, toluene, THF, methanol, or mixtures thereof. Add 0.039 mmol of an N-terminal modified aspartic acid derivative, such as intermediate L136, 0.041 mmol of a standard coupling agent, such as HATU, EDCI/HOBT, BEP, etc., and 0.11 mmol of a standard base, such as N,N-diisopropylethylamine, triethylamine, 4-methylmorpholine, etc. After stirring at RT for 5 min, acidify the mixture with 2 drops of trifluoroacetic acid and concentrate. Purify the residue by preparative HPLC. Concentrate the appropriate fraction under reduced pressure and lyophilize the residue from acetonitrile/water.

When the N-terminal modification of the attached tripeptide derivative is a protecting group, it can subsequently be removed by known methods, for example, preferably by hydrogenolysis to remove the Z protecting group, by acid cleavage to remove the Boc protecting group, by alkaline cleavage to remove the Fmoc protecting group, or by fluoride or by zinc chloride to remove the Teoc group.

Finally, the released amino groups can be acylated or alkylated, for example, with amine reactive groups such as active esters, acyl chlorides, isocyanates, etc., or by coupling with carboxylic acid derivatives in the presence of standard coupling agents such as HATU, EDCI/HOBT, BEP, etc., and standard bases such as N,N-diisopropylethylamine, triethylamine, 4-methylmorpholine, etc., to improve the distribution of properties. If they are still present, other protecting groups in the molecule can be removed in the final step.

Option 1a:

[a) HATU, DMF, N,N-diisopropylethylamine, RT or EDCI, HOBT, N,N-diisopropylethylamine, DMF, RT; b) _H₂ , 10% Pd-C, MeOH, RT; c) _Z₁ -COOH, EDCI, HOBT, N,N-diisopropylethylamine, DMF, RT or _Z₁- COOH, HATU, N,N-diisopropylethylamine, DMF, RT or _Z₁- COOSu, N,N-diisopropylethylamine, DMF, RT]

Option 1b:

[a): ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester, DMF, N,N-diisopropylethylamine, RT; b) _ZnCl2 , trifluoroethanol, 50°C; c) _Z1- COOH, EDCI, HOBT, N,N-diisopropylethylamine, DMF, RT or _Z1- COOH, HATU, N,N-diisopropylethylamine, DMF, RT or _Z1- COOSu, N,N-diisopropylethylamine, DMF, RT]

Furthermore, other intermediates according to schemes 2a and 3a can be converted into ADC precursors that are cleavable by podase:

Option 2a:

[a): HATU, DMF, N,N-diisopropylethylamine, RT or EDCI, HOBT, N,N-diisopropylethylamine, DMF, RT; or, acylation may also be carried out in DMF in the presence of N,N-diisopropylethylamine with ^N2 -[(benzyloxy)carbonyl]-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester; b) _H2 , 10% Pd-C, MeOH, RT; c) 1,1'-[(1,5-dioxolane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, N,N-diisopropylethylamine, DMF, RT] Scheme 3a:

[a): HATU, DMF, N,N-diisopropylethylamine, RT or EDCI, HOBT, N,N-diisopropylethylamine, DMF, RT; or, acylation may also be carried out in DMF in the presence of N,N-diisopropylethylamine with ^N2 -[(benzyloxy)carbonyl]-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester; b) _H2 , 10% Pd-C, MeOH, RT; c) 1,1'-[(1,5-dioxolane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, N,N-diisopropylethylamine, DMF, RT]

As an alternative to the benzyloxycarbonyl group shown in schemes 1-3, other protecting groups established in peptide chemistry can be used, and they can be removed using corresponding methods known to the art. The choice of protecting group strategy is based on the requirement of compatibility with other structural elements present in the molecule, as known to those skilled in the art. If they still exist, other protecting groups in the molecule can be removed in a final step.

The synthesis can also be arbitrarily rearranged according to its sequence.

Furthermore, the protein reactive groups in the context of the connector structures L1-L2 can vary within the scope of the claims.

intermediate S1

N ¹ -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]amino}ethyl)amino]-1-oxobut-2-yl}-N ² -(pyridin-4-ylacetyl)-L-asparagine

5 mg (0.0062 mmol) of intermediate F104 was dissolved in 2 mL of DMF and coupled with 1.9 mg (0.0074 mmol) of intermediate L136 in the presence of 3.5 mg (0.0093 mmol) of HATU and 3 μl of N,N-diisopropylethylamine. After stirring at room temperature for 16 hours and purification by preparative HPLC, 2.8 mg (49% of the theoretical value) of the title compound was obtained.

LC-MS (Method 1): R _{_t} = 0.86 min; MS (ESIpos): m/z = 926(M+H) ⁺ .

intermediate S2

^N2 -acetyl- ^N1 -{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]amino}ethyl)amino]-1-oxobut-2-yl}-L-asparagine

In the presence of 3 μl N,N-diisopropylethylamine, a solution of 6 mg (0.0065 mmol) of intermediate C116 in 2 mL of DMF was coupled with 2 mg (0.013 mmol) of 1-acetoxypyrrolidine-2,5-dione. After purification by preparative HPLC, 5 mg (90% of the theoretical value) of the title compound was obtained. LC-MS (Method 1): R_{_t} = 0.95 min; MS (ESIpos): m/z = 849 (M+H)^⁺ .

intermediate S3

[(2S)-4-amino-1-({(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]amino}ethyl)amino]-1-oxobut-2-yl}amino)-1,4-dioxobut-2-yl]benzyl carbamate

In the presence of 5.7 mg (0.0149 mmol) HATU and 9 μl N,N-diisopropylethylamine, a DMF solution of 10 mg (0.0124 mmol) of intermediate F104 was coupled with 3.6 mg (0.0136 mmol) of ^N₂ -[(benzyloxy)carbonyl]-L-asparagine. After stirring at room temperature for 30 min and purification by preparative HPLC, 6 mg (51% of the theoretical value) of the title compound was obtained.

LC-MS (Method 12): R _{_t} = 2.06 min; MS (ESIneg): m/z = 939 (MH) ^- .

intermediate S4

N-[2-({(2S)-2-[(N ² -acetyl-L-asparaginyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-D-α-glutamine

In the presence of 8 μl N,N-diisopropylethylamine, 15 mg (0.016 mmol) of intermediate C123 in 3 mL DMF was coupled with 7.4 mg (0.047 mmol) of 1-acetoxypyrrolidine-2,5-dione. After preparative HPLC purification, 10 mg (64% of theoretical value) of the protected intermediate was obtained. The tert-butyl ester and Boc protecting group were then separated by stirring with a solution of 6 equivalents of zinc chloride in trifluoroethanol at 50 °C for 3 h. In the final step, 6 mg (0.006 mmol) of the resulting intermediate was converted to the title compound by coupling to 1.9 mg (0.008 mmol) of 1-{2-[(2,5-dioxopyrrolidine-1-yl)oxy]2-oxoethyl}-1H-pyrrole-2,5-dione in DMF. After purification by preparative HPLC, 3 mg (49% of the theoretical value) of the title compound was obtained.

LC-MS (Method 1): R _{_t} = 0.91 min; MS (ESIpos): m/z = 978(M+H) ⁺ .

intermediate S5

N-(2-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-{[N ^2- (pyridin-4-ylacetyl)-L-asparaginyl]amino}butyryl]amino}ethyl)-N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-D-α-glutamine

In the presence of 7.2 mg (0.019 mmol) HATU and 14 μl N,N-diisopropylethylamine, 15 mg (0.016 mmol) of intermediate C123 in 5.6 mL DMF was coupled with 3.3 mg (0.019 mmol) pyridin-4-ylacetic acid hydrochloride (1:1). After preparative HPLC purification, 11 mg (63% of theoretical value) of the protected intermediate was obtained. The tert-butyl ester and Boc protecting group were then separated by stirring with a solution of 6 equivalents of zinc chloride in trifluoroethanol at 50 °C for 3 h. In the final step, 6.5 mg (0.006 mmol) of the intermediate was converted to the title compound by coupling 1.2 mg (0.008 mmol) of 1-{2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in 2 mL of DMF in the presence of 3.3 μl N,N-diisopropylethylamine. After purification by preparative HPLC, 3 mg (49% of the theoretical value) of the title compound was obtained.

LC-MS (Method 1): R _{_t} = 0.82 min; MS (ESIpos): m/z = 1055(M+H) ⁺ .

intermediate S6

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(N ^2- {5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-asparaginamide)amino]butyryl}-β-alanyl-D-glutamic acid

The title compound was prepared starting from compound C121 by first coupling ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-ester in DMF in the presence of N,N-diisopropylethylamine. The Boc protecting group was then removed by stirring with a solution of 6 equivalents of zinc chloride in trifluoroethanol at 50 °C for 1 hour.

In the next step, the benzyl ester was removed by hydrogenation in ethanol at room temperature under standard hydrogen pressure over 10% palladium/activated carbon, and then the deprotected intermediate was converted into the title compound by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)dipyrrolidine-2,5-dione] in DMF in the presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.92 min; MS (ESIpos): m/z = 10³⁹ [M+H] ^⁺ .

intermediate S7

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({N ^2- [6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]-L-asparaginamide}amino)butyryl]-β-alanyl-D-glutamic acid

The title compound was prepared starting from compound C121 by first coupling ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-ester in DMF in the presence of N,N-diisopropylethylamine. The Boc protecting group was then removed by stirring with a solution of 6 equivalents of zinc chloride in trifluoroethanol at 50 °C for 1 hour.

In the next step, the benzyl ester was removed by hydrogenation on 10% palladium/activated carbon in ethanol at room temperature under standard hydrogen pressure, and then the deprotected intermediate was converted into the title compound by reaction in DMF with 1.5 equivalents of 1-{6-[(2,5-dioxopyrrolidone-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione in the presence of 3 equivalents of N,N-diisopropylethylamine.

LC-MS (Method 12): R _{_t} = 1.81 min; MS (ESIneg): m/z = 10¹⁹ [MH] ^- .

intermediate S8

N ¹ -{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]propyl}-N ² -{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-asparagine

The title compound was prepared starting from Example 98 described in WO 2015096982:

First, the compound of Example 98 was coupled with 1.8 equivalents of ^N2 -[(benzyloxy)carbonyl]-L-aspartic acid 4-nitrobenzene ester in DMF in the presence of 2 equivalents of N,N-diisopropylethylamine. The Z protecting group was then removed by hydrogenation over 10% palladium/activated carbon in ethanol at room temperature under standard hydrogen pressure. In the final step, the title compound was prepared by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in DMF in the presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 1.03 min; MS (ESIpos): m/z = 795[M+H] ⁺ .

intermediate S9

(8S,11S)-11-(2-amino-2-oxoethyl)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]ethyl}-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2,7,10,13-tetraoxo-3,6,9,12-tetraazahexadecane-16-oleic acid

First, intermediate C136 was coupled to 1.4 equivalents of dihydrofuran-2,5-dione in the presence of 3 equivalents of N,N-diisopropylethylamine. In the next step, benzyl ester and the Z protecting group were removed by hydrogenation on 10% palladium/activated carbon in ethanol-DCM at room temperature under standard hydrogen pressure. In the final step, the title compound was obtained by reaction with 1-{2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in the presence of 3 equivalents of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.93 min; MS (ESIpos): m/z = 907[M+H] ⁺ .

Intermediate S10

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({N2-[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-L-asparaginyl}-amino)butyryl]-β-alanyl-D-glutamic acid

Similar to the preparation of the title compound from intermediate S6, the difference is that, in the final step, instead of 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, coupling is performed with 1.5 equivalents of 1-{2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in the presence of 3 equivalents of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.90 min; MS (ESIpos): m/z = 965 [M+H] ^⁺ .

Intermediate S11

N-(2-{[2-({(2S)-2-[(N ² -acetyl-L-asparaginyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]sulfonyl}ethyl)-N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-D-α-glutamine

First, intermediate C137 was coupled with 1.3 equivalents of ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester in the presence of 3 equivalents of N,N-diisopropylethylamine. The Boc group was then removed by stirring at 50°C for 3 hours with a solution of 6 equivalents of zinc chloride in trifluoroethanol. The reaction product was coupled to 2 equivalents of 1-acetoxypyrrolidine-2,5-dione in the presence of 3 equivalents of N,N-diisopropylethylamine. The Z protecting group was then removed by hydrogenation on 10% palladium/activated carbon in ethanol at room temperature under standard hydrogen pressure. In the final step, the title compound was given by reaction with 1-{2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in the presence of 3 equivalents of N,N-diisopropylethylamine.

LC-MS (Method 12): R _{_t} = 1.66 min; MS (ESIpos): m/z = 1070 [M+H] ⁺ .

Intermediate S12

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-{[N ^2- (bromoacetyl)-L-asparaginyl]amino}butyryl]-β-alanyl-D-glutamic acid

Similar to the preparation of the title compound from intermediate S10, the difference is that the reaction in the final step is carried out in the presence of 2 equivalents of N,N-diisopropylethylamine with 3 equivalents of 1-(2-bromoacetoxy)pyrrolidine-2,5-dione instead of 1-{2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione.

LC-MS (Method 1): R _{_t} = 0.93 min; MS (ESIpos): m/z = 948 and 950 [M+H] ^⁺ .

Intermediate S13

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({N ^2- [1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3-azaoctadecyl-18-yl]-L-asparaginamide}amino)butyryl]-β-alanyl-D-glutamic acid

The title compound was prepared starting from compound C121 by first coupling it to ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester in DMF in the presence of N,N-diisopropylethylamine. The Boc protecting group was then removed by stirring with a solution of 6 equivalents of zinc chloride in trifluoroethanol at 50 °C for 1 hour.

In the next step, the resulting intermediate was coupled with 3-oxo-1-phenyl-2,7,10,13,16-penta-4-azadecane-19-oleic acid in DMF in the presence of 1.2 equivalents of HATU and 3 equivalents of N,N-diisopropylethylamine. The benzyl ester and Z protecting group were then removed by hydrogenation at room temperature in methanol/DCM 1:1 over 10% palladium/activated carbon at standard hydrogen pressure. The deprotected intermediate was then converted to the title compound by reaction with 1-{1,2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in DMF in the presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.89 min; MS (ESIpos): m/z = 1212[M+H] ⁺ .

Intermediate S14

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-({N2-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,22-dioxo-3,10,13,16,19-pentoxa-7-azacodosadecan-22-yl]-L-asparagineyl}amino]-β-alanyl-D-glutamic acid

Similar to the preparation of the title compound from intermediate S13, the difference lies in the fact that the reaction in the final step is performed in the presence of 3 equivalents of N,N-diisopropylethylamine, with 3 equivalents of 1-(2-{3-[(2,5-dioxopyrrolidone-1-yl)oxy]-3-oxopropoxy}ethyl)-1H-pyrrole-2,5-dione replacing 1-{2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione.

LC-MS (Method 1): R _{_t} = 0.89 min; MS (ESIpos): m/z = 1270 [M+H] ⁺ .

Intermediate S15

N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-L-asparaginamide-N-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]sulfonyl}ethyl)-D-α-glutamine trifluoroacetate

The synthesis of the title compound began with the coupling of intermediate C138 with ^N2 -[(benzyloxy)carbonyl]-L-aspartic acid 4-nitrobenzene ester in DMF in the presence of 2 equivalents of N,N-diisopropylethylamine. The Z protecting group was then removed by hydrogenation at room temperature in DCM/methanol 1:1 over 10% palladium/activated carbon for 2 h at standard hydrogen pressure. The reaction was then carried out in DMF in the presence of 3 equivalents of N,N-diisopropylethylamine with 1.2 equivalents of 1-{2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione. In the final step, the title compound was obtained by stirring at 50 °C with 6 equivalents of zinc chloride in trifluoroethanol solution for 2 h. LC-MS (Method 1): R _{_t} = 0.79 min; MS (ESIpos): m/z = 1028 [M+H] ^⁺ .

Intermediate S16

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-{[N ^2- (18-bromo-17-oxo-4,7,10,13-tetraoxa-16-azaoctadecyl-1-acyl)-L-asparagine]amino}butyryl]-β-alanyl-D-glutamic acid

First, the title compound was synthesized by coupling intermediate C139 with intermediate L138 in DMF in the presence of 1.2 equivalents of HATU and 3 equivalents of N,N-diisopropylethylamine. The tert-butyl ester group was then removed by stirring at 50 °C with an 18 equivalent solution of zinc chloride in trifluoroethanol for 1 hour.

LC-MS (Method 1): R _{_t} = 0.91 min; MS (ESI-neg): m/z = 1193 and 1195 [MH] ^- .

Intermediate S17

S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[N2-(pyridin-4-ylacetyl)-L-asparaginyl]amino}propyl)amino]-2-oxoethyl}-N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3-azaoctadecyl-18-yl]-L-cysteine

4-methylmorpholine (2.8 μl, 26 μmol) was added to a DMF (1 mL) solution of N-(15-amino-4,7,10,13-tetraoxapentadecano-1-acyl)-S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[N2-(pyridin-4-ylacetyl)-L-asparaginamide]amino}propyl)amino]-2-oxoethyl}-L-cysteine trifluoroacetate (10.0 mg, 8.57 μmol) and 1-{2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethyl}-1H-pyrrolo-2,5-dione (2.38 mg, 9.42 μmol), and the reaction was stirred at room temperature for 18 hours. One drop of acetic acid was added to the reaction mixture, and the mixture was purified by preparative RP-HPLC (flow rate: 50 mL/min, MeCN/water, 0.1% TFA). The solvent was evaporated under reduced pressure, and the residue was lyophilized. 2.2 mg of the title compound was given.

LC-MS (Method 12): R _{_t} = 1.62 min; MS (ESIpos): m/z = 1190 [M+H] ⁺

Intermediate S18

S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(3-{[N2-(pyridin-4-ylacetyl)-L-asparaginamide]amino}propyl)amino]-2-oxoethyl}-N-{21-[(2,5-dioxopyrrolidine-1-yl)oxy]-17,21-dioxo-4,7,10,13-tetraoxa-16-azacotetraane-1-acyl}-L-cysteine

Add 1,1'-[(1,5-dioxolane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione (7.41 mg, 22.7 μmol) and N,N-diisopropylethylamine (6.3 μl, 36 μmol) to a DMF (1 ml) solution of N-(15-amino-4,7,10,13-tetraoxapentane-1-acyl)-S-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-yl]-2,2-dimethylpropyl}(3-{[N2-(pyridin-4-ylacetyl)-L-asparaginamide]amino}propyl)amino]-2-oxoethyl}-L-cysteine trifluoroacetate (10.6 mg, 9.08 μmol) and N,N-diisopropylethylamine (6.3 μl, 36 μmol). Stir the reaction mixture at room temperature until conversion is complete. The mixture was combined with water and 0.1% TFA, filtered, and purified directly by preparative HPLC (elution: ACN/water + 0.1% TFA, gradient). 1.2 mg of the target compound was obtained.

LC-MS (Method 1): R _{_t} = 0.89 min; MS (ESIpos): m/z = 1263 [MH] ⁺

Intermediate S19

N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}({[(2R)-2-carboxyl-2-({N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-L-asparaginamide}amino)ethyl]thioalkyl}acetyl)amino]butyryl}-β-alanyl-D-glutamic acid trifluoroacetate

To N-[(2S)-4-[({[(2R)-2-(L-asparagine)-3-tert-butoxy-3-oxopropyl]thioalkyl}acetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butyryl]-β-alanyl-D-glutamic acid tri... Di-tert-butyl fluoroacetate (12.0 mg, 8.84 μmol, intermediate C146) and 1-{2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (2.45 mg, 9.72 μmol) in DMF (1.0 mL) were reacted with N,N-diisopropylethylamine (6.2 μl, 35 μmol), and the mixture was stirred at room temperature for 2 hours. Subsequently, 1-{2-[(2,5-dioxopyrrolidone-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (2.45 mg, 9.72 μmol) were added, and the mixture was stirred again at room temperature for 2 hours. The mixture was then mixed with 1 mL of water + 0.1% TFA and purified directly by preparative HPLC (elution: ACN/water + 0.1% TFA, gradient). Subsequently, the protecting group was removed by stirring at 50°C with a 12-equivalent solution of zinc chloride in trifluoroethanol for 4 hours.

LC-MS (Method 1): R _{_t} = 0.83 min; MS (ESIpos): m/z = 1068 [M+H] ⁺

Intermediate S20

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-(ethanolyl)amino]-2-[(N ^2- {5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-asparagineyl)-amino]butyryl}-β-alanyl-L-asparagine

First, intermediate C141 was coupled with 1.8 equivalents of ^N2 -[(benzyloxy)carbonyl]-L-aspartic acid 4-nitrobenzene ester in DMF in the presence of 12 equivalents of N,N-diisopropylethylamine. The Z protecting group was then removed by hydrogenation over 10% palladium/activated carbon in DCM/methanol 1:1 at room temperature under standard hydrogen pressure. In the final step, the title compound was prepared by reacting it with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-diketone in the presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.91 min; MS (ESIpos): m/z = 1024 [M+H] ^⁺ .

Intermediate S21

N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}-(ethanolyl)amino]-2-({N ² -[(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetyl]-L-asparagineyl}-amino)butyryl]-β-alanyl-L-asparagine

First, intermediate C141 was coupled with 1.8 equivalents of ^N2 -[(benzyloxy)carbonyl]-L-aspartic acid 4-nitrobenzene ester in DMF in the presence of 12 equivalents of N,N-diisopropylethylamine. The Z protecting group was then removed by hydrogenation over 10% palladium/activated carbon in DCM/methanol 1:1 at room temperature under standard hydrogen pressure. In the final step, the title compound was prepared by reacting 2.5 equivalents of 1-{2-[(2,5-dioxopyrrolidine-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione in DMF in the presence of 4 equivalents of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.90 min; MS (ESIpos): m/z = 950 [M+H] ⁺ .

intermediate S22

N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-2-[(N ^2- {16-[(2,5-dioxopyrrolo-1-yl)oxy]-16-oxo-4,7,10,13-tetraoxa-hexadecane-1-yl}-L-asparaginamide)amino]butyryl}-β-alanyl-D-glutamic acid

The title compound was prepared from compound C121 by first coupling to ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester in DMF in the presence of N,N-diisopropylethylamine. The Boc protecting group was then removed by stirring in a solution of 6 equivalents of zinc chloride in trifluoroethanol at 50 °C for 1 hour. In the next step, the benzyl ester was removed by hydrogenation on 10% palladium/activated carbon in DCM/methanol 1:1 at room temperature under standard hydrogen pressure, followed by conversion of the deprotected intermediate to the title compound by reaction with 3 equivalents of 1,1'-[(1,19-dioxo-4,7,10,13,16-pentaenodecane-1,19-diyl)bis(oxy)]pyrrolidine-2,5-diketone in DMF in the presence of 3 equivalents of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.96 min; MS (ESIpos): m/z = 1245 [M+H] ⁺ .

B: Preparation of antibody-drug conjugates (ADCs)

B-1. General methods for antibody production

The protein sequence (amino acid sequence) of the antibody used is converted into a DNA sequence encoding the protein using methods well known to those skilled in the art, and inserted into an expression vector suitable for transient mammalian cell culture. Examples of antibodies include TPP-2090, TPP-2658, TPP-5442, TPP-8825, TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, TPP-10337, TPP-1015, TPP-7510, TPP-7511, TPP-8382, and TPP-8567 (as described in Tom et al., Methods Express: Expression Systems, Chapter 12, edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007).

B-2. General methods for expressing antibodies in mammalian cells

Antibodies such as TPP-2090, TPP-2658, TPP-5442, TPP-8825, TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, TPP-10337, TPP-1015, TPP-7510, TPP-7511, TPP-8382, and TPP-8567 were prepared in transient mammalian cell cultures, as described in Tom et al., Methods Express: Expression Systems, Chapter 12, edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007.

B-3. General methods for purifying antibodies from cell supernatants

Antibodies such as TPP-2090, TPP-2658, TPP-5442, TPP-8825, TPP-7006, TPP-7007, TPP-10334, TPP-10335, TPP-10336, TPP-10337, TPP-1015, TPP-7510, TPP-7511, TPP-8382, and TPP-8567 were obtained from cell culture supernatant. The cell supernatant was clarified by centrifugation. The cell supernatant was then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) column. For this purpose, the column was equilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant was added, and the column was washed with approximately 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride. The antibody was eluted in 50 mM sodium acetate at pH 3.5 and 500 mM sodium chloride, and then further purified by gel filtration chromatography in DPBS at pH 7.4 on a Superdex 200 column (GE Healthcare).

Commercially available antibodies were purified using standard chromatographic methods (protein A chromatography, preparative gel filtration chromatography (SEC-size exclusion chromatography)).

B-4. General methods for coupling with cysteine side chains

Use the following antibodies in the coupling reaction:

Example a: Cetuximab (anti-EGFR AK)

Example e: TPP-1015 (Anti-Her2 AK)

Example k: Anti-TWEAKR AK (TPP-7007)

Example k: Anti-TWEAKR AK (TPP-2658).

The coupling reaction is usually carried out under argon gas.

Two to five equivalents of tris(2-carboxyethyl)phosphonium hydrochloride (TCEP) dissolved in PBS buffer are added to a solution of the appropriate antibody in PBS buffer, at a concentration ranging from 1 mg/ml to 20 mg/ml, preferably from about 10 mg/ml to 15 mg/ml, and the mixture is stirred at RT for 30 min to 1 h. For this purpose, solutions of the respective antibodies used can be used at the concentrations described in the working examples, or optionally diluted with PBS buffer to about half of the starting concentration to achieve the preferred concentration range. Then, depending on the intended loading, 2 to 20 equivalents, preferably about 5 to 10 equivalents, of the maleimide or halide precursor compound to be coupled are added as a solution in DMSO. For higher DAR, 15-20 equivalents can also be used. Here, the amount of DMSO should not exceed 10% of the total volume. In the case of maleimide precursors, the mixture is stirred at RT for 60–240 min; in the case of halide precursors, it is stirred at RT for 8–24 h. The mixture is then applied to a PBS-equilibrated PD10 column (G-25, GE Healthcare) and eluted with PBS buffer. Typically, unless otherwise specified, reduction and subsequent conjugation are performed using 5 mg of the antibody in PBS buffer. Thus, purification is performed on a PD10 column in each case to obtain a solution of the corresponding ADC in 3.5 ml of PBS buffer. The sample is then concentrated by ultracentrifugation and optionally rediluted with PBS buffer. If necessary, ultrafiltration concentration is repeated after redilution with PBS buffer for better removal of low molecular weight components. For biological assays, the final ADC sample concentration is optionally adjusted to the range of 0.5–15 mg/ml by redilution, if desired. The concentrations of each protein in the ADC solution as described in the working examples are determined. Furthermore, the antibody loading (drug/mAb ratio) is determined using the method described in B-7.

Depending on the connector, the ADC shown in the examples may also be present in lower or higher amounts as a hydrolyzed open-chain succinate linked to the antibody.

In particular, KSP-I-ADCs linked to the thiol groups of antibodies via the following linker structures can also be selectively prepared by buffering again after coupling and stirring at pH 8 for about 20-24 h according to schemes 4 and 9.

#1 represents the sulfur bridge with the antibody, and #2 represents the linking site with the modified KSP inhibitor.

Such ADCs can also be selectively prepared by the following exemplary method, wherein the linker is linked to the antibody via hydrolyzed open-chain succinamide.

Small-scale coupling:

Add 2 to 5 equivalents of tris(2-carboxyethyl)phosphonium hydrochloride (TCEP) dissolved in PBS buffer to a solution of 2-5 mg of the appropriate antibody in PBS buffer, at a concentration ranging from 1 mg/ml to 20 mg/ml, preferably from about 5 mg/ml to 15 mg/ml, and stir the mixture at RT for 30 min to 1 h. Then, depending on the desired loading, add 2 to 20 equivalents, preferably about 5 to 10 equivalents, of the maleimide precursor compound to be conjugated as a solution in DMSO. For higher DAR, 15-20 equivalents can also be used. Here, the amount of DMSO should not exceed 10% of the total volume. Stir the mixture at RT for 60-240 min, then dilute with PBS buffer to a volume of 2.5-7.5 ml, wherein the PBS buffer has been pre-adjusted to pH 8, and then flow through a PD10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, and elute with PBS buffer at pH 8. The eluent was stirred overnight under RT and argon. The solution was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2).

Medium-scale coupling:

Under argon atmosphere, add 2-5 equivalents, preferably 3 equivalents, of TCEP in PBS buffer (c ~ 0.2-0.8 mg/ml, preferably 0.5 mg/ml) to 20-200 mg of the antibody in PBS buffer (c ~ 5-15 mg/ml). Stir the mixture at RT for 30 min, then add 2-20 equivalents, preferably 5-10 equivalents, of the maleimide precursor compound dissolved in DMSO. For higher DAR, 15-20 equivalents can also be used. After further stirring at RT for 1.5-2 h, dilute the mixture with PBS buffer pre-adjusted to pH 8.

The solution was then applied to a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8 and eluted with PBS buffer at pH 8. The eluent was diluted with PBS buffer at pH 8 to a concentration of 1–7 mg/mL. The solution was stirred overnight at RT under argon. If necessary, the solution was then reburied to pH 7.2. The ADC solution was concentrated by ultracentrifugation, rediluted with PBS buffer (pH 7.2), and optionally concentrated again to a concentration of approximately 10 mg/mL.

In the working examples, other potential hydrolysis-sensitive thianyl succinimide bridges with antibodies comprise the following linker structures, where #1 represents a thioether link with the antibody, and #2 represents a linking site with a modified KSP inhibitor:

These adapter structures represent linking units with antibodies and have (in addition to other adapter compositions) a significant influence on the structure and characterization of metabolites formed in tumor cells.

In the structural formula shown, AK ₁ has the following meanings

Example a: Cetuximab (partially reduced) - S§ ¹

Example e: Anti-HER2 AK (TPP-1015 partially reduced) - S§ ¹

Example k: Anti-TWEAKR AK (partially reduced from TPP-7007) - S§ ¹

Example 11: Anti-TWEAKR AK (TPP-2658 partially reduced) - S§ ¹

in

§ ¹ represents the linking bond with a succinimide group or with any isomerized hydrolyzed open-chain succinamide or alkylene group produced therefrom.

and

S represents the sulfur atom of the cysteine residue in the partially reduced antibody, B-5. General method for coupling with the lysine side chain.

Use the following antibodies in the coupling reaction:

Example a: Cetuximab (anti-EGFR AK)

Example e: TPP-1015 (Anti-Her2 AK)

Example k: Anti-TWEAKR antibody (TPP-7007)

Example k: Anti-TWEAKR antibody (TPP-2658).

The coupling reaction is usually carried out under argon gas.

Add 2 to 10 equivalents of the precursor compound to be coupled as a solution in DMSO to the antibody solution in PBS buffer, at a concentration ranging from 1 mg/ml to 20 mg/ml, preferably about 10 mg/ml, depending on the intended loading. After stirring at RT for 30 min to 6 h, add the same amount of precursor compound in DMSO again. Here, the amount of DMSO should not exceed 10% of the total volume. After further stirring at RT for 30 min to 6 h, apply the mixture to a PD 10 column (G-25, GE Healthcare) equilibrated with PBS and elute with PBS buffer. Typically, unless otherwise specified, 5 mg of the antibody in PBS buffer is used for coupling. Thus, after purification on the PD 10 column in each case, a solution of each ADC in 3.5 ml of PBS buffer is obtained. The sample is then concentrated by ultracentrifugation and optionally rediluted with PBS buffer. If necessary, the ultrafiltration concentration is repeated after redilution with PBS buffer for better removal of low molecular weight components. For biological assays, if necessary, the concentration of the final ADC sample may be adjusted to the range of 0.5–15 mg/ml by re-dilution.

The concentrations of each protein in the ADC solution as described in the working examples were determined. Additionally, the antibody load (drug/mAb ratio) was determined using the method described in B-7.

In the structural formula shown, AK ₂ has the following meanings.

Example a: Cetuximab-NH§ ²

Example e: Anti-HER2 AK (TPP-1015)-NH§ ²

Example k: Anti-TWEAKR antibody (TPP-7007) - NH§ ²

Example k: Anti-TWEAKR antibody (TPP-2658) - NH§ ²

in

§ ² represents the linkage with the carbonyl group.

and

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

B-5a. General methods for ADC synthesis using bacterial transglutaminase

In conjugation reactions involving bacterial transglutaminase, the following antibodies can be used (the antibody-HC-N297Z designation below refers to an antibody in which amino acid N297 (Kabat number) has been replaced with amino acid Z in both heavy chains; the TPP-xxxx-HC-Q295N-HC-N297Q designation refers to an antibody containing TPP-XXXX, where amino acid Q295 (Kabat number) has been replaced with amino acid N and amino acid N297 (Kabat number) has been replaced with amino acid Q in both heavy chains. The antibody name of the original antibody can be reported as a name (e.g., trastuzumab) or TPP-XXXX (antibody with TPP number XXXX):

AK3a: Anti-TWEAKR antibody (TPP-2658) (corresponding to TPP-2090-HC-N297A)

AK3b: Anti-TWEAKR antibody (TPP-5442) (corresponding to TPP-2090-HC-N297Q)

AK3c: Anti-TWEAKR antibody (TPP-8225) (corresponding to TPP-2090-HC-Q295N-HC-N297Q)

AK3d: Anti-HER2 antibody (TPP-7510) (corresponding to TPP-1015-HC-N297A)

AK3e: Anti-HER2 antibody (TPP-7511) (corresponding to TPP-1015-HC-N297Q).

A general method to achieve a maximum DAR of 2:

Add 20 μl (6 equivalents) of a solution of the appropriate virulence linker precursor (e.g., intermediates R50 and R51; 10 mM in DMSO) to a solution of 5 mg of the corresponding aglycone antibody variant (HC-N297A) in DPBS pH 7.4 (c ~ 5-15 mg/ml). After incubation at 37 °C for 5 min, add 50 μl of a solution of recombinant bacterial transglutaminase (from Zedira GmbH, Darmstadt, Germany, product number T001) (25 U/ml) and continue incubation at 37 °C for another 24 h. The reaction mixture was then diluted to a total volume of 2.5 ml with DPBS pH 7.4 and gel filtered through a DPBS-equilibrated PD10 column (G-25, GE Healthcare), followed by elution with DPBS buffer at pH 7.4. The ADC solution was then concentrated by centrifugation (Millipore) using an Amicon Ultracel-30K and rediluted with DPBS to approximately 2.5 ml. Finally, 0.00625 μmol of the β-transglutaminase inhibitor Zedira C100 in 12.5 μl of DPBS was added to this solution. The protein concentrations of the ADC solution as described in the working examples were determined. Furthermore, the antibody load (drug/mAb ratio) was determined using the method described in B-7.

A general method to achieve a maximum DAR of 4:

Add 16–24 equivalents of a solution of the appropriate virulence linker precursor (e.g., intermediates R50 and R51; 10 mM in DMSO) to a solution of 5 mg of the corresponding sugar-free antibody variant (HC-N297Q) in DPBS pH 7.4 (c ~ 5–15 mg/ml). After incubation at 37 °C for 5 min, add 400 μl (10 U) of an aqueous solution of recombinant bacterial transglutaminase (from Zedira GmbH, Darmstadt, Germany, product number T001) (25 U/ml) and continue incubation at 37 °C for another 24 h. The reaction mixture was then diluted to a total volume of 2.5 ml with DPBS pH 7.4 and gel filtered through a DPBS-equilibrated PD 10 column (G-25, GE Healthcare), eluting with DPBS buffer at pH 7.4. The ADC solution was then concentrated by centrifugation (Millipore) using an Amicon Ultracel-30K and rediluted with DPBS to approximately 2.5 ml. Finally, 0.1 μmol of the β-transglutaminase inhibitor Zedira C100 in 200 μl of DPBS was added to this solution. The protein concentrations of the ADC solution as described in the working examples were determined. Furthermore, the antibody load (drug/mAb ratio) was determined using the method described in B-7.

General methods for obtaining larger-scale transglutaminase-mediated coupling with a maximum DAR of 2:

To a solution of 30 mg of the non-glycosylated variant (HC-N297A) of the specific antibody in DPBS pH 7.4 (c ~ 5-15 mg/ml), 6 equivalents of a suitable cluster adapter precursor solution (10 mM in DMSO) were added. After incubation at 37°C for 5 min, 200 μl (7.5 U) of an aqueous solution of recombinant bacterial transglutaminase (from Zedira GmbH, Darmstadt, Germany, product number T001) (25 U/ml) was added, and incubation was continued at 37°C for another 24 h. The reaction mixture was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to separate the small molecule and transglutaminase from the ADC. The ADC solution was then concentrated to a final concentration of 5-25 mg/ml using Amicon Ultracel-30K centrifuge tubes (Millipore). The solution was then aseptically filtered.

Determine the concentrations of the ADC solutions reported in the working examples. Determine the loading using the methods described in Chapter B7. The characteristics of the ADC batches are as shown in the working examples.

General methods for obtaining larger-scale transglutaminase-mediated coupling with a maximum DAR of 4:

To a solution of 30 mg of the non-glycosylated variant of the specific antibody (HC-N297Q) in DPBS pH 7.4 (c ~ 5-15 mg/ml), 16-24 equivalents of a suitable cluster adapter precursor solution (10 mM in DMSO) were added. After incubation at 37°C for 5 min, 2400 μl (60 U) of an aqueous solution of recombinant bacterial transglutaminase (from Zedira GmbH, Darmstadt, Germany, product number T001) (25 U/ml) was added, and incubation was continued at 37°C for another 24 h. The reaction mixture was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to separate the small molecule and transglutaminase from the ADC. The ADC solution was then concentrated to a final concentration of 5-25 mg/ml using Amicon Ultracel-30K centrifuge tubes (Millipore). The solution was then aseptically filtered.

Determine the concentrations of the ADC solutions reported in the working examples. Determine the loading using the methods described in Chapter B7. The characteristics of the ADC batches are as shown in the working examples.

In each context, AK ₃ has the following meanings:

AK3a: Anti-TWEAKR antibody (TPP-2658) (corresponding to TPP-2090-HC-N297A)-CO-§2

AK3b: Anti-TWEAKR antibody (TPP-5442) (corresponding to TPP-2090-HC-N297Q)-CO-§2

AK3c: Anti-TWEAKR antibody (TPP-8825) (corresponding to TPP-2090-HC-Q295N-HC-N297Q)-CO-§2

AK3d: Anti-HER2 antibody (TPP-7510) (corresponding to TPP-1015-HC-N297A)-CO-§2

AK3e: Anti-HER2 antibody (TPP-7511) (corresponding to TPP-1015-HC-N297Q)-CO-§2

in

§ ² represents the linkage bond with the amino group of the toxic cluster precursor.

and

CO represents the carbonyl group on the side chain of the glutamine group of the antibody.

Potentially suitable substrates for bacterial transglutaminase used for the purposes of this application are:

B-6a. General method for preparing closed succinimide-cysteine adducts:

In one exemplary embodiment, 10 μmol of the above-mentioned maleimide precursor compound was dissolved in 3-5 mL of DMF, and 2.1 mg (20 μmol) of L-cysteine was added. The reaction mixture was stirred at RT for 2 h to 24 h, then concentrated under reduced pressure, and then purified by preparative HPLC.

B-6aa. General method for preparing isomer-opening succinamide-cysteine adducts:

In one exemplary embodiment, 68 μmol of the above-described maleimide precursor compound was dissolved in 15 mL of DMF, and 36 mg (136 μmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine was added. The reaction mixture was stirred at RT for ~20 h, then concentrated under reduced pressure, and then purified by preparative HPLC. Suitable fractions were combined and the solvent was evaporated under reduced pressure, and the residue was then dissolved in 15 mL of THF/water 1:1. 131 μl of 2M lithium hydroxide aqueous solution was added and the mixture was stirred at RT for 1 h. The reaction was then neutralized with 1M hydrochloric acid, the solvent was evaporated under reduced pressure, and the residue was purified by preparative HPLC. This yielded a protected intermediate with approximately 50% regioisomeric content, representing the theoretical value of a colorless foam.

In the final step, 0.023 mmol of the hydrolysis products of these regioisomers were dissolved in 3 mL of 2,2,2-trifluoroethanol. 12.5 mg (0.092 mmol) of zinc chloride was added, and the reaction mixture was stirred at 50 °C for 4 h. Then, 27 mg (0.092 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid was added, and the solvent was evaporated under reduced pressure. The residue was purified by preparative HPLC. Suitable fractions were concentrated and the residue was lyophilized from acetonitrile/water to give hydrolyzed ring-opening thioalkyl succinamides as a mixture of regioisomers.

Further purification and characterization of the conjugates of the present invention

Following the reaction, in some cases, the reaction mixture is concentrated, for example by ultrafiltration, and then desalted and purified by chromatography, for example using G-25. Elution is performed, for example, with phosphate-buffered saline (PBS). The solution is then sterilely filtered and frozen. Alternatively, the conjugate can be lyophilized.

B-7. Determine the ratio of antibody, virulence load, and open-ring cysteine adduct.

For protein identification, in addition to molecular weight determination after deglycosylation and/or denaturation, trypsin digestion is performed, and the protein is confirmed by the trypsin peptides found after denaturation, reduction and derivatization.

The following determination of the toxic cluster loading in the PBS buffer solution obtained from the conjugates described in the working examples is as follows:

The toxicity loading of lysine-linked ADCs was determined by mass spectrometry, measuring the molecular weight of individual conjugate species. Here, the antibody conjugates were first deglycosylated with PNGase F, the sample was acidified, and after HPLC separation/desalting, analysis was performed by mass spectrometry using an ESI-MicroTof _Q (Bruker Daltonik). All spectra of the signals in the TIC (Total Ion Chromatography) were added, and the molecular weights of different conjugate species were calculated based on MaxEnt deconvolution. The DAR (Drug/Antibody Ratio) was then calculated after integrating the signals from different species. For this purpose, the sum of the weighted integrals of all species by the toxicity counts was divided by the sum of the simple weighted integrals of all species.

The toxicity loading of cysteine-linked conjugates was determined by reversed-phase chromatography of reduced and denatured ADCs. A solution of guanidine hydrochloride (GuHCl) (28.6 mg) and DL-dithiothreitol (DTT) (500 mM, 3 μl) was added to the ADC solution (1 mg/mL, 50 μl). The mixture was incubated at 55 °C for 1 h and analyzed by HPLC.

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

The detection peaks are specified by comparing their retention times with those of the light chain (L0) and heavy chain (H0) of the unconjugated antibody. Peaks detected only in conjugated samples are specified as light chain (L1) with one toxic cluster and heavy chain (H1, H2, H3) with one, two, and three toxic clusters.

The average load of the antibody with toxic clusters is calculated from the peak area determined by integrating twice the sum of the HC load and LC load. The LC load is calculated by dividing the sum of the weighted integrals of the toxic cluster counts of all LC peaks by the sum of the simple weighted integrals of all LC peaks, and the HC load is calculated by dividing the sum of the weighted integrals of the toxic cluster counts of all HC peaks by the simple weighted integrals of all HC peaks. In some cases, due to co-elution of certain peaks, the toxic cluster load may not be accurately determined.

When HPLC cannot adequately separate the light and heavy chains, the toxicity load of cysteine-linked conjugates is determined by mass spectrometry to measure the molecular weight of individual conjugate species in the light and heavy chains.

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

For DAR assays, all spectra were added to the signal in the TIC (Total Ion Chromatography), and the molecular weights of different conjugate species in the light and heavy chains were calculated based on MaxEnt deconvolution. The average loading of the antibody with toxic clusters was determined by the peak area, which was calculated by integrating the sum of the HC loading and LC loadings. Here, the LC loading was calculated by dividing the sum of the cluster-count-weighted integrals of all LC peaks by the sum of the simple weighted integrals of all LC peaks, and the HC loading was calculated by dividing the sum of the cluster-count-weighted integrals of all HC peaks by the sum of the simple weighted integrals of all HC peaks.

In the case of open-ring constructs, to determine the proportion of open-ring cysteine adducts, the molecular weight area ratio (molecular weight δ18 Daltons) of the closed to open-ring cysteine adducts of all single-conjugated light and heavy chain variants is determined. The average of all variants yields the proportion of open-ring cysteine adducts.

The toxicity loading of glutamine-linked conjugates was determined by reversed-phase chromatography of reduced and denatured ADCs. A solution of guanidine hydrochloride (GuHCl) (28.6 mg) and DL-dithiothreitol (DTT) (500 mM, 3 μl) was added to the ADC solution (1 mg/mL, 50 μl). The mixture was incubated at 55 °C for 1 h and analyzed by HPLC.

HPLC analysis was performed at 220 nm using an Agilent 1260 HPLC system. A Polymer Laboratories PLRP-S polymer reversed-phase column (catalog number PL1912-3802) (2.1 x 150 mm, 8 μm particle size) was used at a flow rate of 1 mL/min, employing the following gradients: 0 min, 31% B; 1 min, 31% B; 14 min, 38% B; 16 min, 95% B. Eluent A consisted of an aqueous solution of 0.05% trifluoroacetic acid (TFA), and eluent B consisted of an acetonitrile solution of 0.05% trifluoroacetic acid.

The detection peaks are specified by comparing their retention times with those of the light chain (L0) and heavy chain (H0) of the unconjugated antibody. Peaks detected only in conjugated samples are specified as heavy chains with one or two toxic clusters (H1, H2).

The average load of the antibody with toxic clusters is calculated from the peak area determined by integrating twice the sum of the HC load and LC load, wherein the LC load is calculated by dividing the sum of the weighted integrals of the number of toxic clusters in all LC peaks by the sum of the simple weighted integrals of all LC peaks, and wherein the HC load is calculated by dividing the sum of the weighted integrals of the number of toxic clusters in all HC peaks by the sum of the simple weighted integrals of all HC peaks.

Alternatively, the toxicity loading of a glutamine-linked ADC can be determined by mass spectrometry, measuring the molecular weight of individual conjugate species. In this case, the sample is acidified and analyzed by mass spectrometry using an ESI-MicroTof _Q (BrukerDaltonik) after HPLC separation/desalting. All spectra of the signal in the TIC (Total Ion Chromatography) are added, and the molecular weights of different conjugate species are calculated based on MaxEnt deconvolution. The DAR (Drug/Antibody Ratio) is then calculated after integrating the signals of different species. For this purpose, the sum of the weighted integrals of the toxicity counts of all species is divided by the sum of the simple weighted integrals of all species.

Alternatively, the toxic cluster load can be determined independently of the binding site by UV absorption (hereinafter referred to as SEC-UV) during size exclusion chromatography (SEC). For this purpose, 50 μl of ADC solution was analyzed by SEC. The analysis was performed on an Agilent 1260 HPLC system at detection wavelengths of 280 nm and 260 nm. A Superdex 200 10/300GL column (lot number: 10194037) (10 × 310 mm, 1 μm particle size) from GE Healthcare was used at a flow rate of 1 ml/min under isocratic conditions. The mobile phase consisted of PBS buffer (pH 7.2). To determine the drug load from the HPLC chromatogram, the ratio R of the peak areas of the monomer peaks at 260 nm and 280 nm was determined. This ratio was used to determine the drug load (DAR) as follows:

In this formula, ε is the molar extinction coefficient of the antibody (Ab) and the drug (D). _{λ for drug} represents a wavelength of 260 nm, and 280 represents 280 nm. The extinction coefficients of the antibody at 280 nm and 260 nm were determined experimentally. The average of these measurements from various antibodies was used for DAR calculation. For the KSP toxicant cluster, the molar extinction coefficients at 280 nm and 260 nm were also determined experimentally. The following wavelengths and extinction coefficients were used for DAR calculation:

The concentration of the ADC was determined by measuring the UV absorbance at 280 nm. The concentration was determined using the molar absorptivity of each antibody. To also account for the absorption of the toxic clusters at 280 nm, the concentration measured at 280 nm was corrected using the following equation:

Concentration = Initial concentration / (1 + DAR _UV * ( _{toxin cluster 280nm} / _{antibody 280nm} ))

In this formula, "initial concentration" represents the concentration calculated using only the absorption coefficient of the antibody, DAR _UV is the DAR of each ADC determined by SEC-UV, and _{280nm for the toxic cluster} and _{280nm for the antibody} are the extinction coefficients of the toxic cluster and the antibody at 280nm respectively.

Confirmation of antigen binding to B-8.ADC

The ability of the binding moiety to bind to the target molecule is examined after conjugation occurs. Various methods are familiar to those skilled in the art for this purpose; for example, the affinity of the conjugate can be examined using ELISA techniques or surface plasmon resonance analysis (BIAcore ^™ measurement). The conjugate concentration can be measured by those skilled in the art using conventional methods, such as protein assays to measure the conjugate concentration of the antibody conjugate. (See also Doronina et al.; Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007; 1102:616-623).

Working examples of APDC and ADC

Depending on the linker and coupling method, the APDC and ADC (coupled to the cysteine side chain of the antibody via a maleimide group) shown in the structural formula of the working examples are primarily present in either an open-ring or closed-ring form as shown in each case. However, the formulation may contain a small subset of their respective other forms.

The coupling reaction was carried out under argon atmosphere.

Example 1

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 12.5 mg/mL) in 0.4 mL PBS. The mixture was stirred at RT for 30 min, and then 0.22 mg (0.00023 mmol) of intermediate S1 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted to 2.5 mL with PBS buffer (pre-adjusted to pH 8) and then passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, followed by elution with PBS buffer at pH 8. The eluent was then stirred overnight at RT with argon atmosphere. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 2

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 12.5 mg/mL) in 0.4 mL PBS. The mixture was stirred at RT for 30 min, and then 0.22 mg (0.00023 mmol) of intermediate S2 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the reaction mixture was diluted to a volume of 2.5 mL with PBS buffer (pre-adjusted to pH 8) and then applied to a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, and eluted with PBS buffer at pH 8. The eluent was stirred overnight at RT with argon atmosphere. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Exemplary Method B:

Under argon atmosphere, a solution of 0.172 mg TCEP in 0.2 mL of PBS buffer was added to 30 mg of the appropriate antibody (c = 11.9 mg/mL) in 2.52 mL of PBS. The mixture was stirred at RT for 30 min, and then 1.2 mg (0.0014 mmol) of intermediate S2 dissolved in 200 μL of DMSO was added. After stirring at RT for another 90 min, the reaction mixture was diluted to a volume of 5 mL with PBS buffer (pre-adjusted to pH 8) and then applied to a PD10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8 and eluted with PBS buffer at pH 8. The eluent was diluted to a volume of 7.5 mL with PBS buffer at pH 8 and stirred overnight at RT under argon atmosphere. The solution was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

For Example 2k*-7007, the percentage of ring-opening was determined to be 87.4% by mass spectrometry.

Example 3

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 10 mg/mL) in 0.5 mL PBS. The mixture was stirred at RT for 30 min, and then 0.22 mg (0.00023 mmol) of intermediate S3 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pre-adjusted to pH 8) and then passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, followed by elution with PBS buffer at pH 8. The eluent was then stirred overnight under argon atmosphere and RT. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 4

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 10 mg/mL) in 0.5 mL PBS. The mixture was stirred at RT for 30 min, and then 0.23 mg (0.00023 mmol) of intermediate S4 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted to a volume of 2.5 mL with PBS buffer (pre-adjusted to pH 8) and then passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, followed by elution with PBS buffer at pH 8. The eluent was then stirred overnight at RT with argon atmosphere. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 5

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 10 mg/mL) in 0.5 mL PBS. The mixture was stirred at RT for 30 min, and then 0.23 mg (0.00023 mmol) of intermediate S5 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the reaction mixture was diluted to a volume of 2.5 mL with PBS buffer (pre-adjusted to pH 8) and then fed to a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, and eluted with PBS buffer at pH 8. The eluent was then stirred overnight under argon atmosphere and RT. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 6

Exemplary method A:

Under argon atmosphere, 5 equivalents (0.18 mg) of intermediate S6 dissolved in 50 μl DMSO were added to 5 mg of the corresponding antibody (c = 12.5 mg/ml) in 0.4 ml PBS. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The reaction mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, concentrated by ultracentrifugation, and then diluted again with PBS (pH 7.2).

Exemplary Method B:

Under argon atmosphere, 5 equivalents (2.8 mg) of intermediate S6 dissolved in 500 μl DMSO were added to 80 mg of the antibody in 5.58 mL of PBS (c = 14.3 mg/mL; concentrations can typically be between 3 and 20 mg/mL). After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another 1 hour. Subsequently, the mixture was diluted to 7.5 mL with PBS buffer (pH 7.2), purified by Sephadex column chromatography, concentrated by ultracentrifugation, and then diluted again with PBS (pH 7.2).

Example 7

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the antibody (c = 12.5 mg/mL) in 0.4 mL PBS buffer (pH 7.2). The mixture was stirred at RT for 30 min, and then 0.24 mg (0.00023 mmol) of intermediate S7 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted with PBS buffer to a total volume of 2.5 mL. The solution was then passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer (pH 7.2) and eluted with PBS buffer (pH 7.2). The mixture was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 8

Exemplary method A:

Under argon atmosphere, 5 equivalents (0.14 mg) of intermediate S8 dissolved in 50 μl DMSO were added to 5 mg of the corresponding antibody (c = 12.5 mg/ml) in 0.4 ml PBS. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The reaction mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, concentrated by ultracentrifugation, and then diluted again with PBS (pH 7.2).

Example 9

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 10 mg/mL) in 0.5 mL PBS. The mixture was stirred at RT for 30 min, and then 0.22 mg (0.00023 mmol) of intermediate S9 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the reaction mixture was diluted to a volume of 2.5 mL with PBS buffer (pre-adjusted to pH 8) and then fed to a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, and eluted with PBS buffer at pH 8. The eluent was then stirred overnight under argon atmosphere and RT. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 10

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the corresponding antibody (c = 12.5 mg/mL) in 0.4 mL PBS (pH 7.2). The mixture was stirred at RT for 30 min, and then 0.22 mg (0.00023 mmol) of intermediate S10 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted with PBS buffer to a total volume of 2.5 mL and then applied to a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, followed by elution with PBS buffer at pH 8. The eluent was then stirred overnight at RT under argon atmosphere. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 11

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 12.5 mg/mL) in 0.4 mL PBS. The mixture was stirred at RT for 30 min, and then 0.23 mg (0.00023 mmol) of intermediate S11 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted to a total volume of 2.5 mL with PBS buffer (pre-adjusted to pH 8) and then passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer (pH 8), and eluted with PBS buffer (pH 8). The eluent was stirred overnight at RT under argon atmosphere. The mixture was then concentrated by ultracentrifugation and diluted again with PBS buffer (pH 7.2).

Example 12

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the antibody (c = 12.5 mg/mL) in 0.4 mL PBS buffer (pH 7.2). The mixture was stirred at RT for 30 min, then 0.22 mg (0.00023 mmol) of intermediate S12 dissolved in 50 μL DMSO was added, and the mixture was stirred at RT overnight. The mixture was then diluted with PBS buffer to a total volume of 2.5 mL. The solution was passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer and eluted with PBS buffer. The mixture was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 13

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the antibody (c = 10 mg/mL) in 0.5 mL PBS buffer (pH 7.2). The mixture was stirred at RT for 30 min, and then 0.28 mg (0.00023 mmol) of intermediate S13 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted with PBS buffer to a total volume of 2.5 mL, and then passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, followed by elution with PBS buffer at pH 8. The eluent was stirred overnight at RT under argon atmosphere. The mixture was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 14

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the antibody (c = 10 mg/mL) in 0.5 mL PBS buffer (pH 7.2). The mixture was stirred at RT for 30 min, and then 0.3 mg (0.00023 mmol) of intermediate S14 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the mixture was diluted to a total volume of 2.5 mL with PBS buffer (adjusted to pH 8). The solution was then passed through a PD10 column (G-25, GE Healthcare) equilibrated with PBS buffer (pH 8), eluted with PBS buffer (pH 8), and stirred at RT overnight. The mixture was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Under these conditions and in the case of the ADC from Example 14 prepared using this adapter, the ring-opening was incomplete. They still contained a relatively large fraction (over 50%), where the linker to the antibody was via a closed-ring succinimide form (see Example 7).

Example 15

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 10 mg/mL) in 0.5 mL PBS buffer. The mixture was stirred at RT for 30 min, then 0.27 mg (0.00023 mmol) of intermediate S15 dissolved in 50 μL DMSO was added, and the mixture was stirred at RT for another 90 min. The solution was then diluted to 2.5 mL with PBS buffer (pre-adjusted to pH 8) and passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, and eluted with PBS buffer at pH 8. The eluent was stirred overnight at RT under argon atmosphere. The mixture was then concentrated by ultracentrifugation and diluted again with PBS buffer (pH 7.2).

Example 16

Example method A (for obtaining higher DAR):

Under argon atmosphere, a solution of 0.067 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 10 mg/mL) in 0.5 mL PBS buffer. The mixture was stirred at RT for 30 min, and then 0.68 mg (0.00057 mmol) of intermediate S16 dissolved in 50 μL DMSO was added. After further stirring overnight under argon atmosphere and RT, the mixture was diluted to a volume of 2.5 mL with PBS buffer. The solution was then passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 7.2 and eluted with PBS buffer at pH 7.2. The mixture was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 17

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.045 mL PBS buffer was added to 5 mg of appropriate antibody (c = 11 mg/mL) in 0.45 mL PBS buffer. The mixture was stirred at RT for 30 min, then 0.30 mg (0.00023 mmol) of intermediate S17 dissolved in 50 μL DMSO was added, and the mixture was stirred at RT for another 90 min. The solution was then diluted to 2.5 mL with PBS buffer (pre-adjusted to pH 8) and passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, and eluted with PBS buffer at pH 8. The eluent was stirred overnight at RT under argon atmosphere. The mixture was then concentrated by ultracentrifugation and diluted again with PBS buffer (pH 7.2).

Example 18

Exemplary method A:

Under argon atmosphere, 5 equivalents (0.12 mg) of intermediate S18 dissolved in 30 μl DMSO were added to 3 mg of the corresponding antibody (c = 10 mg/ml) in 0.3 ml PBS. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The reaction mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, concentrated by ultracentrifugation, and then diluted again with PBS (pH 7.2).

Example 19

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the appropriate antibody (c = 11 mg/mL) in 0.45 mL PBS. The mixture was stirred at RT for 30 min, then 0.28 mg (0.00023 mmol) of intermediate S19 dissolved in 50 μL DMSO was added, and the mixture was stirred at RT for another 90 min. The solution was then diluted to 2.5 mL with PBS buffer (pre-adjusted to pH 8) and passed through a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, and eluted with PBS buffer at pH 8. The eluent was stirred overnight at RT under argon atmosphere. The mixture was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 20

Exemplary method A:

Under argon atmosphere, 5 equivalents (0.17 mg) of intermediate S20 dissolved in 50 μl DMSO were added to 5 mg of the corresponding antibody (c = 10 mg/ml) in 0.5 ml PBS. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another 1 hour. The mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, concentrated by ultracentrifugation, and then diluted again with PBS (pH 7.2).

Example 21

Exemplary method A:

Under argon atmosphere, a solution of 0.029 mg TCEP in 0.05 mL PBS buffer was added to 5 mg of the associated antibody (c = 10 mg/mL) in 0.5 mL PBS buffer (pH 7.2). The mixture was stirred at RT for 30 min, and then 0.22 mg (0.00023 mmol) of intermediate S21 dissolved in 50 μL DMSO was added. After stirring at RT for another 90 min, the reaction mixture was diluted with PBS buffer to a total volume of 2.5 mL and then applied to a PD 10 column (G-25, GE Healthcare) equilibrated with PBS buffer at pH 8, followed by elution with PBS buffer at pH 8. The eluent was stirred overnight at RT under argon atmosphere. The eluent was then concentrated by ultracentrifugation and further diluted with PBS buffer (pH 7.2).

Example 22

Exemplary method A:

Under argon atmosphere, 5 mg of the corresponding antibody (c = 10 mg/ml) in 0.5 ml PBS was mixed with 5 equivalents (0.22 mg) of intermediate S22 dissolved in 50 μl DMSO. After stirring at room temperature for 1 hour, the same amount was added again, and the mixture was stirred at room temperature for another hour. The reaction mixture was then diluted to 2.5 ml with PBS buffer (pH 7.2), purified on a Sephadex column, concentrated by ultracentrifugation, and then diluted again with PBS (pH 7.2).

Working examples of metabolites

In its various embodiments, metabolites formed in tumors by the ADC of the present invention are prepared and characterized for comparative purposes. Some of these have already been described in earlier disclosures of other ADCs.

Metabolites, for example, formed by Examples 2, 3, and 9

The synthesis of four isomeric cysteine metabolites that can be formed from Examples 1, 2, 3, and 9 is described in WO2016/096610, wherein they are characterized as Examples M13, M14, M15, and M16.

Metabolites M1 and M2, for example, can be formed from Examples 4 and 5.

Example M1

N-(3-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-3-carboxypropionyl)glycyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-D-α-glutamine trifluoroacetate

Regioisomer 1, a mixture of epiisomers

Triethylamine (10 mL, 73 mmol) was added to a solution of L-cysteine methyl ester hydrochloride (1:1) (5.00 g, 29.1 mmol) in 1,4-dioxane (200 mL), followed by 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (8.31 g, 32.0 mmol). The reaction was stirred at room temperature for 20 hours. Subsequently, the solid was filtered off and the filtrate was concentrated under high vacuum. The residue was purified by preparative HPLC.

To a DMF (6.5 mL) solution of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine methyl ester (130 mg, 465 μmol) and 3-bromo-4-methoxy-4-oxobutyric acid (393 mg, 1.86 mmol), 210 μl (1.4 mmol) of 1,8-diazabicyclo(5.4.0)undec-7-ene was added, and the reaction was stirred at room temperature for 10 min. The mixture was then concentrated under reduced pressure, and the residue was purified by preparative HPLC. The solvent was evaporated under reduced pressure, and the residue was dried under high vacuum.

In the presence of HATU, the resulting intermediate was coupled to intermediate C119 using conventional peptide chemistry methods. Subsequently, the methyl ester was hydrolyzed by treatment with a lithium hydroxide THF/water (1:1) solution.

In the final step, 22 mg of the obtained intermediate was dissolved in 10 mL of 2,2,2-trifluoroethanol. 34 mg (0.252 mmol) of zinc chloride was added, and the mixture was stirred at 50 °C for 1 hour. Subsequently, 74 mg (0.252 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid, 10 mL of water, and 500 μL of TFA were added. The mixture was filtered, and the solvent was evaporated under reduced pressure. The residue was purified by preparative HPLC. After concentrating the appropriate fraction and freeze-drying the residue from acetonitrile/water, 13 mg (72% of the theoretical value) of the title compound was given.

LC-MS (Method 5): Rt = 2.44 min; MS (ESIneg): m/z = 959 [MH ^]

Example M2

N-(2-{[(2R)-2-amino-2-carboxyethyl]thioalkyl}-3-carboxypropionyl)glycyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]butyryl}amino)ethyl]-D-α-glutamine trifluoroacetate

Regioisomer 2, a mixture of epiisomers

The title compound M2 was prepared as a mixture of epimers in a similar manner to Example M1:

801 μl (5.4 mmol) of 1,8-diazabicyclo(5.4.0)undec-7-ene was added to a DMF (40 mL) solution of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine methyl ester (1000 mg, 3.58 mmol) and 2-bromo-4-ethoxy-4-oxobutyric acid (926 mg, 4.11 mmol), and the reaction was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure, and the residue was purified by preparative HPLC.

In the presence of HATU and methylmorpholine, the resulting intermediate was coupled to intermediate C119 using conventional peptide chemistry methods. Subsequently, the methyl and ethyl esters were hydrolyzed by treatment with a lithium hydroxide THF/water (1:1) solution.

In the final step, 48 mg of the intermediate was dissolved in 5 mL of 2,2,2-trifluoroethanol. 75 mg (0.550 mmol) of zinc chloride was added, and the mixture was stirred at 50 °C for 3 hours. Subsequently, 160 mg (0.550 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid, 2 mL of water, and 20 μL of TFA were added. The solvent was concentrated under reduced pressure, and the residue was purified by preparative HPLC. After concentrating the appropriate fraction and freeze-drying the residue from acetonitrile/water, 14 mg (39% of the theoretical value) of the title compound was given.

LC-MS (Method 5): Rt = 2.41 min; MS (ESIneg): m/z = 959 [MH ^]

Metabolites M3, for example, can be formed from Examples 6, 7, 10, 12, 13, 14, 16 and 22.

Example M3

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

Intermediate C121 was converted into the title compound by hydrogenation in ethanol on 10% palladium/activated carbon at room temperature under standard hydrogen pressure for 1 hour.

LC-MS (Method 1): R _{_t} = 1.78 min; MS (ESIpos): m/z = 714[M+H] ⁺ .

Metabolite M4, for example, can be formed from Example 8.

Examples of metabolite M4:

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

The preparation and characterization of metabolite M4 are described as metabolite M9 in WO2016/096610. This metabolite, formed from the ADC of the present invention in Example 8, exhibits characteristics different from other metabolites M1, M2, and M3 in terms of transporter substrate properties and cytotoxicity.

Reference Examples: APDC and Small Molecules

First, for comparative purposes, APDC R1e and ADC R6k were each prepared using tripeptide sequences as substrates for podase.

In addition, to examine the cleavage mediated by podase, small molecule RM-A and RM-B cleavable prodrugs of podase were prepared for comparative purposes.

Reference embodiment R1e with TPP1015

An ADC with TPP1015 was prepared similarly to that in Example 1.

Protein concentration: 1.7 mg/ml

Drug/mAb ratio: 3.3

The precursor was prepared by coupling intermediate F104 with intermediate L103 in the presence of HATU and N,N-diisopropylethylamine, similar to intermediate S1.

LC-MS (Method 1): R _{_t} = 0.83 min; MS (ESIpos): m/z = 1068(M+H) ⁺ .

Reference embodiment R6k with TPP7007

An ADC with TPP7007 was prepared similarly to that in Example 6.

Protein concentration: 2.11 mg/ml

Drug/mAb ratio: 5.3

The precursor was prepared from compound C121, similar to intermediate S6, by first coupling it to ^N2- (tert-butoxycarbonyl)-L-aspartic acid 2,5-dioxopyrrolidine-1-yl ester in DMF in the presence of N,N-diisopropylethylamine. The Boc protecting group was then removed by stirring in a trifluoroethanol solution of 6 equivalents of zinc chloride at 50°C for 1 hour. In the next step, the resulting intermediate was coupled to N-(tert-butoxycarbonyl)-L-alanyl-L-alanine in DMF in the presence of HATU and N,N-diisopropylethylamine. The Boc protecting group was then removed again by stirring in a trifluoroethanol solution of 6 equivalents of zinc chloride at 50°C for 2 hours. In the next step, benzyl ester was removed by hydrogenation in ethanol at room temperature under standard hydrogen pressure on 10% palladium/activated carbon, and then the deprotected intermediate was converted into the title compound by reaction with 1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)dipyrrolidine-2,5-dione in DMF in the presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R _{_t} = 0.90 min; MS (ESIpos): m/z = 1181[M+H] ⁺ .

RM-A (Model Compound A)

N-(pyridin-4-ylacetyl)-L-alanyl-L-alanyl-N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-(methylamino)-1-oxobut-2-yl]-L-asparagine

First, trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-methylbutyramide (1:1) (Example 99) was prepared as described in WO 2015096982 A1. Then, the title compound was prepared using this intermediate by coupling it 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] ^⁺ . RM-B (Model compound B)

N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-(methylamino)-1-oxobut-2-yl]-N ^2- (pyridin-4-ylacetyl)-L-asparagine

First, trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-methylbutyramide (1:1) (Example 99) was prepared as described in WO 2015096982 A1. Then, the title compound was prepared using this intermediate by coupling it with intermediate L136 in DMF in the presence of HATU and N,N-diisopropylethylamine.

LC-MS (Method 12): R _{_t} = 1.57 min; MS (ESIpos): m/z = 760 [M+H] ⁺ .

RM-C (Model Compound C)

N ¹ -[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-(methylamino)-1-oxobut-2-yl]-N ^2- (pyridin-4-ylacetyl)-L-glutamine

First, trifluoroacetic acid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrolo-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-N-methylbutyramide (1:1) (Example 99) was prepared as described in WO 2015096982 A1. Then, the title compound was prepared using this intermediate by coupling it with intermediate L137 in DMF in the presence of HATU and N,N-diisopropylethylamine.

LC-MS (Method 12): R _{_t} = 1.56 min; MS (ESIpos): m/z = 774 [M+H] ^⁺ .

C: Evaluation of biological efficacy

The biological activity of the compounds of this invention can be demonstrated in the following assays:

C-1a determines the cytotoxic effects of ADCs

Cytotoxicity of ADCs was analyzed using multiple different cell lines:

NCI-H292: Human lung mucoepidermoid carcinoma cells, ATCC-CRL-1848, standard culture medium: RPMI 1640 (Biochrom; #FG1215, stable glutamine) + 10% FCS (Sigma; #F2442), TWEAKR-positive; EGFR-positive.

BxPC3: Human pancreatic cancer cells, ATCC-CRL-1687, standard culture medium: RPMI 1640 (Biochrom; #FG1215, stable glutamine) + 10% FCS (Sigma; #F2442), TWEAKR-positive.

LoVo: Human colorectal cancer cells, ATCC No. CCL-229. Culture for MTT assay: Standard medium: Kaighn's + L-glutamine (Invitrogen 21127) + 10% heat-inactivated FCS (from Gibco, No. 10500-064). Culture for CTG assay: RPMI 1640 (Biochrom; #FG1215, stable glutamine) + 10% FCS (Sigma #F2442). TWEAKR positive.

KPL4: Human breast cancer cell line, Bayer Pharma AG (individual assay and validation at DSMZ on July 19, 2012), standard culture medium: RPMI 1640 (from Gibco);

#21875-059, stable L-glutamine) + 10% heat-inactivated FCS (from Gibco, No. 10500-064); HER2-positive.

SK-HEP-1: Human hepatocellular carcinoma cell line, ATCC No. HTB-52, standard culture medium: MEM, containing El balanced salts + Glutamax I (Invitrogen 41090) + 10% heat-inactivated FCS (from Gibco, No. 10500-064); EGFR-positive, TWEAKR-positive.

KU-19-19: Human bladder cancer cells, DMSZ, standard culture medium: RPMI 1640 (Biochrom; #FG1215, stable glutamine) + 10% FCS (Sigma; #F2442), TWEAKR-positive.

U251: Human glioblastoma cells, standard culture medium: RPMI 1640 (Biochrom; #FG1215, glutamine) + 10% FCS (Biochrom; #S0415); B7H3 positive.

As described by the American Tissue Culture Collection (ATCC) or the Leibniz Institute of Microbiology and Cell Culture (DSMZ), cells are cultured using standard methods for the corresponding cell lines.

CTG determination

Cells were cultured using standard methods on the growth medium specified at C-1a. Cells were separated, pelleted, resuspended in medium, counted, and seeded into 96-well white-bottomed plates (Costar#3610) (75 μl/well, cell counts per well as: NCI-H292: 2500 cells/well, BxPC3: 2500 cells/well, LoVo: 3000 cells/well) and incubated at 37°C in a 5% CO2 incubator for testing. After 24 h, the antibody-drug conjugate was added to the cells in 25 μl of medium (four-fold concentrated) to obtain a final antibody-drug conjugate concentration of 3 x ^10⁻⁷ M to ³ x ^10⁻¹¹ M (in triplicate). Cells were then incubated in an incubator at 37°C and 5% CO2. Cell viability at the start of drug treatment (day 0) was determined on parallel plates using a CellTiter Glow (CTG) chemiluminescence cell viability assay (Promega #G7573 and #G7571). For this purpose, 100 μl of substrate was added to each batch of cells, the plate was covered with aluminum foil, shaken at 180 rpm for 2 minutes on a plate shaker, allowed to stand on a lab bench for 8 minutes, and then measured using a chemiluminometer (Victor X2, PerkinElmer). The substrate was used to detect ATP content in live cells, which produce a luminescent signal, with the signal intensity proportional to cell viability. After incubation with the antibody-drug conjugate for 72 h, the viability of these cells was determined using the CellTiter Glow chemiluminescence cell viability assay as described above. From the measured data, the _IC50 of growth inhibition compared to day 0 was calculated using a DRC (dose-response curve) analysis spreadsheet and a 4-parameter fit. The DRC analysis spreadsheet is a biobook spreadsheet developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook Suite platform (IDBS: ID BusinessSolutions Ltd., Guildford, UK).

MTT assay

Cells were cultured using standard methods with the growth medium specified under C-1a. Cells were isolated, clumped, resuspended in medium, counted, and seeded into 96-well white-background plates (from Costar #3610) (NCI H292: 2500 cells/well; SK-HEP-1: 1000 cells/well; KPL4: 1200 cells/well; total volume 100 μl) for testing. Cells were then incubated at 37°C in a 5% CO2 incubator. After 48 hours, the medium was changed. Then, antibody-drug conjugates at concentrations of ^10⁻⁵ M to ^10⁻¹³ M in 10 μl of medium were pipetted into the cells (in triplicate), and the mixture was incubated at 37°C in a 5% CO2 incubator. Ninety-six hours later, cell proliferation was assessed using an MTT assay (ATCC, Manassas, Virginia, USA, catalog number 30-1010K). For this, cells were incubated with the MTT reagent for 4 hours, followed by overnight lysis with detergent. The resulting dye (Infinite M1000 pro, Tecan) was detected at 570 nm. The measurements were used to calculate the _IC50 for growth inhibition using a DRC (dose-response curve). Cell proliferation of cells that were not treated with the test substance but were otherwise treated was defined as 100%.

The table below lists the IC ₅₀ values for representative working examples of these measurements:

Table 1a

Table 1b below lists the IC ₅₀ values for these measurements in reference examples:

Table 1b

The reported activity data pertain to the working examples described in this experimental section, indicating the drug/mAB ratio. These values may vary for different drug/mAB ratios. The _IC50 value is an average of several independent experiments or individual values. The antibody-drug conjugate exhibits selectivity for the corresponding isotype control containing the respective linker and toxicant cluster.

C-1b determined the inhibitory effect of the active metabolite formed from the selected embodiment on kinesin spindle protein KSP/Eg5.

The motor domain of human kinin spindle protein KSP/Eg5 (tebu-bio/Cytoskeleton Inc, No. 027EG01-XL) was incubated at 10 nM with microtubules (bovine or porcine, tebu-bio/Cytoskeleton Inc) stabilized with 50 μg/ml paclitaxel (Sigma No. T7191-5MG) at RT for 5 min in 15 mM PIPES, pH 6.8 (5 mM _MgCl2 and 10 mM MTT, Sigma). The freshly prepared mixture was aliquoted into 384 MTP (from Corning). The target inhibitor and ATP (final concentration 500 μM, Sigma) were then added at concentrations ranging from 1.0 x 10⁻⁶ M to 1.0 x 10⁻¹³ M. The mixture was incubated at RT for 2 h. ATPase activity was detected by measuring inorganic phosphate formed using malachite green (Biomol). After adding the reagent, incubate at RT for 50 min, then detect the absorbance at 620 nm. Positive controls used were monostyrene (Sigma, M8515-1 mg) and Espin (AdooQ Bioscience A10486). Individual data for dose-activity curves were obtained from eight assays. The _IC50 value is the average of two independent experiments. The 100% control was the sample not treated with the inhibitor.

Table 2 below lists the _IC50 values of the active metabolites formed from representative working examples from the assays and summarizes the corresponding cytotoxicity data (MTT assays):

Table 2

The activity data reported relate to the working examples described in this experimental section.

C-1c enzymatic assay and stability study

First, the cleavage of the small molecule prodrugs RM-A and RM-B was tested under various different conditions:

a: Legume protease assay

The rh podase assay was performed using recombinant human enzymes. The rh podase enzyme solution (catalog number 2199-CY, R&D Systems) was diluted to the desired concentration in 50 mM sodium acetate buffer/100 mM NaCl, pH 4.0 and pre-incubated at 37°C for 2 hours. The rh podase was then adjusted to a final concentration of 1 ng/μl in 50 mM MES buffer, 250 mM NaCl, pH 5.0. For each podase prodrug to be detected, a mixture was prepared in a microreaction vessel (0.5 ml, from Eppendorf). For this purpose, the substrate solution was adjusted to the desired concentration (double concentration) in 50 mM MES buffer, 250 mM NaCl, pH 5.0. For kinetic measurements of the enzymatic reaction, 250 μl of podase solution was initially added, followed by the addition of 250 μl of substrate solution (final concentration: double concentration). 50 μl samples were taken at different time points. The sample was immediately mixed with 100 μl of ice-cold methanol to terminate the enzymatic reaction, and then frozen at -20 °C. Sampling was performed at 0.5 h, 1 h, 3 h, and 24 h. The samples were then analyzed by RP-HPLC and LC-MS. The determination of the released toxic clusters allowed for the determination of the half-life t _{_1/2} of the enzymatic reaction (Figure 1).

Model compound A (RM-A) was cleaved into the target compound under the above-mentioned podase assay conditions, with a half-life of 0.2 hours.

Model compound B (RM-B) was cleaved into the target compound under the above-mentioned podase assay conditions, with a half-life of approximately 10 hours.

Model compound C (RM-C) was not cleaved into the target compound under the above-mentioned podase assay conditions.

b: Assay used to determine stability in rat plasma

To test the stability of compounds A and B, 1 ml of rat plasma in a 1.5 ml Eppendorf tube was heated to 37 °C on an Eppendorf shaker in each case. Stock solutions of compounds A and B at a concentration of 100 μg/ml (9 acetonitrile/1 DMSO) were prepared. 10 μl of each stock solution was pipetted into 1 ml of heated rat plasma to obtain a concentration of 1 μg/ml.

The samples were incubated at 450 rpm and 37 °C for 24 h. At each sampling time of 0, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 24 h, 50 μl of the sample was taken and pipetteed into 150 μl of methanol. The internal standard was included in the initial methanol feed at a concentration of 0.05 μg/mL. After a brief vortexing, 300 μl of 10 mM ammonium acetate buffer (pH 6.8) was added, and the sample was centrifuged at 1881 g for 10 min. The samples were then analyzed by RP-HPLC and LC-MS.

c: Assay used to determine stability in rat liver lysosomes

To determine the lysosomal stability of compounds RM-A and RM-B, lysosomal enzymes were isolated from rat hepatocytes. Compounds A and B were added separately to the lysosomal extract to test their stability under lysosomal conditions. The proteolytic enzyme podocyte protease is expressed in rat liver lysosomes in very small amounts—if any—(Chen, J-M. et al., 1997). To monitor the enzymatic activity of the lysosomal enzymes, cathepsin-specific substrates were added.

First, fresh rat livers were harvested, weighed, and immediately placed on ice in homogenization medium (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES, pH 7). The livers were pulverized, and the medium was replaced. The rat livers were homogenized in Potter (B. Braun) at 750 rpm with a volume four times their weight in the homogenate. The homogenate was centrifuged at 1000 g for 10 min, and the supernatant was filtered. In the next step, the "light mitochondrial fraction" (LMF) was separated from the supernatant by ultracentrifugation at 26,500 g for 20 min. Lysosomes were also present in the clumps in addition to mitochondria. The supernatant was discarded, and the clumps were resuspended in 0.8 ml/g homogenization medium.

To separate the lysosomal fractions from other cellular components of LMF, six Optiprep density gradients with sucrose contents of 8%, 12%, 16%, 19%, 22.5%, and 27% were prepared in Optiprep buffer (100 mM MOPS, 20 mM EDTA, 0.5% EtOH, pH 7.6). Sucrose was added in specific percentages from a 2.3 M stock solution. An additional 2.5 ml of the separated LMF was added to the 19% sucrose concentration density gradient. The density gradients were then separated into layers in 10 ml centrifuge tubes and centrifuged at 48,500 g for 17 h. Fractions 1-8 were placed in the top 5.6 ml of the gradient and discarded. Fractions 9 and 10 were placed in the bottom 1.6 ml, removed from the gradient, and lysed on ice for 5 min with 1.6 ml of lysis buffer (25 mM HEPES, 150 mM NaCl, 0.1% Triton X100, pH 5). Lysosomes are present in fractions 9 and 10. To monitor lysis, the protein content of the lysosomal fractions is monitored using a BCA assay (Pierce BCA Protein Assay Kit).

To determine the lysosomal stability of compounds RM-A and RM-B, 6 μl of a 100 μg/mL stock solution (9 acetonitrile/1 DMSO) was added to 290 μl of 90 mM citrate buffer and 300 μl of lysosomal extract, and incubated at 37 °C on an Eppendorf shaker. At 0 h, 1 h, 2 h, 6 h, 24 h, and 48 h, 50 μl of the incubation solution was taken each time and transferred to 150 μl of MeOH. An internal standard of 0.05 μg/mL was included in the initial feed. For RP-HPLC-LCMS analysis, the sample was diluted with 300 μl of 10 mM ammonium acetate buffer (pH 6.8) and analyzed.

Under the conditions of lysosomal stability assays, compound A (reference example RM-A), with a half-life of approximately 6 hours, cleaved to approximately 80% within 24 hours, while compound B (reference example RM-B) cleaved to approximately 13% within the same timeframe. Therefore, RM-B was significantly more stable than RM-A in the lysosomal stability assay, implying a reduced degree of formation of active metabolites in a healthy liver.

C-2 internalization assay

Internalization is a crucial process that enables the specific and efficient delivery of cytotoxic payloads to antigen-expressing cancer cells via antibody-drug conjugates (ADCs). This process was monitored by fluorescent labeling of the specific antibody and an allotype control antibody. First, a fluorescent dye was conjugated to the lysine residue of the antibody. Conjugation was performed at pH 8.3 using a two-molar excess of CypHer 5E monoNHS ester (batch 357392, GE Healthcare). Following conjugation, 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 remove excess dye and adjust the pH. Protein solutions were concentrated using a VIVASPIN 500 column (Sartorius stedim biotec). The dye loading of the antibody was determined by spectrophotometric analysis (NanoDrop) and subsequent calculations.

(D:P＝A _{dyeε protein} :(A ₂₈₀ -0.16A _dye )ε _dye ).

The dye loads of the antibodies and isotype controls detected here were on comparable orders of magnitude. Cell binding assays confirmed that conjugation did not cause any change in antibody affinity.

Labeled antibodies were used for internalization assays. Prior to treatment, cells (2 x ^10⁴ /well) were seeded in 100 μl of 96-well MTP (fat, black, clear substrate, No. 4308776, from Applied Biosystems) medium. After incubation at 37°C/5% _CO₂ for 18 h, the medium was replaced and different concentrations (10, 5, 2.5, 1, 0.1 μg/ml) of labeled antibody were added. The same treatment protocol was applied to labeled isotype controls (negative controls). Incubation times were selected as 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6 h, and 24 h. Fluorescence measurements were performed using an InCellAnalyzer 1000 (from GE Healthcare). Kinetics were then assessed by measuring the particle count/cell and total particle intensity/cell parameters.

After binding to the receptor, the antibody's internalization ability was assessed. For this purpose, cells with different receptor expression levels were selected. Target-mediated specific internalization was observed with the antibody described in the context of this invention, while no internalization was observed in the isotype control.

C-3 is used in vitro tests to determine cell permeability.

Cellular permeability of substances can be studied in flux assays using Caco-2 cells in vitro [MD.Troutman and DR.Thakker, Pharm.Res.20(8), 1210-1224(2003)]. For this purpose, cells were cultured in 24-well plates for 15-16 days. To determine permeability, the corresponding test substance was applied to the cells in HEPES buffer from the top (A) or base (B) and incubated for 2 hours. Samples were taken from the cis and trans compartments at 0 and 2 hours. Samples were separated by HPLC (Agilent 1200, Germany) using a reversed-phase column. The HPLC system was coupled to a triple quadrupole mass spectrometer API 4000 (AB SCIEX Deutschland GmbH, Darmstadt, Germany) via a Turbo ion spray interface. Permeability is assessed based on the _Papp value, which is calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. Substances are classified as actively transported when the ratio of _Papp (BA) to _Papp (AB) (external displacement ratio) is >2 or <0.5.

Crucial to the intracellular release of toxic clusters are the permeability from B to A [Papp(B-A)] and the ratio of Papp(B-A) to Papp(A-B) (efflux ratio): the lower this permeability, the slower the active and passive transport of the substance through the monolayer of Caco-2 cells. Furthermore, if the efflux ratio does not indicate any active transport, the substance can remain in the cell for a longer period after intracellular release. Therefore, more time is available for interaction with biochemical targets (in this case, kinesiology, KSP/Eg5).

Table 4 below lists the permeability data from representative working examples of this measurement:

Table 4

Metabolites M1, M2, and M3 formed from the ADCs of the present invention from several different embodiments exhibit very low levels of cellular transport and low efflux ratios. For example, metabolite M4 exhibits distinct characteristics.

C-4 is an in vitro assay used to determine the substrate properties of P-glycoprotein (P-gp).

Many tumor cells express drug transporters, which is often accompanied by the development of resistance to cytotoxic inhibitors. Therefore, substances that are not substrates of such transporters, such as P-glycoprotein (P-gp) or BCRP, can exhibit an improved activity profile.

The substrate properties of substances for P-gp (ABCB1) were determined by flux assay using LLC-PK1 cells (L-MDR1 cells) overexpressing P-gp [AHSchinkel et al., J. Clin. Invest. 96, 1698-1705 (1995)]. For this purpose, LLC-PK1 cells or L-MDR1 cells were cultured in 96-well plates for 3–4 days. To measure permeability, the corresponding test substance, alone or in the presence of an inhibitor (e.g., ivermectin or verapamil), was applied to the cells from the top (A) or base (B) in HEPES buffer and incubated for 2 hours. Samples were taken from the cis and trans compartments at 0 and 2 hours. Samples were separated by HPLC using a reversed-phase column. The HPLC system was coupled to a triple quadrupole mass spectrometer API 4000 (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo ion spray interface. Permeability is assessed based on the P _-app value, which is calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. Substances are classified as P-gp substrates when the efflux ratio of P- _app (BA) to P- _app (AB) is >2.

As a further criterion for assessing the properties of P-gp substrates, the efflux ratios in L-MDR1 and LLC-PK1 cells, or the efflux ratios with and without inhibitors, can be compared. If these values differ by more than a factor of 2, the corresponding substance is a P-gp substrate.

C-5 Pharmacokinetics

C5a: Identification of ADC metabolites after in vitro internalization

Method description:

Internalization studies were performed using immunoconjugates to analyze intracellularly formed metabolites. For this purpose, human lung tumor cells NCIH292 (3 x ^10⁵ /well) were seeded in 6-well plates and incubated overnight (37°C, 5% _CO₂ ). Cells were treated with 10 μg/ml (66 nM) of the target ADC. Internalization was performed at 37°C and 5% _CO₂ . Cell samples were collected at different time points (0, 4, 24, 48, 72 h) for further analysis. First, the supernatant (approximately 5 ml) was harvested, centrifuged (2 min, RT, 1000 rpm Heraeus Variofuge 3.0R), and stored at -80°C. Cells were washed with PBS and separated with Accutase to determine cell count. Following another wash, a defined number of cells (2 x ^10⁵ ) were treated with 100 ml of lysis buffer (Mammalian Cell Lysis Kit (Sigma-MCL1)) and incubated in protein LoBind tubes (Eppendorf, catalog number 0030108.116) with continuous shaking (Thermomixer, 15 min, 4 °C, 650 rpm). After incubation, the lysate was centrifuged (10 min, 4 °C, 12000 g, Eppendorf 5415R) and the supernatant was collected. The obtained supernatant was stored at -80 °C. All samples were then analyzed as follows.

After precipitating proteins with methanol or acetonitrile, compounds in culture supernatants or cell lysates were measured by high-performance liquid chromatography (HPLC) coupled to a triple quadrupole mass spectrometer (MS).

To post-process 50 μl of culture supernatant/cell lysate, add 150 μl of precipitation reagent (methanol) and shake the mixture for 10 seconds. The precipitation reagent contains an appropriate concentration (typically in the range of 20-100 μg/L) of internal standard (ISTD). After centrifuging at 1881 g for 10 min, transfer the supernatant to an autosampler vial, fill with 300 μl of buffer matched with the eluent, shake again, and centrifuge at 1881 g for 10 min.

Finally, cell lysates and supernatant samples were analyzed using an HPLC coupled with an API4500 triple quadrupole mass spectrometer from AB SCIEX Deutschland GmbH.

For calibration, mix blank lysates or blank supernatants at appropriate concentrations (0.1–1000 μg/L). The limit of detection (LLOQ) is approximately 0.2 μg/L.

Quality controls for testing effectiveness included 4 and 40 μg/l.

C5b: Identification of ADC metabolites in vivo

Following intravenous administration of 10 mg/kg of the various conjugates of the present invention to xenograft mice, plasma, tumor, liver, and kidney concentrations of antibodies and potential metabolites can be measured 24 h after administration. More specific methods for xenograft models are described below C-6. The concentrations of metabolites from the conjugates of the present invention are discussed here. The measurements of metabolites in the matrix mentioned also provide information relating to the degree of metabolite loading in plasma, kidney, and liver compared to the load in the tumor.

Analysis for quantifying potential metabolites

Proteins are typically precipitated with methanol, and then compounds in plasma, tumors, liver, and kidneys are analyzed by high-performance liquid chromatography (HPLC) coupled to a triple quadrupole mass spectrometer (MS).

To post-process 50 μl of plasma, add 150 μl of precipitation reagent (usually methanol) and shake the mixture for 10 seconds. The precipitation reagent contains an internal standard (ISTD) at an appropriate concentration (usually in the range of 20-100 μg/L). After centrifuging at 1881 g for 10 minutes, transfer the supernatant to an autosampler vial, fill with 300 μl of buffer matched with the eluent, and shake again.

In the post-processing of tumor or organ materials, the specific material is mixed with 3-20 times its volume of extraction buffer. The extraction buffer contains 50 ml of tissue protein extraction reagent (Pierce, Rockford, IL), two total protease inhibitor cocktail pellets (Roche Diagnostics GmbH, Mannheim, Germany), and benzoyl sulfonyl fluoride (Sigma, St. Louis, MO), to a final concentration of 1 mM. Depending on the tissue type (hard: tumor; soft: liver, kidney), the lysis and homogenization program of the Prescellys 24 lysis and homogenization system (Bertin Technologies) (www.prescellys.com) is selected. The homogenized sample is incubated overnight at 4°C. 50 μl of homogenate is transferred to an autosampler vial, and 150 μl of methanol containing ISTD is added. The vial is shaken for 10 seconds and then incubated for 5 min. 300 μl of ammonium acetate buffer (pH 6.8) is added, and after a brief shake, the sample is centrifuged at 1881 g for 10 min.

For calibration, plasma samples and corresponding blank matrices from tissue samples are mixed at concentrations ranging from 0.6 to 1000 μg/L. The limit of detection (LOQ) ranges from 1 to 20 μg/L, depending on the sample or tissue type.

Finally, plasma and matrix samples were analyzed using an API 6500 triple quadrupole mass spectrometer coupled with HPLC from AB SCIEX Deutschland GmbH.

The quality controls used to test effectiveness include 4, 40, and 400 μg/l.

C-6 in vivo activity test

For example, the activity of the conjugates of the present invention can be tested using a xenograft model. Those skilled in the art are familiar with existing methods for testing the activity of the compounds of the present invention (see, for example, WO 2005/081711; Polson et al., Cancer Res. 2009 Mar 15; 69(6):2358-64). For this purpose, for example, a tumor cell line expressing the target molecule of the binding moiety is implanted into rodents (e.g., mice). The conjugate of the present invention, an allotype antibody control conjugate, a control antibody, or an isotonic saline solution is then administered to the implanted animal. Administration may occur once or multiple times. After several days of incubation, tumor size is determined by comparing the animals treated with the conjugate with the control group. The animals treated with the conjugate exhibit smaller tumor sizes.

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

Human tumor cells expressing the antigen of an antibody-drug conjugate are subcutaneously inoculated into the flank of immunosuppressed mice, such as NMRi nude mice or SCID mice. One million to ten million cells are isolated from the cell culture, centrifuged, and resuspended in culture medium or medium/Matrix gel. The cell suspension is then injected subcutaneously into the mice.

Within a few days, the tumor grows. Treatment begins after the tumor has established itself, at which point the tumor size is approximately 40 ^mm² . To assess the impact on larger tumors, treatment may be initiated only when the tumor size is between 50 and 100 ^mm² .

APDC and ADC were administered via intravenous (i.v.) administration into the tail vein of mice. ADC was administered at a volume of 5 ml/kg.

Treatment regimens depend on the pharmacokinetics of the antibody. As a standard, treatment is administered three times consecutively every fourth day. In cases of slow tumor growth, weekly treatment may be used. For rapid evaluation, a single-drug regimen may also be used. However, treatment can also be continued, or a second cycle of three treatment days can be initiated at a later time.

As a standard procedure, eight animals were used in each treatment group. In addition to the groups that received the active substance, one group served as a control group and was treated with buffer solution only, following the same protocol.

During the trial, tumor area was measured periodically in two dimensions (length/width) using calipers. Tumor area was determined as length x width. The ratio of the mean tumor area in the treatment group to the mean tumor area in the control group was expressed as T/C area.

At the end of treatment, when all experimental groups were simultaneously terminated, the tumors could be removed and weighed. The ratio of the average tumor weight in the treatment group to the average tumor weight in the control group is expressed as T/C weight.

Efficacy of C-6b anti-TWEAKR APDC in various xenograft models

Tumor cells (e.g., KU-19-19, NCI-H292, SCC4) were subcutaneously inoculated into the flank of female NMRI-nude mice or NOD.SCID mice (Janvier). When the tumor size reached approximately 40 ^mm² , intravenous treatment with an antibody-drug conjugate was administered. Tumor growth was monitored post-treatment, if appropriate.

sequence list

<110> Bayer Pharmaceuticals Co., Ltd.

<120> Prodrugs of cytotoxic agents with enzymatically cleavable groups

<130> CP1190421P

<160> 167

<170> PatentIn version 3.5

<210> 1

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 1

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 2

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 2

Asn Tyr Gly Val His

1 5

<210> 3

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 3

Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser

1 5 10 15

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 4

Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr

1 5 10

<210> 5

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 5

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 6

Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His

1 5 10

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 7

Tyr Ala Ser Glu Ser Ile Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 8

Gln Gln Asn Asn Asn Trp Pro Thr Thr

1 5

<210> 9

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 9

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 10

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 10

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 11

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 12

Asp Thr Tyr Ile His

1 5

<210> 13

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 13

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 14

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr

1 5 10

<210> 15

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 16

Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 10

<210> 17

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 17

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 18

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 18

Gln Gln His Tyr Thr Thr Pro Pro Thr

1 5

<210> 19

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 20

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 21

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 21

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 22

Pro Tyr Pro Met Ile

1 5

<210> 23

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 23

Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 24

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 24

Gly Gly Asp Thr Tyr Tyr Phe Asp Tyr Phe Asp Tyr

1 5 10

<210> 25

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 26

Arg Ala Ser Gln Ser Ile Ser Gly Tyr Leu Asn

1 5 10

<210> 27

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 27

Gln Ala Ser Ser Leu Gln Ser

1 5

<210> 28

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 28

Gln Gln Ser Tyr Thr Ser Pro Phe Ile Thr

1 5 10

<210> 29

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 29

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 30

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 31

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 31

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 32

Pro Tyr Pro Met Ile

1 5

<210> 33

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 33

Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 34

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 34

Gly Gly Asp Thr Tyr Tyr Phe Asp Tyr Phe Asp Tyr

1 5 10

<210> 35

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 35

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 36

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 36

Arg Ala Ser Gln Ser Ile Ser Gly Tyr Leu Asn

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 37

Gln Ala Ser Ser Leu Gln Ser

1 5

<210> 38

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 38

Gln Gln Ser Tyr Thr Ser Pro Phe Ile Thr

1 5 10

<210> 39

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 39

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 40

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 41

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 41

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 42

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 42

Pro Tyr Pro Met Ile

1 5

<210> 43

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 43

Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 44

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 44

Gly Gly Asp Thr Tyr Tyr Phe Asp Tyr Phe Asp Tyr

1 5 10

<210> 45

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 46

Arg Ala Ser Gln Ser Ile Ser Gly Tyr Leu Asn

1 5 10

<210> 47

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 47

Gln Ala Ser Ser Leu Gln Ser

1 5

<210> 48

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 48

Gln Gln Ser Tyr Thr Ser Pro Phe Ile Thr

1 5 10

<210> 49

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 49

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 50

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 51

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 51

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 52

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 52

Asp Phe Ile Ile Ala

1 5

<210> 53

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 53

Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe Arg

1 5 10 15

Gly

<210> 54

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 54

Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr

1 5 10

<210> 55

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 55

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 56

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 56

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr

1 5 10 15

<210> 57

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 57

Gln Met Ser Asn Leu Ala Ser

1 5

<210> 58

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 58

Ala His Asn Leu Glu Leu Pro Trp Thr

1 5

<210> 59

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 59

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 60

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 60

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 61

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 62

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 62

Asp Phe Ile Ile Ala

1 5

<210> 63

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 63

Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe Arg

1 5 10 15

Gly

<210> 64

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 64

Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr

1 5 10

<210> 65

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 65

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 66

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 66

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr

1 5 10 15

<210> 67

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 67

Gln Met Ser Asn Leu Ala Ser

1 5

<210> 68

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 68

Ala His Asn Leu Glu Leu Pro Trp Thr

1 5

<210> 69

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 69

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 70

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 70

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 71

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 71

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 72

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 72

Asp Thr Tyr Ile His

1 5

<210> 73

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 73

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 74

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 74

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr

1 5 10

<210> 75

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 75

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 76

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 76

Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 10

<210> 77

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 77

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 78

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 78

Gln Gln His Tyr Thr Thr Pro Pro Thr

1 5

<210> 79

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 79

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 80

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 80

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 81

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 81

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 82

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 82

Asp Thr Tyr Ile His

1 5

<210> 83

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 83

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 84

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 84

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr

1 5 10

<210> 85

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 85

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 86

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 86

Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 10

<210> 87

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 87

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 88

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 88

Gln Gln His Tyr Thr Thr Pro Pro Thr

1 5

<210> 89

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 89

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 90

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 90

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 91

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 91

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Leu Thr Gly Thr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 92

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 92

Ser Tyr Ala Met Ser

1 5

<210> 93

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 93

Ser Ile Ser Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 94

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 94

Leu Thr Gly Thr Ser Phe Asp Tyr

1 5

<210> 95

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 95

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser Ser Leu

85 90 95

Lys Lys Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 96

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 96

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Pro Val Asn

1 5 10

<210> 97

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 97

Ser Asn Asn Gln Arg Pro Ser

1 5

<210> 98

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 98

Gln Ser Phe Asp Ser Ser Leu Lys Lys Val

1 5 10

<210> 99

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 99

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Leu Thr Gly Thr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 100

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 100

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser Ser Leu

85 90 95

Lys Lys Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 101

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 101

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Leu Thr Gly Thr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 102

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 102

Ser Tyr Ala Met Ser

1 5

<210> 103

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 103

Ser Ile Ser Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 104

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 104

Leu Thr Gly Thr Ser Phe Asp Tyr

1 5

<210> 105

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 105

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser Ser Leu

85 90 95

Lys Lys Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 106

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 106

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Pro Val Asn

1 5 10

<210> 107

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 107

Ser Asn Asn Gln Arg Pro Ser

1 5

<210> 108

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 108

Gln Ser Phe Asp Ser Ser Leu Lys Lys Val

1 5 10

<210> 109

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 109

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Leu Thr Gly Thr Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 110

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 110

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser Ser Leu

85 90 95

Lys Lys Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 111

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 111

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 112

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 112

Pro Tyr Pro Met Ile

1 5

<210> 113

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 113

Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 114

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 114

Gly Gly Asp Thr Tyr Tyr Phe Asp Tyr Phe Asp Tyr

1 5 10

<210> 115

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 115

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 116

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 116

Arg Ala Ser Gln Ser Ile Ser Gly Tyr Leu Asn

1 5 10

<210> 117

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 117

Gln Ala Ser Ser Leu Gln Ser

1 5

<210> 118

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 118

Gln Gln Ser Tyr Thr Ser Pro Phe Ile Thr

1 5 10

<210> 119

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 119

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

Pro Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Thr Tyr Phe Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Asn Tyr Gln Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 120

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 120

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Phe

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 121

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 121

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 122

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 122

Asp Phe Ile Ile Ala

1 5

<210> 123

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 123

Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe Arg

1 5 10 15

Gly

<210> 124

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 124

Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr

1 5 10

<210> 125

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 125

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 126

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 126

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr

1 5 10 15

<210> 127

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 127

Gln Met Ser Asn Arg Ala Ser

1 5

<210> 128

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 128

Ala His Asn Leu Glu Leu Pro Trp Thr

1 5

<210> 129

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 130

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 130

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 131

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 131

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 132

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 132

Asp Phe Ile Ile Ala

1 5

<210> 133

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 133

Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe Arg

1 5 10 15

Gly

<210> 134

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 134

Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr

1 5 10

<210> 135

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 135

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Asn Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 136

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 136

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Ile Asn Tyr Leu Tyr

1 5 10 15

<210> 137

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 137

Gln Met Ser Asn Arg Ala Ser

1 5

<210> 138

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 138

Ala His Asn Leu Glu Leu Pro Trp Thr

1 5

<210> 139

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 139

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 140

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 140

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Asn Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 141

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 141

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 142

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 142

Asp Phe Ile Ile Ala

1 5

<210> 143

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 143

Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe Arg

1 5 10 15

Gly

<210> 144

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 144

Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr

1 5 10

<210> 145

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 145

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Trp Ile Asn Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 146

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 146

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Trp Ile Asn Tyr Leu Tyr

1 5 10 15

<210> 147

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 147

Gln Met Ser Asn Arg Ala Ser

1 5

<210> 148

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 148

Ala His Asn Leu Glu Leu Pro Trp Thr

1 5

<210> 149

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 149

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 150

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 150

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Trp Ile Asn Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 151

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 151

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 152

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 152

Asp Phe Ile Ile Ala

1 5

<210> 153

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 153

Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe Arg

1 5 10 15

Gly

<210> 154

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 154

Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr

1 5 10

<210> 155

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 155

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Trp Ile Asn Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 156

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 156

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Trp Ile Asn Tyr Leu Tyr

1 5 10 15

<210> 157

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 157

Gln Gly Ser Asn Arg Ala Ser

1 5

<210> 158

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 158

Ala His Asn Leu Glu Leu Pro Trp Thr

1 5

<210> 159

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 159

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Ile Ile Ala Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Thr Gly Arg Thr Tyr Tyr Ser Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Thr Ile Tyr Tyr Asp Tyr Asp Gly Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 160

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody sequence

<400> 160

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Trp Ile Asn Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala His Asn

85 90 95

Leu Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 161

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> c(RGDfK)

<400> 161

His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln

1 5 10 15

Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn

20 25

<210> 162

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> HER2-targeting peptide

<400> 162

Ala Thr Glu Pro Arg Lys Gln Tyr Ala Thr Pro Arg Val Phe Trp Thr

1 5 10 15

Asp Ala Pro Gly

20

<210> 163

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> HER2-targeting peptide

<400> 163

Leu Gln Trp Arg Arg Asp Asn Val His Asn Phe Gly Val Trp Ala

1 5 10 15

Arg Tyr Arg Leu

20

<210> 164

<211> 129

<212> PRT

<213> Homo sapiens

<400> 164

Met Ala Arg Gly Ser Leu Arg Arg Leu Leu Arg Leu Leu Val Leu Gly

1 5 10 15

Leu Trp Leu Ala Leu Leu Arg Ser Val Ala Gly Glu Gln Ala Pro Gly

20 25 30

Thr Ala Pro Cys Ser Arg Gly Ser Ser Ser Trp Ser Ala Asp Leu Asp Lys

35 40 45

Cys Met Asp Cys Ala Ser Cys Arg Ala Arg Pro His Ser Asp Phe Cys

50 55 60

Leu Gly Cys Ala Ala Ala Pro Pro Ala Pro Phe Arg Leu Leu Trp Pro

65 70 75 80

Ile Leu Gly Gly Ala Leu Ser Leu Thr Phe Val Leu Gly Leu Leu Ser

85 90 95

Gly Phe Leu Val Trp Arg Arg Cys Arg Arg Arg Glu Lys Phe Thr Thr

100 105 110

Pro Ile Glu Glu Thr Gly Gly Glu Gly Cys Pro Ala Val Ala Leu Ile

115 120 125

Gln

<210> 165

<211> 534

<212> PRT

<213> Homo sapiens

<400> 165

Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala

1 5 10 15

Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln

20 25 30

Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu

35 40 45

Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

50 55 60

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala

65 70 75 80

Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe

85 90 95

Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

100 105 110

Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp

115 120 125

Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

130 135 140

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr

145 150 155 160

Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val

165 170 175

Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr

180 185 190

Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu

195 200 205

Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Tyr Ser Cys Leu Val Arg Asn

210 215 220

Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln

225 230 235 240

Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val

245 250 255

Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro

260 265 270

Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr

275 280 285

Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly

290 295 300

Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln

305 310 315 320

Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly

325 330 335

Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val

340 345 350

Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu

355 360 365

Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser

370 375 380

Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln

385 390 395 400

Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu

405 410 415

Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala

420 425 430

Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp

435 440 445

Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro

450 455 460

Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu

465 470 475 480

Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys

485 490 495

Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly

500 505 510

Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp

515 520 525

Asp Gly Gln Glu Ile Ala

530

<210> 166

<211> 1255

<212> PRT

<213> Homo sapiens

<400> 166

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln

610 615 620

Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro

1130 1135 1140

Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro

1145 1150 1155

Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

1160 1165 1170

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly

1175 1180 1185

Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala

1190 1195 1200

Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp

1205 1210 1215

Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro

1220 1225 1230

Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr

1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255

<210> 167

<211> 1210

<212> PRT

<213> Homo sapiens

<400> 167

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Ser Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn

1040 1045 1050

Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg

1055 1060 1065

Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln

1100 1105 1110

Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln

1160 1165 1170

Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys

1175 1180 1185

Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

1190 1195 1200

Ser Ser Glu Phe Ile Gly Ala

1205 1210

Claims (39)

1. Compounds of general formula (Ia) or its salt, hydrate or salt of hydrate, wherein La is a self-extinguishing connector, and n is 0 or 1; D is -D ₁ -(Lb) _o -(LIG) _p , in _D1 is a cytotoxic agent. LIG is an antibody or its antigen-binding fragment. After binding to receptors on tumor cells, it is internalized by the tumor cells and processed by lysosomes within the cells. Lb is the connector, and o and p are each independently 0 or 1; R is LIG-(L _c ) _e -, where LIG is an antibody or its antigen-binding fragment, which, after binding to receptors on tumor cells, is internalized by the tumor cells and processed intracellularly by lysosomes. Lc is a linker, wherein the linker is not an amino acid linker, and e is 0 or 1; or R is Z ₁ -(C＝O)q-, q is 0 or 1. Z ₁ is a _C1-10 -alkyl, C5-10 -aryl, _C6-10 -aryl-C1-6 -alkyl, _C3-10 -heteroalkyl, _C1-10 -alkyl- _OC6-10 -aryl, _C5-10 -heterocyclic alkyl, heteroaryl, heteroarylalkyl, _C5-10 -heteroarylalkoxy, C1-10 -alkoxy, _C6-10 -aryloxy, _C6-10 -aryl- _C1-6 -alkoxy-, _C5-10 -heteroalkoxy, _{C1-10 -alkyl} - _OC6-10 -aryloxy-, or _{C5-10 -heterocyclic} alkoxy, which may be monosubstituted or polysubstituted by the following groups, either the same or different: _-NH2 , -NH _- alkyl, -N(alkyl) ₂ -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2- NH2} , -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or –OH, or -H or - _{( CH2} ) _0-1 - _Ox- ( _CH2CH2O ) _v - ^R11 or _{–Ox- ( CH2CH2O} ) _v - ^R11 , in x is 0 or 1. v is a number from 1 to 20, and R ¹¹ is -H, -alkyl, -CH ₂ -COOH, -CH ₂ -CH ₂ -COOH or -CH ₂ -CH ₂ -NH ₂ ; Where if p is 0, then R is LIG-(L _c ) _e -; and if p is 1, then R is Z ₁ -(C＝O)q-; The self-deactivating linker La is one of the following groups: And among them # ₁ is the bond attached to the carbonyl group in formula (Ia), and # ₂ is a bond attached to a hydroxyl or amine group of the cytotoxic agent _D1 . 2. The compound of general formula (Ia) as claimed in claim 1, wherein q is 1. 3. The compound of general formula (Ia) according to claim 1, wherein _Z1 is _C1-10 -alkyl, _C6-10 -aryl- _C1-6 -alkyl, _C5-10 -heteroaryl- _C1-6 -alkyl, _C6-10 -aryl- _C1-6 -alkoxy- or _C5-10 -heteroarylalkoxy, which may be monosubstituted or polysubstituted by the following groups, either the same or different: _-NH2 , -NH-alkyl, -N(alkyl) ₂ , -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2 -} NH2, -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or –OH. 4. The compound of general formula (Ia) according to claim 1, wherein _Z1 is _C1-3 -alkyl, _C6-7 -aryl- _C1-6 -alkyl, _C5-6 -heteroaryl- _C1-3 -alkyl or _C6-7 -aryl- _C1-6 -alkoxy, which may be monosubstituted or polysubstituted by the same or different of the following groups: -COOH, -C(＝O)- or –OH. 5. The compound of general formula (Ia) of claim 1, wherein the linker -Lb- or –Lc- is bonded to a cysteine side chain or cysteine residue in the antibody or its antigen-binding fragment and has a universal basic structure (i) or (ii). (i)–(C＝O) _m –L1-(L2) _n - or (ii)–(C＝O) _m –L1-SG-L2- in In the general infrastructures (i) and (ii), m and n are each independently 0 or 1. In the general basic structure (ii), SG is a chemically or enzymatically cleavable group in vivo, which is a disulfide, hydrazone, acetal, or acetalamine; or a 2-8-oligopeptide group cleavable by podase, cathepsin, or plasmin. In the general basic structure (i) or (ii), L2 is a single bond or is one of the following groups: # ¹ -S-# ² , in # ¹ indicates the bonding site with the sulfur atom in the bonded portion. # ² indicates the bonding site with the L1 group. In the general infrastructure (i) or (ii), L1 is –(NR ¹⁰ ) _n -(G1)o-G2-, ^R10 in group L1 is –H or _C1 - _C3 -alkyl. The G1 in the group L1 is –NH-C(＝O)-, -C(＝O)-NH-, or –(CH ₂ ) _O-3 -C(＝O)-NH-. In the group L1, n is 0 or 1. The o in the group L1 is 0 or 1. G2 in group L1 is a bond or a straight or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NH-, -C(＝O)-, -CH(COOH)-, -CH( _CH2 -C(＝O) _-NH2 )-, -NMe-, -NHNH-, -S(＝O) _2- NHNH-, -NH-C(＝O)-, -C(＝O)-NH-, -C(＝O)-NHNH-, having up to 4 heteroatoms selected from N, O, and S, or 5- to 10-membered aromatic or non-aromatic heterocycles of -S(＝O)- or -S(＝O) _2- , wherein the side chains in the branched carbon chain may be –NH-C(＝O) _-NH2- if present. Substitution with -COOH, -OH, _-NH₂ , -NH- _CNNH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid, or representing one of the following groups: in Rx is -H, _C1 - _C3 -alkyl, or phenyl. 6. The compound of general formula (Ia) of claim 1, wherein the linker -Lb- or –Lc- is bonded to a lysine side chain or lysine residue in the antibody or its antigen-binding fragment and has a universal basic structure (iii) or (iv). (iii)–(C＝O) _m –(L1) _n -(L4) _n -(C＝O)-§§ or (iv)–(C＝O) _m –L1-SG-L4-(C＝O)-§§ in In the general infrastructure (iii) and (iv), m and n are independently 0 or 1. In the general basic structure (iv), SG is a chemically or enzymatically cleavable group in vivo, which is a disulfide, hydrazone, acetal, or acetalamine; or a 2-8-oligopeptide group cleavable by podase, cathepsin, or plasmin. In the general infrastructure (iii) or (iv), L1 is –(NR ¹⁰ ) _n -(G1) _o -G2-, ^R10 in group L1 is –H or _C1 - _C3 -alkyl. The G1 in the group L1 is –NH-C(＝O)-, -C(＝O)-NH-, or –(CH ₂ ) _O-3 -C(＝O)-NH-. In the group L1, n is 0 or 1. The o in the group L1 is 0 or 1. In the group L1, G2 is a bond or a straight or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NH-, -C(＝O)-, -CH(COOH)-, -CH( _CH2 -C(＝O) _-NH2 )-, -NMe-, -S(＝O) _2- NHNH-, -NH-C(＝O)-, -C(＝O)-NH-, -C(＝O)-NHNH-, having up to 4 heteroatoms selected from N, O, and S, or 5- to 10-membered aromatic or non-aromatic heterocycles of -S(＝O)- or -S(＝O) _2- , and wherein the side chains in the branched carbon chain may be –NH-C(＝O) _-NH2- if present. Substitution with -COOH, -OH, _-NH₂ , -NH- _CNNH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid, or representing one of the following groups: in Rx is -H, _C1 - _C3 -alkyl, or phenyl. In the general basic structure (iii) or (iv), L4 is a single bond or a group. –(C＝O) _y -G4-, y is 0 or 1, and G4 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following or separated more than once by the same or different groups selected from the following: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NH-, -C(＝O)-, -NH-C(＝O)-, -C(＝O)-NH-, -NMe, -S(＝O) _2- NHNH-, -C(＝O)-NHNH-, -CH(COOH)-, and 5- to 10-membered aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O, and S, -S(＝O)-, or -S(＝O) _2- , wherein the side chains in the branched carbon chains may be substituted, if present, with –NH-C(＝O) _-NH2 , -COOH, -OH, -NH-CN-, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid. The §§ in the general basic structure (iii) or (iv) is the binding site of the nitrogen atom of the lysine residue in the antibody or its antigen-binding fragment. 7. The compound of general formula (Ia) as claimed in claim 5 or 6, wherein L1 is a group listed in the table below, and r is a number from 0 to 20: 8. The compound of general formula (Ia) as claimed in claim 7, wherein r is a number from 0 to 15. 9. The compound of general formula (Ia) as claimed in claim 7, wherein r is a number from 0 to 10. 10. The compound of general formula (Ia) according to claim 1, wherein the linker Lb or Lc is bonded to a cysteine side chain or cysteine residue in the antibody or its antigen-binding fragment and has the following formula (v): (v)§-(C＝O) _m -L1-(L2) _n -§§ in In equation (v), m is either 0 or 1. In equation (v), n is either 0 or 1. In formula (v), § represents the bond connecting the drug molecule or the cleavable group of the podase protease, and In equation (v), §§ represents the bond linking the antibody or its antigen-binding fragment. In formula (v), -L2 represents the following groups: # ¹ -S-# ² , Or is a key, in which # ¹ indicates the binding site of the sulfur atom to the antibody or its antigen-binding fragment. # ² indicates the bonding point with L1. In equation (v), L1 is –(NR ¹⁰ ) _n -(G1) _o -G2-, ^R10 in group L1 is –H or _C1 - _C3 -alkyl. In the group L1, G1 is –NH-C(＝O)-, -C(＝O)-NH-, or –(CH ₂ ) _O-3 -C(＝O)-NH-, and n in the group L1 is 0 or 1. The o in the group L1 is 0 or 1. In the group L1, G2 is a bond or a straight or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NH-, -C(＝O)-, -CH(COOH)-, -CH( _CH2 -C(＝O) _-NH2 )-, -NMe-, -NHNH-, -S(＝O) _2- NHNH-, -NH-C(＝O)-, -C(＝O)-NH-, -C(＝O)-NHNH-, having up to 4 heteroatoms selected from N, O, and S, or 5- to 10-membered aromatic or non-aromatic heterocycles of -S(＝O)- or -S(＝O) _2- , and wherein the side chains in the branched carbon chain may be –NH-C(＝O) _-NH2- Substitution with -COOH, -OH, _-NH₂ , -NH- _CNNH₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid, or representing one of the following groups: in Rx is -H, _C1 - _C3 -alkyl, or phenyl. 11. The compound of general formula (Ia) of claim 1, wherein the linkers Lb and/or Lc are bonded to a lysine side chain or lysine residue in the antibody or its antigen-binding fragment and have the following formula (vi): (vi)§-(C＝O) _m -L1-(L4) _n -C＝O-§§ in In equation (vi), m is either 0 or 1. In equation (vi), n is either 0 or 1. In formula (vi), § represents the bond connecting the drug molecule or the cleavable group of the podase protease, and In formula (vi), §§ represents the bond that links the nitrogen atom in the antibody or its antigen-binding fragment. In equation (vi), L1 is –(NR ¹⁰ ) _n -(G1) _o -G2-, ^R10 in group L1 is –H or _C1 - _C3 -alkyl. The G1 in the group L1 is –NH-C(＝O)-, -C(＝O)-NH-, or –(CH ₂ ) _O-3 -C(＝O)-NH-. In the group L1, n is 0 or 1. The o in the group L1 is 0 or 1. In the group L1, G2 is a bond or a straight or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NH-, -C(＝O)-, -CH(COOH)-, -CH( _CH2 -C(＝O) _-NH2 )-, -NMe-, -S(＝O) _2- NHNH-, -NH-C(＝O)-, -C(＝O)-NH-, -C(＝O)-NHNH-, having up to 4 heteroatoms selected from N, O, and S, or 5- to 10-membered aromatic or non-aromatic heterocycles of -S(＝O)- or -S(＝O) _2- , and wherein the side chains in the branched carbon chain may be –NH-C(＝O) _-NH2- if present. Substitution with -COOH, -OH, -NH-CNNH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, or representing one of the following groups: in Rx is -H, _C1 - _C3 -alkyl, or phenyl. In formula (vi), L4 is a single bond or a group. –(C＝O) _y -G4-, y is 0 or 1, and G4 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following groups or separated more than once by the same or different groups selected from the following groups: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NH-, -C(＝O)-, -NH-C(＝O)-, -C(＝O)-NH-, -NMe-, -S(＝O) _2- NHNH-, -C(＝O)-NHNH-, -CH(COOH)-, and 5- to 10-membered aromatic or non-aromatic heterocycles having up to 4 heteroatoms selected from N, O and S, -S(＝O)- or -S(＝O) _2- , wherein the side chains in the branched carbon chain may be substituted, if present, with –NH-C(＝O) _-NH2 , -COOH, -OH, -NH-CN- _NH2 , sulfonamide, sulfone, sulfoxide or sulfonic acid. 12. Compounds of general formula (III') or its salt, hydrate or salt of hydrate, wherein n, e, LIG, La, Lc, and _D1 have the definitions given in claim 1. 13. Compounds of general formula (IV') or its salts, hydrates, or salts of hydrates, wherein n, o, LIG, La, Lb, and _D1 have the definitions given in claim 1, and R is Z ₁ -(C＝O)q-, q is 0 or 1. Z ₁ is a _C1-10 alkyl, C5-10 aryl, _C6-10 aryl- _C1-6 alkyl, _C3-10 heteroalkyl, _C1-10 alkyl-OC6-10 aryl, _C5-10 heterocycloalkyl, heteroaryl, _C5-10 heteroaryl- _C1-6 alkyl, _C5-10 heteroarylalkoxy, C1-10 alkoxy, _C6-10 aryloxy, _C6-10 aryl- _{C1-6 alkoxy, C5-10 heteroalkoxy} , _{C1-10 alkyl} - _{OC6-10 aryloxy} , or _C5-10 heterocycloalkoxy group, which may be monosubstituted or polysubstituted by the following groups, either the same or different: _-NH2 , -NH- _alkyl , -N(alkyl) ₂ -NH-C(=O)-alkyl, -N(alkyl)-C(=O)-alkyl, -S(=O) ₃ -H, -S(=O) _{2- NH2} , -S(=O) ₂ -N(alkyl) ₂ , -COOH, -C(=O)-, -C(=O) _NH2 , -C(=O)-N(alkyl) ₂ or -OH, or -H or - _{( CH2} ) _0-1 - _Ox- ( _CH2CH2O ) _v - ^R11 or _-Ox- ( _CH2CH2O ) _v ^-R11 _group , x is 0 or 1. v is a number from 1 to 20, and R ¹¹ is -H, -alkyl, -CH ₂ -COOH, -CH ₂ -CH ₂ -COOH or -CH ₂ -CH ₂ -NH ₂ . 14. The compound of any one of claims 1, 12, or 13, wherein the cytotoxic agent _D1 is a drug selected from: mitomycin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-aminocamptothecin, n8-acetylspermine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, tamethasone, cytarabine, etoposide, camptothecin, paclitaxel, esperamycin, podophyllotoxin, serpentine, vincristine, vinblastine, morpholine-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, pyruvic acid, bromopyrimidine, etc. Restatin, monomethyloritine, dolalastatin, tubulin inhibitors, maytansine, spirulina cyclopeptide, amanita, indole-benzodiazepine, pyrrolo-benzodiazepine derivatives, calcimycin, daunorubicin, essanotecan, or kinin-spindle protein inhibitors, wherein when n=1, the drug is bonded to the La group of the compound via its hydroxyl or amino group, or when n=0, the drug is bonded to the carbonyl group of the compound via its hydroxyl or amino group. 15. The compound of any one of claims 1, 12 or 13, wherein the cytotoxic agent _D1 is a kinesin spindle protein inhibitor. 16. The compound of claim 15, having a structure represented by formula (IIa): Where R, La, and n have the same definitions as in general formula (Ia) of claim 1. _X1 is N, X ₂ is N, and X ₃ is C, or _X1 is N, X ₂ is C, and X ₃ is N, or _X1 is CH or CF. X ₂ is C, and X ₃ is N, or _X1 is NH, X ₂ is C, and X ₃ is C, or _X1 is CH, X ₂ is N, and X ₃ is C, A is -C(=O)-, -S(=O)-, -S(=O) ₂ - or -S(=O) ₂ -NH-, ^R1 is -H, –L-#1, –MOD, or -( _CH2 ) _0-3Z . The Z in group ^R1 is -H, ^-NHY3 , -OY3, ^-SY3 , ^halogen , -C(=O) ^-NY1Y2 , or ^-C (=O) ^-OY3 ; the ^Y1 and ^Y2 in group ^R1 are independently -H, _-NH2 , -( _CH2CH2O ) _O-3- ( _CH2 )O- _3Z ', or -CH( _CH2W )Z'; and the ^Y3 in group ^R1 is -H or -( _{CH2 ) O-} 3Z'. The Z' in the group ^R1 is -H, _-NH2 , -S(=O) _3H , -COOH, -NH-C(=O) _{-CH2- CH2-} CH( _NH₂ )COOH or -(C(＝O)-NH- ^CHY₄ ) _¹- ³COOH, The W in the group ^R1 is -H or -OH. ^Y4 in group ^R1 is a linear or branched _C1-6 alkyl group optionally substituted with -NH-C(=O) _-NH2 , or an aryl or benzyl group optionally substituted with _–NH2 . ^R2 is -H, –L-#1, -MOD, -C(=O) ^-CHY4 - ^NHY5 , or -( _CH2 ) _0-3Z . The Z in group ^R2 is -H ^{, halogen, -OY3, -SY3, -NHY3, -C(=O)-NY1Y2 , or -C} (=O) ^-OY3 , and the ^Y1 and ^Y2 in group ^R2 are independently -H, _-NH2 , or -( _CH2 ⁾ _O- 3Z'. The ^Y3 in the group ^R2 is -H or -( _CH2 ) _O- 3Z'. The Z' in the group ^R2 is -H, -S(=O) _3H , _-NH2 , or –COOH. ^Y4 in the group ^R2 is a linear or branched _C1-6 alkyl- optionally substituted with –NHC(=O) _-NH2 , or an aryl or benzyl optionally substituted with _–NH2 . The ^Y5 in the group ^R2 is -H or –C(＝O) ^-CHY6 - _NH2 . The ^Y6 in group ^R2 is a linear or branched _C1-6 -alkyl group. R ³ is -MOD, –L-#1, or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl, which may be 1 to 3 OH atoms, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl atoms, 1 to 3 -O-alkyl atoms, 1 to 3 -SH atoms, 1 to 3 -S-alkyl atoms, 1 to 3 -OC(=O)-alkyl atoms, 1 to 3 -OC(=O)-NH-alkyl atoms, 1 to 3 -NH-C(=O)-alkyl atoms, 1 to 3 -NH-C(=O)-NH-alkyl atoms, 1 to 3 -S-alkyl, -S(=O)-alkyl, or -S(=O) ₂ -alkyl atoms, 1 to 3 -S(=O) _2- NH-alkyl atoms, 1 to 3 -NH-alkyl atoms, 1 to 3 -N(alkyl) ₂ , 1 to 3 NH2 _atoms , or 1 to 3 -( _CH2 ) _{0-3 atoms.} Z-group substitution The Z in group ^R3 is -H ^{, halogen, -OY3, -SY3, -NHY3, -C(=O)-NY1Y2 , or -C} (=O) ^-OY3 , and ^{the Y1} and ^Y2 in group ^R3 are independently -H, _-NH2 , or -( _CH2 ) _O- 3Z'. The ^Y3 in the group ^R3 is -H, -( _CH2 ) _O-3 -CH(NHC(=O) _CH3 )Z', -( _CH2 ) _O-3- CH( _NH2 )Z', or -( _CH2 ) _{O- 3Z} '. The Z' in the group ^R3 is -H, -S(=O) _3H , _-NH2 , or –COOH. ^R5 can be -H, _-NH2 , _-NO2 , halogen, -CN, _CF3 , _-OCF3 , _-CH2F , _-CH2F , or -SH. ^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl, fluoro- _C2-10 -ynyl, hydroxyl, _-NO2 , _-NH2 , -COOH, or halogen. ^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl, fluoro- _C2-10 -alkenyl, _C2-10 -ynyl or fluoro-C2-10-ynyl, or _C4-10 -cycloalkyl, fluoro _{-C4-10 -} cycloalkyl or -( _CH2 ) _0-2- ( ^HZ2 ). HZ ² is a 4- to 7-membered heterocycle having at most two heteroatoms selected from N, O, and S, which can be substituted with -OH, –COOH, or -NH ₂ . R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ . –L-#1 is -(Lb) _o -(LIG) _p , LIG is an antibody or its antigen-binding fragment. After binding to receptors on tumor cells, it is internalized by the tumor cells and processed by lysosomes. _Lb is a linker, wherein the linker is not an amino acid linker. In the group -L-#1, o and p are independently 0 or 1. –MOD is –(NR ¹⁰ )n-(G1)o-G2-G3, The ^R10 in the MOD group is -H or _C1 - _C3 -alkyl. The G1 in the MOD group is –NH-C(=O)-, -C(=O)-NH-, or… The n in the MOD group is 0 or 1; The o in the MOD group is 0 or 1, and The G2 in the MOD group is a straight-chain or branched hydrocarbon chain with 1-10 carbon atoms, which may be selected from the following groups, spaced one or more times: -O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , -NR ^y C(＝O)-, -C(＝O)NR ^y- , -NR ^y NR ^y- , -S(＝O) _2- NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain may be substituted with –NH-C(＝O)-NH2, -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid. The R ^_y in the MOD group is -H, phenyl, C1-C10-alkyl, C2-C10-alkenyl, or C2-C10-alkynyl, each of which can be substituted with –NH-C(＝O)-NH₂, -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid. The Rx in the MOD group is -H, C1-C3-alkyl, or phenyl. The G3 in the MOD group is –H or –COOH, and –MOD has at least one –COOH group, and one amino group in –MOD can be acylated by a podase-cleavable group. 17. The compound of claim 16, wherein MOD has two –COOH groups. 18. The compound of claim 16, wherein _X1 is CH, X ₂ is C, and X ₃ is N, A is –C(＝O)-, R ¹ is –L-#1, -H, or –MOD ^R2 is -H, R ³ is -MOD, –L-#1, or optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl, which may be 1 to 3 OH groups, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl groups, 1 to 3 -O-alkyl groups, 1 to 3 -SH- groups, 1 to 3 -S-alkyl groups, 1 to 3 -OC(=O)-alkyl groups, 1 to 3 -OC(=O)-NH-alkyl groups, 1 to 3 -NH-C(=O)-alkyl groups, 1 to 3 -NH-C(=O)-NH-alkyl groups, 1 to 3 -S-alkyl, -S(=O)-alkyl, or -S(=O) ₂ -alkyl groups, 1 to 3 –S(=O) _2- NH-alkyl groups, 1 to 3 -NH-alkyl groups, 1 to 3 -N(alkyl) ₂ , 1 to 3 _NH2 groups, or 1 to 3 -( _CH2 ) groups. _0-3 Z group substitution, The Z in group ^R3 is -H ^{, halogen, -OY3, -SY3, -NHY3, -C(=O)-NY1Y2 , or -C} (=O) ^-OY3 , and ^{the Y1} and ^Y2 in group ^R3 are independently -H, _-NH2 , or -( _CH2 ) _O- 3Z'. The ^Y3 in the group ^R3 is -H, -( _CH2 ) _O-3 -CH(NHC(=O) _CH3 )Z', -( _CH2 ) _O-3- CH( _NH2 )Z', or -( _CH2 ) _{O- 3Z} '. The Z' in the group ^R3 is -H, -S(=O) _3H , _-NH2 , or –C(=O)-OH. R ⁵ is -H, ^R6 and ^R7 are fluorine. ^R8 is tert-butyl. R ⁹ is -H, –L-#1 is -(Lb) _o -(LIG) _p , LIG is an antibody or its antigen-binding fragment. After binding to receptors on tumor cells, it is internalized by the tumor cells and processed by lysosomes within the cells. Lb is a linker, wherein the linker is not an amino acid linker. In the group -L-#1, o and p are independently 0 or 1. –MOD is the group –(NR ¹⁰ )n-(G1)o-G2-G3, The ^R10 in the MOD group is -H or _C1 - _C3 -alkyl-. In the MOD group, n is either 0 or 1. In the MOD group, G1 is –NH-C(＝O)- or -C(＝O)-NH-. The o in the MOD group is 0 or 1, and The G2 in the MOD group is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be selected from the following groups, spaced once or spaced more than once by the same or different groups selected from the following: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , NR ^y C(＝O)-, -C(＝O)NR ^y- , NR ^y NR ^y- , -S(＝O) _2- NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain may be monosubstituted by the following groups or multisubstituted by the same or different groups of the following: –NH-C(＝O)-NH ₂ , -C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, and the R in the MOD group ^y is -H, phenyl-, _C1 - _C10 -alkyl-, _C2 - _C10 -alkenyl-, or _C2 - _C10 -alkynyl-, each of which can be substituted with –NH-C(=O) _-NH2 , -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid. The Rx in the MOD group is -H, _C1 - _C3 -alkyl- or phenyl-, and The G3 in the MOD group is –H or –COOH. 19. The compound of claim 17 or 18, wherein _X1 is CH, X ₂ is C, and X ₃ is N, A is –C(＝O)-, R ¹ is either -H or –MOD –MOD is the group –(NR ¹⁰ ) _n -(G1) _o -G2-G3, The ^R10 in the MOD group is -H or _C1 - _C3 -alkyl-. The n in the MOD group is 0. The G1 in the MOD group is -C(＝O)-NH-. The o in the MOD group is 1, and The G2 in the MOD group is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following or separated more than once by the same or different groups selected from the following: -O-, -S-, -NRy- or -C(=O)-, and the straight-chain or branched hydrocarbon chain may be monosubstituted by the following groups or multisubstituted by the same or different groups of the following: -C(=O) _-NH2 , -COOH, and the ^Ry in the MOD group is –H or _C1 - _C10 -alkyl-, which may be substituted by –NH-C(=O) _-NH2 , -COOH, -OH, _-NH2 . The G3 in the MOD group is –COOH. ^R2 is -H, R ³ is –L-#1, R ⁵ is -H, ^R6 and ^R7 are fluorine. ^R8 is tert-butyl. R ⁹ is -H, –L-#1 is -(Lb) _o -(LIG) _p , LIG is an antibody or its antigen-binding fragment. After binding to receptors on tumor cells, it is internalized by the tumor cells and processed by lysosomes within the cells. Lb is a linker, wherein the linker is not an amino acid linker. In the group –L-#1, o and p are independently 0 or 1. 20. The compound of claim 15, having a structure represented by formula (IIa’). in R is LIG-(L _c ) _e -, where LIG is an antibody or its antigen-binding fragment, which, after binding to receptors on tumor cells, is internalized by the tumor cells and processed intracellularly by lysosomes. Lc is a linker, wherein the linker is not an amino acid linker, and e is 0 or 1; La has the definition given in claim 1, and n is 0 or 1; x is 1 or 2; in _X1 is N, X ₂ is N, and X ₃ is C, or _X1 is N, X ₂ is C, and X ₃ is N, or _X1 is CH or CF. X ₂ is C, and X ₃ is N, or _X1 is NH, X ₂ is C, and X ₃ is C, or _X1 is CH, X ₂ is N, and X ₃ is C, G2 of formula (IIa') is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following groups or separated more than once by the same or different groups selected from the following groups: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , -NR ^y C(＝O)-, -C(＝O)NR ^y- , -NR ^y NR ^y- , -S(＝O) ₂ -NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain may be monosubstituted by the following groups or multisubstituted by the same or different groups of the following groups: –NH-C(＝O)-NH ₂ , -C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, In formula (IIa'), R ^_y in group G2 is -H, phenyl-, C _₁-C_₁₀-alkyl-, C_₂-C_₁₀-alkenyl-, or C_₂-C_₁₀-alkynyl-, each of which can be substituted with –NH-C(=O)-NH_₂, -COOH, -OH, -NH_₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid. In formula (IIa'), Rx in group G2 is -H, _C1 - _C3 -alkyl-, or phenyl-. In formula (IIa’), G3 is –H or –COOH. ^R5 can be -H, _-NH2 , _-NO2 , halogen, -CN, _CF3 , _-OCF3 , _-CH2F , _-CH2F , or -SH. ^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-, fluoro- _C2-10 -alkenyl-, C2-10-ynyl-, fluoro- _C2-10 -ynyl-, hydroxyl-, _-NO2 , _-NH2 , _-COOH , or halogen. ^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-, fluoro- _C2-10 -alkenyl-, _C2-10 -ynyl-, or fluoro- _C2-10 -ynyl-, or a _C4-10 -cycloalkyl- or fluoro- _C4-10 -cycloalkyl-. R ⁹ is -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ ; In formula (IIa'), R ¹⁰ is -H or _C1 - _C3 -alkyl. 21. The compound of claim 20, wherein _X1 is CH, X ₂ is C, and X ₃ is N, x is 1 or 2. In formula (IIa'), ^R10 is -H or _C1 - _C3 -alkyl. G2 of formula (IIa') is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following groups or separated more than once by the same or different groups selected from the following groups: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , -NR ^y C(＝O)-, -C(＝O)NR ^y- , -NR ^y NR ^y- , -S(＝O) ₂ -NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain may be monosubstituted by the following groups or multisubstituted by the same or different groups of the following groups: –NH-C(＝O)-NH ₂ , -C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, In formula (IIa'), R ^_y in group G2 is -H, phenyl-, C _₁-C_₁₀-alkyl-, C_₂-C_₁₀-alkenyl-, or C_₂-C_₁₀-alkynyl-, each of which can be substituted with –NH-C(=O)-NH_₂, -COOH, -OH, -NH_₂ , sulfonamide, sulfone, sulfoxide, or sulfonic acid. In formula (IIa'), Rx in group G2 is -H, _C1 - _C3 -alkyl-, or phenyl-. In formula (IIa’), G3 is –H or –COOH. R ⁵ is –H, ^R6 and ^R7 are fluorine. ^R8 is tert-butyl, and R ⁹ is -H. 22. The compound of claim 15, having a structure represented by formula (IIa”). in R is LIG-(L _c ) _e -, where LIG is an antibody or its antigen-binding fragment, which, after binding to receptors on tumor cells, is internalized by the tumor cells and processed intracellularly by lysosomes. Lc is a linker, wherein the linker is not an amino acid linker, and e is 0 or 1; La has the definition given in claim 1, and n is 0 or 1; x is 1 or 2; _X1 is N, X ₂ is N, and X ₃ is C, or _X1 is N, X ₂ is C, and X ₃ is N, or _X1 is CH or CF. X ₂ is C, and X ₃ is N, or _X1 is NH, X ₂ is C, and X ₃ is C, or _X1 is CH, X ₂ is N, and X ₃ is C, A is -C(=O)-, -S(=O), -S(=O) ₂ - or -S(=O) ₂ -NH-, In formula (IIa”), G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following groups or separated more than once by the same or different groups selected from the following groups: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , -NR ^y C(＝O)-, -C(＝O)NR ^y- , -NR ^y NR ^y- , -S(＝O) _2- NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain may be monosubstituted by the following groups or multisubstituted by the same or different groups of the following groups: –NH-C(＝O)-NH ₂ , -C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, wherein R in the group G2 ^y is -H, phenyl-, _C1 - _C10 -alkyl-, _C2 - _C10 -alkenyl-, or _C2 - _C10 -alkynyl-, each of which can be substituted with –NH-C(=O) _-NH2 , -COOH, -OH, _-NH2 , sulfonamide, sulfone, sulfoxide, or sulfonic acid. Rx in the group G2 is -H, _C1 - _C3 -alkyl-, or phenyl-. R ³ is an optionally substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl, or heterocycloalkyl group, which may be substituted with 1 to 3 OH groups, 1 to 3 halogen atoms, 1 to 3 monohalogenated, dihalogenated, or trihalogenated alkyl groups, 1 to 3 –O-alkyl groups, 1 to 3 –SH- groups, 1 to 3 –S-alkyl groups, 1 to 3 –OC(=O)-alkyl groups, 1 to 3 –OC(=O)-NH-alkyl groups, 1 to 3 –NH-C(=O)-alkyl groups, 1 to 3 –NH-C(=O)-NH-alkyl groups, 1 to 3 –S-alkyl, –S(=O)-alkyl, or –S(=O) ₂ -alkyl groups, 1 to 3 –S(=O) _2- NH-alkyl groups, 1 to 3 –NH-alkyl groups, 1 to 3 –N(alkyl) ₂ , ₁ to 3 NH2, or 1 to 3 –( _CH2 ) _O- 3Z groups. The Z in group ^R3 is -H ^{, halogen, -OY3, -SY3, -NHY3, -C(=O)-NY1Y2 , or -C} (=O) ^-OY3 , and ^{the Y1} and ^Y2 in group ^R3 are independently -H, _-NH2 , or -( _CH2 ) _O- 3Z'. The ^Y3 in the group ^R3 is -H, -( _CH2 ) _O-3 -CH(NHC(=O) _CH3 )Z', -( _CH2 ) _O-3- CH( _NH2 )Z', or -( _CH2 ) _{O- 3Z} '. The Z' in the group ^R3 is -H, -S(＝O) _3H , _-NH2 , or –COOH. ^R5 can be -H, _-NH2 , _-NO2 , halogen, -CN, _CF3 , _-OCF3 , _-CH2F , _-CH2F , or -SH. ^R6 and ^R7 are independently -H, -CN, _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-, fluoro- _C2-10 -alkenyl-, C2-10-ynyl-, fluoro- _C2-10 -ynyl-, hydroxyl-, _-NO2 , _-NH2 , _-COOH , or halogen. ^R8 is a straight-chain or branched _C1-10 -alkyl, fluoro- _C1-10 -alkyl, _C2-10 -alkenyl-, fluoro- _C2-10 -alkenyl-, _C2-10 -ynyl-, or fluoro- _C2-10 -ynyl-, or a _C4-10 -cycloalkyl- or fluoro- _C4-10 -cycloalkyl-. R ⁹ can be -H, -F, -CH ₃ , -CF ₃ , -CH _2F , or -CHF ₂ . 23. The compound of claim 22, wherein _X1 is CH, X ₂ is C, and X ₃ is N, A is –C(＝O)-, G2 is a straight-chain or branched hydrocarbon chain having 1-100 carbon atoms, which may be separated once by a group selected from the following groups or separated more than once by the same or different groups selected from the following groups: –O-, -S-, -S(＝O)-, -S(＝O) _2- , -NR ^y- , -NR ^y C(＝O)-, -C(＝O)NR ^y- , -NR ^y NR ^y- , -S(＝O) ₂ -NR ^y NR ^y- , -C(＝O)-NR ^y NR ^y- , -C(＝O)-, -CR ^x ＝NO-, and the straight-chain or branched hydrocarbon chain may be monosubstituted by the following groups or multisubstituted by the same or different groups of the following groups: –NH-C(＝O)-NH ₂ , -C(＝O)-NH ₂ , -COOH, -OH, -NH ₂ , sulfonamide, sulfone, sulfoxide or sulfonic acid, R ^_y in group G2 is -H, phenyl-, C_₁-C_₁₀-alkyl-, C_₂-C_₁₀-alkenyl-, or C _₂-C_₁₀-alkynyl-, each of which can be substituted with –NH-C(=O)-NH_₂, -COOH, -OH, -NH_₂, sulfonamide, sulfone, sulfoxide, or sulfonic acid. Rx in the group G2 is -H, _C1 - _C3 -alkyl-, or phenyl-. ^R3 is –CH2 _- OH, R ⁵ is –H, ^R6 and ^R7 are fluorine. ^R8 is tert-butyl, and R ⁹ is –H. 24. The compound of any one of claims 1, 12 or 13, wherein the antibody or antigen-binding fragment is bonded via a cysteine residue, a lysine residue or a glutamine residue. 25. The compound of any one of claims 1, 12, or 13, having the following structure: in AK ₁ and AK ₂ are antibody or antigen-binding fragments, and n is a number from 1 to 50. 26. The compound of claim 25, wherein n is a number from 1 to 20. 27. The compound of claim 25, wherein n is a number from 2 to 8. 28. The compound of claim 25, wherein n is a number from 2 to 6. 29. The compound of any one of claims 1, 12 or 13, wherein the antibody or antigen-binding fragment binds to an extracellular cancer target molecule. 30. The compound of any one of claims 1, 12 or 13, wherein the antibody or antigen-binding fragment is internalized by the target cell after binding to an extracellular target molecule on its target cell. 31. The compound of any one of claims 1, 12 or 13, wherein the antibody is an anti-HER2 antibody, an anti-EGFR antibody, an anti-B7H3 antibody, an anti-TWEAKR antibody, or an antigen-binding fragment of such antibodies. 32. The compound of claim 31, wherein the anti-TWEAKR antibody is selected from TPP-7006, TPP-7007 and TPP-10337, the anti-B7H3 antibody is selected from TPP-8382 and TPP-8567, the anti-EGFR antibody is cetuximab TPP-981 and the anti-HER2 antibody is selected from trastuzumab and TPP-1015. 33. The compound of claim 24, wherein the antibody (i) is an anti-EGFR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1) as shown in SEQ ID NO:2, a heavy chain variable CDR2 sequence (H-CDR2) as shown in SEQ ID NO:3, and a heavy chain variable CDR3 sequence (H-CDR3) as shown in SEQ ID NO:4, and the light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1) as shown in SEQ ID NO:6, a light chain variable CDR2 sequence (L-CDR2) as shown in SEQ ID NO:7, and a light chain variable CDR3 sequence (L-CDR3) as shown in SEQ ID NO:8. (ii) is an anti-HER2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1) as shown in SEQ ID NO:12, a heavy chain variable CDR2 sequence (H-CDR2) as shown in SEQ ID NO:13, and a heavy chain variable CDR3 sequence (H-CDR3) as shown in SEQ ID NO:14, and the light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1) as shown in SEQ ID NO:16, a light chain variable CDR2 sequence (L-CDR2) as shown in SEQ ID NO:17, and a light chain variable CDR3 sequence (L-CDR3) as shown in SEQ ID NO:18. (iii) An anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:22, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:23, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:24; and the light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:26, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:27, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:28. (iv) An anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1) as shown in SEQ ID NO:32, a heavy chain variable CDR2 sequence (H-CDR2) as shown in SEQ ID NO:33, and a heavy chain variable CDR3 sequence (H-CDR3) as shown in SEQ ID NO:34, and the light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1) as shown in SEQ ID NO:36, a light chain variable CDR2 sequence (L-CDR2) as shown in SEQ ID NO:37, and a light chain variable CDR3 sequence (L-CDR3) as shown in SEQ ID NO:38. (v) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1) as shown in SEQ ID NO:42, a heavy chain variable CDR2 sequence (H-CDR2) as shown in SEQ ID NO:43, and a heavy chain variable CDR3 sequence (H-CDR3) as shown in SEQ ID NO:44, and the light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1) as shown in SEQ ID NO:46, a light chain variable CDR2 sequence (L-CDR2) as shown in SEQ ID NO:47, and a light chain variable CDR3 sequence (L-CDR3) as shown in SEQ ID NO:48. (vi) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:52, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:53, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:54; and the light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:56, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:57, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:58. (vii) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:62, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:63, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:64. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:66, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:67, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:68. (viii) is an anti-HER2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:72, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:73, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:74. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:76, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:77, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:78. (ix) is an anti-HER2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:82, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:83, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:84. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:86, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:87, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:88. (x) is an anti-B7H3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:92, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:93, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:94. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:96, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:97, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:98. (xi) is an anti-B7H3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:102, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:103, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:104. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:106, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:107, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:108. (xii) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:112, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:113, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:114. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:116, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:117, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:118. (xiii) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:122, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:123, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:124. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:126, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:127, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:128. (xiv) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:132, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:133, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:134. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:136, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:137, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:138. (xv) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:142, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:143, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:144. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:146, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:147, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:148. (xvi) is an anti-TWEAKR antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region (VH) comprises a heavy chain variable CDR1 sequence (H-CDR1), as shown in SEQ ID NO:152, a heavy chain variable CDR2 sequence (H-CDR2), as shown in SEQ ID NO:153, and a heavy chain variable CDR3 sequence (H-CDR3), as shown in SEQ ID NO:154. The light chain variable region (VL) comprises a light chain variable CDR1 sequence (L-CDR1), as shown in SEQ ID NO:156, a light chain variable CDR2 sequence (L-CDR2), as shown in SEQ ID NO:157, and a light chain variable CDR3 sequence (L-CDR3), as shown in SEQ ID NO:158. Or it could be the antigen-binding fragment of these antibodies. 34. The compound of claim 24, wherein the antibody (i) is an anti-EGFR antibody comprising a heavy chain variable region (VH) as shown in SEQ ID NO:1 and a light chain variable region (VL) as shown in SEQ ID NO:5. (ii) is an anti-HER2 antibody comprising a heavy chain variable region (VH) as shown in SEQ ID NO:11 and a light chain variable region (VL) as shown in SEQ ID NO:15. (iii) is an anti-TWEAKR antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:21 and a light chain variable region (VL) as shown in SEQ ID NO:25. (iv) is an anti-TWEAKR antibody 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. (v) is an anti-TWEAKR antibody containing 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. (vi) is an anti-TWEAKR antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:51 and a light chain variable region (VL) as shown in SEQ ID NO:55. (vii) is an anti-TWEAKR antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:61 and a light chain variable region (VL) as shown in SEQ ID NO:65. (viii) is an anti-HER2 antibody containing a heavy chain variable region (VH) as shown in SEQ ID NO:71 and a light chain variable region (VL) as shown in SEQ ID NO:75. (ix) is an anti-HER2 antibody containing a heavy chain variable region (VH) as shown in SEQ ID NO:81 and a light chain variable region (VL) as shown in SEQ ID NO:85. (x) is an anti-B7H3 antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:91 and a light chain variable region (VL) as shown in SEQ ID NO:95. (xi) is an anti-B7H3 antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:101 and a light chain variable region (VL) as shown in SEQ ID NO:105. (xii) is an anti-TWEAKR antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:111 and a light chain variable region (VL) as shown in SEQ ID NO:115. (xiii) is an anti-TWEAKR antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:121 and a light chain variable region (VL) as shown in SEQ ID NO:125. (xiv) is an anti-TWEAKR antibody, which contains a heavy chain variable region (VH) as shown in SEQ ID NO:131 and a light chain variable region (VL) as shown in SEQ ID NO:135. (xv) is an anti-TWEAKR antibody containing a heavy chain variable region (VH) as shown in SEQ ID NO:141 and a light chain variable region (VL) as shown in SEQ ID NO:145, or (xvi) is an anti-TWEAKR antibody containing a heavy chain variable region (VH) as shown in SEQ ID NO:151 and a light chain variable region (VL) as shown in SEQ ID NO:155. Or it could be the antigen-binding fragment of these antibodies. 35. The compound of claim 24, wherein the antibody (i) is an anti-EGFR antibody comprising a heavy chain region as shown in SEQ ID NO:9 and a light chain region as shown in SEQ ID NO:10. (ii) is an anti-HER2 antibody, which contains a heavy chain region as shown in SEQ ID NO:19 and a light chain region as shown in SEQ ID NO:20. (iii) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:29 and a light chain region as shown in SEQ ID NO:30. (iv) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:39 and a light chain region as shown in SEQ ID NO:40. (v) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:49 and a light chain region as shown in SEQ ID NO:50. (vi) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:59 and a light chain region as shown in SEQ ID NO:60. (vii) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:69 and a light chain region as shown in SEQ ID NO:70. (viii) is an anti-HER2 antibody, which contains a heavy chain region as shown in SEQ ID NO:79 and a light chain region as shown in SEQ ID NO:80. (ix) is an anti-HER2 antibody, which contains a heavy chain region as shown in SEQ ID NO:89 and a light chain region as shown in SEQ ID NO:90. (x) is an anti-B7H3 antibody, which contains a heavy chain region as shown in SEQ ID NO:99 and a light chain region as shown in SEQ ID NO:100. (xi) is an anti-B7H3 antibody, which contains a heavy chain region as shown in SEQ ID NO:109 and a light chain region as shown in SEQ ID NO:110. (xii) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:119 and a light chain region as shown in SEQ ID NO:120. (xiii) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:129 and a light chain region as shown in SEQ ID NO:130. (xiv) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:139 and a light chain region as shown in SEQ ID NO:140. (xv) is an anti-TWEAKR antibody containing a heavy chain region as shown in SEQ ID NO:149 and a light chain region as shown in SEQ ID NO:150, or (xvi) is an anti-TWEAKR antibody, which contains a heavy chain region as shown in SEQ ID NO:159 and a light chain region as shown in SEQ ID NO:160. Or it could be the antigen-binding fragment of these antibodies. 36. A pharmaceutical composition comprising a compound of any one of claims 1-35 and an inert, non-toxic, pharmaceutically suitable excipient. 37. Use of the compound of any one of claims 1-35 in the preparation of a medicament for treating proliferative and/or angiogenic diseases. 38. Use of the compound of any one of claims 1-35 in the preparation of a medicament for treating cancer and tumors. 39. Use of a compound of any one of claims 1-35 in combination with one or more treatment methods for cancer immunotherapy or one or more medicaments targeting molecular targets derived from cancer immunotherapy in the preparation of a medicament for treating cancer and tumors.