Ligand drug conjugates with improved pharmacokinetic and drug release properties - Patents.com

Abstract

The present invention relates to ligand drug conjugates for the treatment of diseases. In particular, the present invention relates to ligand drug conjugates comprising a linker system that allows improved delivery of drugs to target cells while retaining the favorable pharmacokinetic properties of antibodies. The present invention also relates to ligand drug conjugates that achieve high drug-antibody ratios and exhibit excellent pharmacokinetic properties, thus significantly improving efficacy. In one aspect, the present invention also relates to ligand drug conjugates for intracellular delivery of cytotoxic drugs to tumor or cancer cells.

Description

[Technical field]

[0001] The present invention relates to ligand drug conjugates (LDCs) for the treatment of diseases. In particular, the present invention relates to ligand drug conjugates comprising linker systems that can improve drug delivery to target cells while retaining the favorable pharmacokinetic properties of antibodies. The present invention also relates to ligand drug conjugates with significantly improved efficacy, exhibiting high drug-antibody ratios (DARs) and superior pharmacokinetic properties. In one aspect, the present invention also relates to ligand drug conjugates for intracellular delivery of cytotoxic drugs to tumor or cancer cells. [Background technology]

[0002] Recently, there has been a great deal of interest in the use of enzyme-triggered drug release systems, such as antibody-drug conjugates (ADCs), for targeted delivery of cytotoxic agents to tumor cells. Antibody-drug conjugates generally consist of three components: an antibody that targets an antigen highly expressed on tumor cells, a cytotoxic agent (termed a "toxin" or "payload"), and a linker system that can release the cytotoxic agent from the antibody upon internalization into cancer cells. Ideally, an antibody-drug conjugate will retain the favorable pharmacokinetic and functional properties of the antibody and be active at the target site with the drug released intact and non-toxic in the systemic circulation (blood) but in sufficient amounts to kill the target cells. Thus, one of the greatest challenges in the development of antibody-drug conjugates is the design of a linker system for antibody-drug conjugation that is non-toxic and stable in the systemic circulation but capable of releasing the drug at high speed and in sufficient amounts within the target cells while retaining the favorable pharmacokinetic properties of the antibody. Many linker systems have been developed for the specific intracellular release of cytotoxic drugs. There are two main families of linkers: cleavable and non-cleavable. Cleavable linkers usually exploit the inherent properties of the target cells, such as protease sensitivity, to selectively release the drug, e.g., the cytotoxic agent, from the conjugate. Non-cleavable linkers usually rely on complete degradation of the antibody after internalization of the conjugate into the target cell. An example of an antibody-drug conjugate using a non-cleavable linker is the humanized anti-HER2 (anti-ErbB2) antibody-maytansine conjugate trastuzumab-emtansine (T-DM1, or Kadcyla®; Non-Patent Document 1), which contains the monoclonal antibody trastuzumab conjugated to the tubulin polymerization inhibitor mertansine (DM1) via a non-cleavable maleimidomethylcyclohexane-1-carboxylate (MCC) linker. Although non-cleavable antibody-drug conjugates such as T-DM1 (Kadcyla®) may be advantageous in terms of stability in the systemic circulation, they may not exhibit the desired efficacy. In particular, the drug may not be released in sufficient amounts to achieve the desired pharmacological effect, for example, due to slow degradation of the antibody moiety, and / or may be released in a modified form that is less potent, for example, as a "linker-drug" construct. For example, lysosomal degradation of the antigen component of T-DM1 releases the lysine-MCC-DM1 moiety to efficiently bind to tubulin, but is limited only in its ability to induce a cytotoxic bystander effect (Non-Patent Document 2). Treatment failure due to intrinsic and acquired resistance to T-DM1 also remains a major clinical challenge (Non-Patent Document 3).

[0003] Peptide linkers have also been proposed due to their good stability in the systemic circulation and rapid intracellular drug release by certain enzymes, such as proteases. In particular, peptide linkers containing valine-citrulline (Val-Cit) dipeptide as a substrate for intracellular cleavage by cathepsin B (CatB) have been described (Non-Patent Documents 4-6). CatB is a lysosomal cysteine ​​protease involved in certain physiological processes, and differs from other cysteine ​​proteases in that it has endopeptidase and exopeptidase (carboxydipeptidase) activity, which means that CatB can remove dipeptide units from the C-terminus of proteins and peptides (Non-Patent Document 7).

[0004] Usually, enzymatic cleavage of the conjugate releases the antibody and the linker-drug conjugate at the target site. The linker must allow rapid release of the drug from the linker-drug conjugate. Therefore, a "self-immolative" spacer between the linker and the drug has been proposed to enhance the drug release rate after enzymatic cleavage. The self-immolative spacer usually releases the drug by a mechanism based on elimination or cyclization. An example of a linker system containing a self-immolative spacer is, for example, the brentuximab-vedotin conjugate Adcetris® (Non-Patent Documents 5 and 8). The PABC linker system used in antibody-drug conjugates utilizes a protease-sensitive Val-Cit-PABC dipeptide linker that can be recognized and cleaved by cathepsin B. A maleimidocaproyl (MC) moiety is usually used to attach the linker unit to the antibody and serves as a spacer between the drug and the antibody to avoid steric clashes in substrate recognition by cathepsin B. After enzymatic cleavage of the citrulline PABC amide bond, the resulting PABC-substituted drug, e.g., monomethyl auristatin E (MMAE), spontaneously undergoes 1,6 elimination, releasing the free drug (e.g., MMAE) as a product. However, the efficacy of the PABC linker system can be limited by delayed intracellular drug release and the propensity of the Val-Cit-PABC moiety to be cleaved in the systemic circulation (Non-Patent Document 9).

[0005] Furthermore, in vivo studies have shown that the pharmacokinetic properties, such as distribution and liver clearance, of Val-Cit-PABC-based conjugates depend on the number of drug molecules attached to the antibody moiety (Non-Patent Document 10). Therefore, an important parameter of antibody-drug conjugates is the drug-antibody ratio (DAR) (or drug load(ing)), which refers to the average number of drug molecules attached to one antibody moiety. The DAR influences the efficacy as well as the pharmacokinetic properties and toxicity of the conjugate. A high DAR increases the tendency of the conjugate molecules to aggregate and / or premature cleavage, and is associated with poorer pharmacokinetic properties and / or higher toxicity.

[0006] To overcome this problem, it has been proposed to use a hydrophilic linker system containing a negatively charged sulfonate group, a polyethylene glycol group, or a pyrophosphate diester group to reduce aggregation of the conjugate. In addition, Patent Document 1 discloses an antibody-drug conjugate based on the combination of a drug such as MMAE with a linker system containing a cleavable Val-Cit-PABC moiety and a hydrophilic untethered polyethylene glycol group. The conjugates disclosed in Patent Document 1 are said to exhibit good pharmacokinetic properties in in vivo models even with high DAR (e.g., <10-8). However, the efficacy of the linker system disclosed in Patent Document 1, for example, may be limited due to non-specific enzymatic cleavage and / or premature cleavage, delayed intracellular drug release, and possibly increased lysosomal trapping. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2015 / 057699 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] LoRusso et al. Clin. Cancer Res. 2011, 17, 6437-6447 [Non-Patent Document 2] Ogitani et al. Cancer Sci. 2016, 107, 1039-1046 [Non-Patent Document 3] Hunter et al. Br. J. Cancer2020, 122(5), 603-612 [Non-Patent Document 4] Lu et al. Int. J. Mol. Sci.2016, 17, 561-582 [Non-Patent Document 5] Jain et al. Pharm. Res.2015, 32(11), 3526-3540 [Non-Patent Document 6] Dubowchik et al. Bioconj. Chem.2002, 13, 855-859 [Non-Patent Document 7] Turk et al. Biochim. Biophys. Acta 2012, 1824(1), 68-88 [Non-Patent Document 8] Younes et al. N. Engl. J. Med. 2010, 363, 1812-1821; Jain et al. Pharm. Res. 2015, 32(11), 3526-3540 [Non-Patent Document 9] Dorywalska et al. Mol. Cancer Ther. 2016, 15(5), 958-970 [Non-Patent Document 10] Strop et al. Chem. & Biol.2013, 20, 161-167 Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need for new compounds that are stable in the systemic circulation and contain linker systems that allow rapid release and delivery of drugs to target cells in sufficient amounts to kill the target cells while retaining favorable pharmacokinetic properties. Thus, the object of the present invention is to provide compounds that are stable in the systemic circulation and contain a linker system that allows rapid release and delivery of drugs to target cells in a traceless manner while retaining favorable pharmacokinetic properties.A further object of the present invention is to provide pharmaceutical compositions that contain said compounds.The present invention also relates to ligand drug conjugates that exhibit high DAR and excellent pharmacokinetic properties, and therefore significantly improved efficacy. It is yet another object of the present invention to provide compounds containing linker systems capable of releasing multiple drug molecules, the individual drug molecules being the same or different, while retaining favorable pharmacokinetic properties. It is a further object of the present invention to provide compounds or compositions that can be used in methods for treating or preventing cancer, autoimmune or inflammatory diseases and / or infectious diseases. [Means for solving the problem]

[0010] The present invention provides a novel hydrophilic cleavable linker system that can be used in ligand drug conjugates. The linker system is preferably characterized by the presence of a C-terminal peptide unit on its side chain with a drug covalently attached to its N-terminus via a specific spacer group and a solubilizing group. The C-terminal peptide unit acts as a highly specific substrate for the exopeptidase (i.e., carboxydipeptidase) activity of cathepsin B, preferably cathepsin B, resulting in improved intracellular cleavage and drug release. The linker system is stable in the systemic circulation and exhibits a high DAR while retaining excellent pharmacokinetic properties, thus significantly improving efficacy.

[0011] That is, the present invention relates to a compound represented by the following general formula (I):

[0012] [ka] (In the formula, D represents a moiety derived from a drug, said drug being selected from a carboxyl-containing drug such as auristatin F (AF), a thiol-containing drug such as mertansine (DM1) or ravtansine (DM4), an amino-containing drug such as monomethylauristatin F (MMAF) or exatecan, and a hydroxyl-containing drug such as Maaa-1181a, or preferably selected from a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug; if more than one (D) is present, each (D) is independently selected from a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug, and the moieties (D) are preferably identical to each other; X represents a divalent group containing 1 to 7, preferably 2 to 6, more preferably 2 to 5 skeletal atoms independently selected from C, N, O, and S, and is covalently bonded to (D) via an atom selected from C, S, N, and O derived from a carboxyl, thiol, amino, or hydroxyl functional group contained in (D); Y is a divalent group containing one or more atoms selected from C, N, O, P and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), and most preferably a divalent group derived from cleaved hydrolyzed maleimide; L represents a linker that can be cleaved by cathepsin B; T represents a (2+n)-valent branched group; S represents a moiety derived from a compound that contains one or more, e.g., two, three or four, solubilizing groups; V represents a moiety derived from a vector group capable of interacting with a target cell; n is an integer of 1 to 4, preferably 1 or 2, and more preferably 1; and m is an integer of 1 to 12, preferably 2 to 10, and more preferably 4 to 8). The present invention also relates to a compound as described herein above or a composition thereof for use in a method for the treatment or prevention of cancer, an autoimmune or inflammatory disease and / or an infectious disease. The present invention specifically includes the following embodiments ("items"): 1. The following general formula (I):

[0013] [ka] (In the formula, D represents a moiety derived from a drug, said drug being selected from a carboxyl-containing drug such as auristatin F (AF), a thiol-containing drug such as mertansine (DM1) or ravtansine (DM4), an amino-containing drug such as monomethylauristatin F (MMAF) or exatecan, and a hydroxyl-containing drug such as Maaa-1181a, or preferably selected from a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug; if more than one (D) is present, each (D) is independently selected from a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug, and the moieties (D) are preferably identical to each other; X represents a divalent group containing 1 to 7, preferably 2 to 6, more preferably 2 to 5 skeletal atoms independently selected from C, N, O, and S, and is covalently bonded to (D) via an atom selected from C, S, N, and O derived from a carboxyl, thiol, amino, or hydroxyl functional group contained in (D); Y is a divalent group containing one or more atoms selected from C, N, O, P and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), and most preferably a divalent group derived from cleaved hydrolyzed maleimide; L represents a linker that can be cleaved by cathepsin B; T represents a (2+n)-valent branched group; S represents a moiety derived from a compound that contains one or more, e.g., two, three or four, solubilizing groups; V represents a moiety derived from a vector group capable of interacting with a target cell; n is an integer of 1 to 4, preferably 1 or 2, and more preferably 1; and m is an integer of 1 to 12, preferably 2 to 10, and more preferably 4 to 8. 2. (L) is represented by the following general formula (II) or (II'):

[0014] [ka] (In the formula, Axx represents a moiety derived from a trifunctional amino acid, with the proviso that Axx in formula (II) is not a moiety derived from an amino acid of the (D) configuration; Ayy represents a moiety derived from an amino acid selected from Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-aminobutyric acid (Abu), ornithine (Orn), Ser, Thr, Leu, and Ile; or Ayy in formula (II) represents a moiety derived from an amino acid selected from homotyrosine (homoTyr), homophenylalanine (homoPhe), beta-phenylalanine (betaPhe) and beta homophenylalanine (betahomoPhe), Tyr(OR1) and homoTyr(OR1), where R1 is -(CH2CHO) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24; provided that Ayy in formula (II') is not a moiety derived from an amino acid of configuration (D); Dxx represents a single covalent bond or a moiety derived from an amino acid with a hydrophobic side chain, preferably a single covalent bond or a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe and Ala, more preferably a single covalent bond or a moiety derived from Phe or Val; Dyy represents a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, preferably a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), more preferably a moiety derived from Arg or Cit; with the proviso that when Dxx is a moiety derived from an amino acid with a hydrophobic side chain, Dyy is a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, and when Dxx is a single covalent bond, Dyy is a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain; Z represents a group covalently attached to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group or a cycloalkyl group, preferably -OH; * indicates a covalent bond to (T); ** indicates a covalent bond to (X). 3. At least one of Axx and Ayy is one of the following: Axx represents a moiety derived from an amino acid selected from Glu, 2-aminopimelic acid (Apa), 2-aminoadipic acid (Aaa), Dap, Dab, Lys, Orn, Ser, Ama, and homolysine (homolysine), preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn, and homolysine, more preferably a moiety derived from Orn or Lys, and most preferably a moiety derived from Lys; Ayy in formula (II) is Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr, homoTyr, Tyr(OR1) and homoTyr(OR1) (wherein R1 is -(CH2CH2O) n1 -R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), preferably a portion derived from Phe, homoPhe, Tyr, homoTyr, Tyr(OR1) and homoTyr(OR1), more preferably a portion derived from Phe or Tyr, and most preferably a portion derived from Tyr; The compound according to item 2, wherein Ayy in formula (II') is defined as representing a moiety derived from an amino acid selected from Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe and Ser, more preferably a moiety derived from Phe or Ser, and most preferably a moiety derived from Phe. 4. (X) represents a divalent carbonyl or thiocarbonyl-containing group, preferably represented by the following formulae (IIIa) to (IIIf):

[0015] [ka] (In the formula, n2 and n3 are each independently selected from 0 to 5, preferably 0, 1 or 2, more preferably 0 or 1; n4, n5 are each selected from 0 or 1; Each A is independently selected from O and S, preferably O; *** represents a covalent bond to (D); Item 4. The compound according to any one of items 1 to 3, wherein **' represents a covalent bond to (L). 5. (X) is represented by the following formulae (IVa) to (IVj'):

[0016] [ka] TIFF2025512064000007.tif248170TIFF2025512064000008.tif90170 (in the formula, *** represents a covalent bond to (D); **' represents a covalent bond to (L); The compound according to any one of items 1 to 3, wherein (D) in formula (I) represents an amino-containing drug or a hydroxyl-containing drug when (X) is represented by formula (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVp), (IVr), (IVt), (IVv), (IVw), (IVx), (IVy) or (IVz); (D) in formula (I) represents an amino-containing drug or a hydroxyl-containing drug when (X) is represented by formula (IVj), (IVq), (IVs) or (IVu); and (D) in formula (I) represents a carboxyl-containing drug when (X) is represented by formula (IVa'), (IVb'), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (IVy') or (IVj').

[0017] 6. The compound (X) is represented by formula (IVb'), (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), preferably formula (IVc) or (IVm). 7. The solubilizing groups contained in (S) are each independently selected from the following: moieties comprising one or more ionic or ionizable groups, such as ammonium, guanidinium, sulfate or sulfonate groups, preferably derived from Arg, (D)-Arg, Dap, (D)-Dap, Dab, (D)-Dab, Orn, (D)-Orn, Lys, D-Lys or carnitine; a sugar moiety selected from monosaccharides, disaccharides and linear or branched oligosaccharides, particularly linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein the monosaccharide units in the monosaccharides, disaccharides and oligosaccharides are each independently selected from glucose, fructose, mannose, ribose and galactose; and Polyalkylene oxide groups, preferably C 2~3 The polyalkylene oxide group is more preferably a C 2~3 is a polyalkylene oxide group; The compound according to any one of items 1 to 6, selected from the group consisting of: 8. (S) is a moiety derived from a compound containing one or more polyethylene oxide groups, preferably the polyethylene oxide groups each independently contain 6 to 200, more preferably 10 to 150, and most preferably 12 to 80 repeat units; Preferably, the following sub-formula (V):

[0018] [ka] (In the formula, n3 is an integer of 6 to 200, preferably 10 to 150, and more preferably 12 to 80; **** indicates a covalent bond to (T); X 1 is selected from a single covalent bond, -(C=O)-, and -N(R)-, where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; X 2 is an alkyl group having 1 to 6 carbon atoms, an acetyl group, -(CH2) n4 Carbonyl-containing groups such as -CO2H groups, thiocarbonyl-containing groups, -(CH2) n4 OR group, -(CH2) n4 -SO3H group, -(CH2) n4 -(C=A)-N(R)2 groups, -(CH2) n4 represents an amino-containing group such as a -N(R) group, where A is O or S, each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group, and n4 is an integer from 1 to 6; X 2 is -CH3, -CH2CH2OH, or the following formula (VI):

[0019] [ka] (In the formula, each A is independently selected from O and S, preferably O; Each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; n5 and n6 each independently represent an integer of 1 to 6, preferably 1 or 2; X 2 is most preferably -CH3; When two or more (S) are present, each (S) is preferably a moiety of the above formula (V). 9. (T) is represented by the following formula (VII):

[0020] [ka] (In the formula, each AA independently represents a moiety derived from a trifunctional amino acid such as a diamino-carboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably derived from an amino acid selected from N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab and Dap, more preferably derived from Lys(Poc), Glu, Orn or Lys, most preferably derived from Lys; α indicates a covalent bond to (Y); When n=1, the side chain derived from a trifunctional amino acid is covalently attached to either (L) or (S) and the C-terminus is covalently attached to the other moiety (S) or (L), respectively; For n=2, 3, or 4: *' indicates a covalent bond to (L); ****' indicates a covalent bond to (S); The compound according to any one of items 1 to 8, wherein n is as defined in claim 1. 10. (T) is represented by the following formula (VIII) or (IX):

[0021] [ka] (In the formula, AA1 and AA2 are each independently a moiety derived from a trifunctional amino acid such as a diamino-carboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn or Lys, most preferably a moiety derived from Lys; α indicates a covalent bond to (Y); In formula (IX), the side chain derived from a trifunctional amino acid is covalently attached to either (L) or (S) and the C-terminus is covalently attached to the other moiety (S) or (L), respectively; The compound according to any one of items 1 to 8, represented by formula (VIII), in which *' represents a covalent bond to (L), and ****' represents a covalent bond to (S).

[0022] 11. (D) The following: (i) Anti-neoplastic agents such as: DNA alkylating agents, such as duocarmycins Topoisomerase inhibitors, e.g. doxorubicin RNA polymerase II inhibitors, e.g. α-amanitin DNA cleaving agents such as calicheamicin Antimitotic or microtubule disrupting agents, such as taxanes, auristatins, or maytansinoids Metabolic antagonists such as gemcitabine derivatives Kinesin spindle protein inhibitors such as Filanesib. Kinase inhibitors such as ipasertib or gefitinib Nicotinamide phosphoribosyltransferase inhibitors Matrix metallopeptidase 9 inhibitors Phosphatase inhibitors such as microcystin-LR (ii) Immunomodulators such as fluticasone (iii) Anti-infective drugs such as rifamycin, clindamycin, or reptamulin; and (iv) radioisotopes, metabolites, pharma- ceutically acceptable salts, and / or prodrugs of any of the foregoing; with the proviso that the drug selected from (i)-(iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, or a hydroxyl-containing drug; and When more than one (D) is present, each said (D) is independently selected from moieties (i)-(iv) above, and the moieties (D) are preferably identical to each other; The compound according to any one of items 1 to 10, wherein the moiety is derived from a drug selected from the group consisting of: 12. The compound according to any one of items 1 to 11, wherein (D) is a moiety derived from a drug selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBDs) and dimers thereof, indilinobenzodiazepines (IBDs) and dimers thereof, or radioisotopes and / or pharma- ceutically acceptable salts thereof; preferably, a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1, and DM4; more preferably, a moiety derived from DM1 or DM4. 13. A compound represented by the following general formula (X) or (X'):

[0023] [ka] (In the formula, Axx in formula (X) and formula (X') represents a moiety derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama and homo-Lys, preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys, more preferably a moiety derived from Lys; In formula (X), Ayy is Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr, homoTyr, Tyr(OR1) and homoTyr(OR1) (wherein R1 is -(CH2CH2O) n1 -R2 (wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), preferably a moiety derived from Phe, homoPhe, Tyr, homoTyr, Tyr(OR1) or homoTyr(OR1), more preferably a moiety derived from Tyr; Ayy in formula (X') represents a moiety derived from an amino acid selected from Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe or Ser, more preferably a moiety derived from Phe or Ser; D, Dxx, Dyy, X, Y, T, S, V, Z, m and n have the meaning as defined in any one of claims 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12; preferably at least one, such as 2, 3, 4, 5, 6, 7 or 8 of D, Dxx, Dyy, X, Y, T, S and Z is as follows: (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (b) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe, and Ala, preferably a moiety derived from Phe or Val; (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit; (d) X is a group represented by formula (III) (wherein n2 is 1 or 2) or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, preferably a group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), more preferably a group derived from cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) The compound according to any one of items 1 to 12, wherein Z is -OH. 14. In formula (X), Dyy-Dxx-Axx-Ayy are each independently Arg-Lys-Phe (wherein Dyy is a covalent bond), Arg-Lys-homoPhe (wherein Dyy is a covalent bond), Arg-Lys-Tyr (wherein Dyy is a covalent bond), Cit-Lys-Phe (wherein Dyy is a covalent bond), Cit-Lys-Tyr (wherein Dyy is a covalent bond), Arg-Lys- is selected from homoTyr (wherein Dyy is a covalent bond), Cit-Lys-homoTyr (wherein Dyy is a covalent bond), Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homoTyr, Phe-Lys-Lys-Phe, homoPhe-Arg-Lys-Phe, homoPhe-Cit-Lys-Tyr; and Item 14. The compound according to item 13, wherein in formula (X'), Dyy-Dxx-Ayy-Axx are each independently selected from Arg-Phe-Lys (wherein Dyy is a covalent bond), Arg-Ser-Lys (wherein Dyy is a covalent bond), Cit-Phe-Lys (wherein Dyy is a covalent bond), Cit-Ser-Lys (wherein Dyy is a covalent bond), Cit-homoPhe-Lys (wherein Dyy is a covalent bond), Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys. 15. The formula:

[0024] [ka] (In the formula, D, X, Y, T, S, V, Z, m and n have the same meaning as defined in claim 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and preferably at least one, for example two, three, four, five or six of D, X, Y, T, S and Z are one of the following: (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (d) X is a group represented by formula (III), wherein n2 is 1 or 2, or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, preferably a group derived from maleimide and its derivatives, for example, a group derived from a cleaved hydrolyzed maleimide derivative, more preferably a group derived from a cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) Z is -OH; The compound according to any one of items 1 to 14, wherein Z is -OH;

[0025] 16. The formula:

[0026] [ka] TIFF2025512064000016.tif175170 (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; The compound according to any one of items 1 to 15, wherein the number of oxyethylene repeating units (17) may be substituted by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide. 17. The compound according to any one of clauses 1 to 16, wherein (V) represents a moiety derived from a vector group capable of interacting with a target cell, said target cell being selected from tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, cells involved in autoimmune diseases, activated cells, bone marrow cells, lymphoid cells, melanocytes, and infectious agents including bacteria, viruses, mycobacteria, fungi, preferably said target cell being selected from lymphoma cells, myeloma cells, bone marrow cells, lymphoid cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, testicular cancer cells, skin cancer cells, pancreatic cancer cells, liver cancer cells, and any cell undergoing unregulated and rapid growth and division that causes cancer. 18. The compound according to any one of items 1 to 17, wherein (V) represents a moiety derived from a vector group selected from an antibody, an antibody fragment, a protein, a peptide and a non-peptide molecule, preferably a moiety derived from an antibody or an antibody fragment, such as a single-chain antibody, a monoclonal antibody, a single-chain monoclonal antibody, a monoclonal antibody fragment, a chimeric antibody, a chimeric antibody fragment, a domain antibody or a fragment thereof, a cytokine, a hormone, a growth factor, a colony-stimulating factor, a neurotransmitter or a nutrient transport molecule. 19. (V) represents a moiety derived from a monoclonal antibody, wherein preferably said monoclonal antibody is selected from the group consisting of adalimumab, aducanumab, alemtuzumab, altumomab pentetate, amivantamab, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, belantamab-mafadotin, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brentuximab-vedotin, brodalumab, catumaxomab, cemiplimab, cetuximab, simpanemab, clivatuzuma ... Lenezumab, tetraxetan, daclizumab, daratumumab, denosumab, dinutuximab, dostarlimab, durvalumab, edrecolomab, elotuzumab, emapalumab, enfortumab, enfortumab vedotin, epcolitab, epratuzumab, epratuzumab-SN-38, etaracizumab, gemtuzumab, gemtuzumab ozogamicin, gemmab, glofitamab, girentuximab, goslanemab, ibritumomab, inebilizumab infliximab, inotuzumab, inotuzumab ozogamicin, ipilimumab, isaximab ixekizumab, J591 PSMA antibody, labetuzumab, lecanemab, roncatuximab tesirin, mogamulizumab, mosunetuzumab, necitumumab, nimotuzumab, natalizumab, naratuximab, naxitamab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, polatuzumab vedotin, prasinezumab, ramucirumab, rituximab, sacituzumab, sacituzumab govitecan, semolinemab, sirtuuki an antibody selected from the group consisting of citamab, solanezumab, tacatuzumab, tafasitamab, teprotumumab, tirabonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, zanolimumab, fragments and derivatives thereof; more preferably, atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab;or an antibody incorporated into an Fc fusion protein, preferably selected from belatacept, aflibercept, divaflibercept, dulaglutide, rilonacept, romiplostim, abatacept and alefacept; 20. The compound or salt according to any one of items 1 to 19, wherein (V) represents a moiety derived from an anti-HER2 antibody, an anti-CD37 antibody, an anti-PDL1 antibody or an anti-EGFR antibody, preferably a moiety derived from trastuzumab, pembrolizumab, naratuximab, atezolizumab, durvalumab, avelumab, panitumumab or cetuximab, more preferably a moiety derived from naratuximab, trastuzumab or cetuximab, most preferably a moiety derived from naratuximab or trastuzumab.

[0027] 21. The compound according to clause 20, wherein (D) is a moiety derived from an anti-neoplastic agent, preferably a moiety derived from a drug selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBD) and dimers thereof, indilinobenzodiazepines (IBD) and dimers thereof, or radioisotopes and / or pharma- ceutically acceptable salts thereof. 22. The compound according to clause 21, wherein (D) represents a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4; more preferably a moiety derived from DM1 or DM4. 23. The formula:

[0028] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups, and / or the maleimide bond to (V) may be substituted with a cleaved hydrolyzed maleimide), and (D) represents a moiety derived from DM1. 24. The formula:

[0029] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups, and / or the maleimide bond to (V) may be substituted with a cleaved hydrolyzed maleimide). 25. A composition comprising a therapeutically effective amount of one or more components selected from the compound according to any one of items 1 to 24 or a pharma- ceutically acceptable salt thereof, a carrier, a diluent and other excipients.

[0030] 26. The compound according to any one of items 1 to 24 or the composition according to item 25 for use in a method for treating or preventing cancer, an autoimmune disease and / or an infectious disease. 27. The compound or composition according to clause 26, wherein the method is a method for treating cancer of the blood and bone marrow, preferably acute myeloid leukemia (AML). 28. The compound or composition according to item 26, wherein the compound is according to any of items 20, 21, 22, 23 and 24, preferably item 23 or 24, most preferably item 24. 29. The compound or composition according to any one of items 26 to 28, administered simultaneously with, prior to or subsequent to one or more other therapeutic agents or therapies, such as chemotherapeutic agents, radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infective agents or other compounds of formula (I), in a method for treating or preventing cancer, an autoimmune disease and / or an infectious disease. 30. A compound of the following general formula (XI):

[0031] [ka] (In the formula, D, X, L, T, S and n have the meanings according to claims 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Y' represents a moiety that contains a conjugate group capable of forming a covalent bond with a molecule capable of interacting with a target cell (V'), such as a monoclonal antibody or antibody fragment incorporated into the Fc fusion protein, and is preferably one of the following: Preferably, an optionally substituted maleimide capable of reacting with one or two thiol groups of (V'), Preferably, an optionally substituted haloacetamide capable of reacting with the thiol group of (V'), an ester, preferably an ester capable of reacting with the side chain of the amino acid of (V'), such as an acyl halide, an N-hydroxysuccinimide ester or a phenol ester; a carbonate, preferably one that can react with the side chain of the amino acid of (V'), such as a haloformate, or a carbonate containing a leaving group, such as N-hydroxysuccinimide or a phenol; Preferably an isocyanate or isothiocyanate capable of reacting with the side chain of the amino acid of (V'); Preferably, an azide capable of reacting with an alkyne group contained in (V'); an alkyne, preferably an alkyne capable of reacting with the azide group contained in (V'); an amino group, preferably an amino group capable of reacting with molecule (V') in the presence of an enzyme such as transglutaminase; wherein when (Y') is replaced with (Y) and m is 1, the compound preferably has the structure shown in item 13 or 15.

[0032] 31. A kit for modifying a molecule capable of interacting with a target cell, comprising the compound according to item 30 and, optionally, a buffer solution, the pH of the buffer solution being preferably 6.0 to 10, more preferably 6.5 to 8.0. 32. A method for modifying a molecule capable of interacting with a target cell, comprising reacting a molecule capable of interacting with a target cell, such as a monoclonal antibody or antibody fragment incorporated into an Fc fusion protein, with a compound described in paragraph 30. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 shows the mechanism of exo-CatB-induced drug release from compounds of formula (I), where L represents a moiety of formula (II). Intracellular exo-CatB cleavage at the N-terminus of the dipeptide Axx-Ayy releases the drug (e.g., the moiety DX-Dxx-Dyy) in the target cell. [Diagram 2] FIG. 1 shows the mechanism of exo-CatB-induced drug release from compounds of formula (I), where L represents the moiety of formula (II'). Intracellular exo-CatB cleavage at N-terminus of the dipeptide Ayy-Axx releases the drug, e.g., the moiety DX-Dxx-Dyy, in the target cell. [Diagram 3] Figure 1 shows reversed-phase liquid chromatography (RPLC) chromatograms of Trastuzumab (Tmab). A: chromatogram with base gradient; B: chromatogram with gradient dev2; C: chromatogram with gradient dev7; D: chromatogram with gradient dev7 / short. [Figure 4] Figure 1 shows RPLC chromatograms of cultured naratuximab: A: chromatogram with basic gradient; B: chromatogram with gradient dev7. [Diagram 5]Figure 1 shows RPLC chromatograms (DAR attributes) of prepared ADCs: A: chromatogram of ADC-1 with base gradient; B: chromatogram of ADC-2 with gradient dev7; C: chromatogram of ADC-3 with gradient dev7 / short; D: chromatogram of ADC-4 with gradient dev7. [Figure 6] Figure 1 shows RPLC chromatograms (DAR attributes) of prepared ADCs: A: chromatogram of ADC-5 with basic gradient; B: chromatogram of ADC-6 with basic gradient; C: chromatogram of ADC-7 with gradient dev7 / short; D: chromatogram of ADC-8 with gradient dev7 / short; E: chromatogram of ADC-9 with gradient dev2. [Figure 7] FIG. 1 shows the results of an in vitro cytotoxicity assay (Example 8.6.1) using trastuzumab and ADC-2 in JIMT-1 (HER2-positive) cells. [Figure 8] FIG. 1 shows the results of an in vitro cytotoxicity assay (Example 8.6.1) using trastuzumab, ADC-2 and ADC-4 in JIMT-1 cells. [Figure 9] FIG. 1 shows the results of an in vitro cytotoxicity assay (Example 8.6.1) using trastuzumab, ADC-2, ADC-1, ADC-3 and ADC-9 in JIMT-1 cells. [Figure 10] FIG. 1 shows the results of an in vitro cytotoxicity assay (Example 8.6.1) using trastuzumab, ADC-2 and ADC-7 in JIMT-1 cells. [Figure 11] FIG. 8 shows the results of an in vitro cytotoxicity assay (Example 8.6.1) using naratuximab-DHL-5 (CD37 positive) cells with culture medium, ADC-5 and ADC-6. [Figure 12] FIG. 1 shows the results of an in vivo efficacy assay (tumor volume) with ADC-2 and ADC-4 (Example 8.6.2). [Figure 13] FIG. 1 shows the results of an in vivo efficacy assay (body weight) with ADC-2 and ADC-4 (Example 8.6.2). [Figure 14] FIG. 1 shows the results of an in vivo efficacy assay (tumor volume) with ADC-1, ADC-2, ADC-3 and ADC-9 (Example 8.6.3). [Figure 15] FIG. 1 shows the results of an in vivo efficacy assay (body weight) with ADC-1, ADC-2, ADC-3 and ADC-9 (Example 8.6.3). [Figure 16] FIG. 1 shows the results of an in vivo efficacy assay (tumor volume) with naratuximab and ADC-4 (Example 8.6.4). [Figure 17] FIG. 1 shows the results of an in vivo efficacy assay (survival) with naratuximab, ADC-2, and ADC-4 (Example 8.6.4). [Figure 18] FIG. 1 shows the results of an in vivo efficacy assay (tumor growth and survival) with naratuximab, ADC-2, and ADC-4 (Example 8.6.4). [Figure 19] FIG. 14 shows the results of an in vivo efficacy assay (tumor growth and survival) using naratuximab emtansine (Debio1562) or ADC-4 (0.3 mg / kg, 1 mg / kg, or 3 mg / kg) (Example 8.6.4). [Figure 20] FIG. 14 shows the results of an in vivo efficacy assay (tumor growth and survival) using naratuximab emtansine (Debio1562) or ADC-4 (0.3 mg / kg, 1 mg / kg, or 3 mg / kg) (Example 8.6.4). [Figure 21]Figure 21a shows cell binding assay results - cell binding of naratuximab antibody to CD37 expressing AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 (Example 8.6.5). Figure 21b shows internalization assay results - internalization of naratuximab antibody in CD37 expressing AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 (Example 8.6.5). Figure 21c shows cytotoxicity assay results - cytotoxicity of ADC-4 against CD37 expressing AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 (Example 8.6.5). [Figure 22] Figure 22a shows the results of a cytotoxicity assay - comparative cytotoxicity of ADC-4, naratuximab, and Debio1562 against the CD37 expressing AML cell line MV-4-11 (Example 8.6.6). Figure 22b shows the results of a cytotoxicity assay - comparative cytotoxicity of ADC-4 and Debio1562 against the CD37 expressing AML cell line THP-1 (Example 8.6.6). [Diagram 23] FIG. 8 shows in vivo efficacy assay results—In vivo tumor growth in NCG mice (inoculated with MV4; Luc AML cells) following treatment with ADC-4 (1 mg / kg), ADC-4 (5 mg / kg) or venetoclax and azacitidine (Example 8.6.7). [Figure 24] Figure 24a shows the results of an in vivo pharmacokinetic assay - total antibody in vivo pharmacokinetic profile (plasma concentrations) following administration of ADC-4 (5 mg / kg) and naratuximab (5 mg / kg) to male Swiss mice (Example 8.6.8). Figure 24b shows the results of an in vivo pharmacokinetic assay - total antibody and total ADC in vivo pharmacokinetic profile (plasma concentrations) following administration of ADC-4 to female CD1 mice (Example 8.6.8). [Diagram 25]Figure 25a shows in vitro plasma stability results - Comparative human and mouse plasma stability (DAR reduction) of ADC-4, Enhertu, and ADCETRIS (Example 8.6.9). Figure 25b shows in vivo pharmacokinetic assay results - Comparative human and mouse plasma stability (DAR reduction) of ADC-2, Enhertu, and ADCETRIS (Example 8.6.9). [Figure 26] FIG. 8 shows in vivo efficacy assay results—In vivo efficacy (% survival) of ADC-4 (1 mg / kg), ADC-4 (1 mg / kg) + rituximab, Debio1562 (10 mg / kg), and Debio1562 (10 mg / kg) + rituximab in female SCID beige mice (inoculated with Farage DLBCL cells) (Example 8.6.11). [Figure 27] FIG. 13 shows CatB cleavage assay results—CatB cleavage of DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH to release DM1-Ac-Cit (Example 8.7.3). [Figure 28] FIG. 13 shows CatB cleavage assay results—CatB cleavage of Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH to release Exatecan-Suc-Phe-Cit (Example 8.7.3). [Figure 29] FIG. 1 shows a scheme for the retro-Michael reaction of LDC with a (closed) maleimide bond. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0035] 1.Definition The term "drug" as used herein characterizes a substance (e.g., a naturally occurring substance or a synthetic substance) that can inhibit or prevent the function of a cell and / or kill a cell. In certain embodiments, the term "drug" should be understood to be synonymous with other terms commonly used in the art, such as "cytotoxic agent," "toxin," or "payload" as used in the field of cancer therapy. A drug may include a group derivable from a functional group that can covalently attach the drug to the remainder of a compound, such as the divalent group (X) in formula (I), e.g., a carboxylic acid, a thiol group, a primary amine, a secondary amine, a hydroxyl group, etc.

[0036] The term "moiety derived from a drug" as used herein characterizes a moiety that contains a group that is identical to the natural drug except for the structural modifications necessary to attach the drug to the remainder of the compound of the invention. Depending on the functional groups available in the natural drug, attachment can be achieved using one of the functional groups already present in the natural drug or by incorporating a new functional group. As a result, the (natural) drug can be used for attachment in unmodified or modified form. That is, the drug can be unmodified (in its natural form) except for the replacement of a hydrogen atom by a covalent bond, or it can be chemically modified to incorporate a functional group (e.g., a group selected from hydroxyl, carboxyl, amino and thiol groups) that can be covalently attached to an adjacent group or moiety, for example, the divalent group (X) in formula (I). However, said adjacent group or moiety remains attached to the drug after cleavage by cathepsin B. Thus, as used herein, the term "moiety derived from a drug" refers to a portion that differs from an unmodified (natural) or modified (to incorporate a functional group) drug only by the covalent bonds necessary to attach it to the remainder of the molecule, and does not include any additional groups, e.g., groups or moieties that remain attached after cleavage by cathepsin B.

[0037] The term "carboxyl-containing drug" characterizes an unmodified (natural) or modified drug that contains a carboxylic acid group that can be covalently bonded to an adjacent group or moiety. The term "thiol-containing drug" characterizes an unmodified (natural) or modified drug that contains a thiol group that can be covalently bonded to an adjacent group or moiety. Non-limiting examples of thiol-containing drugs include mertansine (DM1) and ravtansine (DM4). The term "amino-containing drug" characterizes an unmodified (natural) or modified drug that contains a primary or secondary amino group that can be covalently bonded to an adjacent group or moiety. Non-limiting examples of amino-containing drugs include monomethyl auristatin F (MMAF), exatecan, and indilinobenzodiazepine (IBD) derivative 2554618-79-8 (shown below). The term "hydroxyl-containing drug" characterizes an unmodified (natural) or modified drug that contains a hydroxyl group that can be covalently bonded to an adjacent group or moiety. An example of a hydroxyl-containing drug is the exatecan derivative Maaa-1181a (shown below).

[0038] [ka] In a similar manner, the term "derivative" is used to characterize moieties bound to adjacent moieties that differ from the molecule from which they are derived only by the structural element responsible for the bond to the adjacent moiety. This may involve a covalent bond formed by an existing functional group, or a covalent bond and an adjacent functional group newly introduced for this purpose. Similarly, unless otherwise specified or the context dictates otherwise, "derived from" (e.g., "derived from a compound"), when used in connection with another group or moiety, is meant to describe a group or moiety that is identical to the referenced compound, etc., except for structural modifications necessary to attach the group or moiety to one or more adjacent groups or moieties, usually by covalent replacement of a hydrogen atom or group of atoms (e.g., replacement of an OH in a carboxyl group by a covalent bond upon formation of an amide bond with an amino group; further examples are shown at the end of the "Divalent Group (X)" section in the Table below).

[0039] The term "natural drug" refers to a compound whose therapeutic efficacy has been established by in vitro and / or in vivo testing. In a preferred embodiment, the natural drug is a compound whose therapeutic efficacy has been established by clinical trials. Most preferably, the natural drug is a drug that is already on the market. The type of therapeutic efficacy to be established and the appropriate tests to be applied will, of course, depend on the type of medical indication to be treated. When referring to a particular class of drug molecules, such as antineoplastic agents, topoisomerase inhibitors, RNA polymerase II inhibitors, DNA cleaving agents, antimitotic or microtubule disrupting agents, antimetabolites, kinase inhibitors, immunomodulators, or anti-infective agents, these terms may be used in any manner, for example, as described in Mosby's Medical Dictionary, Mosby, Elsevier 10 th ed. (2016) or Oxford Textbook of Oncology, David J. Kerr, OUP Oxford 3 rd It is intended that these have meanings that are generally accepted in the medical field, as reflected in The American Medical Association (AHA). Thus, the drug used in the ligand drug conjugates of the present invention may be a native drug (e.g., a drug that naturally contains one or more functional groups that can be covalently attached to a conjugate) or may be a drug that has been chemically modified to incorporate a functional group (e.g., a group selected from hydroxyl, carboxyl, amino and thiol groups), provided that the modified drug is pharmacologically active. In this context, pharmacological activity means at least 20%, preferably at least 50%, and more preferably at least 80% of the pharmacological activity of the native drug.

[0040] Furthermore, a modified drug may be referred to as "intradrug" if the drug is released in modified form after cleavage by cathepsin B as long as the group or moiety is attached to it, e.g., released as the moiety DX or DX-Dxx-Dyy. In some instances, the remaining groups X, Dxx and Dyy may then be removed via other mechanisms, e.g., by hydrolysis of an ester bond that may be present between D and X. Bonds that are not cleaved by other mechanisms are advantageous if the remaining modified drug DX or DX-Dxx-Dyy is pharmacologically active. Pharmacological activity in this context means that the released modified drug, e.g., the moiety DX or DX-Dxx-Dyy, retains at least 20%, preferably at least 50%, more preferably at least 80% of the pharmacological activity of the native drug when released intracellularly by the ADC. To ensure realistic conditions, the test for activity should not be performed via a cytotoxicity comparison of the released modified drug with the native drug, since such conditions require the entry of the modified drug into the cell, which may introduce bias in cell permeability. The difference in permeability between these two entities is not relevant here due to the intracellular release of the modified drug. The activity of the modified and native drugs may be compared in cell-free binding assays to determine the Ki value (binding affinity) for the drug's appropriate target receptor. If the Ki value cannot be determined, the IC50 of cytotoxicity in HER2+ cell lines of two trastuzumab ADCs with the exact same linker system (e.g., Val-Cit-PAB), one designed to release the modified drug and the other designed to release the native drug, may be compared.

[0041] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds (including reactive conjugates) where the parent compound is modified by making acid or base salts thereof. Pharmaceutically acceptable salts include, for example, non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids or bases. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 thed., Mack Publishing Company, Easton, PA, 1985, page 1418; SM Berge, LM Bighley, and DC Monkhouse, "Pharmaceutical Salts" J. Pharm. Sci. 1977, 66(1), 1-19; PH Stahl and CG Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zuerich, Wiley-VCH, 2008; and AK Bansal et al., Pharmaceutical Technology, 3(32), 2008. Pharmaceutical salts can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. For reactive conjugates, this can be done before or after the drug moiety is incorporated into the compound of the invention. Unless the context requires otherwise, all references to the compounds of the invention (conjugates, modified antibodies, etc.) should also be understood as references to pharma- ceutically acceptable salts of the respective compounds.

[0042] The term "backbone" refers to the connected link of atoms that runs the length of a molecule. Thus, a "divalent group containing X-Y backbone atoms" defines a group in which atoms, e.g., C, N, O, S, are covalently bonded to each other to form a connected chain of atoms, which is covalently bonded at its ends to adjacent groups or moieties. The divalent group may include pendant atoms or groups attached to the backbone moiety. Hydrogen atoms that saturate the valences of backbone atoms do not count as backbone atoms. When cyclic groups are present, the backbone is specified as the shortest connection between the ends.

[0043] The term "functional group" refers to a group that can be attached to another functional group by forming at least one covalent bond without breaking a C-C or C-H covalent bond.

[0044] "Can be cleaved by cathepsin B" characterizes any compound (or moiety that can be incorporated into a compound) that is cleaved when contacted with cathepsin B (CatB) under suitable conditions, for example as described in WO 2019 / 096867. In a preferred embodiment, said cleavage (a) is rapid and / or cleavage (b) is via the exopeptidase activity of CatB. Said embodiment (b) regarding a compound or moiety "capable of being cleaved by the exopeptidase activity of CatB" is defined in more detail in the following paragraph. The above "fast" cleavage of embodiment (a) typically refers to a compound of interest that is faster than the cleavage rate T of the corresponding unconjugated compound (i.e. a compound that lacks the vector group V and is quenched with a conjugate group, e.g. a cysteine ​​covalently attached to the maleimide conjugate group). 1 / 2 is 25 minutes or less, preferably 20 minutes or less, more preferably 18 minutes or less, even more preferably 16 minutes or less, and most preferably 14 minutes or less. There is no particular lower limit. However, since solubilizing groups (such as PEG) tend to reduce the cleavage rate, the cleavage rate T 1 / 2 It is realistic to expect a delay of at least 1 minute, usually at least 2 minutes, and more usually at least 5 minutes.

[0045] As used herein, the term "can be cleaved by the exopeptidase activity of CatB" indicates that each portion of the compound, particularly the linker, e.g., the C-terminal peptide unit, can be specifically recognized and cleaved by the exopeptidase (i.e., carboxydipeptidase) of cathepsin B. Said cleavage results in rapid release of the drug (or modified drug, "intradrug," which has a group or moiety that remains attached thereto after cleavage by cathepsin B) into the target cell. Cleavage of the linker, e.g., the C-terminal peptide unit, via the exopeptidase activity of CatB can be assessed in an in vitro enzyme cleavage assay using recombinant human CatB and UHPLC-MS / MS analysis, as further described below. Given that the exopeptidase activity of CatB is usually associated with a higher cleavage rate compared to the endopeptidase activity of CatB, in some embodiments, a "compound that can be cleaved by the exopeptidase activity of CatB" can be confirmed by a high CatB cleavage rate. In this embodiment, a "compound that can be cleaved by the exopeptidase activity of CatB" is defined as a compound that satisfies the following criteria: the cleavage rate T of the corresponding unconjugated compound (i.e., a compound that lacks the vector group V and is quenched with a conjugate group, e.g., a compound with a cysteine ​​covalently attached to a maleimide conjugate group). 1 / 2 is 25 minutes or less, preferably 20 minutes or less, more preferably 18 minutes or less, even more preferably 16 minutes or less, and most preferably 14 minutes or less. There is no particular lower limit. However, since solubilizing groups (such as PEG) tend to reduce the cleavage rate, the cleavage rate T 1 / 2 It is realistic to expect a reaction time of 1 minute or more, usually 2 minutes or more, and even more usually 5 minutes or more. In one embodiment, a "compound that can be cleaved by the exopeptidase activity of CatB" is one of the following:

[0046] [ka] wherein the ratio (T 1 / 2 Compound / T 1 / 2(see, for example) is 0.6 or less, preferably 0.4 or less, more preferably 0.2 or less. Again, there is no particular lower limit to this range, but typically the cleavage ratio is 0.001 or more, more usually 0.01 or more, especially 0.05 or more.

[0047] The term "solubilizing group" or "solubilizing moiety" as used herein refers to a hydrophilic group or moiety that can enhance (improve) the water solubility of the moiety or compound to which it is attached. The solubilizing group may be, for example, a polyalkylene oxide group, such as a polyethylene oxide (PEO) group or a polypropylene oxide (PPO) group, a saccharide group, or a moiety that contains one or more ionic or ionizable groups, i.e., functional groups that are charged (anionic or cationic) at physiological pH (7.4), such as moieties derived from amino acids, such as Lys, Glu, Asp, His, Arg, diaminopropionic acid (Dap), diaminobutyric acid (Dab), 2-aminoadipic acid (Aad), carnitine, Orn. ​​The ionic or ionizable groups may be ammonium, guanidinium, sulfate, phosphate, phosphonate, and sulfonate groups. Examples of saccharide groups include monosaccharides, disaccharides and linear or branched oligosaccharides, especially linear or branched oligosaccharides with 3 to 10 monosaccharide units linked by glycosidic bonds, where the monosaccharide units in the monosaccharides, disaccharides and oligosaccharides are each independently selected from glucose, fructose, mannose, ribose and galactose. The term "solubilizing moiety" according to the present invention refers to a moiety derived from a compound that contains one or more, e.g., two, three or four, solubilizing groups. In further embodiments, the solubilizing moiety may consist of one or more solubilizing groups, e.g., amino acids, PEO groups.

[0048] As used herein, the term "polyalkylene oxide" (or "polyalkylene glycol", "polyoxyalkylene") refers to a compound having the general structure HO-(XO) n-H material, where X represents an alkylene group having 2 or 3 carbon atoms, and n represents the number of repeating units, for example, 6 to 200, 10 to 150, or 12 to 80 repeating units, for example, 16 or 40 repeating units, for example, 17, 18, 20 or 24 PEO repeating units. Thus, the term "polyalkylene oxide group" refers to a material of the formula *-O-(XO) n It should be understood as a divalent group of -**, where X and n are as defined above, and * and ** indicate a covalent bond to the adjacent moiety. In an embodiment, the term "polyalkylene oxide" may be polyethylene oxide (or polyethylene glycol, C2 polyalkylene oxide), or polypropylene oxide (or polypropylene glycol, C3 polyalkylene oxide). It may also provide a polyalkylene oxide group in which two or more different alkylene groups as defined above are arranged randomly or in blocks.

[0049] The term "peptide" as used herein refers to a compound comprising a consecutive sequence of at least two amino acids linked together via peptide bonds. The terms "dipeptide", "tripeptide" and "tetrapeptide" refer to compounds comprising a consecutive sequence of 2, 3 and 4 amino acids linked together via peptide bonds, respectively. The term "peptide bond" in this context is meant to encompass (backbone) amide bonds as well as modified bonds that can be obtained when non-natural amino acids are introduced into a peptide sequence. In this case, the modified bond replaces the (backbone) amide bond formed in the consecutive peptide sequence by reacting the amino and carboxyl groups of two amino acid residues. For example, the modified bond may be an ester, thioester, carbamide, thiocarbamide or triazole bond. Preferably, the amino acids forming the consecutive peptide sequence are linked together via backbone amide bonds. The peptides may be linear or branched. In a preferred embodiment, the peptide is a linear di-, tri-, tetra-peptide, more preferably a linear tri- or tetra-peptide.

[0050] The term "amino acid" as used herein refers to a compound that contains or is derived from a compound that contains at least one amino group and at least one acidic group, preferably a carboxyl group. The distance between the amino group and the acidic group is not particularly limited. α-, β-, and γ-amino acids are suitable, with α-amino acids, especially α-amino carboxylic acids, being particularly preferred. The term "amino acid" includes both naturally occurring amino acids, such as naturally occurring proteinogenic amino acids, as well as synthetic amino acids not found in nature. Hereinafter, reference to amino acids may be made by the three letter amino acid code (Arg, Phe, Ala, Cys, Gly, Gln, etc.) or by the one letter amino acid code (R, F, A, C, G, Q, etc.). Amino acid sequences are written hereinafter from N-terminus to C-terminus (left to right). Unless otherwise specified or dictated otherwise by context, all connections between adjacent amino acid groups are formed by peptide (amide) bonds.

[0051] As used herein, the term "amino acid of the (D) configuration" refers to the (D)-isomer of any naturally occurring or synthetic amino acid. It applies to α-amino acids as well as β- and γ-amino acids. As used herein, the term "amino acid of the (D) configuration" is not meant to encompass non-chiral amino acids such as glycine or other non-chiral amino acids such as aminoisobutyric acid.

[0052] As used herein, the term "side chain of an amino acid" may be the moiety attached to the α-carbon of an amino acid. For example, the side chain of Ala is methyl, the side chain of Phe is phenylmethyl, the side chain of Cys is thiomethyl, the side chain of Tyr is 4-hydroxyphenylmethyl, etc. Both naturally occurring and non-naturally occurring side chains are included in this definition.

[0053] The term "trifunctional" as used herein refers to a compound or moiety that has three functional groups that can form or have formed three covalent bonds to adjacent moieties. Thus, the term "trifunctional amino acid" refers to a compound that contains or is derived from at least an amino group, an acid group (e.g., a carboxyl group), and a compound that contains another functional group such as an amino group or a carboxyl group. Non-limiting examples of trifunctional amino acids include Ser, Cys, Tyr, N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab, and Dap.

[0054] The term "C-terminus" as used herein refers to the C-terminus of an amino acid (peptide) chain. Attachment to the "C-terminus" means that a covalent bond is formed between an acid group in the backbone of the amino acid residue and the binding pair. For example, attachment of a group "X" to the C-terminus of an amino acid residue "Axx" results in an ester or amide type structural element *-C(O)-X, where the carbonyl group is derived from the acid group of Axx and (*) indicates the bond to the backbone. The term "C-terminal peptide unit" as used herein is used to characterize a peptide sequence of 2, 3 or 4 amino acids, where the C-terminal amino acid forms the C-terminus of the peptide sequence.

[0055] The term "N-terminus" as used herein refers to the N-terminus of an amino acid (peptide) chain. Attachment to the "N-terminus" means that a covalent bond is formed between the amino group in the main chain (backbone) of the amino acid residue and the bond pair (replacing one hydrogen atom). For example, attachment of the group "X" to the N-terminus of the amino acid residue "Axx" results in the structural element X-NH-*, where the amino group is derived from Axx and (*) indicates the attachment to the main chain.

[0056] The term "hydrophobic" as used herein is used to characterize compounds, groups or moieties that have no affinity for water. For example, the term "amino acid with hydrophobic side chain" is used to characterize amino acids with hydrophobic or partially hydrophobic aliphatic side chains or amino acids with aromatic side chains (e.g., Phe, Leu, Ile, Val, Tyr, Trp, Ala). Of course, any other amino acid with the same or higher hydrophobicity should also be treated as hydrophobic in the sense of the present invention. Comparison of the degree of hydrophobicity can be performed by determining the n-octanol / water partition coefficient (25°C and pH 7): if the ratio of the concentration of the other amino acid in n-octanol / water is equal to or greater than the ratio of one or more of the amino acids Phe, Leu, Ile, Val, Tyr, Trp, Ala, the other amino acid should be treated as a hydrophobic amino acid.

[0057] As used herein, the term "amino acid with a basic side chain" is used to characterize natural or unnatural amino acids that contain one or more ionizable groups with a side chain pKa value of 6 or greater. Examples of natural amino acids with basic side chains include Arg (guanidino group, pKa=12.5), Lys (amino group, pKa=10.5), and His (imidazole group, pKa=6). Examples of unnatural amino acids with basic side chains include citrulline (Cit), ornithine (Orn), 2,3-diamino-propionic acid (Dap), and 2,4-diamino-butyric acid (Dab).

[0058] The term "alkyl group" as used herein means a linear or branched hydrocarbon group having 1 to 20 carbon atoms, preferably 1 to 5, more preferably a methyl group or an ethyl group, or a cycloalkyl group having 3 to 20 carbon atoms, preferably 5 to 8. The cycloalkyl group may be a single ring or two or more condensed rings.

[0059] As used herein, the term "divalent maleimide derivative" (or, e.g., "divalent group derived from a compound selected from maleimides") refers to a divalent moiety (e.g., a succinimide moiety) derived from maleimide in which the double bond has been hydrogenated and two hydrogen atoms have been replaced by two covalent bonds capable of binding to an adjacent moiety. For example, a divalent maleimide derivative can have the following structure:

[0060] [ka] where R and R' represent the adjacent moieties to which the maleimide derivative is attached. The moiety contains a chiral carbon atom (i.e., the atom at which the sulfur atom resides). Unless otherwise specified, reference to a divalent maleimide derivative is to be understood as a reference to the pure stereoisomers as well as any mixtures thereof, in particular racemic mixtures thereof. The term "divalent maleimide derivative" or "divalent group derived from a compound selected from maleimides" should be further understood to include any derivative of maleimide (as described above) that is further substituted at positions other than the 2- and 3-positions, as well as ring-opened hydrolyzed maleimide derivatives. Divalent maleimide-type disulfide bridges (e.g., of the formula -SX 2 -S- / -SX 3 -S(where X 2 / X 3 represents a divalent radical derived from maleimide) can be obtained, for example, by side chain-side chain cyclization in the presence of 2,3-dibromomaleimide or other suitable reagents as described in Kuan et al. in Chem. Eur. J. 2016, 22, 17112-17129.

[0061] "Cleaved hydrolyzed maleimide derivative" in the context of the present invention refers to a divalent moiety derived from maleimide (as described above) in which the maleimide ring has been cleaved by hydrolysis. For example, hydrolysis of the divalent maleimide derivative RXS-R' (wherein X represents an unsubstituted divalent group derived from maleimide, R / R' represents adjacent groups or moieties, and the maleimide double bond at X is no longer present) leads to a ring-opened hydrolyzed maleimide derivative of formula R-NH-C(=O)-CH(S-R')-CH2-COOH, or R-NH-C(=O)-CH2-CH(S-R')-COOH, or mixtures thereof. Ring hydrolysis can be carried out, for example, under basic conditions. The following conditions are particularly suitable: upon completion of the cysteine-maleimide conjugation reaction (e.g., after reaction of the maleimide moiety (Y') with the side chain (V') of the cysteine ​​residue contained in the molecule capable of interacting with the target cell), the pH is adjusted to pH 8 by addition of 10x pH8 DPPS (0.2-0.5 reaction volumes), excess reactive drug linker and reducing agent (TCEP) are removed via gel filtration using a column suitable for gel filtration (e.g., PF column, elution with pH 8 buffer), and the eluate is stirred overnight for 16 hours to complete the release before a final buffer exchange with DPBS into an Amicon concentration unit; unless otherwise specified, reference to "cleaved hydrolyzed maleimide derivative" should be understood as a reference to one of the structures alone or to any mixture of these structures. Furthermore, the carbon bearing the sulfur atom is chiral. Unless otherwise specified, reference to "cleaved hydrolyzed maleimide derivative" should be understood as a reference to the pure stereoisomers as well as any mixtures thereof, in particular racemic mixtures thereof. Furthermore, the term "maleimide bond" in the context of the present invention refers to a divalent moiety derived from the above maleimide, which contains two covalent bonds that can be attached to adjacent groups or moieties. For example, in a maleimide derivative of the formula RXS-R', where R / R' represent adjacent groups or moieties, X represents a maleimide bond (a divalent group derived from a maleimide where the maleimide double bond is no longer present). Thus, the term "maleimide bond" is synonymous with "maleimide derivative bond." Similarly, a "ring-opened hydrolyzable bond" in the context of the present invention refers to a divalent moiety derived from the maleimide described above that contains two covalent bonds that can be attached to adjacent groups or moieties. For example, in a cleaved hydrolyzed maleimide derivative of the formula RXS-R', where R / R' represent adjacent groups or moieties, X represents a cleaved hydrolyzed maleimide bond. Thus, the term "cleaved hydrolyzed bond" is synonymous with "cleaved hydrolyzed derivative bond."

[0062] Reference to a "divalent radical derived from a compound selected from triazoles" is meant to characterize a divalent radical resulting from a 3+2 cycloaddition of an alkyne and an azide. The divalent radical typically has the following structure:

[0063] [ka] where the single bond characterizes the bond to the adjacent group, such that there is no particular restriction as to which adjacent group is bonded to the nitrogen atom and which adjacent group is bonded to the carbon atom. In the context of the group Y, a divalent group may be formed by reacting an alkyne group bonded to V with an azide group bonded to T, or vice versa.

[0064] Reference to a "divalent radical derived from a compound selected from hydrazones" is meant to characterize a divalent radical of CH=N-NH by condensation of a hydrazine group with a carbonyl. In the context of the group Y, the divalent radical may be formed by reacting a carbonyl group attached to V with a hydrazine group attached to T, or vice versa. Reference to a "divalent group derived from a compound selected from carbonyl-containing compounds" is meant to characterize the divalent group -C(=O)-X (wherein X represents O, S or NH) resulting from the reaction of an (activated) carbonyl with a nucleophilic group, for example the formation of an amide, ester or thioester group. In the context of the group Y, the divalent group may be formed by reacting a carbonyl-containing group (e.g. -C(=O)-Cl) attached to V with a nucleophilic group (e.g. -NH2) attached to T, or vice versa. In the above divalent groups, the term "derivatives thereof" means that any hydrogen atom may be replaced by a substituent as defined hereinafter, provided that such substitution does not prevent the formation of the divalent group.

[0065] The term "leaving group" as used herein refers to an atom or group (which may be charged or uncharged) that leaves an atom or molecule that is considered to be the remaining or major part of the molecule involved in a particular reaction, such as a nucleophilic substitution reaction (Pure Appl. Chem. 1994, 66, 1134). Examples of leaving groups include thiophenolates, phenolates, carboxylates, and sulfonates.

[0066] As used herein, the term "moiety derived from a vector group" indicates that the vector group may be in unmodified or modified form, i.e., the vector group may be unmodified (in its native form) except for the replacement of hydrogen atoms by covalent bonds, or may be chemically modified to introduce one or more functional groups (e.g., groups selected from hydroxyl, carboxyl, thiol and / or amino groups) that can be covalently attached to the vector group to T, provided that the modifications do not interfere to any significant extent with the interaction between the vector group and the target cell.

[0067] As used herein, the term "capable of interacting with a target cell" indicates that a vector group can bind to, complex with, or react with a moiety, such as a protein or receptor, that is exposed on the surface of the target cell. Said interaction can result in a targeting effect (i.e., a local increase in the concentration of the vector-carrying compound in the vicinity of the target cell) and / or internalization of the vector-carrying compound of the invention into the target cell.

[0068] The term "antibody" (synonymously "immunoglobulin" (Ig)) as used herein includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), veneered antibodies, and small immune proteins. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. A target antigen generally has multiple binding sites, also called epitopes, that are recognized by complementarity determining regions on multiple antibodies. The structure of each antibody that specifically binds to a different epitope is different. Thus, one antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a target antigen or a portion thereof of interest. The antibody may be an IgG, e.g., IgG1, IgG2, IgG3, IgG4. Preferably, the antibody is an IgG protein, more preferably an IgG1, IgG2 or IgG4 protein. Most preferably, the antibody is an IgG1 protein. The antibody may be human or derived from another species. Preferably, the antibody is a human antibody. The term "monoclonal antibody" as used herein characterizes antibodies that are produced by one type of immune cell and are all identical because they are clones of a single parent cell. The term "antibody fragment" as used herein refers to a molecule that contains at least one polypeptide chain derived from an antibody that is not full-length.

[0069] The term "Fc fusion protein" as used herein refers to a protein comprising at least an Fc-containing antibody fragment (i.e., an immunoglobulin-derived portion comprising at least one Fc region) and a portion derived from a second non-immunoglobulin protein. The Fc-containing antibody fragment forms part of the Fc fusion protein and is incorporated into the Fc fusion protein. The Fc-containing antibody fragment may be derived from an antibody as described herein above, in particular IgG, such as IgG1, IgG2, IgG3, IgG4. Preferably, the Fc-containing portion is derived from an IgG1 protein, more preferably from a human IgG1 protein. The non-Ig protein may be a therapeutic protein, such as erythropoietin (EPO), thrombopoietin (THPO), e.g., THPO-binding peptide, growth hormone, an interferon (IFN), e.g., IFNα, IFNβ, or IFNγ, a platelet-derived growth factor (PDGF), an interleukin (IL), e.g., IL1α or IL1β, a transforming growth factor (TGF), e.g., TGFα or TGFβ, or a tumor necrosis factor (TNF), or derived from a receptor, in particular a ligand-binding fragment of the extracellular domain of a receptor. For example, the therapeutic protein may be derived from cluster of differentiation 2 (CD2), CD4, CD8, CD11, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD58 (LFA3), CD80, CD86, CD147, CD164, IL2 receptor, IL4 receptor, IL6 receptor, IL12 receptor, epidermal growth factor (EGF) receptor, vascular endothelial growth factor (VEGF) receptor, epithelial cell adhesion molecule (EpCAM), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4). Examples of Fc fusion proteins include belatacept (Nulojix®), aflibercept (Eyla®), rilonacept (Arcasit®), romiplostim (NPlate®), abtacept (Orencia®), alefacept (Amevine®), and etanercept (Enbrel®).

[0070] The term "cancer" as used herein refers to a physiological condition in mammals characterized by unregulated cell growth. A tumor comprises one or more cancer cells. Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, and lymphoid malignancies. Further examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma, etc.), non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma, small cell lung cancer, non-small cell lung cancer, acute myeloid leukemia (AML), lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, gastrointestinal stromal tumor, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, thyroid cancer, and liver cancer.

[0071] The term "drug-antibody ratio" (or "DAR") as used herein refers to the average number of drug molecules (or molecules) bound to one (e.g., antibody) moiety (V). DAR is also referred to in the art as "drug loading" or "drug loading". The DAR in the compounds of the present invention can be calculated by multiplying n and m in formula (1). When n=1, m in formula (1) represents the DAR. However, it is also understood that when DAR is used to describe a sample containing many molecules, it is often an average value due to some degree of heterogeneity usually associated with the conjugation process. The average DAR can range, for example, from about 1 to about 10, and can be about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. In some embodiments, the DAR may be about 3 to about 8, and typically may be about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9. In some embodiments, the DAR may be about 4. In some embodiments, the DAR may be about 8. In some embodiments, a DAR of "about n" means that the measured DAR is within ±20% of n (e.g., between 80% of n and 120% of n).

[0072] As used herein, the term "emtansine" refers to a conjugate formed by covalent attachment of the non-reducing linker N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", designated "MCC" after conjugation to an antibody, for example) to the antimitotic agent mertansine (DM1). Emtansine is used in the known antibody drug conjugate trastuzumab emtansine (Kadcyla®).

[0073] The term "patient" as used herein refers to a subject to which a compound of the invention, i.e., a Ligand Drug Conjugate, is administered. In the context of the present invention, a patient is a mammal, preferably a human being (male or female). Where the specification refers to "preferred" embodiments / features, combinations of said "preferred" embodiments / features should also be considered as disclosed, insofar as said combinations of "preferred" embodiments / features make technical sense. Hereinafter, the terms "containing" and "comprising" in the present specification and claims should be understood so that in addition to the mentioned elements, further unmentioned elements may be present. However, the terms should also be understood as disclosing the term "consisting of" as a more limited embodiment, so that there may be no further unmentioned elements as long as they are technically meaningful. For example, a "divalent carbonyl-containing group" includes a divalent group consisting of carbonyl (-CO-) as a preferred embodiment. Furthermore, "at least one of X and Y" should be broadly understood to disclose one or both of X and Y, i.e., equivalent to the expression "at least one selected from the group of X and Y". Unless otherwise specified or unless the context dictates otherwise, reference to a "substituted" or "optionally substituted" group is to be understood as a reference to the presence (or optional presence) of at least one substituent selected from F, Cl, Br, I, CN, NO2, NH2, NH-C 1-6 -Alkyl, N(C 1-6-alkyl)2, -XC 1-6 -Alkyl, -XC 2-6 -Alkenyl, -XC 2-6 -Alkynyl, -XC 6-14 -aryl, -X-(5-14 membered heteroalkyl having 1-3 heteroatoms selected from N, O, S), where X represents a single bond, -(CH2)-, -O-, -S-, -S(O)-, -S(O)2-, -NH-, -CO-, or any combination thereof including, for example, -C(O)-NH-, -NH-C(O)-. The number of substituents is not particularly limited and can range from 1 to the maximum valence number that can be saturated with the substituents. It is usually 1, 2 or 3, usually 1 or 2, and most usually 1. Unless otherwise specified, all valences of the individual atoms of the compounds or moieties described herein are saturated. In particular, they are saturated by the bond pairs shown. If no bond pairs are shown or the number of bond pairs is too small, the remaining valences of each atom are saturated by the corresponding number of hydrogen atoms. Unless otherwise specified, chiral compounds and moieties can be present in the form of pure stereoisomers or in the form of mixtures of stereoisomers, including a 50:50 racemate. In the context of the present invention, a reference to a particular stereoisomer should be understood as a reference to a compound or moiety in which the specified stereoisomer is present in at least 90% enantiomeric excess (ee), more preferably at least 95% ee, and most preferably 100% ee, where % ee is defined as (IR-SI) / (R+S)*100%, where R and S represent the molar amounts of each enantiomer. Unless the context requires otherwise and / or an alternative meaning is expressly provided herein, all terms are defined as having the same meaning as those listed in the IUPAC Gold Book (status of st Aug. 2020), or the Dictionary of Chemistry, Oxford, 6 th This is the meaning generally accepted in the industry as reflected in Ed.

[0074] 2. Overview The present invention includes a linker, e.g., a C-terminal peptide unit, with (1) a drug covalently attached thereto via a specific spacer group and (2) a solubilizing group. The linker, e.g., a C-terminal peptide unit, acts as a highly specific substrate for fast cleavage by CatB and / or cleavage by the exopeptidase (i.e., carboxydipeptidase) activity of CatB, resulting in improved (fast) cleavage and release of the drug into target cells. The linker system is less susceptible to premature cleavage and remains stable in the systemic circulation (blood). Thus, the linker system of the present invention can be used in ligand drug conjugates (LDCs). CatB is a lysosomal cysteine ​​protease of the papain superfamily that acts in intracellular protein turnover and various physiological and pathological processes. Extensive structural and functional data are now available, making this protease a versatile tool in the current state of intracellular drug delivery. The papain fold is composed of two domains, the left (L) and right (R) domains. The L domain contains three α-helices, whereas the R domain forms a β-barrel, as described in (2002) J. Biol. 143:1311-1320. The interface of the two domains opens at the top and forms the active site cleft of the enzyme. The active site cleft is centered on residues Cys25 (N-terminal to the central helix, L domain) and His163 (within the β-barrel residue, R-domain). These two catalytic residues form a thiolate-imidazolium ion pair that is essential for the proteolytic activity of the enzyme. The substrate binds in an extended conformation along the active site groove, making alternating contacts with the L and R domains, as described in (2002) J. Biol. 143:1311-1320. Most cysteine ​​cathepsins exhibit mainly endopeptidase activity (F, L, K, S, V), whereas CatX and C only exhibit exopeptidase activity. In contrast, CatB exhibits both endopeptidase and exopeptidase activity. X-ray analysis reveals that exopeptidase / carboxydipeptidases such as CatB contain additional structural features, namely an additional ("occluded") loop that modifies the active site cleft and provides the rationale for substrate binding in endopeptidase and exopeptidase activity. In particular, the closed loop provides the structural basis for the predominant activity of exo vs. endo CatB, as shown by Renko et al. (FEBS Journal 2010, 277, 4338-4345).

[0075] The CatB cleavable linker system described in the prior art (e.g., the Val-Cit-PABC linker system) is mainly based on the endopeptidase activity of CatB. On the other hand, the linker system of the present invention is specifically designed to meet the structural requirements for rapid cleavage by CatB and / or to act as a specific substrate for the exopeptidase activity of CatB. The linker system also allows multiple drug molecules to be released intracellularly, where each drug molecule can be the same or different. When the drug is a cytotoxic agent, the linker system allows multiple drug molecules to be released intracellularly, which can be multiple molecules of the same drug or multiple molecules of different drugs (e.g., two or more different drugs).

[0076] Moreover, it has been surprisingly found that any effect on CatB binding due to the presence of sterically demanding solubilizing moieties is fully compensated for by rapid cleavage by CatB, and thus sterically demanding solubilizing groups can be incorporated into ADCs to improve ADC PK and manufacturability without affecting cytotoxicity.Cleavage by CatB, particularly by the exopeptidase activity of CatB, is rapid for many substrates, and this high cleavage rate compensates for some of the slowdown in cleavage rate resulting from the incorporation of solubilizing moieties, so that the resulting cleavage rate can still be reasonably fast.Without wishing to be bound by any theory, it is believed that the solubilizing moieties can achieve high DAR because they are directed outside the CatB binding groove, resulting in superior selectivity and cleavage rate by CatB, for example, via the exopeptidase activity of CatB. Furthermore, it has been found that the attachment of the drug to the linker via a specific spacer group and the presence of a solubilizing moiety can provide surprising improved efficacy and overcome resistance problems that may occur with other conjugates.Without wishing to be bound by any theory, it is believed that the spacer group and the solubilizing moiety work together in such a way that after cleavage by CatB, the drug can engage its molecular target(s) in the cytoplasm and avoid lysosomal capture, diffusing to nearby cells to induce bystander effects, such as cytotoxic bystander effects.

[0077] 3. Compound of formula (I) The present invention relates to a compound having the following general formula (I):

[0078] [ka] The present invention relates to a compound represented by the formula: The compounds of formula (I) include a linker (L), e.g., a C-terminal peptide unit, that serves as a substrate for specific recognition and cleavage by cathepsin B, in particular for rapid cleavage and / or cleavage by the exopeptidase activity of Cat B. The linker is covalently attached to a branching group (T) as well as a divalent group (X). Furthermore, in formula (I), D represents a moiety derived from a drug, and the drug is selected from carboxyl-containing drugs such as auristatin F (AF), thiol-containing drugs such as mertansine (DM1) or ravtansine (DM4), amino-containing drugs such as monomethyl auristatin F (MMAF), exatecan or 2554618-79-8, and hydroxyl-containing drugs such as Maaa-1181a. The drug is covalently bonded to the divalent group (X) via a functional group contained therein, i.e., a carboxyl, thiol, amino, or hydroxyl functional group. Preferably, the drug is selected from thiol-containing drugs, amino-containing drugs, and hydroxyl-containing drugs. More preferably, the drug is a thiol-containing drug such as DM1 or DM4. When more than one (D) is present in the conjugate molecule (n>1 and / or m>1), at least one, preferably each (D) is independently selected from a carboxyl-, thiol-, amino-, and hydroxyl-containing drug, preferably a thiol-, amino-, and hydroxyl-containing drug, and the multiple moieties (D) are preferably identical to one another. X represents a divalent group containing 1 to 7, preferably 2 to 6, more preferably 2 to 5, for example 2, 3, 4, or 5 skeletal atoms independently selected from C, N, O, and S; X is covalently bonded to (D) via an atom selected from C, S, N, and O derived from a carboxyl, thiol, amino, or hydroxyl functional group contained in (D). Y represents a divalent group containing one or more atoms selected from C, N, O, P, and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives, for example, a cleaved hydrolyzed maleimide derivative, and most preferably a divalent group derived from a cleaved hydrolyzed maleimide. T represents a (2+n)-valent branched group. S represents a moiety derived from a compound that contains one or more, for example, 2, 3, 4 or 5, solubilizing groups. V represents a moiety derived from the vector group that is capable of interacting with a target cell. n is an integer of 1 to 4, preferably 1 or 2, and more preferably 1; m is an integer of 1 to 12, preferably 2 to 10, and more preferably 4 to 8. In one embodiment, n is 1 and m is an integer of 1-12, preferably 2-10, more preferably 4-8, for example 5-8 or 6-8.

[0079] 3.1 Cleavable Linker (L) The compounds of formula (I) contain a linker (L), e.g. a C-terminal peptide unit that serves as a substrate for specific recognition and cleavage by cathepsin B, in particular rapid cleavage and / or cleavage by the exopeptidase activity of Cat B. The linker allows for the rapid (fast) release of the drug-containing moiety, e.g. the moiety DX or DX-Dxx-Dyy, from the compound into the target cell. In one embodiment of the invention, the linker (L) has the following general formula (II) or (II′):

[0080] [ka] In the general formula (II) or (II'), Axx represents a moiety derived from a trifunctional amino acid. However, Axx may be a moiety derived from any natural or unnatural trifunctional amino acid, provided that Axx in formula (II) is not a moiety derived from an amino acid of the (D) configuration. Examples of trifunctional amino acids include amino-dicarboxylic acids and diamino-carboxylic acids such as α-aminoadipic acid (Aaa), diaminopropionic acid (Dap), diaminobutyric acid (Dab), and aminomalonic acid (Ama). Further suitable trifunctional amino acids include Glu, 2-aminopimelic acid (Apa), Lys, Orn, Ser, and homo-lysine (homo-Lys). In one embodiment, Axx represents a moiety derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama, and homolysine (homoLys). In a preferred embodiment, Axx represents a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn, and homoLys. More preferably, Axx represents a moiety derived from Orn or Lys, most preferably from Lys. Ayy represents a moiety derived from an amino acid selected from Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-aminobutyric acid (Abu), ornithine (Orn), Ser, Thr, Leu, and Ile; or Ayy in formula (II) represents a moiety derived from an amino acid selected from homotyrosine (homoTyr), homophenylalanine (homoPhe), beta-phenylalanine (betaPhe) and beta homophenylalanine (betahomoPhe), Tyr(OR1) and homoTyr(OR1), where R1 is -(CH2CHO) n1-R2, where R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24, e.g., an integer of 5 to 20 or 8 to 16; provided that Ayy in formula (II') is not a moiety derived from an amino acid of the (D) configuration. Without being bound by theory, it is believed that Ayy provides the compounds of the present invention with structural features for specific recognition and cleavage by the exopeptidase activity of CatB. As a result, the compounds may release the drug at a significantly higher rate than compounds that are cleaved by the endopeptidase activity of CatB, e.g., compounds that include the Val-Cit-PABC linker system. In one embodiment, Ayy in formula (II) represents a moiety derived from an amino acid selected from Phe, homoPhe, Ala, Trp, Leu, Tyr, Phg, Met, Abu, Val, Lys, Cit, Tyr(OR1) and homoTyr(OR1), preferably a moiety derived from Phe, homoPhe, Ala, Trp, Leu, Val, Tyr, homoTyr, Tyr(OR1) or homoTyr(OR1), where R1 is as specified above, in particular a moiety derived from Phe or Tyr, most preferred Ayy in formula (II') is a moiety derived from Phe, homoPhe, Ala, Ser, Thr, Leu, Val, Tyr, homoTyr, Tyr(OR1) or homoTyr(OR1), preferably a moiety derived from an amino acid selected from Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Ile and Arg, more preferably a moiety derived from Phe, homoPhe or Ser, particularly preferably a moiety derived from Phe or Ser, and most preferably a moiety derived from Phe. In relation to formulae (II) and (II') above, Dxx represents a single covalent bond or a moiety derived from an amino acid with a hydrophobic side chain, preferably a single covalent bond or a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe and Ala, more preferably a single covalent bond or a moiety derived from Phe or Val; Dyy represents a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, preferably a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, with the proviso that when Dxx is a moiety derived from an amino acid with a hydrophobic side chain, Dyy is a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, and when Dxx is a single covalent bond, Dyy is a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, and Dyy more preferably represents a moiety derived from Arg or Cit. In the above formulas (II) and (II'), Z represents a group covalently bonded to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R) (wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group), preferably -OH. In the above formulas (II) and (II'), * indicates a covalent bond to (T) and ** indicates a covalent bond to (X).

[0081] 3.2 Divalent Group (X) The compounds of formula (I) represent a divalent group (X) comprising 1 to 7, preferably 2 to 6, more preferably 2 to 5, e.g., 2, 3, 4, or 5 skeletal atoms independently selected from C, N, O, and S, and X is covalently bonded to (D) via an atom selected from C, S, N, and O derived from a carboxyl, thiol, amino, or hydroxyl functional group contained in (D). The divalent group (X) remains covalently attached to (D) after cleavage of the linker (L) by CatB, resulting in the release of the modified drug (intra-drug) (e.g., moiety DX) into the target cell. In the present invention, it has surprisingly been found that the modified drug exhibits improved efficacy, e.g., cytotoxic efficacy, compared to the same drug without the group (X) attached thereto. Without wishing to be bound by any theory, it is believed that the spacer group (X) is of suitable size, e.g., 1-7 backbone atoms, to not adversely affect the pharmacokinetic properties of the conjugate even at high DAR (DAR>4), while contributing to the membrane permeability properties of the released drug moiety, e.g., due to a suitable level of hydrophobicity. As a result, upon CatB-induced rapid cleavage, the drug engages its molecular target(s) in the cytoplasm and can also diffuse to proximal cells to induce bystander effects, e.g., cytotoxic bystander effects.

[0082] In one embodiment, (X) represents a divalent carbonyl or thiocarbonyl-containing group, preferably represented by the following formulae (IIIa)-(IIIf):

[0083] [ka] (In the formula, n2 and n3 are each independently selected from 0 to 5, preferably 0, 1 or 2, more preferably 0 or 1; n4, n5 are each selected from 0 or 1; Each A is independently selected from O and S, preferably O; *** represents a covalent bond to (D); **' represents a covalent bond to (L) In one embodiment, (X) is represented by the following formulae (IVa) to (IVj'):

[0084] [ka] TIFF2025512064000028.tif207170 (in the formula, *** represents a covalent bond to (D); **' represents a covalent bond to (L); However, when (X) is represented by formula (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVp), (IVr), (IVt), (IVv), (IVw), (IVx), (IVy) or (IVz), (D) in formula (I) represents an amino-containing drug or a hydroxyl-containing drug, when (X) is represented by formula (IVj), (IVq), (IVs) or (IVu), (D) in formula (I) represents an amino-containing drug or a hydroxyl-containing drug, and when (X) is represented by formula (IVa'), (IVb'), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (IVy') or (IVj'), (D) in formula (I) represents a carboxyl-containing drug). In a preferred embodiment, (X) is represented by formula (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt). Preferably, (X) is represented by formula (IVc) or (IVm).

[0085] It is advantageous to select a structure of X that can be conveniently bonded to the functional group of (D). In one embodiment, when (D) has a carboxyl group, (X) preferably has an amino group that can be bonded to (D) via the formation of an amide bond (linkage); when (D) has an amino group, (X) preferably has a carbonyl (or thiocarbonyl) group that can be bonded to (D) via the formation of an amide bond; when (D) has a thiol group, (X) preferably has a methylene group that can be bonded to (D) via the formation of a thioether bond; when (D) has a hydroxyl group, (X) preferably has a methylene group or a carbonyl (or thiocarbonyl) group that can be bonded to (D) via the formation of an ether or ester (or thioester) bond, respectively. Further adaptations of the structural groups and the resulting bonds are also possible. The following table provides an overview of the available options.

[0086] [Table 1] In the above table, R1 characterizes the remainder of the group (X), while R2 characterizes the remainder of the drug (D).

[0087] 3.3 Solubilized part (S) The compound of formula (I) contains a solubilizing moiety (S), which is a moiety derived from a compound containing one or more, e.g., 2, 3, 4 or 5 solubilizing groups. The presence of the solubilizing moiety reduces (or prevents) the tendency of the conjugated molecule to aggregate, and excellent pharmacokinetic properties, e.g., biodistribution, liver clearance, can be achieved even at high DARs (DAR>4, e.g., DAR=8). In some instances, aggregation of the conjugated molecule can be completely suppressed even at high DARs. The inventors have surprisingly found that cleavage by CatB is possible even in the presence of sterically demanding solubilizing moieties. Preferably, rapid cleavage of the linker by CatB can be achieved, preferably via the exopeptidase mechanism of CatB. Without being bound by any theory, it is believed that the solubilizing moiety is directed outside the CatB binding groove, allowing for excellent selectivity and cleavage rates, e.g., via the exopeptidase mechanism. Moreover, it has surprisingly been found that the solubilizing moiety can compensate for the potential hydrophobicity of the drug moiety DX, such that excellent pharmacokinetic properties can be retained even when multiple drug moieties are attached to the linker (e.g., n>1). In one embodiment, S represents a moiety derived from a compound that contains one or more, e.g., two, three or four, solubilizing groups, each of the solubilizing groups in (S) is independently selected from the following: moieties comprising one or more ionic or ionizable groups, such as ammonium, guanidinium, sulfate or sulfonate groups, preferably derived from Arg, (D)-Arg, Dap, (D)-Dap, Dab, (D)-Dab, Orn, (D)-Orn, Lys, D-Lys or carnitine; a sugar moiety selected from monosaccharides, disaccharides and linear or branched oligosaccharides, particularly linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein the monosaccharide units in the monosaccharides, disaccharides and oligosaccharides are each independently selected from glucose, fructose, mannose, ribose and galactose; and Polyalkylene oxide groups, preferably C 2~3 Polyalkylene oxide groups, more preferably C 2~3 polyalkylene oxide groups each independently containing 6 to 200, preferably 10 to 150, more preferably 12 to 80 repeating units; There is no particular restriction on the general arrangement of the solubilizing groups in the solubilizing moiety.Thus, the solubilizing moiety may be, for example, a linear structure in which several solubilizing groups are arranged randomly or in a block manner, a cyclic structure, or a branched structure in which, for example, several solubilizing groups are attached to a core molecule such as pentaerythritol or glycerol in a graft or dendrimer manner.The solubilizing moiety may also comprise several blocks, each of which independently has a linear or branched structure.

[0088] In one embodiment, the solubilizing moiety comprises one or more solubilizing groups arranged in a linear block manner. For example, the solubilizing moiety may have the following formula: -(So 1 )-(So 2 )-[...]-(So n )(where each So 1 ~So n represents a solubilizing group, e.g., a polyalkylene oxide group, e.g., a PEO group having 6-200 repeat units, or a moiety containing one or more ionic or ionizable groups, e.g., Arg, and n' is an integer from 1 to 20, e.g., 1 to 10, with the proviso that directly attached polyalkylene oxide groups of the same structure are considered to be multiple repeat units of the same solubilizing group (and are not considered to be adjacent So groups). That is, to be treated as separate So groups, adjacent polyalkylene oxide groups must be of different structure and / or be attached via a functional group such as -C(O)-O.

[0089] In other embodiments, the solubilizing moiety comprises one or more solubilizing groups attached to a core molecule, such as pentaerythritol or glycerol, in an unlinked, grafted or dendrimeric manner. For example, the solubilizing moiety may include a -((-Y'-X'(So m’ )) n’ There may be a graft structure represented by -H.

[0090] [ka] wherein X' is an (m'+2)-valent group, e.g., a trivalent or tetravalent group, Y' is a divalent group, each So is independently selected to be a solubilizing group such as a polyalkylene oxide group, e.g., a PEO group having 4-600 repeating units, or a moiety containing one or more ionizable groups, m' is 1, 2, 3 or more, preferably 1 or 2, and n' is an integer from 1 to 20, e.g., 1 to 10; or a tree-like dendrimer structure represented by -X'(So)n' as exemplified in more detail below:

[0091] [ka] (Wherein, X' is an n-valent (branched) group, and each So 1 ~So n are each independently selected to be a solubilizing group as described above, such as a polyalkylene oxide group, e.g., a PEO group having 4-600 repeating units, or a moiety containing one or more ionic groups, and n' is an integer from 1 to 20, e.g., 1 to 10.

[0092] In one embodiment, (S) is a moiety derived from a compound containing one or more polyethylene oxide groups, and preferably the polyethylene oxide groups each independently contain 6 to 200, more preferably 10 to 150, and most preferably 12 to 80 repeating units. In a preferred embodiment, (S) is preferably of the following formula (V):

[0093] [ka] (In the formula, n3 is an integer of 6 to 200, preferably 10 to 150, and more preferably 12 to 80; **** indicates a covalent bond to (T); X 1 is selected from a single covalent bond, -(C=O)-, and -N(R)-, where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; X 2 is an alkyl group having 1 to 6 carbon atoms, an acetyl group, -(CH2) n4 Carbonyl-containing groups such as -CO2H groups, thiocarbonyl-containing groups, -(CH2) n4 OR group, -(CH2) n4 -SO3H group, -(CH2) n4 -(C=A)-N(R)2 groups, -(CH2) n4 represents an amino-containing group such as a -N(R) group, where A is O or S, each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group, and n4 is an integer from 1 to 6; X 2 is -CH3, -CH2CH2OH, or the following formula (VI):

[0094] [ka] (In the formula, Each A is independently selected from O and S, preferably O; Each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; n5 and n6 each independently represent an integer of 1 to 6, preferably 1 or 2; X 2 is most preferably -CH3). When more than one (S) is present, each (S) is preferably a moiety of formula (V) above.

[0095] 3.4 Branching Group (T) T represents a (2+n)-valent, for example, trivalent, tetravalent, pentavalent, hexavalent branching group. The branching group links the vector group (V), the solubilizing moiety (S) and one or more (n) linker moieties (L), thereby forming a branched structure. Preferably, T is a trivalent (n=1) or tetravalent (n=2) branching group. More preferably, T is a trivalent branching group. In one embodiment, the branching group is a group comprising at least one moiety derived from a trifunctional amino acid (A), e.g., Lys. The branching group may comprise, in addition to the above trifunctional amino acids, further (any) linker and / or amino acid, provided that the further linker does not contain a solubilizing group, such as a polyalkylene oxide group, and / or the further amino acid is not a trifunctional amino acid or a moiety comprising one or more ionic or ionizable groups. Such further amino acids may be selected, for example, from homoPhe (hPHe) and Phe. In some aspects, the branching group consists of a moiety derived from a trifunctional amino acid (i.e., does not comprise further linkers and / or amino acids).

[0096] In one embodiment, (T) is represented by the following formula (VII):

[0097] [ka] (In the formula, each AA independently represents a moiety derived from a trifunctional amino acid such as a diamino-carboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably derived from an amino acid selected from N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab and Dap, more preferably derived from Lys(Poc), Glu, Orn or Lys, most preferably derived from Lys; α indicates a covalent bond to (Y); When n=1, the side chain derived from a trifunctional amino acid is covalently attached to either (L) or (S) and the C-terminus is covalently attached to the other moiety (S) or (L), respectively; For n=2, 3, or 4: *' indicates a covalent bond to (L); ****' indicates a covalent bond to (S); and n is as defined in formula (I).

[0098] In a further embodiment, (T) is represented by the following formula (VIII) or (IX):

[0099] [ka] (In the formula, AA1 and AA2 are each independently a moiety derived from a trifunctional amino acid such as a diamino-carboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn or Lys, most preferably a moiety derived from Lys; α indicates a covalent bond to (Y); In formula (IX), the side chain derived from a trifunctional amino acid is covalently attached to either (L) or (S) and the C-terminus is covalently attached to the other moiety (S) or (L), respectively; In formula (VIII), *' represents a covalent bond to (L), and ****' represents a covalent bond to (S).

[0100] 3.5 Divalent Group (Y) The compound of formula (I) contains a divalent group (Y) containing one or more atoms selected from C, N, O, P and S. The divalent group links the vector group (V) to the branching group (T). The divalent group is usually attached to the side chain of an amino acid contained in the vector, such as Cys. Preferably, Y is a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof. More preferably, Y is a divalent group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide derivatives). Most preferably, Y is a divalent group derived from cleaved hydrolyzed maleimide. Hydrolysis of the maleimide bond to V is usually carried out under basic conditions as the final step of the conjugation of maleimide derivatives to V, as per general procedures 1-3 described in the Examples herein. The following conditions are particularly suitable: at the end of the cysteine-maleimide conjugation reaction, the pH is adjusted to pH 8 by adding 10x pH 8 DPPS (0.2-0.5 reaction volumes) and the excess reactive drug linker and reducing agent (TCEP) are removed by gel filtration using a column suitable for gel filtration (PF column, eluted with pH 8 buffer). The eluate is then stirred overnight for 16 h to complete the release before a final buffer exchange with DPBS into an Amicon concentration unit. Thus, in compounds described herein in which a group (R) is linked to a vector (V) via a maleimide (as shown below, on the left), when m is at least 2, hydrolysis may be performed such that compounds of the invention may contain both a ring-closed maleimide bond (A) and a ring-opening hydrolyzed maleimide bond (B) (as shown below, on the left) to V.

[0101] [ka] In a preferred embodiment, in compounds of formula (I) where m is at least 2, at least 50% of the Y bonds to V are ring-opened hydrolyzed maleimide bonds (B) and the remaining bonds are ring-closed maleimide bonds (A). In certain examples, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, preferably at least 98% of the Y bonds to V are cleaved hydrolyzed maleimide bonds (B). The presence of one or more cleaved, hydrolyzable maleimide bonds (even when present together with one or more ring-closed maleimide bonds) may contribute to the stability and therapeutic efficacy of the compounds of the invention. Without being bound by any theory, it is believed that the cleaved, hydrolyzable maleimide bonds prevent, for example, the retro-Michael reaction (illustrated in FIG. 29) that liberates reactive maleimides in the circulation and ultimately transfers the linker-payload to other thiol-containing molecules in the body, such as albumin. The cleaved maleimides may cooperate with the divalent group X and the solubilizing moiety S to improve stability and therapeutic efficacy.

[0102] In a preferred embodiment, Y is a divalent group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), preferably represented by the following formulae (XIIIa) to (XIIIc):

[0103] [ka] (In the formula, R 3 is -(CH2) n7 -(C=A) n9 -or-(CH2CH2O) n8 -(C=A) n9 -, preferably -(CH2) n7 -(C=A) n9 -, where n7 is 1 to 6, preferably 1 or 2, and more preferably 1; n8 is 1 to 6, preferably 1; n9 is 0 or 1, preferably 1, and A is O or S, preferably O; wherein the methylene carbon atom is covalently bonded to the nitrogen atom of formula (XIIIa)-(XIIIc) and the carbonyl or thiocarbonyl-carbon is covalently bonded to T β indicates a covalent bond to V; and α' represents a covalent bond to T. In a more preferred embodiment, Y is represented by formula (XIIIb) or (XIIIc), wherein R 3 is preferably of the formula -(CH2) n7 -(C=A) n9 -, where n7 is 1 or 2, n9 is 1 and A is O. Most preferably, R 3 is -CH2-C=O-.

[0104] 3.6 Part (D) The compound of formula (I) contains a moiety derived from a drug, the drug being selected from a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug. Preferably, the drug is selected from a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug. More preferably, (D) is a moiety derived from a thiol-containing drug, such as DM1 or DM4. When more than one (D) is present in a compound of formula (I) (n>1 and / or m>1), each (D) is independently selected from a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug. Nevertheless, it is preferred that the moieties (D) are identical to each other. The drug may be unmodified (in its natural form, except for the replacement of hydrogen atoms by covalent bonds) or may be chemically modified to incorporate one or more functional groups (e.g., one or more groups selected from hydroxyl, carboxyl, amino and thiol groups) that can be covalently attached to the divalent group (X), and the drug moiety, e.g., the moiety DX or the moiety DX-Dxx-Dyy, is preferably pharmacologically active upon release from the conjugate. In one embodiment, the drug moiety, e.g., the moiety DX or the moiety DX-Dxx-Dyy, that is released from the conjugate is pharmacologically active in the sense that it retains at least 20%, preferably at least 35%, more preferably at least 50%, and even more preferably at least 70% of the pharmacological activity of the corresponding unmodified (natural) drug.

[0105] In certain aspects of the invention, the drug-derived moieties each independently represent, for example, a prodrug group when found in a compound of formula (I), which is not pharmacologically active in the conjugated form, but becomes pharmacologically active upon release from the conjugate. In one embodiment, the moieties derived from the drug are each independently one of the following: (i) Anti-neoplastic agents such as: DNA alkylating agents, such as duocarmycins Topoisomerase inhibitors, e.g. doxorubicin RNA polymerase II inhibitors, e.g. α-amanitin DNA cleaving agents such as calicheamicin Antimitotic or microtubule disrupting agents, such as taxanes, auristatins, or maytansinoids Metabolic antagonists such as gemcitabine derivatives Kinesin spindle protein inhibitors such as Filanesib. Kinase inhibitors such as ipasertib or gefitinib Nicotinamide phosphoribosyltransferase inhibitors Matrix metallopeptidase 9 inhibitors Phosphatase inhibitors such as microcystin-LR (ii) Immunomodulators such as fluticasone (iii) Anti-infective drugs such as rifamycin, clindamycin, or reptamulin, and (iv) radioisotopes, metabolites, pharma- ceutically acceptable salts, and / or prodrugs of any of the foregoing; with the proviso that the drug selected from (i)-(iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, or a hydroxyl-containing drug; When more than one (D) is present, each (D) is independently selected from moieties (i)-(iv), where each drug selected from (i)-(iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, or a hydroxyl-containing drug. , moieties (D) are preferably identical to each other. The following are exemplary drugs that may be used in the ligand drug conjugates of the present invention, provided that the drug is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, or a hydroxyl-containing drug. (i) Anti-neoplastic agents include: (a) alkylating agents, such as nitrogen mustard analogues (e.g., cyclophosphamide, chlorambucil, melphalen, chlormethine, ifosfamide, trofosfamide, prednimustine, bendamustine, chlornaphazine, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitolactol, novemustine, phenesterine, uracil mustard); alkyl sulfonates (e.g., busulfan, treosulfan, mannosulfan, improsulfan and piposulfan; ethylenimines (e.g. thiotepa, triaziquone, carboquone); nitrosoureas (e.g. carmustine, lomustine, semustine, streptozocin, chlorozotocin, fotemustine, nimustine, ranimustine); epoxides (e.g. etoglucide); other alkylating agents (e.g. mitobronitol, pipobroman, temozolomide, dacarbazine); (b) Alkaloids, such as vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine, navelbine, vinflunide, vintafolide); taxanes (e.g., paclitaxel, docetaxel, paclitaxel poliglumex, cabazitaxel) and analogs thereof, maytansinoids (e.g., DM1, DM2, DM3, DM4, maytansine, and ansamitocins) and analogs thereof, cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); epothilones, eleutherobin, discodermolide, bryostatin, dolostatin, autafolide, eu ... Listatins (e.g., monomethylauristatin E, monomethylauristatin F), tubulysin, cephalostatin; panclastatin; sarcodictyin; spongistatin; demecolcine; epipodophyllins (e.g., 9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan and its metabolites, e.g., SN-38, mitoxantrone, novantrone, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS2000)); mitomycins (e.g., mitomycin C); (c) DHFR inhibitors (e.g., methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or other folic acid analogues such as raltitrexed, pemetrexed, pralatrexate); IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine); pyrimidine analogues (e.g., cytarabine, fluorouracil, 5-fluorouracil and its metabolites, tegafur) , carmofur, gemcitabine, capecitabine, azacitidine, decitabine, fluorouracil combinations, tegafur combinations, trifluridine combinations, cytosine arabinoside, ancitabine, floxuridine, doxifluridine), uracil analogues (e.g., 6-azauridine, deoxyuridine); cytosine analogues (e.g., enocitabine); purine analogues (e.g., azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine, cladribine, clofarabine, nelarabine); metabolic antagonists such as folic acid supplements such as folinic acid; (d) endocrine therapy, such as estrogens, progestogens, gonadotropin releasing hormone analogues, antiestrogens, antiandrogens, aromatase inhibitors, etc., used specifically in the treatment of neoplastic diseases; (e)BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, afatinib, vemurafenib, crizotinib, regorafenib, masitinib, dabrafenib, trametinib, ibrutinib, ceritinib, lenvatinib, nintedanib, cediranib, palvocidib, osimer Kinase inhibitors such as tinib, alectinib, alectinib, rociletinib, cobimetinib, midostaurin, olmutinib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, tivozanib, ispinesib, temsirolimus, everolimus, and ridaforolimus; (f) Others, such as duocarmycins (including synthetic analogs: adozelesin, carzelesin, bizelesin, KW-2189, CBI-TMI), benzodiazepine dimers (dimers of pyrrolobenzodiazepines or tomaymycins, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines); platinum-containing compounds (e.g. carboplatin, cisplatin, oxaliplatin, satraplatin, polyplatylene); aziridines, such as benzodopa, meturedopa, uredopa; altretamine, methylmelamines, including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide, and trimethylolmelamine; dynemycin, esperamicin, kedarcidin, maduropeptin, aclacinomycin, actinomycin, autramycin, aza Serine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin; chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; polyketides (e.g., acetogenins); gemcitabine, epoxomicins (e.g., carfilzomib). (ii) Immunomodulators include immunostimulants, immunosuppressants, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquinone, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone, danazol, dexamethasone, prednisone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, hydroxychloroquine, meloxicam, methotrexate, mofetil, mycophenylate, sirolimus, tacrolimus, everolimus, fingolimod, and ibrutinib. (iii) Anti-infective agents include antibacterial agents, antimitotic agents, antimycobacterial agents, and antiviral agents. A non-limiting example of an antibiotic for use in an antibiotic antibody drug conjugate is a rifalog, which is a derivative of rafamycin.

[0106] As used herein, a drug also includes its radioisotope. Examples of radioisotopes (radionuclides) include, for example, 3 H, 11 C. 14 C. 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, 213 Bi or 225 Radioisotope-labeled drugs can be used in targeted imaging experiments or targeted treatments (Wu et al Nat. Biotech. 2005, 23, 1137-1146).

[0107] As used herein, a drug also includes pharma- ceutically acceptable salts, acids or derivatives thereof. In preferred embodiments, the drug-derived moieties are each independently a moiety derived from a drug selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBDs) and dimers thereof, indilinobenzodiazepines (IBDs) and dimers thereof, or radioisotopes and / or pharma- ceutically acceptable salts thereof; preferably, a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1, and DM4; more preferably, a moiety derived from DM1 or DM4.

[0108] 3.7 Compounds of formula (I) containing a linker of formula (II) / (II') The compounds of formula (I) may, in some cases, include a linker of formula (II) or (II') as described above. Thus, the compounds of the present invention have the following general formula (X) or (X'):

[0109] [ka] (In the formula, Axx in formula (X) and formula (X') represents a moiety derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama and homo-Lys, preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys, more preferably a moiety derived from Lys; In formula (X), Ayy is Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr, homoTyr, Tyr(OR1) and homoTyr(OR1) (wherein R1 is -(CH2CH2O) n1-R2 (wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer of 2 to 24), preferably a moiety derived from Phe, homoPhe, Tyr, homoTyr, Tyr(OR1) or homoTyr(OR1), more preferably a moiety derived from Tyr; Ayy in formula (X') represents a moiety derived from an amino acid selected from Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe or Ser, more preferably a moiety derived from Phe or Ser; D, Dxx, Dyy, X, T, S, V, Z, m and n in general formulae (X) and (X') have the same meanings as above; preferably, at least one of D, Dxx, Dyy, X, Y, T, S and Z, for example 2, 3, 4, 5, 6, 7 or 8, is as follows: (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (b) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe, and Ala, preferably a moiety derived from Phe or Val; (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit; (d) X is a group represented by formula (III) (wherein n2 is 1 or 2) or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, preferably a group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), more preferably a group derived from cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) Z is -OH;

[0110] In a preferred embodiment, at least one of D, Dxx, Dyy, X, Y, T, S, V and Z, for example, two, three, four, five, six, seven, eight or nine, are selected from the following: (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (b) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe, and Ala, preferably a moiety derived from Phe or Val; (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit; (d) X is a group represented by formula (III) (wherein n2 is 1 or 2) or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, preferably a group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), more preferably a group derived from cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) V is a moiety derived from naratuximab; (i) Z is defined as -OH; In a further preferred embodiment, in formula (X), Dyy-Dxx-Axx-Ayy are each independently selected from the group consisting of Arg-Lys-Phe (wherein Dyy is a covalent bond), Arg-Lys-homoPhe (wherein Dyy is a covalent bond), Arg-Lys-Tyr (wherein Dyy is a covalent bond), Cit-Lys-Phe (wherein Dyy is a covalent bond), Cit-Lys-Tyr (wherein Dyy is a covalent bond), A wherein Dyy is a covalent bond, Cit-Lys-homoTyr (wherein Dyy is a covalent bond), Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homoTyr, Phe-Lys-Lys-Phe, homoPhe-Arg-Lys-Phe, homoPhe-Cit-Lys-Tyr; and In formula (X'), each Dyy-Dxx-Ayy-Axx is independently selected from Arg-Phe-Lys (wherein Dyy is a covalent bond), Arg-Ser-Lys (wherein Dyy is a covalent bond), Cit-Phe-Lys (wherein Dyy is a covalent bond), Cit-Ser-Lys (wherein Dyy is a covalent bond), Cit-homoPhe-Lys (wherein Dyy is a covalent bond), Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys.

[0111] In one embodiment, the compound of the present invention has the following formula:

[0112] [ka] (In the formula, D, X, Y, T, S, V, Z, m and n have the same meanings as above). In a preferred embodiment, at least one, for example two, three, four, five or six of D, X, Y, T, S and Z are the following: (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (d) X is a group represented by formula (III), wherein n2 is 1 or 2, or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, preferably a group derived from maleimide and its derivatives, for example, a group derived from a cleaved hydrolyzed maleimide derivative, more preferably a group derived from a cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) Z is defined as -OH;

[0113] In a preferred embodiment, at least one, for example two, three, four, five, six or seven of D, X, Y, T, S, V and Z are one of the following: (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (d) X is a group represented by formula (III), wherein n2 is 1 or 2, or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, preferably a group derived from maleimide and its derivatives, for example, a group derived from a cleaved hydrolyzed maleimide derivative, more preferably a group derived from a cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) V is a moiety derived from naratuximab; (i) Z is defined as -OH;

[0114] In one embodiment, the compound of the present invention has the following formula:

[0115] [ka] TIFF2025512064000041.tif217170TIFF2025512064000042.tif217170TIFF2025512064000043.tif212170TIFF2025512064000044.tif186170TIFF2025512064000045.tif243170TIFF2025512064000046.tif228170TIFF2025512064000047.tif187170 (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8). Although, as is evident from formula (I), in the above formula, the vector group (V) should be understood to be outside the brackets, so that the symbol m does not apply to (V), while all other depicted structural elements should be understood to be within the brackets, and therefore occur m times in the molecule. In one embodiment, when the compound solubilizing portion (S) contains a C2 polyoxyalkylene oxide group, the number of oxyethylene repeat units (17) may be substituted with 12 to 22, preferably 15 to 19, oxyethylene groups. In one embodiment, in the above compound, the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide bond. In examples where m is at least 2, the compound may comprise a mixture of (ring-closed) and ring-opened hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the bonds to V are ring-opened hydrolyzed maleimide bonds.

[0116] In one preferred embodiment, the compound (LDC) has the following formula:

[0117] [ka] TIFF2025512064000049.tif238170TIFF2025512064000050.tif64170 (wherein m is an integer of 1 to 12, preferably 2 to 10, and more preferably 4 to 8) As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), while all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule.

[0118] In one embodiment, in the above compound in which the solubilizing moiety (S) comprises a C2 polyoxyalkylene oxide group, the number of oxyethylene repeating units (17 or 24) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups. In one embodiment, in the above compound, the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide bond. In examples where m is at least 2, the compound may comprise a mixture of (ring-closed) and ring-opened hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the bonds to V are ring-opened hydrolyzed maleimide bonds.

[0119] In one preferred embodiment, the compound (LDC) has the following formula:

[0120] [ka] TIFF2025512064000052.tif233170 (in the formula, V is as defined in formula (I); Y is maleimide and its derivatives, for example, cleaved hydrolyzed maleimide derivatives, preferably groups derived from cleaved hydrolyzed maleimide. m is an integer of 1 to 12, preferably 2 to 10, and more preferably 4 to 8). As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), whereas all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule. In one embodiment, in the above compound in which the solubilizing moiety (S) comprises a C2 polyoxyalkylene oxide group, the number of oxyethylene repeating units (17 or 24) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups.

[0121] In a preferred embodiment, in the above compound, Y is represented by the following formulae (XIIIa) to (XIIIc):

[0122] [ka] (In the formula, R 3 is -(CH2) n7 -(C=A) n9 -or-(CH2CH2O) n8 -(C=A) n9 -, preferably -(CH2) n7 -(C=A) n9 - represents n7 is 1 to 6, preferably 1 or 2, and more preferably 1; n8 is 1 to 6, preferably 1; n9 is 0 or 1, preferably 1; A is O or S, preferably O; wherein the methylene carbon atom is covalently bonded to the nitrogen atom of formula (XIIIa)-(XIIIc) and the carbonyl or thiocarbonyl-carbon is covalently bonded to the nitrogen atom of T (to the amino group derived from the Lys residue of T); β indicates a covalent bond to V; α' represents a covalent bond to T (to the amino group derived from the Lys residue of T). In a more preferred embodiment, Y is represented by formula (XIIIb) or (XIIIc), wherein R 3 is preferably of the formula -(CH2) n7 -(C=A) n9 -, where n7 is 1 or 2, n9 is 1 and A is O. Most preferably, R 3 is -CH2-C=O-.

[0123] 3.8 Vector Group (V) The vector group (V) in formulae (I), (X) and (X') represents a moiety derived from the vector group that can interact with a target cell. As used herein, the term "can interact with a target cell" indicates that the vector group can bind to, form a complex with or react with a moiety on the surface of the target cell, such as an antigen or a receptor. Such interaction with the target cell can be experimentally verified by methods known in the art, for example, by providing a compound of formula (I) with a label (such as a fluorescent marker), contacting the compound with a target cell-containing tissue, and detecting the distribution of the fluorescent marker in the tissue (e.g., by fluorescent microscopy). An increase in the fluorescence intensity in the target cell indicates an interaction with the target cell according to the present invention. In some preferred embodiments, the vector group can also cause or contribute to the internalization of the target drug conjugate (i.e., the compound of formula (I)) into the target cell. In one embodiment, (V) represents a moiety derived from a vector group selected from an antibody, an antibody fragment, a protein, a peptide, and a non-peptide molecule. In one embodiment, (V) represents a moiety derived from an antibody or antibody fragment, such as a single chain antibody, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment, a chimeric antibody, a chimeric antibody fragment, a domain antibody or fragment thereof, a cytokine, a hormone, a growth factor, a colony stimulating factor, a neurotransmitter, or a nutrient transport molecule.

[0124] In a preferred embodiment, (V) is one of the following: Monoclonal antibodies, preferably the following: adalimumab, aducanumab, alemtuzumab, altumomab pentetate, amivantamab, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, belantamab mapadotin, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brentuximab vedotin, brodalumab, catumaxomab, cemiplimab, cetuximab, simpanemab, clivatuzumab, crenezumab, tetraxetan, da Crizumab, Daratumumab, Denosumab, Dinutuximab, Dostarlimab, Durvalumab, Edrecolomab, Elotuzumab, Emapalumab, Enfortumab, Enfortumab vedotin, Epcolitab, Epratuzumab, Epratuzumab-SN-38, Etaracizumab, Gemtuzumab, Gemtuzumab ozogamicin, Genmab, Glofitamab, Girentuximab, Goslanemab, Ibritumomab, Inebilizumab infliximab, Inotuzumab, Inotuzumab ozogamicin, Ipilimumab, Isaximab ixekizumab, J591 PSMA antibody, labetuzumab, lecanemab, roncatuximab tesirin, mogamulizumab, mosunetuzumab, necitumumab, nimotuzumab, natalizumab, naratuximab, naxitamab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, polatuzumab vedotin, prasinezumab, ramucirumab, rituximab, sacituzumab, sacituzumab govitecan, semolinemab, siltuximab an antibody selected from the group consisting of tsmab, solanezumab, tacatuzumab, tafasitamab, teprotumumab, tirabonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, zanolimumab, fragments and derivatives thereof; more preferably, atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab; or It represents a portion derived from an antibody incorporated into an Fc fusion protein, preferably selected from belatacept, aflibercept, divaflibercept, dulaglutide, rilonacept, romiplostim, abatacept and alefacept. In a preferred embodiment, (V) represents a moiety derived from an anti-HER2 antibody, an anti-CD37 antibody, an anti-PDL1 antibody or an anti-EGFR antibody, preferably a moiety derived from trastuzumab, pembrolizumab, naratuximab, atezolizumab, durvalumab, avelumab, panitumumab and cetuximab, more preferably a moiety derived from naratuximab, trastuzumab and cetuximab, most preferably a moiety derived from naratuximab and trastuzumab.

[0125] As used herein, the term "naratuximab" (also referred to herein as K7153A) refers to huCD37-3 (version 1.0) as described in WO 2019 / 229677, which is incorporated herein by reference. In other aspects, naratuximab is characterized as monoclonal antibody huCD37-3v1.0 in WO 2011 / 112978, which specifically describes its heavy chain (SEQ ID NO: 90) and light chain (SEQ ID NO: 107). In still further aspects, the antibody comprises the CDRs represented by SEQ ID NOs: 2-7 in Tables 1 and 2, VH of SEQ ID NO: 8 in Table 3, and VL of SEQ ID NO: 10 in Table 4 of WO 2019 / 229677. In one embodiment, naratuximab is selected from the group consisting of: full length light chain of SEQ ID NO: 11 in Table 5 of WO 2019 / 229677; A full-length heavy chain according to SEQ ID NO: 12 in Table 6 of WO 2019 / 229677; A full length light chain according to SEQ ID NO: 11 in Table 5 and a full length heavy chain according to SEQ ID NO: 12 in Table 6 of WO 2019 / 229677; a light chain or light chain variable region having an amino acid sequence identical to the amino acid sequence encoded by recombinant plasmid DNA phuCD37-3LC (ATCC deposit no. PTA-10722, deposited with the ATCC on March 18, 2010); a heavy chain or heavy chain variable region comprising the same amino acid sequence as the amino acid sequence encoded by recombinant plasmid DNA phuCD37-3HCv.1.0 (ATCC deposit no. PTA-10723, deposited with the ATCC on March 18, 2010); a light chain or light chain variable region comprising the same amino acid sequence as the amino acid sequence encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722) and a heavy chain or heavy chain variable region comprising the same amino acid sequence as the amino acid sequence encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (PTA-10723); (i) a VL-CDR that comprises the same amino acid sequence as the VL-CDR encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722), and (ii) a VH-CDR that comprises the same amino acid sequence as the VH-CDR encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (PTA-10723). In one embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or an anti-HER2 antibody, preferably trastuzumab.

[0126] In other embodiments, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or a moiety derived from an anti-HER2 antibody, preferably trastuzumab, and (D) represents a moiety derived from an anti-neoplastic agent, preferably a drug selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBDs) and dimers thereof, indilinobenzodiazepines (IBDs) and dimers thereof, or radioisotopes and / or pharma- ceutically acceptable salts thereof. In a preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or a moiety derived from an anti-HER2 antibody, preferably trastuzumab, and (D) represents a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4. More preferably, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or a moiety derived from an anti-HER2 antibody, preferably trastuzumab, and (D) represents a moiety derived from DM1 or DM4.

[0127] In another preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or an anti-HER2 antibody, preferably trastuzumab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0128] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide). is represented by one of As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), whereas all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule.

[0129] In one embodiment, in the above compound, the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide bond. In examples where m is at least 2, the compound may comprise a mixture of (ring-closed) and ring-opened hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the bonds to V are ring-opened hydrolyzed maleimide bonds.

[0130] In yet another preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or an anti-HER2 antibody, preferably trastuzumab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0131] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; Y is represented by any of formulae (XIIIa) to (XIIIc), preferably formula (XIIIb) or (XIIIc); and / or the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups). In a more preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or an anti-HER2 antibody, preferably trastuzumab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0132] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide). It is represented by: In an even more preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably naratuximab, or an anti-HER2 antibody, preferably trastuzumab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0133] [ka] (In the formula, m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; Y is represented by any one of formulas (XIIIa) to (XIIIc), preferably formula (XIIIb) or (XIIIc); and / or the number of oxyethylene repeating units (17) may be 12 to 30, preferably 14 to 25, more preferably 15 to 19, oxyethylene groups.) It is represented by: As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), whereas all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule. In one embodiment, in the above compound, the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide bond. In examples where m is at least 2, the compound may comprise a mixture of (ring-closed) and ring-opened hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the bonds to V are ring-opened hydrolyzed maleimide bonds. In other preferred embodiments, (V) represents a moiety derived from a peptide capable of interacting with a target of interest. Non-limiting examples of peptides include somatostatin or analogs thereof (e.g., octreotide, Angiopep-2, gastrin releasing peptide, transferrin derived peptides, derivatives of neuropeptide Y, RGD peptides, α-melanocyte stimulating hormone peptide analogs, vasoactive intestinal peptide, neurotensin, and luteinizing hormone releasing hormone (LHRH) analogs). In yet other preferred embodiments, (V) represents a moiety derived from a non-peptide molecule, such as folic acid, hyaluronic acid, neurotensin receptor 1 (NRT1) antagonists (such as SR 142948A derivatives), and ligands of prostate specific membrane antigen (PSMA) (such as PSMA-617 and PSMA-11).

[0134] In one embodiment, the target cell is selected from a tumor cell, a virus-infected cell, a microorganism-infected cell, a parasite-infected cell, a cell involved in an autoimmune disease, an activated cell, a myeloid cell, a lymphocytic cell, a melanocyte, and an infectious agent including bacteria, viruses, mycobacteria, and fungi. In a preferred embodiment, the target cell is any tumor cell from a solid or liquid tumor, including, but not limited to, lymphoma cells, myeloma cells, bone marrow cells, lymphoid cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, testicular cancer cells, skin cancer cells, pancreatic cancer cells, liver cancer cells, and any cell whose unregulated and rapidly growing and dividing causes cancer.

[0135] 4. Pharmaceutical Compositions The compounds of the invention may be provided in the form of pharmaceutical compositions for human or animal use in human and veterinary medicine, which typically comprise a therapeutically effective amount of an LDC according to the invention or a pharma- ceutically acceptable salt thereof, together with one or more components selected from carriers, diluents and other excipients. In one embodiment, the pharmaceutical composition comprises a mixture of LDCs according to the invention. In a more specific embodiment, the pharmaceutical composition comprises a mixture of LDCs according to the invention, said LDCs (compounds of formula I) comprising (closed) maleimide and / or open hydrolyzable maleimide bonds to V (as described above). The ratio of the (closed) maleimide derivative (A) bound to V to the open hydrolyzed maleimide derivative (B) (with respect to the total maleimide bonds in the composition) is A:B / 0-50:50-100%, preferably A:B / 10-40:60-90%, more preferably A:B / 15-35:65-85%, and most preferably about A:B / 30:70%. The respective proportions of closed maleimide derivatives and open hydrolyzed maleimide derivatives (and thereby closed (A) and open (B) maleimide bonds) in the composition may be determined by MS techniques such as Tof or Orbitrap analysis of reduced LDC (compound of formula (I)). Detailed examples of protocols are available in chapter 6d of Chem. Eur. J. 2019, 25, 8208-8213.

[0136] Suitable carriers, diluents and other excipients for use in pharmaceutical compositions are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (Gennaro AR, 1985). Carriers, diluents and / or other excipients may be selected with regard to the intended route of administration and pharmaceutical practice. The pharmaceutical composition may include a diluent and / or other excipient as a carrier, or may further include any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent. The therapeutically effective amount can be routinely determined by a physician. The specific dose level and dosing frequency for any particular subject / patient can vary and depend on a variety of factors, including the activity of the specific drug compound used, the metabolic stability and duration of action of the compound, the patient's age, weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of the particular condition, and the individual being treated. These factors are taken into account by a physician when determining a therapeutically effective dose.

[0137] 5. Use of LDC or compositions thereof in methods for preventing or treating disease The compounds of the present invention can be used to treat diseases. Treatment can be therapeutic and / or prophylactic treatment aimed at preventing, reducing or stopping undesirable physiological changes or disorders. In some embodiments, treatment can prolong the survival of a subject compared to expected survival in the absence of treatment. The disease treated by LDC can be any disease that benefits from treatment, including chronic and acute disorders or diseases, as well as pathological conditions that predispose to disorders. In some embodiments, the disease is a neoplastic disease, such as cancer, that can be treated through targeted destruction of tumor cells. Non-limiting examples of cancers that can be treated include benign and malignant tumors (either solid or liquid); lymphoid malignancies, such as non-Hodgkin's lymphoma (NHL), such as diffuse large B-cell lymphoma (DLBCL), as well as breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer, and bone and bone marrow cancers, such as acute myeloid leukemia (AML). The disease can be a disease of neurons, glia, astrocytes, hypothalamus or other glands, macrophages, epithelium, stroma and blastocoel; or an inflammatory, angiogenic or immunological disease. Exemplary diseases are solid malignancies, and other exemplary diseases are liquid malignancies. In one embodiment, the compounds of the invention or compositions thereof are used in a method for treating or preventing cancer, an autoimmune or inflammatory disease, and / or an infectious disease, e.g., by administering a therapeutically effective amount of a compound of the invention or a composition thereof to a patient in need thereof. Non-limiting examples of autoimmune, inflammatory, and / or infectious diseases include rheumatoid arthritis, multiple sclerosis, type 1 diabetes, idiopathic inflammatory myopathy, systemic lupus erythematosus (SLE), myasthenia gravis, Graves' disease, dermatomyositis, polymyositis, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's disease, asthma, psoriasis, psoriatic arthritis, dermatitis, generalized edema and sclerosis, inflammatory bowel disease (IBD), respiratory distress syndrome, meningitis, encephalitis, uveitis, itching, and bronchitis. Glomerulonephritis, eczema, atherosclerosis, leukocyte adhesion deficiency, Raynaud's syndrome, Schogen's syndrome, Reiter's disease, Behçet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, atopic dermatitis, pemphigus vulgaris, blepharoclonus-myoclonus syndrome, pure red cell aplasia, mixed clinoplasty, Cryoglobulinemia, ankylosing spondylitis, Hepatitis C-associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranoproliferative glomerulonephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, neuromyelitis optica, Graves' disease, bullous pemphigoid, membranoproliferative glomerulonephritis, Churg-Strauss syndrome, juvenile-onset diabetes mellitus, hemolytic anemia, atopic dermatitis, systemic sclerosis, Schogen's syndrome and thread. Glomerulonephritis, dermatomyositis, ANCA, aplastic anemia, autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, Castleman syndrome, Goodpasture syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), autoimmune hepatitis, lymphocytic interstitial pneumonia, bronchiolitis obliterans (non-transplant), Guillain-Barre syndrome, large vessel vasculitis, giant cell Takayasu arteritis, medium vessel vasculitis, Kawasaki disease, polyarteritis nodosa. Wegener's granulomatosis, microscopic polyangiitis (MPA), Omenn's syndrome, chronic renal failure, acute infectious mononucleosis, HIV, hepatitis, herpes, and bacterial infections. For further information, see WO2012 / 135740, for example, paragraphs

[0004] and

[0039] .

[0138] In one embodiment, the compound of the invention or a composition thereof is used in a method for treating or preventing cancer of the bone and bone marrow, preferably acute myeloid leukemia (AML).Preferably, the compound is a compound of the invention, wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab. In certain other embodiments, the compound of the invention or a composition thereof is used in a method for treating or preventing lymphoma, preferably NHL, more preferably DLBCL. Preferably, said compound is a compound of the invention, wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab. In a more specific embodiment, the compound of the invention or composition thereof is a compound of the invention, wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) is a moiety derived from an anti-neoplastic agent, preferably selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBD) and dimers thereof, indilinobenzodiazepines (IBD) and dimers thereof, or radioisotopes and / or pharma- ceutically acceptable salts thereof. In an even more specific embodiment, the compound of the invention or a composition thereof is used in a method for treating or preventing osteosarcoma and bone marrow cancer, preferably acute myeloid leukemia (AML), said compound being a compound of the invention, wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4; more preferably, a moiety derived from DM1 or DM4.

[0139] In a preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing cancer of the bone marrow, preferably acute myeloid leukemia (AML), wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0140] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or the maleimide bond to (V) may be substituted by a cleaved hydrolyzed maleimide).

[0141] Preferably, the compound has the following formula:

[0142] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or the maleimide bond to (V) may be substituted by a cleaved hydrolyzed maleimide). As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), whereas all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule. In a more preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing osteosarcoma and bone marrow cancer, preferably acute myeloid leukemia (AML), wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0143] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8, and the number of oxyethylene repeating units (17) may be 12 to 30, preferably 14 to 25, more preferably 15 to 19, oxyethylene groups.) As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), whereas all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule. In one embodiment, in the above compound, the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide bond. In examples where m is at least 2, the compound may comprise a mixture of (ring-closed) and ring-opened hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the bonds to V are ring-opened hydrolyzed maleimide bonds.

[0144] In a preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing cancer of the bone marrow, preferably acute myeloid leukemia (AML), wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0145] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; Y is represented by any of formulae (XIIIa) to (XIIIc), preferably formula (XIIIb) or (XIIIc); and / or the number of oxyethylene repeating units (17) may be 12 to 30, preferably 14 to 25, more preferably 15 to 19, oxyethylene groups.)

[0146] In a more preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing osteosarcoma and bone marrow cancer, preferably acute myeloid leukemia (AML), wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0147] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; Y is represented by any of formulae (XIIIa) to (XIIIc), preferably formula (XIIIb) or (XIIIc); and / or the number of oxyethylene repeating units (17) may be 12 to 30, preferably 14 to 25, more preferably 15 to 19, oxyethylene groups.) As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), whereas all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule.

[0148] The compounds / molecules of the present invention may be administered to a subject (e.g., a patient) in any therapeutically effective dose. The therapeutically effective amount may depend on the characteristics of the patient in question, such as size and weight. The molecules may be administered to a subject (e.g., a patient) over one or more courses of treatment. Depending on the type and severity of the disease, between about 0.1 μg / kg and 1 mg / kg of drug may be used as an initial candidate dosage for initial administration in first-in-human trials, e.g., by single or multiple separate administrations or by continuous infusion. Typical once-daily, once-weekly (QW), once-every-two-weeks (Q2W), once-every-three-weeks (Q3W) or once-monthly doses may range from about 0.1 mg / kg to 50 mg / kg or more, or from about 0.5 to about 25 mg / kg of patient body weight. The subject to whom the compound / molecule is administered may be a patient in need thereof, i.e., a patient in need of treatment, e.g., a patient suffering from a disease such as cancer, e.g., AML, an autoimmune disease or an infectious disease. When treating cancer, the therapeutic effect observed may be a reduction in the number of cancer cells; a reduction in tumor size; inhibition or delay of cancer cell invasion into peripheral organs; inhibition of tumor growth; and / or alleviation of one or more symptoms associated with the cancer. Routes of administration (delivery) include one or more of the following: oral (e.g., tablets, capsules, ingestible solutions), topical, mucosal (e.g., nasal sprays, aerosols for inhalation), nasal, parenteral (e.g., injectable forms), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intraventricular, intracerebral, subcutaneous, ocular (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual, etc. In a preferred embodiment, the compounds of the invention are administered by injection, such as parenteral, intravenous, subcutaneous, intramuscular, transdermal, etc. In a further embodiment, the compounds of the invention are used in a method for treating or preventing cancer, an autoimmune or inflammatory disease and / or an infectious disease and are administered simultaneously with one or more other therapeutic agents, such as chemotherapeutic agents, radiotherapy agents, immunotherapy agents, autoimmune disorder agents, anti-infective agents, or one or more other compounds of formula (I). The other therapeutic agents may be administered before or after the compounds of the invention.

[0149] In a more specific embodiment, the compounds of the invention are used in a method for treating or preventing cancer, preferably lymphoma or blood and bone marrow cancer, wherein said compound of the invention is administered simultaneously with an anti-CD20 antibody, preferably rituximab, wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) represents a moiety derived from DM1, said compound having the following formula:

[0150] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; the maleimide bond to V may be substituted with a cleaved hydrolyzed maleimide bond, and preferably, the lymphoma is DLBCL, and the bone and bone marrow cancer is AML).

[0151] In a preferred embodiment, the compound of the invention is used in a method for treating or preventing cancer, preferably lymphoma or blood and bone marrow cancer, wherein said compound of the invention is administered simultaneously with an anti-CD20 antibody, preferably rituximab, wherein (V) represents a moiety derived from an anti-Cd37 antibody, preferably naratuximab, and (D) represents a moiety derived from DM1, the compound having the following formula:

[0152] [ka] (wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8; Y is represented by any of formulae (XIIIa) to (XIIIc), preferably formula (XIIIb) or (XIIIc); and / or the number of oxyethylene repeating units (17) may be substituted with 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; preferably, the lymphoma is DLBCL, and the bone and bone marrow cancer is AML). The compound of the present invention is represented by the following formula: As is evident from formula (I), in the above compounds, the vector group (V) is to be understood as being outside the brackets, so that the symbol m does not apply to (V), whereas all other depicted structural elements are to be understood as being within the brackets, and therefore occurring m times in the molecule.

[0153] As used herein, the term "anti-CD20 antibody" or "antibody that binds to CD20" refers to an antibody that can bind to CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeted to CD20. The extent to which an anti-CD20 antibody binds to unrelated, non-CD20 proteins may be less than about 10% of the antibody's binding to CD20 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that binds to CD20 is ≦1 μM, ≦1100 nM, ≦110 nM, ≦11 nM, or ≦1.1 nM. Similarly, the terms "anti-CD37 antibody," "anti-HER2 antibody," "anti-PDL1 antibody," and "anti-EGFR antibody" (or "antibody that binds CD37," "antibody that binds HER2," "antibody that binds PDL1," and "antibody that binds EGFR") as used herein refer to an antibody that can bind to CD37, HER2, PDL1, or EGFR, respectively, with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting the antigens CD37, HER2, PDL1, or EGFR, respectively. The extent of binding of an anti-CD37, anti-HER2, anti-PDL1, or anti-EGFR antibody, respectively, to an unrelated non-CD37, non-HER2, non-PDL1, or non-EGFR protein may be less than about 10% of the binding of the antibody to CD37, HER2, PDL1, or EGFR, e.g., as measured by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that binds to any of CD37, HER2, PDL1, and EGFR is ≦1 μM, ≦1100 nM, ≦110 nM, ≦11 nM, or ≦1.1 nM. In one embodiment, in the above compound, the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide bond. In examples where m is at least 2, the compound may comprise a mixture of (ring-closed) and ring-opened hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the bonds to V are ring-opened hydrolyzed maleimide bonds. Rituximab may be administered before, after, or simultaneously with the compounds of this invention.

[0154] 6. Compounds of formula (XI), kits for the modification of molecules capable of interacting with target cells, and methods for the modification of molecules capable of interacting with target cells In one aspect, the present invention relates to a compound that can be used to modify vector molecules, such as antibodies, that can interact with the above-mentioned target cells. That is, the present invention relates to a compound having the following general formula (XI):

[0155] [ka] (wherein D, X, L, T, S and n have the same meanings as defined above for compounds of formula (I); and Y' represents a moiety that contains a conjugate group capable of forming a covalent bond with a molecule capable of interacting with a target cell (V'), such as a monoclonal antibody or antibody fragment optionally incorporated into an Fc fusion protein. The present invention relates to a compound represented by the formula:

[0156] In one embodiment, Y′ in formula (XI) is: Preferably, an optionally substituted maleimide capable of reacting with one or two thiol groups of (V'), said thiol groups of (V') each independently being optionally substituted or protected, for example with a monomethoxytrityl group, Preferably, an optionally substituted haloacetamide capable of reacting with the thiol group of (V') An ester preferably reactive with the side chain of an amino acid of (V') (e.g., Lys), such as an acyl halide, an N-hydroxysuccinimide ester (structure shown below, left hand side, σ indicates a covalent bond to the remainder of the compound of formula (XI)) or a phenol ester (structure shown below, right hand side, σ indicates a covalent bond to the remainder of the compound of formula (XI)), where each R is individually selected from H, F, NO2 and CN.

[0157] [ka] a carbonate, preferably a carbonate capable of reacting with the side chain of an amino acid of (V') (e.g. Lys), such as a haloformate, or a carbonate containing a leaving group, such as N-hydroxysuccinimide or a phenol derivative; Preferably, an isocyanate or isothiocyanate capable of reacting with the side chain of an amino acid of (V') (e.g., Lys), an azide capable of reacting with an alkyne group preferably contained in molecule (V'); an alkyne, preferably an alkyne capable of reacting with the azide group contained in molecule (V'); and an amino group, e.g., a primary or secondary amino group, preferably capable of reacting with a molecule (V'), e.g., an antibody, in the presence of an enzyme such as transglutaminase; is a moiety that includes a linking group selected from In a preferred embodiment, the compound has the following general formula (XII) or (XII'):

[0158] [ka] (wherein Axx has the same meaning as Axx in the above formulae (X) and (X'); Ayy has the same meaning as Ayy in the above formula (X); Ayy has the same meaning as in the above formula (X'); and Y' has the same meaning as above with respect to formula (XI); D, Dxx, Dyy, X, T, S, Z and n have the same meanings as described above for the Ligand Drug Conjugate. There is a structure represented by: In a preferred embodiment, at least one, for example two, three, four, five, six or seven of D, Dxx, Dyy, X, T, S and Z in formulae (XII) and (XII') are as follows: (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (b) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe and Ala, preferably Phe or Val; (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably selected from an amino acid selected from Arg or Cit; (d) X is a group represented by formula (III) (wherein n2 is 1 or 2) or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) T is a group of formula (VII), (VIII) or (IX); (f) S is a moiety of formula (V); (g) Z is defined as -OH; In a further preferred embodiment, Dyy-Dxx-Axx-Ayy in formula (XII) are each independently Arg-Lys-Phe (wherein Dyy is a covalent bond), Arg-Lys-homoPhe (wherein Dyy is a covalent bond), Arg-Lys-Tyr (wherein Dyy is a covalent bond), Cit-Lys-Phe (wherein Dyy is a covalent bond), Cit-Lys-Tyr (wherein Dyy is a covalent bond), Arg-Lys-homoTyr (wherein Dyy is a covalent bond), Cit-Lys-homoTyr (wherein Dyy is a covalent bond), Phe-Cit-Phe-Lys, homoPhe- each Dyy-Dxx-Axx-Ayy in formula (XII') is independently selected from Arg-Phe-Lys (wherein Dyy is a covalent bond), Arg-Ser-Lys (wherein Dyy is a covalent bond), Cit-Phe-Lys (wherein Dyy is a covalent bond), Cit-Ser-Lys (wherein Dyy is a covalent bond), Cit-homoPhe-Lys (wherein Dyy is a covalent bond), Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys.

[0159] In yet another preferred embodiment, the compound has the following formula:

[0160] [ka] (In the formula, D, X, T, S, Y', Z and n have the same meanings as above.) There is a structure represented by one of: In a preferred embodiment, at least one, such as two, three, four or five of D, X, T, S and Z in the above formula are as follows: (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (d) X is a group represented by formula (III), wherein n2 is 1 or 2, or a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) T is a group of formula (VII), (VIII) or (IX); (f) S is a moiety of formula (V); (g) Z is defined as -OH;

[0161] In one embodiment, the compound has the following formula:

[0162] [ka] TIFF2025512064000070.tif249170TIFF2025512064000071.tif249170TIFF2025512064000072.tif213170TIFF2025512064000073.tif254170TIFF2025512064000074.tif254170TIFF2025512064000075.tif228170TIFF2025512064000076.tif115170

[0163] In one aspect, the present invention relates to a kit comprising a compound as herein above (i.e., a compound according to formula (XI), (XII) or (XII')) and a buffer, which can be used for the engineering of a vector molecule capable of interacting with a target cell, e.g., an antibody or a fragment thereof, said antibody fragment optionally being incorporated into an Fc fusion protein, particularly for the engineering of therapeutic antibodies. The compounds and buffers (together forming a kit) can be presented individually, for example, in separate primary containers (which can be shipped to the customer in a single box), and can be stored for long periods of time without degradation. The compounds and buffers can be formulated and combined for a given amount of antibody or fragment thereof to be modified. In some embodiments, the compounds of the present invention are presented as solids (e.g., as lyophilized powders, or non-covalently adsorbed or covalently attached to a solid phase matrix as further described below), or as solutions in a suitable solvent, such as a water-miscible polar aprotic solvent (e.g., DMF, DMSO), which can be mixed with the buffer immediately prior to antibody or antibody fragment modification. The buffer solution used in the kit of the present invention is not particularly limited. The pH of the buffer solution is preferably 6.0 to 10, more preferably 6.5 to 8.0. The buffer solution may be selected from, for example, 2-bis(2-hydroxyethyl)aminoacetic acid (Bicine), carbonate-bicarbonate, tris(hydroxylmethyl)methylaminopropanesulfonic acid (TAPS), and 4-(2-hydroxylethyl)-1-piperazineethanesulfonic acid (HEPES).

[0164] In one embodiment, the compounds of the present invention can be used in a method for the modification of vector molecules, such as antibodies, capable of interacting with target cells. Thus, the method produces a ligand drug conjugate of formula (I) above. In one embodiment, the method includes a step of reacting (contacting) a molecule capable of interacting with a target cell, for example, a monoclonal antibody or antibody fragment, optionally incorporated into an Fc fusion protein. The reaction mixture may be purified by techniques known in the art, such as dialysis techniques or gel permeation chromatography, using a suitable solvent. Examples of suitable stationary phases for isolating the pure conjugate include polyacrylamide gels (e.g., Bio-Gel® P-30) and cross-linked dextran (e.g., Sorbadex®, Zetadex® or Sephadex®). The method may be applied to any molecule capable of interacting with a target cell. Preferably, the method is applied to a molecule selected from antibodies, antibody fragments, proteins, peptides and non-peptide molecules.In one embodiment, the method comprises the step of administering to the patient a monoclonal antibody (mAb) or an antibody fragment incorporated into an Fc fusion protein, preferably an antibody selected from the group consisting of adalimumab, aducanumab, alemtuzumab, altumomab pentetate, amivantamab, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, belantamab mafadotin, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brentuximab vedotin, brodalumab, catumaxomab, cemiplimab, cetuximab, simpanemab, clivatuzuma ... , crenezumab, tetraxetan, daclizumab, daratumumab, denosumab, dinutuximab, dostarlimab, durvalumab, edrecolomab, elotuzumab, emapalumab, enfortumab, enfortumab vedotin, epcolitab, epratuzumab, epratuzumab-SN-38, etaracizumab, gemtuzumab, gemtuzumab ozogamicin, gemmab, glofitamab, girentuximab, goslanemab, ibritumomab, inebilizumab infliximab, inotuzumab, inotuzumab ozogamicin, ipilimumab, isaximab ixekizumab, J591 PSMA antibody, labetuzumab, lecanemab, roncatuximab tesirin, mogamulizumab, mosunetuzumab, necitumumab, nimotuzumab, natalizumab, naratuximab, naxitamab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, polatuzumab vedotin, prasinezumab, ramucirumab, rituximab, sacituzumab, sacituzumab The present invention relates to an antibody selected from the group consisting of tuzumab govitecan, semolinemab, siltuximab, solanezumab, tacatuzumab, tafasitamab, teprotumumab, tirabonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, zanolimumab, fragments and derivatives thereof.

[0165] In one embodiment, the method is applied to moieties derived from anti-HER2, anti-CD37, anti-PDL1 or anti-EGFR antibodies, preferably trastuzumab, pembrolizumab, naratuximab, atezolizumab, durvalumab, avelumab, panitumumab and cetuximab, more preferably naratuximab, trastuzumab and cetuximab, most preferably naratuximab and trastuzumab.

[0166] 7. Preparation of Compounds of the Invention The following provides methods for the preparation of linkers, drug linkers and ligand drug conjugates. The compounds of the present invention can be synthesized by relying on standard organic chemistry reactions or Fmoc-based solid phase peptide synthesis (SPPS), including peptide coupling and convergent strategies in solution and on resin. The introduction of various maleimide derivatives and their subsequent chemoselective linking to moieties derived from vector groups are also illustrated below. The general strategies and methodologies that can be used to prepare the compounds of the present invention are well known to those skilled in the art. EXAMPLES

[0167] 8. Working Example 8.1 List of Abbreviations Used in the Examples Ac: Acetyl ADC: Antibody-drug conjugate ACN: Acetonitrile AMAS: N-α-maleimidoacet-oxysuccinimide ester AML: Acute myeloid leukemia Arg: arginine Bn: Benzyl Boc: tert-butyloxycarbonyl Bu: butyl Cit: Citrulline DAR: Drug-to-Antibody Ratio DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene DCM: dichloromethane DIEA: Diisopropylethylamine DLBCL: Diffuse large B-cell lymphoma DM1: Mertansine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide DOC: Degree of conjugation DPBS: Dulbecco's phosphate buffered saline EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Et: Ethyl eq.: equivalent FA: Formic acid Fmoc: 9-fluorenylmethoxycarbonyl g: grams Gly: glycine Glu: glutamic acid h: time HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide HPLC: High-performance liquid chromatography HRMS: High resolution mass spectrometry IR: Infrared L: Liters Lys: Lysine m: millimeter ma: Maleimidoacetic acid mAb: Monoclonal antibody Me: Methyl min:minutes Mtt: 4-methyltrityl mole: mole MS: Mass spectrometry m / z: specific mass / charge NHS: N-hydroxysuccinimide Nmab: Naratuximab Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PBS: Phosphate-buffered saline PEG: Polyethylene glycol Peg16 or Peg16: CO-CH2CH2O-(CH2CH2O) 16 -CH3 PF: CentriPure PF filtration column PFP: Pentafluorophenyl pH: Potential relative to hydrogen Phe: Phenylalanine quant.: quantitative rt: room temperature Rt: retention time SMCC: succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Suc: succinic acid TCEP: Tris(2-carboxyethyl)phosphine TFA: Trifluoroacetic acid Tmab: trastuzumab Tyr: Tyrosine UPLC: Ultra-high performance liquid chromatography UV: Ultraviolet light μ: Micro 8.2 Starting Materials and Chemicals: The main starting materials and chemicals used in the following examples are listed below. >Synthesis solvents and deprotection reagents were obtained from Merck or Fischer Scientific AG (Switzerland). > TFA, DIEA, N-hydroxysuccinimide, DBU, DPBS, 10xDPBS and H-Glu(OtBu)-OBn (Sigma-Aldrich, Switzerland); > Fmoc-Cit-OPFP, H-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, H-Tyr(OtBu)-OtBu, Fmoc-Lys(Mtt)-OH, Fmoc-Cit-OH, Fmoc-Phe-OH and Boc-Cit-OH, manufactured in Bachem (Switzerland); > AMAS is manufactured by Astatech Inc (USA). > Solvents and chemicals for high performance liquid chromatography (HPLC) and ultra performance liquid chromatography mass spectrometry (UPLC-MS) were from Biosolve (France). > PEG 24-amine from BroadPharm (USA); > HATU made by Combi-Blocks (USA); > Exatecan mesylate, Angel Pharmatech (China) > LiOH·H2O, manufactured by VWR (Switzerland); > DM 1-SMCC from Enovation Chemicals (USA); > Boc-Gly-OH and dihydrofuran-2,5-dione from Fluka (USA); > EDC·HCl, manufactured by Apollo Chemical (USA); > DM1 is manufactured by Angel Pharmatech, Ltd. (USA). > TCEP·HCl, Boc-Lys-OH, manufactured by Fluorochem (UK); > BrCH2COOH, H-Tyr-OMe and Pd / C (from Acros Organics, Belgium); > H-Cit-Lys(PEG5-ma)-Tyr-OH, manufactured by Ambiopharm (USA). > Herceptin® (trastuzumab) manufactured by Roche (Switzerland); > Naratuximab - the antibody huCD37-3 (version 1.0) described in WO 2019 / 229677; > mPEG16-NHS ester, PurePEG (mPEG16-OCH2CH2COO-NHS ester, m=methyl), from LLC (USA); > N-Succinimidyl-4-(maleimidomethyl)-cyclohexanecarboxylate (SMCC) from Sigma Aldrich Fine Chemical.

[0168] 8.3 Method: The following methods were used to evaluate the compounds and conjugates of the invention. 8.3.1 Purity determination The purity of the compounds was determined by a UPLC-MS system. Method 1: Waters Acquity UPLC system coupled to a Waters SQD mass spectrometer equipped with a CSHC18 column (130 Angstroms, 1.7 μm, 2.1 mm×50 mm) heated at 40° C. using solvent systems A (water+0.1% FA) and B (ACN+0.1% FA) with a flow rate of 0.9 mL / min and a gradient of 5-100% of B over 2.7 min. Method 2: Waters Acquity UPLC system coupled to a Waters SQD mass spectrometer equipped with a CSH fluoro-phenyl column (130 Angstroms, 1.7 μm, 2.1 mm×50 mm) heated at 40° C. using solvent systems A (water+0.1% FA) and B (ACN+0.1% FA) with a flow rate of 0.9 mL / min and a gradient of 5-100% of B over 2.9 min. Method 3: Waters Acquity UPLC system coupled to a Waters SQD mass spectrometer, a BEHC18 1.7 μm 50×2.1 mm column (available from Waters) heated to 40° C. and fitted with a 2 μm insert filter precolumn, and solvent systems A1 (water+0.1% FA) and B1 (ACN+0.1% FA) at a flow rate of 0.9 mL / min and a 5-100% gradient of B1 over 2.9 min. Method 4: Waters Acquity UPLC system coupled to a Waters SQD mass spectrometer equipped with a CSHC18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm) using solvent systems A (water + 0.1% FA) and B (ACN + 0.1% FA) with a flow rate of 0.6 mL / min and a gradient of 5-85% of B over 5 min heated at 40 °C. Method 5: Instrument: Shimadzu LCMS2020 mass spectrometer. Column: HALOC18 2.7 μm, 3.0 mm x 30 mm. Mobile phase: ACN (0.05% TFA)-water (0.05% TFA). Gradient: 5% to 95% MeCN over 1.4 min, hold for 0.6 min, total run time was 2.5 min, flow rate: 1.8 mL / min. Column temperature: 50° C. Wavelength: 214 and 254 nm PDA. Method 6: Instrument: Shimadzu LCMS2020 mass spectrometer. Column: Kinetex C18 2.6 μm, 4.6 mm x 50 mm; Mobile phase: ACN (0.05% TFA)-water (0.05% TFA); Gradient: 5% to 95% MeCN over 1.8 min, hold for 0.7 min, total run time was 3.0 min, flow rate: 1.8 mL / min. Column temperature: 50° C. Wavelength: 214 and 254 nm PDA.

[0169] 8.3.2 Aggregates: Size Exclusion Chromatography (SEC) The aggregate content of the conjugates was determined using the following method. Apparatus 1 UPLC Waters Acquity Apparatus 2 UPLC Waters Acquity H-Class plus Bio Detector: Tunable UV detector (TUV) Detector cell Titanium Precolumn Agilent Advance Bio SEC 300 Angstrom 2.7 μm 4.6*50 mm Column: Agilent Advance Bio SEC 300 Angstrom 2.7 μm 4.6*150 mm Mobile phase Potassium phosphate 50 pH 6.8 / 250mM KCl Wavelength 280nm Injection volume 10μL Column temperature Ambient Sample manager temperature 25℃ Runtime 10 minutes Flow 0.35mL / min Isocratic Mode Weak wash H2O / ACN(90 / 10v / v) Powerful cleaning H2O / ACN(10 / 90v / v) Preparation of mobile phase: Potassium phosphate 50 mM pH 6.8 / 250 mM KCl. Weigh 3.61 g KH2PO4 and 4.09 g K2HPO4 into a 1 L volumetric flask. Complete with milliQ water. Adjust pH with HCl or NaOH 1 mol / L if necessary. Add 18.64 g KCl. Sample preparation: Prepare 1-2.5 mg / mL ADC solution in milliQ water.

[0170] 8.3.3 DAR: Reversed Phase Liquid Chromatography (RPLC) Equipment UPLC Waters Acquity Detector: Tunable UV detector (TUV) Detector cell Titanium Column Waters Bioresolve 2.1*150 2.7um part number 186008946. Mobile phase A water + TFA0.1% (v / v) Mobile phase B ACN+TFA0.1%(v / v) Wavelength 280nm Injection volume 10μL Column temperature: 90℃ Sample manager temperature 25℃ Runtime 10 minutes Flow 0.35mL / min Weak wash H2O / ACN(90 / 10v / v) Powerful cleaning H2O / ACN(10 / 90v / v)

[0171] [Table 2] Preparation of mobile phase: ACN + 0.1% TFA (V / V): Measure 1000 mL of ACN in a graduated cylinder and pour into a 1 L glass bottle. Add 1 mL of TFA with a 1 mL pipette and shake vigorously. Water + 0.1% TFA (V / V): Measure 1000 mL of ultrapure water in a graduated cylinder and pour into a 1 L glass bottle. Add 1 mL of TFA with a 1 mL pipette and shake vigorously. Sample preparation: mAb: Prepare a 2.5 mg / mL mAb solution in milliQ water. Dispense 45 μL of this solution and 5 μL of 1 mol / L DTT solution into a vial. Incubate at 45° C. for 30 minutes. ADC: Prepare a 2.5 mg / mL ADC solution in milliQ water. Dispense 45 μL of this solution and 5 μL of 0.1 mol / L DTT solution into a vial. Incubate at 30° C. for 1 hour.

[0172] 8.3.4 DAR:MS method Sample preparation: Samples were diluted twice with 50 mM ammonium acetate and 25 μg of each sample was injected. UPLC: Separation was performed using a MAbPac™ SEC-1 column (Thermo Scientific) and 50 mM ammonium acetate, pH 7, as the mobile phase at 0.3 mL / min. MS: MS was performed using a QExactive HF Orbitrap operated in the high mass range. MS spectra were acquired from 1800 to 8000 m / z with the resolution set at 15k, SID50 eV. Mass spectra were deconvoluted using ProteinDeconvolution (Thermo Scientific).

[0173] 8.3.5 Concentration: UV method Device: UV spectrophotometer BioTek Synergy HT Preparation of buffers: PBS pH 7.4: Weigh 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4.2H2O and 0.24 g KH2PO4 into a 1 L volumetric flask. Add 900 mL milliQ water. Mix it when all salts are dissolved and adjust the pH to 7.35-7.44 with HCl 1 mol / L. Make up to 1 L with milliQ water. Sample preparation: Prepare a 1 mg / mL ADC solution in PBS pH 7.4. Measure absorbance at 252 and 280 nm using a UV reader. Calculation formula:

[0174]

number

[0175] 8.4 Preparation of linker payload : Linker-payloads were prepared using standard chemical methods and convergent strategies. The linker-payloads prepared in Examples 8.4.1-8.4.9 are shown in Table 1 below.

[0176] [Table 3] TIFF2025512064000080.tif253170Table 1: Linker payload Example 8.4.1: Preparation of DM1-MCC-Cit-Lys(ma-PEG5)-Tyr-OH

[0177] [ka] H-Cit-Lys(polyethylene glycol 5-ma)-Tyr-OH was purchased from Ambiopharm and prepared according to the following general procedure. Peptide synthesis was performed on 2-CTC resin following the general Fmoc / tBu strategy of solid-phase peptide synthesis, with carboxyl group activation achieved by diisopropylcarbodiimide / HOBT. Each amino acid was then coupled to the peptide chain as an active ester, starting from the C-terminal amino acid. The last amino acid in the sequence was coupled with an N-terminal protecting Boc group. Lys derivatives were incorporated with the side chain amino group protected by ivDde, which was removed with 2% hydrazine in DMF. After ivDde removal, the side chain was derivatized with maleimide-PEG 5-OH using the activated ester. The peptide was subsequently treated with a TFA-based acidolysis cocktail to cleave from the resin and deprotect the side chain groups. The peptide was then purified by liquid chromatography (RP-HPLC). The purified peptide TFA salt was lyophilized and obtained as a white to off-white powder. DIEA (20 μL, 0.11 mmol, 1.0 equiv) was added to a solution of H-Cit-Lys(PEG5-ma)-Tyr-OH (100 mg, 0.11 mmol, 1.0 equiv) and DM1-SMCC (131.8 mg, 0.12 mmol, 1.1 equiv) in DMF (1 mL) at room temperature. After stirring for 4 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-80% ACN + 0.1% TFA in water + 0.1% TFA) afforded DM1-MCC-Cit-Lys(ma-PEG5)-Tyr-OH (138.5 mg, 74.8 μmol, 100% UV purity, 67% yield) as a white powder after lyophilization. UPLC-MS (method 4): Rt = 2.58 and 2.63 min, m / z = 1854 [M+H] +, 1852 [MH] Example 8.4.2: Preparation of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH

[0178] [ka] TIFF2025512064000083.tif252170 Step 1: DIEA (0.84 mL, 4.82 mmol, 5.0 equiv) was added to a mixture of mPEG16-NHS ester (900 mg, 0.96 mmol, 1.0 equiv) and Boc-Lys-OH (300 mg, 1.16 mmol, 1.2 equiv) in DMF (12 mL) at room temperature. After stirring for 16 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-80% ACN + 0.1% TFA in water + 0.1% TFA) afforded Boc-Lys(PEG16)-OH (990 mg, 0.95 mmol, 100% UV purity, 99% yield) as a colorless oil after lyophilization. UPLC-MS (method 1): Rt = 1.16 min, m / z = 1038 [M+H] + Step 2: TFA (6 mL) was added to a solution of Boc-Lys(PEG16)-OH (990 mg, 0.95 mmol, 1.0 equiv) in DCM (6 mL) at room temperature. After stirring for 30 min at room temperature, the reaction mixture was concentrated under vacuum. The residue was dissolved in a mixture of ACN / water (1:1, 16 mL) and then lyophilized to give H-Lys(PEG16)-OH.TFA (1.00 g, 0.95 mmol, 100% UV purity, quantitative) as a colorless oil. UPLC-MS (method 1): Rt = 0.89 min, m / z = 938 [M+H] + , 936 [MH] - Step 3: DIEA (0.15 mL, 0.85 mmol, 4.0 equiv) was added to a mixture of H-Lys(PEG16)-OH (200 mg, 0.21 mmol, 1.0 equiv) and AMAS (56.5 mg, 0.22 mmol, 1.05 equiv) in DMF (2.5 mL) at room temperature. After stirring for 1 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10-40% ACN + 0.1% TFA in water + 0.1% TFA) afforded ma-Lys(PEG16)-OH (220 mg, 0.20 mmol, 100% UV purity, 96% yield) as a colorless oil after lyophilization. UPLC-MS (method 4): Rt = 1.74 min, m / z = 1075 [M+H] + , 1073 [MH] - Step 4: DIEA (0.6 mL, 3.44 mmol, 3.7 equiv) was added to a mixture of Fmoc-Cit-OPFP (520.7 mg, 0.92 mmol, 1.0 equiv) and H-Lys(Boc)-OH (218.1 mg, 0.89 mmol, 0.96 equiv) in a mixture of DMF (6 mL) and water (3 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added dropwise until an acidic pH was reached. Purification by C18 flash column chromatography (20-80% ACN + 0.1% TFA in water + 0.1% TFA) afforded Fmoc-Cit-Lys(Boc)-OH (593.3 mg, 0.92 mmol, 97% UV purity, quantitative) as a white powder after lyophilization. UPLC-MS (method 3): Rt = 1.80 min, m / z = 626 [M+H] + , 526 [MBoc+H] + , 624 [MH] - Step 5: H-Tyr-OMe (139.2 mg, 0.71 mmol, 1.1 equiv.) was added to a solution of Fmoc-Cit-Lys(Boc)-OH (400 mg, 0.64 mmol, 1.0 equiv.) in DMF (10 mL) at room temperature, followed by HATU (334.2 mg, 0.88 mmol, 1.4 equiv.) and DIEA (0.5 mL, 2.87 mmol, 4.5 equiv.). After stirring for 2 h at room temperature, TFA was added until an acidic pH was reached. Purification by C18 flash column chromatography (20-80% ACN + 0.1% TFA in water + 0.1% TFA) afforded H-Cit-Lys(Boc)-Tyr-H (513.3 mg, 0.57 mmol, UV purity 89%, yield 89%) as a white powder after lyophilization. UPLC-MS (method 3): Rt = 1.83 min, m / z = 803 [M+H] + , 703 [M-Boc+H] + , 847 [M+FA-H] - .

[0179] Step 6: Lithium hydroxide monohydrate (158.5 mg, 3.78 mmol, 4.2 equiv) was added to a solution of Fmoc-Cit-Lys(Boc)-Tyr-OMe (730.6 mg, 0.91 mmol, 1.0 equiv) in THF (8 mL) and water (5 mL) at room temperature. After stirring for 40 min at room temperature, TFA was added until a slightly acidic pH was reached, and the reaction mixture was then concentrated in vacuo. Purification by preparative HPLC (15-40% ACN+0.1% TFA in water+0.1% TFA) afforded H-Cit-Lys(Boc)-Tyr-OH (510 mg, 0.90 mmol, 100% UV purity, 99% yield) as a white powder after lyophilization. UPLC-MS (method 1): Rt = 0.80 min, m / z = 567 [M+H] + , 565 [MH] - Step 7: DIEA (0.28 mL, 1.63 mmol, 6.0 equiv) was added to a solution of bromoacetic acid (64.8 mg, 0.47 mmol, 1.7 equiv) and DM1 (200 mg, 0.27 mmol, 1.0 equiv) in DMF (2 mL) at room temperature. After stirring at room temperature for 1 h, a mixture of HATU (113.3 mg, 0.30 mmol, 1.1 equiv) and 1-hydroxypyrrolidine-2,5-dione (34.3 mg, 0.30 mmol, 1.1 equiv). After stirring at room temperature for 30 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30-60% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-Ac-NHS ester (134.7 mg, 0.12 mmol, 80% UV purity, 45% yield) as a white powder after lyophilization. UPLC-MS (method 2): Rt = 1.57 min, m / z = 891 [MH] - Step 8: DIEA (0.22 mL, 1.24 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (347 mg, 0.31 mmol, 1.0 equiv) and H-Cit-Lys(Boc)-Tyr-OH (222.1 mg, 0.33 mmol, 1.05 equiv) in DMF (4 mL) at room temperature. After stirring for 1 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30-60% ACN + 0.1% TFA in water + 0.1% TFA) afforded DM1-Ac-Cit-Lys(Boc)-Tyr-OH (417 mg, 0.31 mmol, 100% UV purity, quantitative) as a white powder after lyophilization. UPLC-MS (method 3): Rt = 1.67 min, m / z = 1344 [MH] - Step 9: A mixture of TFA (0.9 mL) and DCM (3.6 mL) was added to DM1 Ac-Cit-Lys(Boc)-Tyr-OH (112.0 mg, 83.3 μmol, 1.0 equiv) at room temperature for 5 min. After stirring, a mixture of ACN / water (1:1, 8 mL) was added to the reaction mixture. DCM was removed by concentration under vacuum. Purification by preparative HPLC (2-40% ACN+0.1% TFA in water+0.1% TFA) gave DM1-Ac-Cit-Lys-Tyr-OH (80 mg, 64.3 μmol, 100% UV purification, 77% yield) as a white powder after lyophilization. UPLC-MS (method 2): Rt = 1.03 min, m / z = 1242 [M+H] + , 1240 [MH] - Step 10: 1-Hydroxypyrrolidine-2,5-dione (2.14 mg, 18.6 μmol, 1.0 equiv) and EDC·HCl (3.57 mg, 18.6 μmol, 1.0 equiv) were added to a solution of ma-Lys(PEG16)-OH (20.0 mg, 18.6 μmol, 1.0 equiv) in DCM (2 mL) at room temperature. After stirring at room temperature for 2.5 h, DM1-Ac-Cit-Lys-Tyr-OH (37.1 mg, 22.3 μmol, 1.2 equiv) and DIEA (13 μL, 74.5 μmol, 4.0 equiv) were added to the reaction mixture at room temperature. After stirring at room temperature for 10 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (5-100% ACN + 0.1% FA in water + 0.1% FA) gave DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (17.2 mg, 7.50 μmol, 100% UV purity, 40% yield) as a white powder after lyophilization. UPLC-MS (method 4): Rt = 2.59 min, m / z = 1173 [M+FA-H] - .

[0180] Example 8.4.3: Preparation of DM1-Ac-Cit-Lys(ma-PEG5)-Tyr-OH

[0181] [ka] DIEA (31 μL, 0.18 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (50.0 mg, 44.8 μmol, 1.0 equiv) and H-Cit-Lys(ma-PEG5)-Tyr-OH.TFA (45.2 mg, 44.8 μmol, 1.0 equiv) in DMF (1 mL) at room temperature. After stirring for 30 min at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-50% ACN + 0.1% TFA in water + 0.1% TFA) afforded DM1-Ac-Cit-Lys(ma-PEG5)-Tyr-OH (74.9 mg, 44.8 μmol, 100% UV purified, quantitative) as a white powder after lyophilization. UPLC-MS (method 1): Rt = 1.31 min, m / z = 1672 [MH] - Example 8.4.4: Preparation of Exatecan-Suc-Phe-Cit-Lys(ma-Lys(PEG16))-Tyr-OH

[0182] [ka] Step 1: DIEA (2.0 mL, 11.8 mmol, 3.0 equiv) was added to a mixture of H-Tyr(OtBu)-OtBu (2.59 g, 7.85 mmol, 1.0 equiv), Fmoc-Lys(Mtt)-OH (2.51 g, 3.94 mmol, 1.0 equiv) and HATU (1.95 g, 5.12 mmol, 1.3 equiv) in DMF (40 mL) at 0-5 °C. After stirring at room temperature for 50 min, the reaction mixture was diluted with EtOAc (300 mL) and washed with half-saturated brine (2 x 200 mL). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give Fmoc-Lys(Mtt)-Tyr(OtBu)-OtBu (7.07 g, 7.69 mmol, UV purity 98%, yield 98%) as a light brown oil. UPLC-MS (method 3): Rt = 2.42 min, m / z = 900 [M+H] + , 944 [M+FA-H] - Step 2: A mixture of DMF / piperidine (9:1, 40 mL) was added to Fmoc. Lys(Mtt)-Tyr(OtBu)-OtBu (7.07 g, 7.69 mmol, 1.0 equiv.) at room temperature. After stirring for 10 min at room temperature, the reaction mixture was concentrated to dryness. The residue was purified by flash chromatography (n-heptane, then DCM, and finally DCM:MeOH 95:5 to 90:10) to give H-Lys(Mtt)-Tyr(OtBu)-OtBu (5.10 g, 7.15 mmol, 95% UV purity, 93% yield) as an orange oil after concentration under vacuum. UPLC-MS (method 3): Rt = 1.58 min, m / z = 678 [M+H] + , 722 [M+FA-H] - Step 3: DIEA (1.73 mL, 9.96 mmol, 3.0 equiv) was added to a solution of H-Lys(Mtt)-Tyr(OtBu)-OtBu (2.50 g, 3.32 mmol, 1.0 equiv), Fmoc-Cit-OH (1.48 g, 3.65 mmol, 1.1 equiv) and HATU (1.70 g, 4.31 mmol, 1.3 equiv) in DMF (20 mL) at 0° C. After stirring at room temperature for 30 min, the reaction mixture was poured into 5% aqueous NaHCO3 (200 mL) and extracted with EtOAc (2×100 mL). The combined organic layers were washed with brine (100 mL), dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash chromatography (heptane:EtOAc 80:20 to plain EtOAc, then 5-10% EtOAc:MeOH 95:5 to 90:10) afforded Fmoc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (3.40 g, 3.05 mmol, 95% UV purity, 92% yield) as a beige gum after concentration in vacuo. UPLC-MS (method 3): Rt = 2.15 min, m / z = 1058 [M+H] + , 1102 [M+FA-H] - Step 4: A mixture of DMF / piperidine (20 mL, 9:1 V / V) was added to Fmoc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (3.40 g, 3.22 mmol, 1.0 equiv) at room temperature. After stirring for 10 min at room temperature, the reaction mixture was concentrated to dryness. Purification by flash chromatography (heptane:EtOAc 80:20 to plain EtOAc, then 5-10% EtOAc:MeOH 95:5 to 90:10, then 5-15% 25% aq. NH3 in ACN) afforded H-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (2.55 g, 2.96 mmol, UV purity 97%, yield 92%) as a colorless oil after concentration under vacuum. UPLC-MS (method 3): Rt = 1.54 min, m / z = 836 [M+H] + , 880 [M+FA-H] - Step 5: DIEA (0.61 mL, 3.48 mmol, 3.0 equiv) was added to a solution of H-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.00 g, 1.16 mmol, 1.0 equiv), Fmoc-Phe-OH (539 mg, 1.39 mmol, 1.2 equiv) and HATU (594 mg, 1.51 mmol, 1.3 equiv) in DMF (15 mL) at 0° C. After stirring at room temperature for 15 min, the reaction mixture was poured into 5% aqueous NaHCO3 (50 mL) and extracted with EtOAc (2×70 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and partially concentrated under reduced pressure. Precipitation with ether gave Fmoc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.33 g, 1.06 mmol, UV purity 96%, yield 92%) as a white solid. UPLC-MS (method 3): Rt = 2.29 min, m / z = 1205 [M+H] + , 1249 [M+FA-H] - .

[0183] Step 6: To a mixture of DMF / piperidine (10 mL, 9:1 V / V) was added Fmoc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.33 g, 1.06 mmol, 1.0 equiv.) at room temperature. After stirring for 10 min at room temperature, the reaction mixture was concentrated to dryness. After concentration under vacuum by purification by flash chromatography (heptane:EtOAc 80:20 to plain EtOAc, then 5-10% EtOAc:MeOH 90:10, then 10-20% 25% aq. NH3 in ACN), H-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (0.93 g, 0.90 mmol, 95% UV purity, 81% yield) was obtained as a white powder. UPLC-MS (method 3): Rt = 1.57 min, m / z = 983 [M+H] + , 1027 [M+FA-H] - Step 7: Dihydrofuran-2,5-dione (1.16 mg, 11.5 μmol, 1.0 equiv) was added to a mixture of exatecan mesylate (5.0 mg, 11.5 μmol, 1.0 equiv) and DIEA (0.02 mL, 0.14 mmol, 12.0 equiv) in DMF (0.5 mL) at room temperature. After stirring for 15 min at room temperature, H-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (11.28 mg, 11.5 μmol, 1.0 equiv) was added followed by HATU (4.37 mg, 11.5 μmol, 1.0 equiv). After stirring for 1 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10-100% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (8.1 mg, 5.30 mmol, 98% UV purity, 46% yield) as a yellow powder after lyophilization. UPLC-MS (method 4): Rt = 3.70 min, m / z = 1501 [M+H] + , 1545 [M+FA-H] - Step 8: A mixture of DCM (5 mL) and TFA (0.1 mL) was added to Exatecan-Suc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (61.0 mg, 40.7 μmol, 1.0 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 1 h and then concentrated in vacuo. Purification by preparative HPLC (20-40% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit-Lys-Tyr(OtBu)-OtBu (6.95 mg, 5.4 μmol, 97% UV purity, 13% yield) as a yellow powder after lyophilization. UPLC-MS (method 4): Rt = 2.82 min, m / z = 1244 [M+H] + , 1288 [M+FA-H] - Step 9: 1-Hydroxypyrrolidine-2,5-dione (9.0 mg, 78 μmol, 2.0 equiv) and EDC·HCl (15.0 mg, 78 μmol, 2.0 equiv) were added to a solution of ma-Lys(PEG16)-OH (41.9 mg, 39 μmol, 1.0 equiv) in DCM (2.0 mL) at room temperature. After stirring at room temperature for 1 h, the reaction mixture was added with Exatecan-Suc-Phe-Cit-Lys-Tyr(OtBu)-OtBu (50.0 mg, 39 μmol, 1 equiv) and DIEA (27 μL, 0.16 mmol, 4.0 equiv) at room temperature. After stirring at room temperature for 20 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30-80% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit-Lys(ma-Lys(Peg16))-Tyr(OtBu)-OtBu (10.0 mg, 4.30 mmol, 100% UV purity, 11% yield) as a white powder after lyophilization. UPLC-MS (method 4): Rt = 3.15 min, m / z = 1151 [M+H] + Step 10: TFA (0.11 mL) was added to a solution of Exatecan-Suc-Phe-Cit-Lys(ma-Lys(Peg16))-Tyr(OtBu)-OtBu (10.0 mg, 4.30 μmol, 1.0 equiv) in DCM (0.11 mL) at room temperature. After stirring for 3 h at room temperature, the reaction mixture was concentrated under vacuum. Purification by preparative HPLC (20-60% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit-Lys(ma-Lys(Peg16))-Tyr-OH (5.3 mg, 2.40 μmol, 100% UV purification, 56% yield) as a white powder after lyophilization. UPLC-MS (method 4): Rt = 2.44 min, m / z = 1094 [M+H] + , 1092 [M+FA-H] - .

[0184] Example 8.4.5: ma-Gly-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-PEG24

[0185] [ka] Step 1: DIEA (1.17 mL, 6.66 mmol, 3.0 equiv) was added to a mixture of Fmoc-Glu(OtBu)-OH·H2O (1.00 g, 2.22 mmol, 1.0 equiv), H-Glu(OtBu)-OBn·HCl (750 mg, 2.22 mmol, 1.0 equiv) and HATU (1.14 g, 2.99 mmol, 1.35 equiv) in DMF (15 mL) at 0 °C. After stirring for 15 min at 0 °C and 1 h at room temperature, the reaction mixture was concentrated to dryness. The resulting brown oil was diluted with EtOAC and washed with 5% aqueous NaHCO3. The organic phase was washed with brine, dried over MgSO4, filtered and concentrated to dryness. Purification by flash chromatography (n-heptane:EtOAc 80:20 to 0:100) afforded Fmoc-Glu(OtBu)-Glu(OtBu)-OBn (1.46 g, 2.09 mmol, 100% UV purity, 94% yield) as a white foam. UPLC-MS (method 3): Rt = 2.62 min, m / z = 701 [M+H] + , 745 [M+FA-H] - Step 2: DBU (0.3 mL, 2.00 mmol, 1.2 equiv) was added to a solution of Fmoc-Glu(OtBu)-Glu(OtBu)-OBn (1.17 g, 1.67 mmol, 1.0 equiv) in DMF (5 mL) at room temperature. After stirring for 5 min at room temperature, the reaction mixture was added dropwise over 3 min to a mixture of Boc-Gly-OH (351 mg, 2.00 mmol, 1.2 equiv), HATU (855.3 mg, 2.17 mmol, 1.3 equiv) and DIEA (0.87 mL, 5.01 mmol, 3.0 equiv) in DMF (12 mL) at room temperature. After stirring for 30 min at room temperature, the reaction mixture was concentrated to dryness. The residue was diluted with EtOAc and then washed with citric acid 10%. The organic layer was dried over MgSO4 and concentrated to dryness. Flash chromatography (DCM:EtOAc 90:10 to 0:100) afforded Boc-Gly-Glu(OtBu)-Glu(OtBu)-OBn (982 mg, 1.47 mmol, UV purity 95%, yield 88%) as a white solid. UPLC-MS (method 3): Rt = 2.22 min, m / z = 636 [M+H] + , 634 [MH] - Step 3: For hydrogenation, palladium (164.4 mg, 0.15 mmol, 0.1 equiv) was added to a 25 mL flask under nitrogen atmosphere. A solution of Boc-Gly-Glu(OtBu)-Glu(OtBu)-OBn (982 mg, 1.54 mmol, 1.0 equiv) in ethanol (9.8 mL) was added. The resulting suspension was stirred at room temperature under 1 bar of hydrogen for 30 min and then filtered through a Celite® pad. The filtrate was concentrated under reduced pressure to give Boc-Gly-Glu(OtBu)-Glu(OtBu)-OH (898 mg, 1.65 mmol, 94% UV purity, quantitative) as a white foam. UPLC-MS (method 3): Rt = 1.73 min, m / z = 546 [M+H] + , 544 [MH] - Step 4: DIEA (48 mL, 0.27 mmol, 3.0 equiv) was added to a mixture of Boc-Gly-Glu(OtBu)-Glu(OtBu)-OH (50.0 mg, 91.6 mmol, 1.0 equiv), PEG24-amine (120.5 mg, 0.11 mmol, 1.2 equiv) and HATU (59.0 mg, 0.15 mmol, 1.6 equiv) in DMF (0.5 mL) at room temperature. After stirring for 10 min at room temperature, the reaction mixture was acidified to pH 4-5 with TFA. Purification by preparative HPLC (10-100% ACN+0.1% TFA in water+0.1% TFA) afforded Boc-Gly-Glu(OtBu)-Glu(OtBu)-PEG24 (100 mg, 61.9 mmol, 100% UV purity, 68% yield) as a white solid after lyophilization. UPLC-MS (method 3): Rt = 1.82 min, m / z = 1614 [MH] - , 1660 [M+FA-H] - Step 5: TFA (0.5 mL) was added to a solution of Boc-Gly-Glu(OtBu)-Glu(OtBu)-PEG24 (100 mg, 61.9 mmol, 1.0 equiv) in DCM (0.5 mL) at room temperature. After stirring for 30 min at room temperature, the reaction mixture was concentrated to dryness to give H-Gly-Glu-Glu-PEG24·TFA (93 mg, 61.3 mmol, 99% yield) as a yellow oil. UPLC-MS (method 1): Rt = 0.94 min, m / z = 1404 [M+H] + , 1402 [MH] - .

[0186] Step 6: DIEA (53 μL, 0.31 mmol, 5.0 equiv) was added to a mixture of H-Gly-Glu-Glu-PEG24.TFA (93.0 mg, 61.3 μmol, 1.0 equiv) and AMAS (21.2 mg, 79.7 μmol, 1.3 equiv) in DMF (0.3 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added (24 μL). Purification by preparative HPLC (20-100% ACN + 0.1% TFA in water + 0.1% TFA) afforded ma-Gly-Glu-Glu-PEG24 (94.0 mg, 58.0 mmol, 95% UV purity, 95% yield) as a colorless oil. UPLC-MS (method 3): Rt = 1.20 min, m / z = 1539 [MH] - Step 7: DIEA (27 μL, 0.16 mmol, 8.0 equiv) was added to a mixture of ma-Gly-Glu-Glu-PEG24 (30.0 mg, 19.5 μmol, 1.0 equiv) and HATU (14.8 mg, 38.9 μmol, 2.0 equiv) in DMF (3 mL) at room temperature. After stirring for 7 min at room temperature, the reaction mixture was added to DM1-Ac-Cit-Lys-Tyr-OH (52.9 mg, 38.9 μmol, 2.0 equiv) at room temperature. After stirring for 15 min at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30-70% ACN + 0.1% TFA in water + 0.1% TFA) afforded ma-Gly-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-PEG24 (15.1 mg, 3.40 μmol, 89% UV purity, 17% yield) as a white powder after lyophilization. UPLC-MS (method 3): Rt = 1.70 min, m / z = 1996 [M-2H] 2- .

[0187] Example 8.4.6: Preparation of DM1-MCC-Cit-Lys(ma-Lys(PEG16))-Tyr-OH

[0188] [ka] Step 1: DIEA (0.7 mL, 3.98 mmol, 3.0 equiv.) was added to a solution of H-Lys(Mtt)-Tyr(OtBu)-OtBu (1.00 g, 1.33 mmol, 1.0 equiv.), HATU (680 mg, 1.73 mmol, 1.3 equiv.) and Boc-Cit-OH (373 mg, 1.33 mmol, 1.0 equiv.) in DMF (8 mL) at 0-5 °C. After stirring at room temperature for 30 min, the reaction mixture was poured into EtOAc (200 mL) and washed with brine (100 mL) and then with half-saturated brine (50 mL). The resulting emulsion was first evaporated under reduced pressure and then lyophilized to give Boc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (2.10 g, 1.80 mmol, 60% pure, quantitative) as a beige solid. UPLC-MS (method 3): Rt = 2.02 min, m / z = 936 [M+H] + , 980 [M+FA-H] - Step 2: A mixture of DCM (65 mL) and TFA (2 mL) was added to Boc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.24 g, 1.33 mmol, 1.0 equiv) at room temperature. After stirring for 10 min at room temperature, the reaction mixture was partially concentrated and then slowly added to cold ether. The oily product obtained by centrifugation was purified by preparative HPLC (25-80% ACN+0.1% TFA in water+0.1% TFA) to give Boc-Cit-Lys-Tyr(OtBu)-OtBu (602 mg, 0.76 mmol, 100% UV purity, 57% yield) as a white powder after lyophilization. UPLC-MS (method 3): Rt = 1.46 min, m / z = 680 [M+H] + , 723 [M+FA-H] - Step 3: DIEA (0.06 mL, 0.37 mmol, 4.0 equiv) was added to a mixture of ma-Lys(PEG16)-OH (100 mg, 93.1 μmol, 1.0 equiv), Boc-Cit-Lys-Tyr(OtBu)-OtBu (81.2 mg, 102.4 μmol, 1.1 equiv) and HATU (47.7 mg, 121 μmol, 1.3 equiv) in DMF (1.5 mL) at 5° C. After stirring for 10 min at 5° C., TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-70% ACN+0.1% TFA in water+0.1% TFA) afforded, after a lyophilization step, Boc-Cit-Lys(ma-Lys(PEG16))-Tyr(OtBu)-OtBu (158 mg, mmol, UV purity 96%, yield 94%) as a white powder. UPLC-MS (method 3): Rt = 1.81 min, m / z = 1736 [M+H] + , 1780 [M+FA-H] - Step 4: A mixture of TFA (2.5 mL) and DCM (2.5 mL) was added to Boc-Cit-Lys(ma-Lys(PEG16))-Tyr(OtBu)-OtBu (158 mg, 87.2 μmol, 1.0 equiv) at room temperature. After stirring at 4 °C for 16 h, the reaction mixture was concentrated under vacuum. Purification by preparative HPLC (10-50% ACN + 0.1% TFA in water + 0.1% TFA) afforded H-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (132 mg, 79.6 μmol, 99% UV purity, 91% yield) as a colorless oil after lyophilization. UPLC-MS (method 3): Rt = 1.04 min, m / z = 1523 [M+H] + , 1521 [MH] - Step 5: DIEA (41 μL, 0.25 mmol, 4.0 equiv) was added to a mixture of DM1-SMCC (68.6 mg, 62.0 μmol, 1.0 equiv) and H-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (103 mg, 62.0 μmol, 1.0 equiv) in DMF (1.5 mL) at room temperature. After stirring at room temperature for 1.5 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-90% ACN+0.1% TFA in water+0.1% TFA) afforded DM1-MCC-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (73.4 mg, 29.4 μmol, 99% UV purity, 47% yield) as a white powder after lyophilization. UPLC-MS (method 3): Rt = 1.60 and 1.62 min, m / z = 827 [M+3H] 3+ , 870 [M+FA-3H] 3- .

[0189] Example 8.4.7: Preparation of DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH

[0190] [ka] Step 1: DIEA (0.14 mL, 0.83 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (185 mg, 0.21 mmol, 1.0 equiv) and citrulline (72.6 mg, 0.41 mmol, 2.0 equiv) in DMSO (3.7 mL) at room temperature. After stirring for 18 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10-40% water + 0.1% TFA / water + 0.1% TFA) afforded DM1-Ac-Cit (130 mg, 0.14 mmol, 100% UV purity, 66% yield) as a white powder after lyophilization. UPLC-MS (method 1): Rt = 1.33 min, m / z = 952 [MH] - . Step 2:

[0191] [ka] Acetyl-L-cysteine ​​(0.32 mg, 2.17 μmol, 1.0 equiv) was added to a solution of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (5.0 mg, 2.17 μmol, 1.0 equiv) in DMF (1 mL) at room temperature. After stirring for 40 min at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (5-100% can+0.1% TFA / water+0.1% TFA) afforded DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH (4.59 mg, 1.86 μmol, 99% UV purity, 94% yield) as a white powder after lyophilization. UPLC-MS (method 4): Rt = 2.41 min, m / z = 1231 [M-2H] 2- .

[0192] Example 8.4.8: Preparation of Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH

[0193] [ka] Step 1: DIEA (0.78 mL, 4.48 mmol, 4.0 equiv) was added to a mixture of citrulline (200 mg, 1.12 mmol, 1.0 equiv) and Boc-Phe-OSu (405 mg, 1.12 mmol, 1.0 equiv) in DMF (10 mL) at room temperature. After stirring for 16 h at room temperature, the reaction mixture was filtered and then concentrated under reduced pressure. A mixture of water / CA N / TFA (1:1:0.5%) was added. The mixture was filtered and then lyophilized. Purification by preparative HPLC (10-60% ACN+0.1% TFA in water+0.1% TFA) gave Boc-Phe-Cit-OH (86 mg, 0.20 mmol, 100% UV purity, 18% yield) as a white powder after lyophilization. UPLC-MS (method3): Rt=1.27 min, m / z=423[M+H]+, 421[MH]-. Step 2: A mixture of DCM (0.9 mL) and TFA (0.9 mL) was added to Boc-Phe-Cit-OH (60 mg, 0.14 mmol, 1.0 equiv) at room temperature. After stirring at room temperature for 20 min, the reaction mixture was concentrated. Water (10 mL) and ACN (10 mL) were added and the mixture was lyophilized to give H-Phe-Cit-OH (30 mg, 83.8 μmol, 90% UV purity, 59% yield) as a white powder. UPLC-MS (Method 3): Rt=0.35 min, m / z=323[M+H]+, 321[MH]-. Step 3: Example B1: Dihydrofuran-2,5-dione (8.4 mg, 83.5 μmol, 1.0 eq.) was added to a mixture of exatecan (36 mg, 83.5 μmol, 1.0 eq.) and DIEA (0.17 mL, 1.00 mmol, 12 eq.) in DMF (0.8 mL) at room temperature. After stirring for 10 minutes at room temperature, 1-hydroxypyrrolidine-2,5-dione (9.6 mg, 83.5 μmol, 1.0 eq.) was added, followed by HATU (31.7 mg, 83.5 μmol, 1.0 eq.). After stirring for 15 minutes at room temperature, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 eq.) was added to the reaction mixture. After stirring at room temperature for 1 h, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 equiv) was added to the reaction mixture. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-60% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit (26.4 mg, 30.8 mmol, UV purity 98%, yield 37%) as a yellow powder after lyophilization. UPLC-MS (method2): Rt=1.39 min, m / z=840[M+H]+, 838[MH]-. Process 4

[0194] [ka] DIEA (1.2 μL, 6.8 μmol, 4.0 equiv.) was added to a mixture of Exatecan-Suc-Phe-Cit-Lys(ma-C1-Lys(Peg16))-Tyr-OH (3.71 mg, 1.7 mmol, 1.0 equiv.) and Acetyl-L-cysteine ​​(0.28 mg, 1.7 μmol, 1.0 equiv.) in DMF (0.6 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-60% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH (2.87 mg, 1.2 μmol, 100% UV purification, 72% yield) as a white powder after lyophilization. UPLC-MS (method 4): Rt = 2.37 min, m / z = 1176 [M+2H]2+, 1174 [M-2H]2-.

[0195] Example 8.4.9: Preparation of DM1-Ac-Cit-Lys(D-Arg-ma)-Tyr-OH

[0196] [ka] Step 1: DIEA (22.0 g, 0.17 mol, 4.0 equiv) was added to a mixture of Boc-Lys(Fmoc)-OH (20.0 g, 43.0 mmol, 1.0 equiv), H-Tyr-OMe (8.30 g, 43.0 mmol, 1.0 equiv) and HATU (21.0 g, 55.0 mmol, 1.3 equiv) in DMF (150 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was poured into water (400 mL) and extracted with EtOAc (3×80 mL). The combined organic phase was dried over sodium sulfate, concentrated and purified by silica gel column chromatography (eluted with MeOH / DCM, 0%-5%) to give Boc-Lys(Fmoc)-Tyr-OMe (26.0 g, 32.6 mmol, 85% UV purification, 76% yield) as a white solid. LC-MS (method5): Rt=0.71 min, m / z=668.3[M+Na]+. Step 2: TFA (0.23 g, 2.00 mmol, 2.0 equiv) was added to a solution of Boc-Lys(Fmoc)-Tyr-OMe (1.00 g, 1.00 mmol, 1.0 equiv) in DCM (10 mL) at 0° C. After stirring at room temperature for 1 h, the reaction mixture was concentrated to give H-Lys(Fmoc)-Tyr-OMe (800 mg, 0.70 mmol, UV purity 85%, yield 70%), which was used directly in the next step without further purification. LC-MS (method 5): Rt=1.11 min, m / z=546.3 [M+H]+.

[0197] Step 3: DIEA (11.3 g, 87.8 mmol, 2.4 equiv.) was added to a mixture of H-Lys(Fmoc)-Tyr-OMe (20.0 g, 36.6 mmol, 1.0 equiv.), Boc-Cit-OH (10.1 g, 36.6 mmol, 1.0 equiv.) and HATU (15.3 g, 40.2 mmol, 1.1 equiv.) in DMF (100 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was poured into water (400 mL) and extracted with EtOAc (3×80 mL). The combined organic phases were dried over sodium sulfate, concentrated and purified by silica gel column chromatography (eluted with MeOH / DCM, 0%-5%) to give Boc-Cit-Lys(Fmoc)-Tyr-OMe (12.6 g, 15.4 mmol, UV purity 90%, yield 42%) as a white solid. LC-MS (method5): Rt=1.25 min, m / z=803.4[M+H]+. Step 4: Diethylamine (1.36 g, 18.6 mmol, 3.0 equiv) was added to a solution of Boc-Cit-Lys(Fmoc)-Tyr-OMe (5.00 g, 6.20 mmol, 1.0 equiv) in DCM (10 mL) at room temperature. After stirring at room temperature for 10 h, the reaction mixture was concentrated, washed with Et2O, and triturated in Et2O to give Boc-Cit-Lys-Tyr-OMe (3.00 g, 4.59 mmol, UV purity 90%, yield 74%), which was used directly in the next step without further purification. LC-MS (method 5): Rt=1.26 min, m / z=581.4 [M+H]+.

[0198] [ka] Step 5: DIEA (0.80 g, 6.20 mol, 2.0 equiv) was added to a mixture of Boc-Cit-Lys-Tyr-OMe (1.98 g, 3.40 mmol, 1.1 equiv), Fmoc-D-Arg(Pbf)-OH (2.00 g, 3.10 mmol, 1.0 equiv) and HATU (1.30 g, 3.40 mmol, 1.1 equiv) in DMF (30 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was poured into water (300 mL) and extracted with EtOAc (3×80 mL). The combined organic phase was dried over sodium sulfate, concentrated and purified by silica gel column chromatography (eluted with MeOH / DCM, 0%-5%) to give Boc-Cit-Lys(Fmoc-D-Arg(Pbf))-Tyr-OMe (2.10 g, 2.26 mmol, UV purity 95%, yield 73%) as a white solid. LC-MS (method 6): Rt=2.08 min, m / z=1211.7[M+H]+. Step 6: LiOH (50 mg, 1.20 mmol, 1.5 equiv) was added to a solution of Boc-Cit-Lys(Fmoc-D-Arg(Pbf))-Tyr-OMe (1.00 g, 0.80 mmol, 1.0 equiv) in THF / water (1:1, 10 mL) at room temperature. After stirring for 10 h at room temperature, the organic solvent was concentrated and the aqueous solution was adjusted to pH 2-3 with citric acid. The precipitate was collected by filtration, washed with water, and purified on a Biotage Isolera One (C18 column, eluted with 5%-95% ACN / water containing 0.1% FA) to give Boc-Cit-Lys(D-Arg(Pbf))-Tyr-OH (0.40 g, 0.37 mmol, UV purity 90%, yield 46%) as a white solid. LC-MS (method5): Rt=0.98 min, m / z=975.4[M+H]+. Step 7: Triethylamine (15 mg, 0.15 mmol, 1.5 equiv) was added to a mixture of Boc-Cit-Lys(D-Arg(Pbf))-Tyr-OH (100 mg, 0.10 mmol, 1.0 equiv), AMAS (28 mg, 0.11 mmol, 1.1 equiv) in DMF (1 mL) at room temperature. After stirring for 30 min at room temperature, TFA was added until a pH of 5-6 was reached. Purification on Biotage Isolera One (C18 column, eluted with 5%-95% ACN / water containing 0.1% TFA) afforded Boc-Cit-Lys(D-Arg(Pbf)-ma)-Tyr-OH (38 mg, 29 µmol, UV purity 88%, yield 29%) as a white solid. LC-MS (method6): Rt=1.70 min, m / z=1112.6[M+H]+. Step 8: Boc-Cit-Lys(D-Arg(Pbf)-ma)-Tyr-OH (10 mg, 9.0 μmol, 1.0 equiv) was dissolved in a mixture of TFA / DCM (1:1, 1 mL) at room temperature. After stirring at room temperature for 30 min, the mixture was concentrated, washed with Et2O, and triturated in Et2O to give H-Cit-Lys(D-Arg-ma)-Tyr-OH (6.0 mg, 6.2 μmol, 90% UV purity, 69% yield), which was used directly in the next step without further purification. LC-MS (method 6): Rt=0.97 min, m / z=758.4 [M+H]+. Step 9: HOSu (1.6 mg, 13.8 μmol, 1.1 equiv) was added to a mixture of DM1-Ac (10 mg, 12.6 μmol, 1.0 equiv) and EDCI (2.6 mg, 13.8 μmol, 1.1 equiv) in DMF (1 mL) at room temperature. After stirring for 1 h at room temperature, the reaction mixture was poured into water (40 mL) and extracted with EA (3×20 mL). The combined organic phases were dried over sodium sulfate and concentrated in vacuo. The residue was dissolved in DMF (1 mL). H-Cit-Lys(D-Arg-ma)-Tyr-OH (11.4 mg, 12.6 μmol, 1.0 equiv) and DIEA (2.44 mg, 18.9 μmol, 1.5 equiv) were added to the latter solution at room temperature. After stirring for 1 h at room temperature, TFA was added until a pH of 5-6 was reached. Purification on Biotage Isolera One (C18 column, eluted with 5%-75% ACN / 0.1% TFA in water) gave DM1-Ac-Cit-Lys(D-Arg-ma)-Tyr-OH (3.0 mg, 2.27 μmol, 88% UV purity, 18% yield) as a white solid. LC-MS (method 5): Rt=1.01 min, m / z=1537.5 [M+H]+.

[0199] 8.5 Preparation and Characterization of Antibody Drug Conjugates (ADCs) : 8.5.1 Preparation of ADCs: General Procedure All linker-payloads prepared in part 8.4 were conjugated to Herceptin® (trastuzumab). Linker-payloads DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH and Exatecan-Suc-Phe-Cit-Lys(ma-Lys(PEG16))-Tyr-OH were also conjugated to naratuximab. General Procedure 1: For DOC 4 Tmab conjugation: A solution of TCEP·HCl (0.32 mg, 1.09 μmol, 2.3 equiv) in DPBS (0.32 mL) was added to a solution of trastuzumab (70 mg, 0.45 μmol, 1.0 equiv) in water (3.33 mL) and DPBS (3.33 mL) at room temperature. The reaction mixture was purged with nitrogen and then stirred at 40 °C. After stirring for 70 min at 40 °C, a solution of linker-payload (3.55 μmol, 7.4 equiv) in DMSO (0.7 mL) was added. The reaction mixture was stirred for 70 min at room temperature and then diluted to V = 10 mL with 10 × pH 8 DPBS. Purification using a PF100 column and DPBS pH 8 as eluent afforded a fraction (14 mL) containing the desired ADC. This fraction was stirred for 16 h at room temperature, then centrifuged (10 min, 4000 rpm) and finally the supernatant was transferred to an Amicon concentration cell (15 mL, 50 kDa). The mixture was concentrated to V=1 mL by centrifugation (4500 rpm, 3800 G), DPBS buffer was added (14 mL), and the mixture was concentrated again to V=1 mL (4500 rpm, 3800 G). DPBS buffer was added (14 mL), and the mixture was concentrated again to V=1 mL (4500 rpm, 3800 G). The final volume was adjusted to V=7.0 mL with DPBS buffer. The solution was filtered using a 25 mm PES 0.22 μm Millex filter, then aliquoted and stored at −80° C.

[0200] General Procedure 2: For DOC 8 Tmab conjugation: A solution of TCEP·HCl (1.10 mg, 3.80 μmol, 8.0 equiv) in DPBS (0.11 mL) was added to a solution of trastuzumab (70 mg, 0.45 μmol, 1.0 equiv) in water (3.33 mL) and DPBS (3.33 mL) at room temperature. The reaction mixture was purged with nitrogen and then stirred at 40 °C. After stirring for 70 min at 40 °C, a solution of linker-payload (9.60 μmol, 20.0 equiv) in DMSO (0.7 mL) was added. The reaction mixture was stirred for 70 min at room temperature and then diluted to V = 10 mL with 10 × pH 8 DPBS. Purification using a PF100 column and DPBS pH 8 as eluent afforded a fraction (14 mL) containing the desired ADC. This fraction was stirred for 16 h at room temperature, then centrifuged (10 min, 4000 rpm) and finally the supernatant was transferred to an Amicon concentration cell (15 mL, 50 kDa). The mixture was concentrated to V=1 mL by centrifugation (4500 rpm, 3800 G), DPBS buffer was added (14 mL) and the mixture was concentrated again to V=1 mL (4500 rpm, 3800 G). DPBS buffer was added (14 mL) and the mixture was concentrated again (4500 rpm, 3800 G) to V=1 mL. The final volume was adjusted to V=7.0 mL with DPBS buffer. The solution was filtered using a 25 mm PES 0.22 μm Millex filter before being divided into aliquots and stored at −80° C.

[0201] General Procedure 3: For DOC 8 Naratuximab Conjugate: A solution of TCEP·HCl (1.10 mg, 3.80 μmol, 8.0 equiv) in DPBS (0.11 mL) was added to a solution of naratuximab (70 mg, 0.48 μmol, 1.0 equiv) in buffer (7.00 mL, 2 M). 50 mM potassium phosphate, 50 mM potassium chloride, 2 mM EDTA, pH 6.5) was added at room temperature. The reaction mixture was purged with nitrogen and then stirred at 40° C. After stirring for 70 min at 40° C., a solution of linker-payload (9.60 μmol, 20.0 equiv.) in DMSO (0.7 mL) was added. The reaction mixture was stirred for 70 min at room temperature and then diluted to V=10 mL with 10× pH 8 DPBS. Purification using a PF100 column and DPBS pH 8 as eluent afforded a fraction (14 mL) containing the desired ADC. This fraction was stirred for 16 h at room temperature, then centrifuged (10 min, 4000 rpm) and finally the supernatant was transferred to an Amicon concentration cell (15 mL, 50 kDa). The mixture was concentrated to V=1 mL by centrifugation (4500 rpm, 3800 G), DPBS buffer was added (14 mL) and the mixture was concentrated again to V=1 mL (4500 rpm, 3800 G). DPBS buffer was added (14 mL) and the mixture was concentrated again (4500 rpm, 3800 G) to V=1 mL. The final volume was adjusted to V=7.0 mL with DPBS buffer. The solution was filtered using a 25 mm PES 0.22 μm Millex filter, then aliquoted and stored at -80°C.

[0202] 8.5.2 Overview of Characterization of Prepared ADCs

[0203] [Table 4] Table 2: Overview of prepared ADCs Full characterization is shown in Table 3 below.

[0204] [Table 5] Table 3: Characterization of the prepared ADCs. The RPLC chromatograms of the prepared ADCs are shown in Figures 3 to 6.

[0205] 8.5.3 Preparation of Naratuximab Emtansine (Debio1562) (Comparative Example) Naratuximab was reacted with the heterobifunctional crosslinker SMCC and the maytansinoid DM1 using the one-step process described in WO2012135517. The DAR was 3.5 (measured by UV at two different wavelengths 280 nm and 252 nm).

[0206] 8.6 In vitro and in vivo studies 8.6.1 In Vitro Cytotoxicity Data In vitro cytotoxicity assay: JIMT-1 (HER2 positive) cells were plated and left overnight (12 hours), after which serial dilutions of drugs (trastuzumab, naratuximab, ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7, or ADC-9) were added to the cells. After 72 hours of incubation, CellTiter-Glo 2.0 (Promega kit (following manufacturer's instructions)) was added to each well, after which luminescence was read from a luminometer. Relative IC50 was calculated using GraphPad Prism. The results of the in vitro cytotoxicity assay are shown in Figures 7-11. 8.6.2 In Vivo Efficacy Data A) Briefly, NMRI mice were inoculated with Jimt-1 breast cancer cells (5 × 10 per mouse). 6 The tumor was 150 mm 3 When mice reached an average size of 100 μg / kg, they were randomized into three groups of 8 (day 0) and treatment began the following day (day 1). Non-control mice received two (100 μl) intravenous injections of 5 mg / kg ADC-2 or ADC-4, one week apart. The vehicle was phosphate buffered saline (PBS). Control mice received two (100 μl) intravenous injections of vehicle. After tumor cell inoculation, animals were monitored periodically as follows: Mean tumor volume and body weight results from the study are shown in Figures 12 and 13. 8.6.3 Periodic monitoring After tumor cell inoculation, animals were checked daily for morbidity and mortality. During regular monitoring, animals were checked for tumor growth and any effects of treatment on behavior, such as motility, food and water consumption, weight gain / loss (body weight was measured twice a week after randomization), eye / hair matting, and any other abnormalities. Mortality and observed clinical signs were recorded in detail for each individual animal. Tumor volumes were measured twice weekly in two dimensions using calipers and volumes are expressed in mm3 using the formula: V=(L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Dosing and tumor and body weight measurements were performed in a laminar flow cabinet. B) Briefly, NOD / SCID mice were inoculated with Jimt-1 breast cancer cells (5x106 cells per mouse). When tumors reached an average size of 150mm3, mice were randomized into 5 groups of 8 mice, and treatment was initiated on the same day (day 0). Non-control mice received two intravenous injections of 5mg / kg ADC-1, ADC-2, ADC-3, or ADC-9, 1 week apart. The vehicle was PBS. Control mice received two intravenous injections of vehicle (100μl). After tumor cell inoculation, animals were monitored periodically as described above. Mean tumor volume and weight results from the study are shown in Figures 14 and 15.

[0207] 8.6.4 In Vivo Efficacy Data NMRI nude mice were inoculated with MV4;11 acute myeloid leukemia cells (5x106 cells per mouse). When tumors reached an average size of 115 mm3, mice were randomized (D0) into groups of 8 mice for vehicle (group 1) or 6 mice for treatment conditions (groups 2 and 3), with treatment starting the next day (D1). Non-control mice received a single intravenous injection of 10 mg / kg naratuximab or ADC-4. The vehicle was PBS. Control mice received a single intravenous injection of vehicle (5 mL / kg). After tumor cell inoculation, animals were monitored periodically as described above. The results of the mean tumor volume are shown in Figure 16. NOD / SCID mice were irradiated with 200 rad Co60 for 24 hours, and then inoculated with THP-1 acute myeloid leukemia cells in the tail to allow tumor seeding (1x107 cells per mouse). Mice were randomized into 4 groups of 10 mice (randomization was performed based on the "matched distribution" method / "stratified" method (StudyDirectorTM software, version 3.1.399.19) / randomized block design) (D0) and treatment was started 7 days later (D7). Non-control mice received a single intravenous injection of 5 mg / kg naratuximab, ADC-2, or ADC-4. The vehicle was PBS. Control mice were injected with vehicle (5 mL / kg). After tumor cell inoculation, animals were monitored periodically as described above. The survival percentage results from the study are shown in Figure 17. NOD / SCID mice were inoculated with MOLM-13 luciferase acute myeloid leukemia cells in the tail to seed tumors (2x106 cells per mouse). Mice were randomized based on total flux values ​​3 days after inoculation in 4 groups of 8 mice (D0), and treatment was started 7 days after inoculation (D4). Non-control mice received a single intravenous injection of 5 mg / kg naratuximab, ADC-2 or ADC-4. Vehicle was PBS. Control mice were injected with vehicle (5 mL / kg). After tumor cell inoculation, animals were monitored regularly as described above, and tumor growth was imaged twice a week after luciferin (using IVIS Spectrum BL from PerkinElmer). Results of tumor growth and % survival are shown in Figures 18a and 18b. Tumors were seeded by inoculating the tails of NOD / SCID mice with MOLM-13 luciferase acute myeloid leukemia cells (2 × 106 cells per mouse). Mice were randomized based on total flux values ​​7 days after inoculation in five groups of eight mice, and treatment was initiated on the day of randomization (D0). Non-control mice received a single intravenous injection of 3 mg / kg ADC-4, 1 mg / kg ADC-4, 0.3 mg / kg ADC-4, or 3 mg / kg naratuximab emtansine (Debio-1562). Vehicle was PBS. Control mice were injected with vehicle (5 mL / kg). After tumor cell inoculation, animals were monitored periodically as above, and tumor growth was imaged twice weekly after luciferin administration (using an IVIS Spectrum BL from PerkinElmer). The results of tumor growth and % survival are shown in Figures 19a, 19b, 20a and 20b.

[0208] 8.6.5 Cell Binding, Internalization and Cytotoxicity in AML Cell Lines CD37 expressing AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 were labeled with Live / Dead near IR (Thermo Fisher, L10119), Fc blocked with HumanTruStain FcX (Biolegend, 422302) and incubated with 1 μg / mL naratuximab-PE (Biolegend, 99341, lot B304638, custom conjugated). Phycoerythrin fluorometric beads (BD, 340495) were used as a reference to estimate bound naratuximab antibody per cell. Cells and beads were finally acquired on an Attune NxT flow cytometer. The CD37 negative ALL cell line MOLT-4 was included as a negative control. The number of bound naratuximab antibody / cell is shown in Figure 21a. CD37-expressing AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 cells were labeled with Live / Dead near IR (Thermo Fisher, L10119) and then incubated with 0.5 μg / mL anti-CD37-Alexa Fluor 488 (clone K7153A) for 30 min or 2 h either on ice or at 37°C. Cells were washed twice and incubated with quenching anti-Alexa Fluor 488 antibody (Thermo fisher, 710369) before fixation (Biolegend, 422101). After fixation, cells were analyzed by flow cytometry. Data are expressed as absolute internalization rate + / - SD (n=3), defined as the fluorescence of the quenched sample corrected for incomplete surface quenching. The CD37-negative ALL cell line MOLT-4 was included as a negative control. Naratuximab absolute internalization rate is shown in Figure 21b. In vitro cytotoxicity assay: MOLT-4, MV-4-11, MOLM-13, HL-60 and THP-1 cells were plated and after overnight rest (12 hours), serial dilutions of ADC-4 (eight 10-fold dilutions, 1 μM to 0.1 pM in triplicate) were added to the cells. After 72 hours of incubation, the plates were inspected under an inverted microscope to ensure control growth and sterile conditions. Luminescence was then read following the manufacturer's instructions after adding 100 μL of CellTiterGlo 2.0 (Promega, G9242) to each well. Relative IC50 was calculated for each cell line using GraphPad Prism. The percentage of viability relative to the control (untreated cells) curve is shown in Figure 21c.

[0209] 8.6.6 In Vitro Cytotoxicity Data MV-4-11 cells were plated and left overnight (12 hours) before serial dilutions of ADC-4, Debio1562 or naratuximab (eight 10-fold dilutions, 1 μM to 0.1 pM in triplicate) were added to the cells. After 72 hours of incubation, plates were inspected under an inverted microscope to ensure control growth and sterile conditions. Luminescence was then read following the manufacturer's instructions after adding 100 μL of CellTiterGlo 2.0 (Promega, G9242) to each well. Relative IC50s were calculated for each cell line using GraphPad Prism. The percentage of viability relative to the control (untreated cells) curve is shown in Figure 22a. THP-1 cells were plated and left overnight (12 hours) before serial dilutions of ADC-4 or Debio1562 (nine 10-fold dilutions, 1 μM to 0.01 pM in triplicate) were added to the cells. After 72 hours of incubation, the plates were inspected under an inverted microscope to ensure control growth and sterile conditions. Luminescence was then read following the manufacturer's instructions after adding 100 μL of CellTiterGlo 2.0 (Promega, G9242) to each well. Relative IC50s were calculated for each cell line using GraphPad Prism. The percentage of viability relative to the control (untreated cells) curve is shown in Figure 22b.

[0210] 8.6.7 In Vivo Efficacy Compared to AML Standard of Care NCG mice were inoculated with MV4;11-Luc acute myeloid leukemia cells via the tail vein to seed tumors (2 × 10 per mouse). 7 Mice were then cultured for 14 days after inoculation to determine the bioluminescence results for each animal (total flux, photons / sec / cm 2) into four groups of eight mice, with treatment starting immediately after randomization (D14). Non-control mice received a single intravenous injection of ADC-4 at 1 mg / kg or 5 mg / kg, or five consecutive daily intravenous injections of 3.5 mg / kg azacitidine in addition to 100 mg / kg venetoclax administered orally on days 1-6 and 9-14. The vehicle was PBS, except for venetoclax, which was formulated in 60% phosal 50 propylene glycol, 30% polyethylene glycol 400, and 10% ethanol. Control mice were injected with vehicle (10 μL / g). After tumor cell inoculation, animals were monitored daily for clinical signs, morbidity, and mortality, and body weights were measured twice weekly. Tumor growth was imaged twice weekly 15 minutes after D-luciferin injection (PerkinElmer, XenoLight D-Luciferin K+Salt) using an IVIS Spectrum BL from PerkinElmer. Tumor growth results in luminescence signal are shown in FIG. 23.

[0211] 8.6.8 Pharmacokinetic profile in mice Three Swiss male mice were treated with an intravenous injection of 5 mg / kg naratuximab or ADC-4 (vehicle was PBS) and blood was collected by microsampling with a K3EDTA microcapillary at 5 min, 1 h, 6 h, 24 h, 48 h and 72 h after treatment. The microcapillary blood samples were centrifuged (2000 g, 5 min, +4° C.) as soon as possible to allow plasma processing. Plasma samples were frozen at −80° C. and further analyzed for the determination of the concentration of total antibody using a qualified analytical immunoassay procedure. Toxicokinetic parameters were estimated using Phoenix pharmacokinetic software. PK parameters and plasma concentrations are shown in FIG. 24a. Three CD1 female mice were treated with an intravenous injection of 5 mg / kg ADC-4 (vehicle was PBS) and blood was collected by microsampling with a K2EDTA microcapillary at 5 min, 1 h, 6 h, 24 h, 48 h and 96 h after treatment. The microcapillary blood samples were centrifuged (1500 g, 10 min, +4° C.) as soon as possible to allow plasma processing. Plasma samples were frozen at −80° C. and further analyzed for the determination of the concentrations of total antibodies (Total Ab) and total ADC using two qualified analytical immunoassay procedures. Toxicokinetic parameters were estimated using Phoenix pharmacokinetic software. PK parameters and plasma concentrations are shown in FIG. 24b and Table 4. Table 4

[0212] [Table 6] 8.6.9 Human and mouse plasma stability ADC-2, ADC-4, Enhertu and ADCETRIS (Enhertu and ADCETRIS were used as controls) were spiked into human and mouse plasma at a concentration of 1 mg / mL. After 0, 24, 48 and 96 hours of incubation, the ADC was captured with streptavidin magnetic beads coated with biotin anti-IgG-Fc (human) conjugate (100 μL conjugate / 250 μL bead slurry). For ADC-2, 40 μL of ADC-spiked plasma was mixed with 60 μL of PBS and incubated with 20 μL of beads at room temperature for 1 hour at 900 rpm (Eppendorf thermomixer). For ADC-4, Enhertu and ADCETRIS, 20 μL of ADC-spiked plasma was mixed with 80 μL of PBS and incubated with 20 μL of beads at room temperature for 1 hour at 900 rpm (Eppendorf thermomixer). The beads were then washed three times with 500 μL PBS, eluted with 2 mM HCl solution, and neutralized with 0.5 M ammonium bicarbonate pH 8. ADC-2 and ADCETRIS were deglycosulated (PNGase F) and ADCETRIS was reduced with DTT. Analysis of the samples was performed by LC-MS (Waters BioAccord). The DAR reduction of the ADCs was measured and is shown in Figure 25a (ADC-2) and Figure 25b (ADC-4).

[0213] 8.6.10 In vitro cytotoxicity against a panel of DLBCL cell lines A3 / KAW, DOHH-2, HBL-1, KARPAS-422, OCI-LY19, OCI-LY3, OCI-LY7, Pfeiffer, U-DHL-2, U-DHL-4, U-DHL-5, U-DHL-6, U-DHL-8, Toledo, U-2932, WSU-DLCL2 and WSU-NHL cells were plated on day 0 and serial dilutions of ADC-4, Debio1562 or naratuximab (nine 10-fold dilutions, 1 μM to 0.01 pM in triplicate) were added to the cells on day 1. After 72 hours of incubation, plates were inspected under an inverted microscope to ensure control growth and sterile conditions. Luminescence was then read following the manufacturer's instructions after adding 50 μL of CellTiterGlo (Promega, G7572). Relative IC50s were calculated using GraphPad Prism. IC50 values ​​for ADC-4, Debio1562 and naratuximab are shown in Table 5. Table 5

[0214] [Table 7] 8.6.11 In vivo efficacy in DLBCL models SCID beige female mice were inoculated with Farage DLBCL cells in the tail to seed tumors (2x107 cells per mouse). Mice were randomized based on body weight 7 days after inoculation into 6 groups of 10 mice, and treatment was initiated on the day of randomization (D0). Non-control mice received a single intravenous injection of 1 mg / kg ADC-4 or 10 mg / kg Debio1562, alone or in combination with three weekly intravenous injections of 10 mg / kg Rituximab. The vehicle was PBS. Control mice were injected with vehicle (5 mL / kg). After tumor cell inoculation, animals were monitored regularly for clinical signs, morbidity and mortality, and body weights were measured twice weekly. Results of survival endpoints are shown in Figure 26.

[0215] 8.6.12 Screening Study with ADC-4 at 5 and 50 mg / kg The objective of this study was to determine the potential toxicity of ADC-4 when administered intravenously (via bolus injection) as a single dose to female mice. Additionally, the toxicokinetic properties of ADC-4 as both a total antibody (TAb) and a total ADC were determined. The study design was as follows.

[0216] [Table 8] Table 6: Experimental design (based on most recent body weight measurements) The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology parameters (hematology and clinical chemistry), toxicokinetic parameters, organ weights, and gross and microscopic examinations. CD-1® IGS female mice were administered a single intravenous injection of either 5 mg / kg or 50 mg / kg ADC-4. All animals administered a single dose of 5 or 50 mg / kg ADC-4 survived to termination. Median tmax of total ADC and TAb were observed at the first sampling time (5 min post-dose). Systemic exposure to total ADC and TAb (mean C0, Cmax, and / or AUCtlast) increased approximately dose-proportionally with increasing doses of ADC-04 from 5 to 50 mg / kg. Plasma exposure to TAb (mean C0, Cmax, and AUCtlast) was slightly higher than total ADC following administration of 5 and 50 mg / kg ADC-4. There were no ADC-4-related clinical signs in animals administered a single dose of 5 mg / kg or 50 mg / kg ADC-4. There were no ophthalmologic findings at either of these dose levels. Ten days after administration, there were increases in reticulocyte counts (with or without increases in leukocyte, neutrophil, eosinophil, monocyte, and lymphocyte counts), as well as increases in triglycerides, total protein, globulin concentrations, and calcium and / or phosphorus levels. At necropsy, ADC-4 was found to induce splenic enlargement with a dose-related incidence, which correlated with increased spleen weight and extramedullary hematopoiesis (EMH). Increased kidney and liver weights were also noted without correlation. There were no significant microscopic findings 10 days after injection, and injection of ADC-4 was well tolerated locally.

[0217] 8.6.13 Dose Ranging (DRF) ADC-4 - Determining Potential Toxicity of ADC-4 ADC-4 was administered intravenously to CD-1® IGS female mice (two separate bolus injections of the same dose (25 mg, 50 mg or 100 mg / kg / adm) given on days 1 and 8 (7 days apart)) to assess reversibility and / or delayed onset of any findings over a 10 day observation period. In addition, the toxicokinetic properties of ADC-4 (total ADC and total antibody) were determined. A control group of CD-1® IGS female mice received the control item: phosphate buffered saline (PBS). The study design was as follows.

[0218] [Table 9] Table 7: Experimental design (based on most recent body weight measurements) The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology parameters (hematology and clinical chemistry), toxicokinetic parameters, organ weights, and gross and microscopic examinations. All animals receiving two injections of 100 mg / kg / adm ADC-4 were euthanized early or found dead on day 10 due to severe weight loss and food consumption accompanied by mature fur, hunched posture, and reduced activity. There were no relevant ADC-4-related clinical signs in animals receiving two injections of 25 mg / kg / adm ADC-4 or two injections of 50 mg / kg / adm ADC-4, and there were no early deaths in these animal groups. In animals receiving two injections of 25 μL ADC-4 or two injections of 50 mg / kg / adm ADC-4, higher mean weight gain was observed between days 1 and 17 (when compared to mean control values) without any effect on food consumption. There were no ophthalmological findings. In mice receiving two injections of 100 mg / kg / adm ADC-4, a significant increase in total white blood cell count was observed (mainly due to an increase in neutrophil and monocyte counts) two days after administration of the second injection. Reductions in red blood cell, reticulocyte and platelet counts, as well as hematocrit, were also noted. Significant increases in enzyme activities (AST, ALT and ALP), moderate increases in total bilirubin, total protein and especially globulin concentrations (and a resulting decrease in the A / G ratio), as well as decreases in cholesterol, triglyceride and glucose concentrations were observed. An increase in phosphorus concentration was also observed. In mice administered two injections of ADC-4 at 25 mg / kg / adm or two injections of ADC-4 at 50 mg / kg / adm, an increase in total white blood cell counts (when compared to mean control values) was observed 10 days after administration of the second injection (primarily due to increased neutrophil, lymphocyte, and monocyte counts). A decrease in platelet counts was observed in animals administered two injections of ADC-4 at 50 mg / kg / adm (this was not observed in animals administered two injections of ADC-4 at 25 mg / kg / adm). Notable changes in clinical chemistry consisted of increases in enzyme activities (AST, ALT, and ALP), total protein (especially albumin concentrations), and decreases in triglyceride concentrations, which were generally more pronounced in animals receiving two injections of 50 mg / kg / adm ADC-4.

[0219] Microscopically, the following findings were noted: In animals treated with two injections of >25 mg / kg / adm, ADC-4-related findings were present in the liver (diffuse hepatocellular hypertrophy, increased extramedullary hematopoiesis and mitosis, correlating with higher body weight and gross hypertrophy), spleen (dose-related extramedullary hematopoiesis and single-cell necrosis, correlating with higher body weight and gross hypertrophy / hypopigmentation), and duodenum (single-cell necrosis of mucosal and submucosal epithelial cells). At the injection site, perivascular subacute inflammation reflected minor local tissue effects of ADC-4. In animals receiving two injections of >50 mg / kg / adm, ADC-4-related findings were present in the bone marrow (single cell necrosis, increased myeloid cellularity, decreased red blood cell cellularity), liver (single cell necrosis in addition to findings observed at 25 mg / kg / adm), heart (myocardial degeneration / necrosis, and extramedullary hematopoiesis), and eye (single cell necrosis in the corneal epithelium). In animals dosed with two injections of 100 mg / kg / adm, ADC-4-related findings were present in the liver (glycogen depletion), spleen (reduced white pulp cellularity), kidney (single cell necrosis of epithelial tubular and glomerular cells), gastrointestinal tract (single cell necrosis in the stomach, jejunum, and ileum, erosions / ulcers in the duodenum, jejunum, and ileum, epithelial hypertrophy / hyperplasia in the duodenum and jejunum), iliac lymph nodes (single cell necrosis and extramedullary hematopoiesis), and skin (extramedullary hematopoiesis). Conclusion: Two 100 mg / kg / adm injections of ADC-4 administered to CD-1® mice (7 days apart) were not tolerated; all animals were euthanized early or found dead on day 10 due to weight loss, significant clinical signs, and decreased appetite. In addition, there was an increase in white blood cell counts, and decreases in red blood cell, reticulocyte, and platelet counts and hematocrit, correlating with necrosis and decreased cellularity in the spleen. Two 25 mg / kg / adm or 50 mg / kg / adm injections of ADC-4 administered (7 days apart) to CD-1 mice were clinically well tolerated. Treatment induced an increase in white blood cell counts and a decrease in platelet counts after 18 days (correlated with extramedullary hematopoiesis in the spleen and liver). There was no effect on food consumption or body weight, and no clinical signs. Target organ effects were observed at all dose levels in the liver, spleen, gastrointestinal tract, kidneys, bone marrow, iliac lymph nodes, heart, eyes and / or skin. The severity and extent of findings were dose-related. Based on the above results, the no observed adverse effect level (NOAEL) for ADC-4 was considered to be 25 mg / kg / adm in mice.

[0220] 8.7 CatB-induced cleavage studies 8.7.1 Preparation of DM1-Ac-Cit and DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH DM1-Ac-Cit

[0221] [ka] DIEA (0.14 mL, 0.83 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (185 mg, 0.21 mmol, 1.0 equiv) and citrulline (72.6 mg, 0.41 mmol, 2.0 equiv) in DMSO (3.7 mL) at room temperature. After stirring for 18 h at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10-40% ACN + 0.1% TFA in water + 0.1% TFA) afforded DM1-Ac-Cit (130 mg, 0.14 mmol, 100% UV purity, 66% yield) as a white powder after lyophilization. UPLC-MS (method1): Rt = 1.33 min, m / z = 952 [MH]-.

[0222] DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH

[0223] [ka] Acetyl-L-cysteine ​​(0.32 mg, 2.17 μmol, 1.0 equiv) was added to a solution of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (5.0 mg, 2.17 μmol, 1.0 equiv) in DMF (1 mL) at room temperature. After stirring for 40 min at room temperature, TFA was added until an acidic pH was reached. Purification by preparative HPLC (5-100% ACN + 0.1% TFA in water + 0.1% TFA) afforded DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH (4.59 mg, 1.86 μmol, 99% UV purity, 94% yield) as a white powder after lyophilization. UPLC-MS (method4): Rt=2.41 min, m / z=1231 [M-2H]2-.

[0224] 8.7.2 Preparation of Exatecan-Suc-Phe-Cit and Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH Exatecan-Suc-Phe-Cit

[0225] [ka] Step 1: DIEA (0.78 mL, 4.48 mmol, 4.0 equiv) was added to a mixture of citrulline (200 mg, 1.12 mmol, 1.0 equiv) and Boc-Phe-OSu (405 mg, 1.12 mmol, 1.0 equiv) in DMF (10 mL) at room temperature. After stirring for 16 h at room temperature, the reaction mixture was filtered and then concentrated under reduced pressure. A mixture of water / CA N / TFA (1:1:0.5%) was added. The mixture was filtered and then lyophilized. Purification by preparative HPLC (10-60% ACN+0.1% TFA in water+0.1% TFA) gave Boc-Phe-Cit-OH (86 mg, 0.20 mmol, 100% UV purity, 18% yield) as a white powder after lyophilization. UPLC-MS (method3): Rt=1.27 min, m / z=423[M+H]+, 421[MH]-. Step 2: A mixture of DCM (0.9 mL) and TFA (0.9 mL) was added to Boc-Phe-Cit-OH (60 mg, 0.14 mmol, 1.0 equiv) at room temperature. After stirring at room temperature for 20 min, the reaction mixture was concentrated. Water (10 mL) and ACN (10 mL) were added and the mixture was lyophilized to give H-Phe-Cit-OH (30 mg, 83.8 μmol, 90% UV purity, 59% yield) as a white powder. UPLC-MS (method 3): Rt=0.35 min, m / z=323[M+H]+, 321[MH]-. Step: Example B1: Dihydrofuran-2,5-dione (8.4 mg, 83.5 μmol, 1.0 eq.) was added to a mixture of exatecan (36 mg, 83.5 μmol, 1.0 eq.) and DIEA (0.17 mL, 1.00 mmol, 12 eq.) in DMF (0.8 mL) at room temperature. After stirring for 10 minutes at room temperature, 1-hydroxypyrrolidine-2,5-dione (9.6 mg, 83.5 μmol, 1.0 eq.) was added, followed by HATU (31.7 mg, 83.5 μmol, 1.0 eq.). After stirring for 15 minutes at room temperature, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 eq.) was added to the reaction mixture. After stirring at room temperature for 1 h, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 equiv) was added to the reaction mixture. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-60% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit (26.4 mg, 30.8 mmol, UV purity 98%, yield 37%) as a yellow powder after lyophilization. UPLC-MS (method2): Rt=1.39 min, m / z=840[M+H]+, 838[MH]-.

[0226] Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH

[0227] [ka] DIEA (1.2 μL, 6.8 μmol, 4.0 equiv.) was added to a mixture of Exatecan-Suc-Phe-Cit-Lys(ma-C1-Lys(Peg16))-Tyr-OH (3.71 mg, 1.7 mmol, 1.0 equiv.) and Acetyl-L-cysteine ​​(0.28 mg, 1.7 μmol, 1.0 equiv.) in DMF (0.6 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20-60% ACN+0.1% TFA in water+0.1% TFA) afforded Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH (2.87 mg, 1.2 mmol, 100% UV purity, 72% yield) as a white powder after lyophilization. UPLC-MS (method 4): Rt = 2.37 min, m / z = 1176 [M+2H]2+, 1174 [M-2H]2-.

[0228] 8.7.3 CatB-induced cleavage CatB-induced cleavage of linker payloads (DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH and Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH) (Ac-Cys quenched linker of the invention) was analyzed using recombinant human cathepsin B (R&D Systems, Bio-Techne AG, cat#.953-CY-010) and UHPLC-MS / MS analysis. The enzyme was reconstituted in 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer adjusted to pH 5.0 with 1 M NaOH solution, and then activated with a 20 nM solution of dithiothreitol (DDT) for at least 15 min at room temperature. In vitro enzyme assays were performed at 37°C with a test compound at a concentration of 10 μM (2.5 μM if the test compound is an antibody drug conjugate) in the presence of 2 μg / mL of activated recombinant human cathepsin B enzyme in 25 mM MES buffer pH 5.0. The enzymatic cleavage reaction was stopped at each defined time point by mixing acetonitrile + 0.1% formic acid (FA) containing an internal standard (0.5 μM warfarin) in a 1 / 10 volume ratio. Analysis was performed using a Waters Acquity UPLC system coupled to a Waters Xevo TQ triple quadrupole mass spectrometer. UHPLC was performed using an HSS T3 1.8 μm 50×2.1 mm column (Waters) heated to 45° C. and fitted with a 2 μm insert filter precolumn (Waters), and solvent systems A1 (HO+0.1% FA) and B1 (acetonitrile+0.1% FA) at a flow rate of 0.6 mL / min and a 5-95% gradient of B1 over 2.0 min. MS / MS was performed using an electrospray ionization (ESI) interface in positive mode with specific MRM transitions for each compound. The results are shown in Figures 27 and 28.

[0229] 8.8 DRF-DM1-Ac-Cit and DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH The purpose of this study was to determine the potential toxicity of Multilink-DM 1-Ac-Cit and DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH when administered intravenously (via bolus injection). DM1-Ac-Cit was prepared as described above in Section 8.7.1.

[0230] [ka] Preparation of DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH Cysteine ​​(9.15 mg, 75.5 μmol, 2.0 equiv.) was added to a solution of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (86.9 mg, 37.8 μmol, 1.0 equiv.) in DMF (7.6 mL) at room temperature. After stirring for 5 h at room temperature, the reaction mixture was filtered using a 33 mm 0.22 μm hydrophobic filter. Purification by preparative HPLC (10-50% ACN + 0.1% TFA in water + 0.1% TFA) afforded DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH (62.4 mg, 25.8 μmol, 100% UV purity, 68% yield) as a white powder after lyophilization. UPLC-MS (method1): Rt=1.34 min, m / z=1210[M-2H]2-. Two injections of the same dose of DM1-Ac-Cit ((Test Item 1) 0.15, 0.68 or 1.4 mg / kg / adm) or two injections of the same dose of DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH ((Test Item 2) 3.5 mg / kg / adm) were administered separately (on days 1 and 8 (7 days apart)) to CD-1® IGS mice and reversibility and / or delayed onset of any findings was assessed during a 10 day observation period. Additionally, the toxicokinetic properties of DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH, DM1-Ac-Cit, and the catabolite DM1 were determined. The control group received vehicle control 1:8% ethanol / 8% polysorbate (Tween®) 80 / PBS 84%. The study design was as follows.

[0231] [Table 10] Table 8: Experimental design (adm = administration, a is based on the most recent body weight measurement). The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology parameters (hematology and clinical chemistry), toxicokinetic parameters, organ weights, and gross and microscopic examinations. There were no effects on DM1-Ac-Citor DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH-associated mortality, clinical observations, or body weight and food consumption. There were no changes in ophthalmological findings or clinical pathology parameters associated with DM1-Ac-Citor DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH. No DM1-Ac-Citor DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH-related organ weight changes were observed, and no macroscopic or microscopic findings were observed. Conclusion: Two intravenous (bolus) injections of DM1-Ac-Cit at 0.15, 0.68, and 1.4 mg / kg / adm given 7 days apart were well tolerated in female CD-1 mice. Based on these results, the no observed adverse effect level (NOAEL) was considered to be 1.4 mg / kg / adm. Similarly, DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH at 3.5 mg / kg / adm was well tolerated.

[0232] Reference example: CatB-induced cleavage study The reference compounds shown in Table 9 below were prepared in the Reference Examples according to the methods described in WO 2019 / 096867.

[0233] [Table 11] Table 9: Reference compounds containing linkers of formula (II) / (II') Peptides were prepared by standard Fmoc-based SPPS using an Activo P-11 automated peptide synthesizer (available from Activotec) and Fmoc-Xxx-Wang resin (Xxx: C-terminal amino acid; loading: 0.60 mmol / g; Bachem). Coupling reactions for amide bond formation were carried out with 3 equivalents of Fmoc-amino acids, Fmoc-NH-PEG4-COOH or Fmoc-NH-PEG5-COOH activated with HBTU (2.9 equivalents) in the presence of DIEA (7 equivalents) for 30 min at room temperature. Fmoc deprotection was carried out with a solution of 20% piperidine in DMF. Selective removal of the Mtt side chain protecting group (Lys) was carried out with DCM / TFA / TIS (94 / 1 / 5, v / v / v). For the synthesis of compounds 1-4 and 8, auristatin F (AF) was coupled after Fmoc removal by fragment condensation (3 equivalents of AF, 2.9 equivalents of HBTU, 7 equivalents of DIEA) for 30 min. For the synthesis of compounds 9 and 10, auristatin Cit (ACit) was coupled after Fmoc removal under the same conditions (3 equivalents of ACit, 2.9 equivalents of HBTU, 7 equivalents of DIEA). For the synthesis of compounds 1-4 and 8-10, after removal of Mtt by DCM / TFA / TIS (94 / 1 / 5, v / v / v), the derivative Mal-PEG4-NHS was added on the resin for 30 min (3 eq. Mal-PEG4-NHS, 7 eq. DIEA). Then, for compounds 1, 3, 8, 9 and 10, the maleimide group on the PEG chain was reacted with acetyl-cysteine ​​(Ac-Cys-OH) on the resin for 20 min via chemoselective coupling (3 eq. Ac-Cys-OH, DIEA, 7 eq.). The peptides were cleaved from the resin with simultaneous deprotection of the side chains by treatment with TFA / TISa / water (95 / 2.5 / 2.5, v / v / v; TIS=triisopropylsilane) for 60 min. After concentration of the cleavage mixture, the crude peptides were precipitated with cold diethyl ether and centrifuged. For the synthesis of compounds 5–7, after Mtt removal with DCM / TFA / TIS (94 / 1 / 5, v / v / v), the derivative Mal-PEG4-NHS was added to the resin for 30 min (3 equiv. Mal-PEG4-NHS, 7 equiv. DIEA). The maleimide residue on the PEG chain was then reacted with acetyl-cysteine ​​(Ac-Cys-OH) on the resin for 20 min via chemoselective ligation between the maleimide and a thiol (3 equiv. Ac-Cys-OH, DIEA, 7 equiv.). The Mal derivative was inserted by adding a Mal-NHS moiety to the N-terminus of Phe after Fmoc deprotection. The peptide was cleaved from the resin with simultaneous side-chain deprotection by treatment with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 60 min. After concentration of the cleavage mixture, the crude peptide was precipitated with cold diethyl ether and centrifuged. Mertansine (DM1, 1.45 equivalents) was then reacted with the terminal maleimide group via chemoselective ligation in PBS buffer at pH 7.4 and acetonitrile (ratio 2:1). For the synthesis of compounds 11-13, after Fmoc removal, the derivative Ma-NHS was added to the resin for 30 min (3 eq. Mal-NHS, 7 eq. DIEA). The maleimide residue was then reacted on the resin with acetyl-cysteine ​​(Ac-Cys-OH) for 20 min via chemoselective coupling (3 eq. Ac-Cys-OH, DIEA, 7 eq.). The peptides were cleaved from the resin with simultaneous deprotection of the side chains by treatment with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 60 min. After concentration of the cleavage mixture, the crude peptides were precipitated with cold diethyl ether and centrifuged. After their purification, the derivative DM1-smcc (1.1 eq.) was reacted to the N-terminus of the linker in solution in DMF and 4-methylmorpholine (6 eq.) for 4 h. Peptides were purified on a Waters Autopurification HPLC system coupled to an SQD mass spectrometer equipped with an XSelect Peptide CSH C18 OBD Prep column (130 Å, 5 μm, 19 mm × 150 mm) using solvent systems A (0.1% TFA in water) and B (0.1% TFA in acetonitrile) at a flow rate of 24 mL / min and a 20-60% gradient of B over 30 min. Appropriate fractions were concentrated and lyophilized. Purity was determined on a Waters Acquity UPLC System coupled to an SQD mass spectrometer equipped with a CSH C18 column (130 Å, 1.7 μm, 2.1 mm×50 mm) using solvent systems A (0.1% FA in water) and B (0.1% FA in acetonitrile) at a flow rate of 0.6 mL / min and a gradient of 5-85% of B over 5 min, or a CSH Floro-Phenyl column (130 Å, 1.7 μm, 2.1 mm×50 mm) using solvent systems A (0.1% FA in water) and B (0.1% FA in acetonitrile) at a flow rate of 0.9 mL / min and a gradient of 5-95% of B over 2.9 min. MS analysis was performed using an electrospray ionization (ESI) interface in positive and negative modes. The analytical results of the compounds obtained in the Reference Examples are shown in Table 10 below.

[0234] [Table 12] Table 10: Analysis of compounds 1-13 The propensity of compounds 1-13 to be cleaved by cathepsin B was assessed according to an in vitro enzymatic cleavage assay using recombinant human cathepsin B and UHPLC-MS / MS analysis, as described below. The reference compounds Cys-MC-Val-Cit-PABC-MMAF and MMAF were used as positive controls. The enzyme was reconstituted in 25 mM MES buffer adjusted to pH 5.0 with 1 M NaOH solution and then activated with a 20 nM solution of DDT for at least 15 min at room temperature. In vitro enzyme assays were performed with test compounds at a concentration of 10 μM (2.5 μM when the test compound was an antibody drug conjugate) in the presence of 2 μg / mL of activated recombinant human cathepsin B enzyme in 25 mM MES buffer pH 5.0 at 37° C. The enzyme cleavage reaction was stopped for each defined time point by mixing an equal volume of acetonitrile + 0.1% FA containing an internal standard (8 μM warfarin). The analysis was carried out using a Waters Acquity UPLC system coupled to a Waters Xevo TQ triple quadrupole mass spectrometer. UHPLC was performed using a BEH C8 1.7 μm 100×2.1 mm or BEH C18 1.7 μm 50×2.1 mm or HSS T3 1.7 μm 50×2.1 mm column heated to 45° C. or 50° C. depending on the test compound and fitted with a 2 μm insert filter precolumn (available from Waters), and solvent systems A1 (HO+0.1% FA) and B1 (acetonitrile+0.1% FA) with a flow rate of 0.6 mL / min and a 10-95% gradient of B1 over 1.9 min. MS / MS was performed using an electrospray ionization (ESI) interface in positive mode, using specific MRM transitions for each test compound. The results are shown in Table 11 below.

[0235] [Table 13] Table 11: CatB-induced cleavage studies of compounds 1, 3, 5, 6-8, 11 and 12 (reference compound: Cys-MC-Val-Cit-PABC-MMAF) From these results, it is clear that exo-CatB cleavage and drug release (AF-Arg, AF-Cit, ACit, DM1-Mal-Phe-Lys, DM1-Mal-Phe-Cit, DM1-Mcc-Phe-Cit) in compounds 1, 3, 5, 6-8, 11 and 12 occurred simultaneously and was extremely rapid. For example, CatB-induced drug release from compound 5 was 20 times more rapid compared to the reference PABC compound Cys-MC-Val-Cit-PABC-MMAF. The rapid cleavage rates in compounds 18 and 11-12 demonstrate the high selectivity and binding affinity of the compounds of the invention containing linkers of formula (II) or (II') for the exopeptidase activity of CatB. Moreover, it was surprisingly found that the presence of an Ac-Cys-PEG4 moiety (corresponding to residue Axx in formula (II) or (II')) on the side chain of the Lys residue does not have a detrimental effect on the binding affinity of the compounds to CatB. These results also show, in contrast, a significantly slower rate of cleavage by an endopeptidase-based mechanism of CatB, as achieved with the PABC linker system (e.g., reference compound Cys-MC-Val-Cit-PABC-MMAF). As particularly notable examples, compounds 11 and 12 are spontaneously cleaved by exo-CatB (T 1 / 2 <1 min), demonstrating highly favorable binding properties of substrates based on linkers of formula (II) / (II'). The favorable interaction between the C-terminal Tyr and the closing loop of CatB is believed to contribute significantly to the rapid cleavage rates observed with the compounds.

Claims

1. The following general formula (I): 【Chemistry 1】 (In the formula, D represents a moiety derived from a drug, said drug being selected from a carboxyl-containing drug such as auristatin F (AF), a thiol-containing drug such as mertansine (DM1) or ravtansine (DM4), an amino-containing drug such as monomethyl auristatin F (MMAF) or exatecan, and a hydroxyl-containing drug such as Maaa-1181a, or preferably selected from a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug; if more than one (D) is present, each (D) is independently selected from a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug, and the moieties (D) are preferably identical to each other; X represents a divalent group containing 1 to 7, preferably 2 to 6, more preferably 2 to 5 skeletal atoms independently selected from C, N, O, and S, and is covalently bonded to (D) through an atom selected from C, S, N, and O derived from a carboxyl, thiol, amino, or hydroxyl functional group contained in (D); Y is a divalent group containing one or more atoms selected from C, N, O, P and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), and most preferably a divalent group derived from cleaved hydrolyzed maleimide; L represents a linker that can be cleaved by cathepsin B; T represents a (2+n)-valent branched group; S represents a moiety derived from a compound that contains one or more, e.g., two, three or four, solubilizing groups; V represents a moiety derived from a vector group capable of interacting with a target cell; n is an integer from 1 to 4, preferably 1 or 2, and more preferably 1; m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8. A compound represented by the formula:

2. (L) is represented by the following general formula (II) or (II′): 【Chemistry 2】 (In the formula, Axx represents a moiety derived from a trifunctional amino acid, with the proviso that Axx in formula (II) is not a moiety derived from an amino acid of the (D) configuration; Ayy represents a moiety derived from an amino acid selected from Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-aminobutyric acid (Abu), ornithine (Orn), Ser, Thr, Leu, and Ile; or Ayy in formula (II) represents a moiety derived from an amino acid selected from homotyrosine (homoTyr), homophenylalanine (homoPhe), beta-phenylalanine (betaPhe) and beta homophenylalanine (beta homoPhe), Tyr (OR1) and homoTyr (OR １ ) (wherein, R １ Ha-(CH ２ CH ２ O) ｎ１ -R ２ where R ２ represents a moiety derived from an amino acid selected from the group consisting of: Dxx represents a single covalent bond or a moiety derived from an amino acid with a hydrophobic side chain, preferably a single covalent bond or a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe and Ala, more preferably a single covalent bond or a moiety derived from Phe or Val; Dyy represents a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, preferably a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), more preferably a moiety derived from Arg or Cit; with the proviso that when Dxx is a moiety derived from an amino acid with a hydrophobic side chain, Dyy is a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, and when Dxx is a single covalent bond, Dyy is a single covalent bond, a moiety derived from Phe, or a moiety derived from an amino acid with a basic side chain; Z represents a group covalently bonded to the C-terminus of Ayy or Axx selected from -OH and -N(H)(R), where R represents a hydrogen atom, an alkyl group or a cycloalkyl group, preferably -OH; * indicates a covalent bond to (T); ** indicates a covalent bond to (X) The compound according to claim 1 ,

3. At least one of Axx and Ayy is one of the following: Axx represents a moiety derived from an amino acid selected from Glu, 2-aminopimelic acid (Apa), 2-aminoadipic acid (Aaa), Dap, Dab, Lys, Orn, Ser, Ama, and homolysine (homolysine), preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn, and homolysine, more preferably a moiety derived from Orn or Lys, and most preferably a moiety derived from Lys; In formula (II), Ayy is Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr, homoTyr, Tyr (OR １ ) and homoTyr(OR １ ) (wherein, R １ Ha-(CH ２ CH ２ O) ｎ１ -R ２ where R ２ is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24), preferably Phe, homoPhe, Tyr, homoTyr, Tyr (OR １ ) and homoTyr(OR １ ), more preferably a moiety derived from Phe or Tyr, most preferably a moiety derived from Tyr; Ayy in formula (II') represents a moiety derived from an amino acid selected from Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe and Ser, more preferably a moiety derived from Phe or Ser, and most preferably a moiety derived from Phe; 3. The compound of claim 2 defined as follows:

4. (X) represents a divalent carbonyl or thiocarbonyl-containing group, preferably represented by the following formulae (IIIa) to (IIIf): 【Chemistry 3】 (In the formula, n2, n3 are each independently selected from 0 to 5, preferably 0, 1 or 2, more preferably 0 or 1; n4, n5 are each selected from 0 or 1; Each A is independently selected from O and S, preferably O; *** represents a covalent bond to (D); **' represents a covalent bond to (L) The compound according to any one of claims 1 to 3, wherein the compound is a group represented by any one of the following formulas:

5. (X) is represented by the following formulas (IVa) to (IVj′): 【Chemistry 4】 【change】 【change】 (In the formula, *** represents a covalent bond to (D); **' represents a covalent bond to (L); However, when (X) is represented by formula (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVp), (IVr), (IVt), (IVv), (IVw), (IVx), (IVy) or (IVz), (D) in formula (I) represents an amino-containing drug or a hydroxyl-containing drug, and (X) is represented by formula (IVj), (IVq) When (X) is represented by formula (IVa'), (IVb'), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (IVy') or (IVj'), (D) in formula (I) represents a carboxyl-containing drug. The compound according to any one of claims 1 to 3, which is one of the compounds represented by the formula:

6. The compound of claim 5, wherein the compound (X) is represented by formula (IVb'), (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), preferably formula (IVc) or (IVm).

7. The solubilizing groups contained in (S) are each independently selected from the following: a moiety comprising one or more ionic or ionizable groups, such as ammonium, guanidinium, sulfate or sulfonate groups, preferably derived from Arg, (D)-Arg, Dap, (D)-Dap, Dab, (D)-Dab, Orn, (D)-Orn, Lys, D-Lys or carnitine; a sugar moiety selected from monosaccharides, disaccharides and linear or branched oligosaccharides, particularly linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein the monosaccharide units in the monosaccharides, disaccharides and oligosaccharides are each independently selected from glucose, fructose, mannose, ribose and galactose; and Polyalkylene oxide groups, preferably C ２～３ A polyalkylene oxide group, more preferably C ２～３ polyalkylene oxide groups each independently containing from 6 to 200, preferably from 10 to 150, more preferably from 12 to 80 repeat units; The compound according to any one of claims 1 to 6, selected from the group consisting of:

8. (S) is a moiety derived from a compound containing one or more polyethylene oxide groups, preferably the polyethylene oxide groups each independently contain from 6 to 200, more preferably from 10 to 150, and most preferably from 12 to 80 repeat units; Preferably the following sub-formula (V): 【Chemistry 5】 (In the formula, n3 is an integer from 6 to 200, preferably from 10 to 150, more preferably from 12 to 80; **** indicates a covalent bond to (T); X １ is selected from a single covalent bond, —(C═O)—, and —N(R)—, where R represents a hydrogen atom, an alkyl group, or a cycloalkyl group; X ２ represents an alkyl group having 1 to 6 carbon atoms, an acetyl group, or -(CH ２ ) ｎ４ -CO ２ H group, a thiocarbonyl-containing group, a -(CH ２ ) ｎ４ OR group, -(CH ２ ) ｎ４ -SO ３ H group, -(CH ２ ) ｎ４ -(C=A)-N(R) ２ group, -(CH ２ ) ｎ４ -N(R) ２ A represents an amino-containing group such as a group, wherein A is O or S, each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group, and n4 is an integer from 1 to 6; X ２ is -CH ３ , -CH ２ CH ２ OH, or the following formula (VI): 【Chemistry 6】 (In the formula, Each A is independently selected from O and S, preferably O; Each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; n5 and n6 each independently represent an integer from 1 to 6, preferably 1 or 2. It is preferable that the group is represented by the following formula: X ２ is most preferably -CH ３ and When more than one (S) is present, each (S) is preferably a moiety of formula (V) above. The compound according to any one of claims 1 to 7, represented by:

9. (T) is represented by the following formula (VII): 【Chemistry 7】 (In the formula, each AA independently represents a moiety derived from a trifunctional amino acid such as a diamino-carboxylic acid, an aminodicarboxylic acid, an azidoamino acid, or an alkyne-containing amino acid, preferably derived from an amino acid selected from N-ε-propargyloxycarbonyl-L-lysine (Lys(Poc)), Asp, GIu, Orn, Lys, Dab, and Dap, more preferably derived from Lys(Poc), GIu, Orn, or Lys, most preferably derived from Lys; a represents a covalent bond to (Y); When n=1, the side chain derived from a trifunctional amino acid is covalently attached to either (L) or (S) and the C-terminus is covalently attached to another moiety (S) or (L), respectively; When n=2, 3, or 4: *' indicates a covalent bond to (L); ****' indicates a covalent bond to (S); n is as defined in claim 1. The compound according to any one of claims 1 to 8, represented by:

10. (T) is represented by the following formula (VIII) or (IX): 【Chemistry 8】 (In the formula, AA1 and AA2 are each independently a moiety derived from a trifunctional amino acid such as a diamino-carboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn or Lys, most preferably a moiety derived from Lys; a represents a covalent bond to (Y); In formula (IX), the side chain derived from a trifunctional amino acid is covalently attached to either (L) or (S) and the C-terminus is covalently attached to the other moiety (S) or (L), respectively; In formula (VIII), *' represents a covalent bond to (L) and ****' represents a covalent bond to (S). The compound according to any one of claims 1 to 8, represented by:

11. (D) is the following: (i) antineoplastic agents such as: DNA alkylating agents, such as duocarmycins Topoisomerase inhibitors, e.g. doxorubicin RNA polymerase II inhibitors, such as α-amanitin DNA cleaving agents such as calicheamicin Antimitotic or microtubule disrupting agents, such as taxanes, auristatins, or maytansinoids Metabolic antagonists such as gemcitabine derivatives Kinesin spindle protein inhibitors such as filanesib. Kinase inhibitors such as ipasertib or gefitinib Nicotinamide phosphoribosyltransferase inhibitors Matrix metallopeptidase 9 inhibitors Phosphatase inhibitors such as microcystin-LR (ii) Immunomodulators such as fluticasone (iii) anti-infective agents such as rifamycin, clindamycin, or reptamulin, and (iv) radioisotopes, metabolites, pharma- ceutically acceptable salts, and / or prodrugs of any of the preceding agents; with the proviso that the drug selected from (i)-(iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, or a hydroxyl-containing drug; and When more than one (D) is present, each said (D) is independently selected from moieties (i)-(iv) above, and the moieties (D) are preferably identical to each other; The compound according to any one of claims 1 to 10, wherein the moiety is derived from a drug selected from:

12. The compound according to any one of claims 1 to 11, wherein (D) is a moiety derived from a drug selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBD) and dimers thereof, indilinobenzodiazepines (IBD) and dimers thereof, or radioisotopes and / or pharma- ceutically acceptable salts thereof; preferably, a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1, and DM4; more preferably, a moiety derived from DM1 or DM4.

13. The following general formula (X) or (X′): 【Chemistry 9】 (In the formula, Axx in formula (X) and formula (X') represents a moiety derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama and homo-Lys, preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys, more preferably a moiety derived from Lys; In formula (X), Ayy is Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr, homoTyr, Tyr (OR １ ) and homoTyr(OR １ ) (wherein, R １ Ha-(CH ２ CH ２ O) ｎ１ -R ２ (In the formula, R ２ is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24), preferably Phe, homoPhe, Tyr, homoTyr, Tyr (OR １ ) or homoTyr(OR １ ), more preferably a moiety derived from Tyr; Ayy in formula (X') represents a moiety derived from an amino acid selected from Phe, homoPhe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe or Ser, more preferably a moiety derived from Phe or Ser; D, Dxx, Dyy, X, Y, T, S, V, Z, m and n have the meaning as defined in any one of claims 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12; preferably at least one, such as 2, 3, 4, 5, 6, 7 or 8 of D, Dxx, Dyy, X, Y, T, S and Z is as follows: (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (b) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homoPhe and Ala, preferably a moiety derived from Phe or Val; (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit; (d) X is a group represented by formula (III) (wherein n2 is 1 or 2), or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound, and derivatives thereof, preferably a group derived from maleimide and its derivatives (e.g., cleaved hydrolyzed maleimide), more preferably a group derived from cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) Z is -OH; (defined as The compound according to any one of claims 1 to 12, represented by:

14. In formula (X), Dyy-Dxx-Axx-Ayy are each independently Arg-Lys-Phe (wherein Dyy is a covalent bond), Arg-Lys-homoPhe (wherein Dyy is a covalent bond), Arg-Lys-Tyr (wherein Dyy is a covalent bond), Cit-Lys-Phe (wherein Dyy is a covalent bond), Cit-Lys-Tyr (wherein Dyy is a covalent bond), Arg-Lys- is selected from homoTyr (wherein Dyy is a covalent bond), Cit-Lys-homoTyr (wherein Dyy is a covalent bond), Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homoTyr, Phe-Lys-Lys-Phe, homoPhe-Arg-Lys-Phe, homoPhe-Cit-Lys-Tyr; and In formula (X'), each Dyy-Dxx-Ayy-Axx is independently selected from Arg-Phe-Lys (wherein Dyy is a covalent bond), Arg-Ser-Lys (wherein Dyy is a covalent bond), Cit-Phe-Lys (wherein Dyy is a covalent bond), Cit-Ser-Lys (wherein Dyy is a covalent bond), Cit-homoPhe-Lys (wherein Dyy is a covalent bond), Phe-Cit-Phe-Lys, homoPhe-Cit-Phe-Lys, and Phe-Arg-Phe-Lys; The compound of claim 13.

15. The formula: 【Chemistry 10】 (In the formula, D, X, Y, T, S, V, Z, m and n have the same meaning as defined in claim 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and preferably at least one, for example two, three, four, five or six of D, X, Y, T, S and Z are one of the following: (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (d) X is a group represented by formula (III), wherein n2 is 1 or 2, or a group represented by any one of formulas (IVa) to (IVz), preferably a group represented by any one of formulas (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, preferably a group derived from maleimide and its derivatives, for example, a cleaved hydrolyzed maleimide derivative, more preferably a group derived from a cleaved hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); (h) Z is -OH; (defined as follows) The compound according to any one of claims 1 to 14, wherein the compound is represented by one of the following formulas:

16. The formula: 【Chemistry 11】 【change】 (In the formula, m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; The number of oxyethylene repeat units (17) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or the maleimide bond to (V) may be replaced by a cleaved hydrolyzed maleimide. The compound according to any one of claims 1 to 15, wherein the compound is represented by one of the following formulas:

17. (V) represents a moiety derived from a vector group capable of interacting with a target cell, said target cell being selected from tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, cells involved in autoimmune diseases, activated cells, bone marrow cells, lymphoid cells, melanocytes, and infectious agents including bacteria, viruses, mycobacteria, fungi, preferably said target cell being selected from lymphoma cells, myeloma cells, bone marrow cells, lymphoid cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, testicular cancer cells, skin cancer cells, pancreatic cancer cells, liver cancer cells, and any cell undergoing unregulated and rapid growth and division that causes cancer, A compound according to any one of claims 1 to 16.

18. 18. The compound according to any one of claims 1 to 17, wherein (V) represents a moiety derived from a vector group selected from an antibody, an antibody fragment, a protein, a peptide and a non-peptide molecule, preferably a moiety derived from an antibody or an antibody fragment, such as a single chain antibody, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment, a chimeric antibody, a chimeric antibody fragment, a domain antibody or fragment thereof, a cytokine, a hormone, a growth factor, a colony stimulating factor, a neurotransmitter or a nutrient transport molecule.

19. (V) is represented by a moiety derived from a monoclonal antibody, wherein preferably said monoclonal antibody is selected from the group consisting of adalimumab, aducanumab, alemtuzumab, altumomab pentetate, amivantamab, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, belantamab mafadotin, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brentuximab vedotin, brodalumab, catumaxomab, cemiplimab, cetuximab, simpanemab, clivatuzuma ... Lenezumab, tetraxetan, daclizumab, daratumumab, denosumab, dinutuximab, dostarlimab, durvalumab, edrecolomab, elotuzumab, emapalumab, enfortumab, enfortumab vedotin, epcolitab, epratuzumab, epratuzumab-SN-38, etaracizumab, gemtuzumab, gemtuzumab ozogamicin, gemmab, glofitamab, girentuximab, goslanemab, ibritumomab, inebilizumab infliximab, inotuzumab, inotuzumab ozogamicin, ipilimumab, isaximab ixekizumab, J591 PSMA antibody, labetuzumab, lecanemab, roncatuximab tesirin, mogamulizumab, mosunetuzumab, necitumumab, nimotuzumab, natalizumab, naratuximab, naxitamab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oregovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, polatuzumab vedotin, prasinezumab, ramucirumab, rituximab, sacituzumab, sacituzumab govitecan, semolinemab, siltuximab an antibody selected from the group consisting of mab, solanezumab, tacatuzumab, tafasitamab, teprotumumab, tirabonemab, tocilizumab, tositumomab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, TS23, ustekinumab, vedolizumab, votumumab, zagotenemab, zalutumumab, zanolimumab, fragments and derivatives thereof; more preferably, atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab; or An antibody incorporated into an Fc fusion protein, preferably selected from belatacept, aflibercept, divaflibercept, dulaglutide, rilonacept, romiplostim, abatacept and alefacept; A compound according to any one of claims 1 to 18, which represents a moiety obtained from

20. 20. The compound or salt according to any one of claims 1 to 19, wherein (V) represents a moiety derived from an anti-HER2 antibody, an anti-CD37 antibody, an anti-PDL1 antibody or an anti-EGFR antibody, preferably a moiety derived from trastuzumab, pembrolizumab, naratuximab, atezolizumab, durvalumab, avelumab, panitumumab and cetuximab, more preferably a moiety derived from naratuximab, trastuzumab and cetuximab, most preferably a moiety derived from naratuximab and trastuzumab.

21. 21. The compound of claim 20, wherein (D) is a moiety derived from an anti-neoplastic agent, preferably a moiety derived from a drug selected from amanitin, duocarmycin, auristatin, auristatin F (AF), monomethylauristatin F (MMAF), maytansine, mertansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, taxol, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepines (PBD) and dimers thereof, indilinobenzodiazepines (IBD) and dimers thereof, or a radioisotope and / or a pharma- ceutically acceptable salt thereof.

22. 22. The compound of claim 21, wherein (D) represents a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4; more preferably a moiety derived from DM1 or DM4.

23. The formula: 【Chemistry 12】 wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; the number of oxyethylene repeat units (17) may be replaced with 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups, and / or the maleimide bond to (V) may be replaced with a cleaved hydrolyzed maleimide. and (D) represents a moiety derived from DM1.

24. The formula: 【Chemistry 13】 wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; the number of oxyethylene repeat units (17) may be replaced with 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups, and / or the maleimide bond to (V) may be replaced with a cleaved hydrolyzed maleimide. The compound according to claim 23, represented by:

25. A composition comprising a therapeutically effective amount of one or more components selected from a compound according to any one of claims 1 to 24 or a pharma- ceutically acceptable salt thereof, carriers, diluents and other excipients.

26. A compound according to any one of claims 1 to 24 or a composition according to claim 25 for use in a method for treating or preventing cancer, an autoimmune disease and / or an infectious disease.

27. 27. The compound or composition of claim 26, wherein the method is a method for treating cancer of the blood and bone marrow, preferably acute myeloid leukemia (AML).

28. 27. A compound or composition according to claim 26, wherein the compound is according to any of claims 20, 21, 22, 23 and 24, preferably according to claim 23 or 24, most preferably according to claim 24.

29. 29. A compound or composition according to any one of claims 26 to 28, administered simultaneously with, prior to or following one or more other therapeutic agents or therapies, such as chemotherapeutic agents, radiotherapy, immunotherapy agents, autoimmune disorder agents, anti-infective agents or other compounds of formula (I), in a method for treating or preventing cancer, autoimmune disease and / or infectious diseases.

30. The following general formula (XI): 【Chemistry 14】 (In the formula, D, X, L, T, S and n have the meanings according to claims 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Y′ represents a moiety that contains a conjugate group capable of forming a covalent bond with a molecule capable of interacting with a target cell (V′), such as a monoclonal antibody or antibody fragment incorporated into the Fc fusion protein, and is preferably one of the following: Preferably, an optionally substituted maleimide capable of reacting with one or two thiol groups of (V'), Preferably, an optionally substituted haloacetamide capable of reacting with the thiol group of (V'), an ester, preferably an ester capable of reacting with the side chain of the amino acid of (V'), such as an acyl halide, an N-hydroxysuccinimide ester or a phenol ester; a carbonate, preferably one that can react with the side chain of the amino acid of (V'), such as a haloformate, or a carbonate that contains a leaving group, such as N-hydroxysuccinimide or a phenol; Preferably, an isocyanate or isothiocyanate capable of reacting with the side chain of the amino acid of (V'); Preferably, an azide capable of reacting with an alkyne group contained in (V'); an alkyne, preferably an alkyne capable of reacting with the azide group contained in (V'); an amino group, preferably an amino group capable of reacting with molecule (V') in the presence of an enzyme such as transglutaminase; is a moiety that includes a conjugated group selected from wherein when (Y') is replaced with (Y) and m is 1, the compound preferably has the structure shown in claim 13 or 15.

31. 31. A kit for modifying a molecule capable of interacting with a target cell comprising a compound according to claim 30 and optionally a buffer, the pH of said buffer being preferably between 6.0 and 10, more preferably between 6.5 and 8.

0.

32. A method for modifying a molecule capable of interacting with a target cell, comprising reacting a molecule capable of interacting with a target cell, such as a monoclonal antibody or antibody fragment incorporated into an Fc fusion protein, with a compound described in claim 30.

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