RU2773881C2 - Selective sulfonation of benzodiazepine derivatives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to new methods for producing conjugates of cell-binding agent-cytotoxic agent, where the cytotoxic agent is an imine-containing cytotoxic agent carrying a maleimide group. In some embodiments, the cell binding agent (hereinafter – CBA) is covalently bound to the cytotoxic agent by means of constructed Cys, such as a constructed Cys in CH3 domain of a heavy chain, at a position corresponding to position 442 (or C442) according to EU/OU numbering in the antibody CBA. The method for producing a conjugate of cell-binding agent-cytotoxic agent includes stages of: (a) interaction of an imine fragment in the imine-containing cytotoxic agent, represented by the following formula:

, or its pharmaceutically acceptable salt, with sulfur dioxide, bisulfite salt or metabisulfite salt in an aqueous solution at pH from 2.9 to 4.0 to form a modified cytotoxic agent containing a modified imine fragment represented by the following formula:

, or its pharmaceutically acceptable salt; and (b) interaction of the modified cytotoxic agent with the cell-binding agent to form the conjugate of cell-binding agent-cytotoxic agent, where the cell-binding agent is an antibody or an antibody fragment. D is an imine-containing benzodiazepine compound; and L is a linker, wherein D is represented by the following formula:

, or its pharmaceutically acceptable salt, where R₁, R₂, R₃, R₄, R₁', R₂', R₃' and R₄' all are H; R₆ is -OMe; X₁' and Y₁' both are -H; A and A' are -O-; L'' and L''' both are H, L'is represented by the following formula -NR₅-P₁-C(=O)-(CR_aR_b)_s-C(=O)- (B1'); -NR₅-P₁-C(=O)-Cy-(CR_aR_b)_s1'-C(=O)-(B2'); -C(=O)-P₁-NR₅-(CR_aR_b)_s-C(=O)-(C1'), or -C(=O)-P₁-NR₅-Cy-(CR_aR_b)_s1'-C(=O)-(C2'), where each of R_a and R_b in each case is independently -H, (C₁-C₃)alkyl, or charged substituent, or ionizable group Q; s is an integer from 1 to 6; s1' is 0 or an integer from 1 to 6; Cy is cyclic alkyl with 5 or 6 ring carbon atoms, optionally substituted with halogen, -OH, (C₁-C₃)alkyl, (C₁-C₃)alkoxy or halogen(C₁-C₃)alkyl; P₁ is an amino acid residue or peptide containing from 2 to 20 amino acid residues; G is -CH- or -N-; R₅ in each case is independently -H or optionally substituted linear or branched alkyl with from 1 to 10 carbon atoms; L is represented by the following structural formula:

, or its pharmaceutically acceptable salt, where s1 is a site covalently bound to D; s2 is a site covalently bound to a maleimide group; R₂₃ and R₂₄ in each case are independently H or optionally substituted alkyl; m' is an integer from 0 to 10; and R^h' is H or optionally substituted alkyl.

EFFECT: obtaining a conjugate of cell-binding agent-cytotoxic agent.

72 cl, 5 dwg, 7 tbl, 8 ex

Description

RELATED APPLICATIONS

This application claims priority by filing date under 35 U.S.C. § 119(e) U.S. Provisional Application No. 62/425,761 filed November 23, 2016; the entire content of which, including all drawings, claims, description, claims and sequence listings, is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Antibody-cytotoxic agent conjugates (or "antibody-drug conjugates" (ADC) and cell-binding agent-drug conjugates are a highly effective class of anticancer agents with efficacy in a number of cancers. Cell-binding conjugates drug agent (such as an ADC) usually consists of three distinct elements: a cell-binding agent ( e.g. , an antibody) a linker and a cytotoxic moiety The cytotoxic moiety can be covalently attached to lysines on an antibody, resulting in conjugates that are heterogeneous mixtures of ADCs bearing varying amounts of drugs attached at different positions on the antibody molecule, alternatively the cytotoxic moiety can be covalently linked to cysteine thiol groups in the antibody via a thiol-reactive group, such as a maleimide group, to form site-specific ADC. Conjugation reactions between antibodies and cytotoxic agents are often carried out in water or an aqueous solution with a small amount of organic solvent necessary to solubilize the cytotoxic agents.

Benzodiazepine compounds, including tricyclic benzodiazepines such as pyrrolobenzodiazepines (PBBs) and tetracyclic benzodiazepines such as indolinobenzodiazepines, have been used as cytotoxic agents in combination with antibodies to produce ADCs that show promising antitumor activity. These benzodiazepine compounds contain imine bonds that can bind to the DNA minor groove and interfere with DNA function, leading to cell death. Benzodiazepine compounds generally have very low water solubility. To dissolve the benzodiazepine compounds in the antibody conjugation reaction, a relatively large amount of organic solvent is required, which can destabilize the antibodies.

Therefore, there is a need to develop new methods for the preparation of conjugates of cell-binding agent and imine-containing benzodiazepine drugs.

SUMMARY OF THE INVENTION

To improve water solubility, imine-containing benzodiazepine compounds can be treated with an imine-reactive reagent, such as a bisulfite salt or a metabisulfite salt, prior to the antibody conjugation reaction to form antibody-benzodiazepine conjugates. Sulfation of the imine group can increase the water solubility of benzodiazepine compounds, resulting in improved conditions for conjugation reactions with CCA, such as antibodies. In addition, nucleophilic additions between nucleophiles and α,β-unsaturated carbonyls are well known in synthetic organic chemistry. The nucleophilic addition of bisulfite to an activated maleimide olefin is one such example of this reaction. Therefore, when imine-containing benzodiazepine compounds bear a maleimide, the reactive group for covalent binding to antibodies, both the maleimide moiety and the imine moiety, can be reacted with a bisulfite salt or a metabisulfite salt to form a sulfonated maleimide and/or a sulfonated imine. Surprisingly, it has been found that the reaction of an imine-containing compound of indolinebenzodiazepine containing a maleimide group with a bisulfite salt or a metabisulfite salt at low pH can selectively and effectively influence the sulfonation of the imine group without significantly sulfonating the maleimide group, thereby increasing the reaction yield for the conjugation reaction between the compound of indolinobenzodiazepine and antibodies. In addition, the sulfonated compound of indoline benzodiazepine has increased water solubility, and as a result, much less organic solvent (eg, DMA) is required in the antibody conjugation reaction. The presence of a large amount of organic solvent in the conjugation reaction can destabilize antibodies.

The present invention provides a novel process for preparing a cell-binding agent-cytotoxic agent conjugate containing an imine-containing cytotoxic agent bearing a maleimide group covalently linked to the cell-binding agent (CBA). In some embodiments, the present invention relates to a method for preparing a cell-binding agent-cytotoxic agent conjugate comprising the steps of:

(a) the interaction of the imine fragment in the imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof with sulfur dioxide, a bisulfite salt, or a metabisulfite salt in an aqueous solution at pH 1.9 to 5.0 to form a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent with a cell-binding agent to form a cell-binding agent-cytotoxic agent conjugate,

where D is an imine-containing cytotoxic compound; and L is a linker.

In some embodiments, D is an imine-containing tricyclic or tetracyclic benzodiazepine compound.

In some embodiments, D is an imine-containing tricyclic benzodiazepine compound.

In some embodiments, D is an imine-containing tetracyclic benzodiazepine compound.

The term "imine-containing tricyclic benzodiazepine compound" as used herein refers to a compound having a monocyclic ring fused to the diazepine moiety of the benzodiazepine core. The monocyclic ring may contain one or more additional heteroatoms such as oxygen, sulfur or nitrogen and substituents such as monocyclic rings or polycyclic rings. Exemplary tricyclic benzodiazepine compounds include, but are not limited to, pyrrolobenzodiazepines (PBBs) such as those described in WO 2010/043880, WO 2011/130616, WO 2009/016516, WO 2013/177481 and WO 2012/112708.

As used herein, the term "imine-containing tetracyclic benzodiazepine compound" refers to a compound having a bicyclic ring fused to the diazepine moiety of the benzodiazepine core. A bicyclic ring is a fused bicyclic ring optionally containing one or more additional heteroatoms such as oxygen, sulfur or nitrogen. Exemplary tetracyclic benzodiazepine compounds include, but are not limited to, indolinobenzodiazepines (IGNs) such as those described in WO/2010/091150 and WO 2012/128868.

In some embodiments, D is an indolinobenzodiazepine.

In some embodiments, D is pyrrolobenzodiazepine (PBB).

The present invention also relates to cell-binding agent-cytotoxic agent conjugates and modified cytotoxic agents prepared by the methods described herein.

It should be understood that any embodiments described herein, including embodiments described in accordance with only one aspect of the invention and not in other aspects, and including embodiments that appear only in the examples, may be combined with any one or more other embodiments, unless explicitly stated to be excluded or unacceptable.

BRIEF DESCRIPTION OF GRAPHICS

FIG. 1 shows an SELC chromatogram (SULC) of a reaction mixture of an imine-containing cytotoxic agent D1 with sodium bisulfite at a pH of 3.3.

FIG. 2 shows the SELC chromatogram of the reaction mixture of the imine-containing cytotoxic agent D1 with sodium bisulfite at pH 4.75.

FIG. 3 shows an SELC chromatogram of a reaction mixture of the pbd-imine-containing cytotoxic agent talirin with sodium bisulfite.

FIG. 4A and 4B show HPLC chromatograms of the reaction mixture of the imine-containing cytotoxic agent D5 with 2.0 (FIG. 4A) or 2.5 (FIG. 4B) equivalents of sodium bisulfite.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the appended structures and claims. Although the invention will be described in connection with the listed embodiments, it should be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined by the claims. One of skill in the art will appreciate a variety of methods and materials, similar or equivalent to those described herein, that may be used in the practice of the present invention.

It should be understood that any of the embodiments described herein, including those described in various aspects of the invention and various parts of the description (including embodiments described in the examples only), may be combined with one or more other embodiments of the invention if no explicit indication of their exclusion or inadmissibility. The combination of embodiments is not limited to these specific combinations claimed by a plurality of dependent claims.

DEFINITIONS

" Alkyl " or " straight or branched alkyl " as used herein refers to a saturated linear or branched monovalent hydrocarbon radical. In preferred embodiments, straight or branched alkyl has thirty or fewer carbon atoms ( eg, C ₁ -C ₃₀ for straight chain alkyl and C ₃ -C ₃₀ for branched alkyl) and more preferably twenty or less carbon atoms. Even more preferably, straight or branched alkyl has ten or fewer carbon atoms ( ie , C ₁ -C ₁₀ for straight chain alkyl and C ₃ -C ₁₀ for branched alkyl). In other embodiments, the straight or branched alkyl has six or fewer carbon atoms ( ie , C ₁ -C ₆ for a straight chain alkyl group or C ₃ -C ₆ for a branched chain alkyl). Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl, 2- methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1 -hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3- pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl and the like. In addition, the term "alkyl" as used in the specification, examples and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl groups having substituents replacing hydrogen on one or more carbon atoms of the hydrocarbon backbone. Used in this document, the term (C _x -C _xx )alkyl or C _x-xx alkyl means a linear or branched alkyl having x-xx carbon atoms.

" Alkenyl " or " straight or branched alkenyl " refers to a monovalent hydrocarbon radical with a straight or branched chain of two to twenty carbon atoms, with at least one unsaturated bond, i.e. a carbon-carbon double bond, where the alkenyl radical includes radicals, having "cis" and "trans" orientations or, alternatively, "E" and "Z" orientations. Examples include, but are not limited to, ethyleneyl or vinyl (-CH=CH ₂ ), allyl (-CH ₂ CH=CH ₂ ) and the like. Preferably the alkenyl has two to ten carbon atoms. More preferably, alkyl has two to four carbon atoms.

" Alkynyl " or " linear or branched alkynyl " refers to a linear or branched monovalent hydrocarbon radical of two to twenty carbon atoms with at least one center of unsaturation, i.e. carbon-carbon triple bond. Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like. Preferably alkynyl has two to ten carbon atoms. More preferably, alkynyl has two to four carbon atoms.

The terms " cyclic alkyl " and " cycloalkyl " may be used interchangeably. In the context of this document, this term refers to the radical of a saturated carbocyclic ring. In preferred embodiments, cycloalkyls have 3 to 10 carbon atoms in their ring structure, and more preferably 5 to 7 carbon atoms in their ring structure. In some embodiments, two cyclic rings may share two or more atoms, e.g. the rings are "fused rings". Suitable cycloalkyls include, but are not limited to, cycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl. In some embodiments, cycloalkyl is a monocyclic group. In some embodiments, cycloalkyl is a bicyclic group. In some embodiments, cycloalkyl is a tricyclic group.

The term " cycloalkyl " refers to an alkyl group as described above that is substituted with a cycloalkyl group.

The term " cyclic alkenyl " refers to a carbocyclic ring radical having at least one double bond in the ring structure.

The term " cyclic alkenyl " refers to a carbocyclic ring radical having at least one triple bond in the ring structure.

The term " aryl " as used herein includes substituted and unsubstituted single ring aromatic groups in which each ring atom is a carbon. Preferably the ring is a 5-7 membered ring, more preferably a 6 membered ring. The aryl group includes, but is not limited to, phenyl, phenol, aniline, and the like. The terms "aryl" also include "polycyclyl", "polycyclic", and "polycyclic" ring systems having two or more rings in which two or more atoms are shared by two adjacent rings, for example , the rings are "fused rings" where at least one of the rings is aromatic, eg other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, or aromatic rings. In some preferred embodiments, the polycycles have 2-3 rings. In some preferred embodiments, the implementation of polycyclic ring systems have two cyclic rings, in which both rings are aromatic. Each of the polycycle rings may be substituted or unsubstituted. In certain embodiments, each polycycle ring has 3 to 10 ring carbon atoms, preferably 5 to 7. For example, aryl groups include, but are not limited to, phenyl (benzene), tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl, and the like. In some embodiments, aryl is a single ring aromatic group. In some embodiments, the implementation of the aryl is an aromatic group with two rings. In some embodiments, the implementation of the aryl is an aromatic group with three rings.

As used herein, the terms " heterocycle ", " heterocyclyl " and " heterocyclic ring " refer to substituted or unsubstituted non-aromatic ring structures of 3-18 membered rings, preferably 3-10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. In certain embodiments, the implementation of the ring structure may have two cyclic rings. In some embodiments, two cyclic rings may share two or more atoms, e.g. the rings are "fused rings". Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), in particular chapters 1, 3, 4, 6, 7 and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular in volumes 13, 14, 16, 19 and 28; and J. Am. Chem. soc. (1960) 82:5566. Examples of heterocyclic rings include, but are not limited to, tetrahydrofuran, dihydrofuran, tetrahydrothien, tetrahydropyran, dihydropyran, tetrahydrothiopyran, thiomorpholine, thioxane, homopiperazine, azetidine, oxetane, thietane, homopiperidine, piperidine, piperazine, pyrrolidine, morpholine, oxepan, thiepan, oxazepine, diazepine, thiazepine, 2-pyrroline, 3-pyrroline, indoline, 2H-pyran, 4H-pyran, dioxane, 1,3-dioxolane, pyrazoline, dithian, dithiolane, dihydropyran, dihydrothien, dihydrofuran, pyrazolidinylimidazoline, imidazolidine, 3-azabicyclo[ 3.1.0]hexane, 3-azabicyclo[4.1.0]heptane and azabicyclo[2.2.2]hexane. Spiro fragments are also included within the scope of this definition. Examples of a heterocyclic group in which ring atoms are substituted by oxo (=O) moieties are pyrimidinone and 1,1-dioxothiomorpholine.

The term " heteroaryl " as used herein refers to substituted or unsubstituted aromatic single ring structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, whose ring structures include at least one heteroatom ( e.g. O, N or S), preferably one to four or one to three heteroatoms, more preferably one or two heteroatoms. When two or more heteroatoms are present in a heteroaryl ring, they may be the same or different. The term "heteroaryl" also includes "polycyclyl", "polycyclic", and "polycyclic" ring systems having two or more cyclic rings, in which two or more ring atoms are shared by two adjacent rings, e.g. , the rings are "fused rings", where at least one of the rings is heteroaromatic, for example , other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaromatic and/or heterocyclic. In some preferred embodiments, polycyclic heteroaryls have 2-3 rings. In some embodiments, preferred polycyclic heteroaryls have two cyclic rings in which both rings are aromatic. In certain embodiments, each polycycle ring contains 3 to 10 ring atoms, preferably 5 to 7 ring atoms. For example, heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline, pyrimidine, indolizine, indole, indazole, benzimidazole, benzothiazol, benzofuran, benzothiophene, cinnoline , phthalazine, quinazoline, carbazole, phenoxazine, quinoline, purine and the like. In some embodiments, heteroaryl is a single ring aromatic group. In some embodiments, heteroaryl is an aromatic group with two rings. In some embodiments, heteroaryl is a tri-ring aromatic group.

Heterocyclic or heteroaryl groups may be carbon-linked (carbon-linked) or nitrogen-linked (nitrogen-linked) where possible. By way of example, and not limitation, carbon-linked heterocycles or heteroaryls are bonded at the 2, 3, 4, 5, or 6 position of pyridine, the 3, 4, 5, or 6 position of pyridazine, the 2, 4, 5, or 6 position of pyrimidine, position 2 , 3, 5 or 6 of pyrazine, 2, 3, 4 or 5 position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, 2, 4 or 5 position of oxazole, imidazole or thiazole, 3, 4 or 5 position of isoxazole, pyrazole or isothiazole, position 2 or 3 of aziridine, position 2, 3 or 4 of azetidine, position 2, 3, 4, 5, 6, 7 or 8 of quinoline or position 1, 3, 4, 5, 6, 7 or 8 of isoquinoline.

By way of example, and not limitation, nitrogen bonded heterocycles or heteroaryls are bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or O-carboline.

Heteroatoms present in heteroaryl or heterocyclyl include oxidized forms such as NO, SO and SO ₂ .

The term " halo " or " halogen " refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

The term " haloalkyl " as used herein refers to alkyl as defined herein that is substituted with one or more halogen groups as defined herein. Haloalkyl may be monohaloalkyl, dihaloalkyl or polyhaloalkyl. Monohaloalkyl may have one fluorine, chlorine, bromine or iodine substituent. The dihaloalkyl or polyhaloalkyl may be substituted with two or more of the same halogen atoms or a combination of different halogen groups. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl.

" Alkoxy " as used herein refers to alkyl-O-, where alkyl is defined herein above. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, t-butoxy, pentyloxy, hexyloxy, and the like.

The alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl described above may be optionally substituted with one or more ( e.g. , 2, 3, 4, 5, 6, or more) substitutes.

Unless specifically stated that they are "unsubstituted", it is understood that references to chemical moieties in this description also include substituted options. For example, reference to an "alkyl" group or moiety is implicitly inclusive of both substituted and unsubstituted variants. Examples of substituents on chemical moieties include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), alkoxyl, alkylthio, acyloxy, phosphoryl, phosphate, a phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl or aryl, or a heteroaryl moiety.

"Optional" or "optional" means that the circumstance described thereafter may or may not occur, so the application includes cases where the circumstance occurs and cases where it does not. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus the application includes structures in which a non-hydrogen substituent is present and structures in which a non-hydrogen substituent is not present.

The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbon, nitrogen, oxygen, or sulfur atoms. It is understood that "substitution" or "substituted" includes the implicit condition that such substitution corresponds to the allowable valency of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., which does not undergo spontaneous transformation such as rearrangement, cyclization, elimination etc. As used herein, the term "substituted" is intended to include all acceptable organic substituents. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and straight, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Allowable substituents may be one or more and the same or different for the respective organic compounds. For the purposes of the invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any valid substituents of the organic compounds described herein that satisfy the valency of the heteroatoms. Substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate , phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro substituent and difluoroalkyl is alkyl substituted with two fluoro substituents. It should be understood that if there is more than one substitution on a substituent, each non-hydrogen substituent may be the same or different (unless otherwise indicated).

If the carbon of a substituent is described as being optionally substituted with one or more of the list of substituents, one or more hydrogen atoms on the carbon atom (if any) may be individually and/or together replaced by an independently selected optional substituent. If the nitrogen of a substituent is described as being optionally substituted by one or more of the list of substituents, one or more hydrogen atoms in the nitrogen (to the extent that they are present) may be replaced by an independently selected optional substituent. One exemplary substituent may be designated as -NR'R'', where R' and R'', together with the nitrogen atom to which they are attached, may form a heterocyclic ring. The heterocyclic ring formed from R' and R'' together with the nitrogen atom to which they are attached may be partially or completely saturated. In some embodiments, the implementation of the heterocyclic ring consists of 3-7 atoms. In other embodiments, the heterocyclic ring is selected from the group consisting of pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl, and thiazolyl.

In this description, the terms " substituent ", " radical ", and " group " are used interchangeably.

Where a group of substituents is collectively described as being optionally substituted by one or more of the list of substituents, the group may include: (1) non-substituted substituents, (2) substituted substituents that are not substituted by optional substituents, and/or (3) substituted substituents that are substituted by one or more optional substituents.

If a substituent is described as being optionally substituted with up to a certain number of non-hydrogen substituents, that substituent may be (1) unsubstituted; or (2) substituted with up to that specified number of non-hydrogen substituents, or up to the maximum number of substituted positions on the substituent, whichever is less. Thus, for example, if a substituent is described as heteroaryl optionally substituted with up to 3 non-hydrogen substituents, then any heteroaryl with less than 3 substituted positions will only be optionally substituted with as many non-hydrogen substituents as the heteroaryl has substituted positions. Such substituents in non-limiting examples can be selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [-NH(C=NH)NH ₂ ], - OR ¹⁰⁰ , NR ¹⁰¹ R ¹⁰² , -NO ₂ , -NR ¹⁰¹ COR ¹⁰² , -SR ¹⁰⁰ , sulfoxide represented by -SOR ¹⁰¹ , sulfone represented by -SO ₂ R ¹⁰¹ , sulfonate -SO ₃ M, sulfate -OSO ₃ M, a sulfonamide represented by -SO ₂ NR ¹⁰¹ R ¹⁰² , cyano azido, -COR ¹⁰¹ , -OCOR ¹⁰¹ , -OCONR ¹⁰¹ R ¹⁰² and a polyethylene glycol unit (-OCH ₂ CH ₂ ) _n R ¹⁰¹ where M is H or a cation (Na ⁺ or K ⁺ ); R ¹⁰¹ , R ¹⁰² and R ¹⁰³ are each independently selected from H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit (-OCH ₂ CH ₂ ) _n -R ¹⁰⁴ where n represents is an integer from 1 to 24, aryl with 6-10 carbon atoms, heterocyclic ring with 3-10 carbon atoms and heteroaryl with 5-10 carbon atoms; and R ¹⁰⁴ is H or linear or branched alkyl having from 1 to 4 carbon atoms, where alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl in the groups represented by R ¹⁰⁰ , R ¹⁰¹ , R ¹⁰² , R ¹⁰³ and R ¹⁰⁴ , optionally substituted with one or more (for example, 2, 3, 4, 5, 6 or more) substituents independently selected from halogen, -OH, -CN, -NO ₂ and unsubstituted linear or branched alkyl having from 1 to 4 atoms carbon. Preferably, the substituents for the optionally substituted alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl described above include halogen, -CN, -NR ¹⁰² R ¹⁰³ , -CF ₃ , -OR ¹⁰¹ , aryl, heteroaryl, heterocyclyl, -SR ¹⁰¹ , -SOR ¹⁰¹ , -SO ₂ R ¹⁰¹ and -SO ₃ M.

The number of carbon atoms in a group may be indicated herein by the prefix "C _x-xx " or "C _x -C _xx ", where x and xx are integers. For example, "C _1-4 alkyl" or "C 1-4 alkyl" is an alkyl group having 1 to 4 carbon atoms.

The terms " compound " or " cytotoxic compound ", " cytotoxic dimer ", and " cytotoxic dimer compound " are used interchangeably. They are intended to include compounds for which the structure or formula, or any derivative thereof, has been described in the present invention, or the structure or formula, or any derivative thereof, which has been incorporated by reference. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts ( eg pharmaceutically acceptable salts) and prodrugs and prodrug salts of the compounds of all the formulas described in the present invention. The term also includes any solvates, hydrates and polymorphs of any of the above. Specific enumeration of "stereoisomers", "geometric isomers", "tautomers", "solvates", "metabolites", "salts", "prodrugs", "prodrug salts", "conjugates", "conjugate salts", "solvate", "hydrate" or "polymorph" in certain aspects of the invention described in this application should not be interpreted as an intentional omission of these forms in other aspects of the invention, where the term "compound" is used without listing these other forms.

As used herein, the term " conjugate " refers to a compound as described herein, or a derivative thereof, that is associated with a cell-binding agent.

The term " binding to a cell-binding agent " as used herein refers to compounds described herein, or derivatives thereof, containing at least one binding group or a precursor thereof suitable for binding these compounds or derivatives thereof to a cell-binding agent.

The term " predecessor " of a given group refers to any group that can lead to the formation of that group by any of deprotection, chemical modification, or coupling reaction.

The term " chiral " refers to molecules that have the mismatch property when mirrored, while the term "achiral" refers to molecules that match when mirrored.

The term " stereoisomer " refers to compounds that have the same chemical structure and connectivity, but different orientations of atoms in space, which cannot interconvert by rotation around single bonds.

" Diastereomer " refers to a stereoisomer with two or more centers of chirality whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high resolution analytical procedures such as crystallization, electrophoresis and chromatography.

" Enantiomers " refers to two stereoisomers of a compound that are not overlapping mirror images of each other.

The stereochemical definitions and conventions used herein generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds,” John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the invention, including but not limited to diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active forms, that is, they are able to rotate the plane of plane polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule with respect to its chiral center(s). The prefixes d and I or (+) and (-) are used to denote the rotation sign of plane polarized light by the compound, where (-) or 1 means that the compound is left-handed. A connection with a (+) or d prefix is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A mixture of enantiomers in a ratio of 50:50 is called a racemic mixture or racemate, which can occur when there was no stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species devoid of optical activity.

The term " tautomer " or " tautomeric form " refers to structural isomers with different energies that are interconvertible across a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include proton migration interconversions such as keto-enol and imine-enamine isomerization. Valence tautomers involve interconversions by rearranging some of the bonding electrons.

As used herein, the term " benzodiazepine " compound is a compound having the structure of a benzodiazepine core. The benzodiazepine core may be substituted or unsubstituted and/or fused to one or more ring structures. It also includes a compound having two benzodiazepine nuclei connected by a linker. The functionality of the imine (-C=N-) in the benzodiazepine core can be reduced.

As used herein, the term " pyrrolobenzodiazepine " (PBB) compound is a compound having the core structure of pyrrolobenzodiazepine. Pyrrolobenzodiazepine may be substituted or unsubstituted. It also includes a compound having two pyrrolobenzodiazepine cores connected by a linker. The functionality of the imine (-C=N-) in the composition of the indolinobenzodiazepine core can be reduced.

, которая может быть необязательно замещенной.In certain embodiments, the pyrrolobenzodiazepine compound contains a core structure represented by

, which may be optionally substituted.

, которая может быть необязательно замещена.In some embodiments, the pyrrolobenzodiazepine compounds contain a core structure represented by

, which may be optionally substituted.

As used herein, the " indolinobenzodiazepine " (IGN) compound is a compound having the core structure of an indolinebenzodiazepine. The indolinobenzodiazepine may be substituted or unsubstituted. It also includes a compound having two indolinobenzodiazepine cores linked by a linker. The functionality of the imine (-C=N-) in the composition of the indolinobenzodiazepine core can be reduced.

, которая может быть необязательно замещена.In some embodiments, the indolinobenzodiazepine compound contains a core structure represented by

, which may be optionally substituted.

, которая может быть дополнительно замещена.In some embodiments, the indolinebenzodiazepine compound contains a core structure represented by

, which can be further substituted.

As used herein, the phrase " pharmaceutically acceptable salt " refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Representative salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate , ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate salts ( for example , 1,1'-methylene-bis- (2-hydroxy-3-naphthanoate)), alkali metal salts ( eg sodium and potassium), alkaline earth metal salts ( eg magnesium) and ammonium salts. A pharmaceutically acceptable salt may include the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge of the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counterions may be present. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

If the compound of the invention is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like, or an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidilic acid such as glucuronic acid or galacturonic acid, alpha hydroxy acid such as citric acid or tartaric acid, amino acid such as aspartic acid or glutamic acid, aromatic acid such as benzoic acid or cinnamic acid, sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid vaya acid, or the like.

If the compound of the invention is an acid, the desired pharmaceutically acceptable salt can be obtained by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary, secondary, or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines such as piperidine, morpholine, and piperazine, and inorganic salts derived from from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

The term " solvate " as used herein means a compound which further comprises a stoichiometric or non-stoichiometric amount of a solvent such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid and dichloromethane, ethanolamine, 2-propanol or the like, bound by non-covalent intermolecular forces. Solvates or hydrates of the compounds are readily prepared by adding at least one molar equivalent of a hydroxyl solvent such as methanol, ethanol, 1-propanol, 2-propanol, or water to the compound to solvate or hydrate the imine moiety.

The phrase " pharmaceutically acceptable " indicates that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients constituting the formulation and/or the mammal being treated with them.

The term " protecting group " or " protective moiety " refers to a substituent that is typically used to block or protect a particular functionality when interacting with other functional groups in a compound, derivative, or conjugate thereof. For example, an " amino protecting group " or " amino protecting moiety " is a substituent attached to an amino group that blocks or protects the amino functionality in a compound. Such groups are well known in the art (see, for example, P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis, Chapter 7, J. Wiley & Sons, NJ) and carbamates such as methyl and ethyl carbamate, FMOC, substituted ethyl carbamates, carbamates cleaved by 1,6-β-elimination (also called "self- cleaving "), ureas, amides, peptides, alkyl and aryl derivatives. Suitable amino protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a general description of protecting groups and their use, see PGM Wuts & TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 2007.

The term " leaving group " refers to a group of charged or uncharged fragments that leave during replacement or displacement. Such leaving groups are well known in the art and include, but are not limited to, halogens, esters, alkoxy, hydroxyl, tosylates, triflates, mesylates, nitriles, azide, carbamate, disulfides, thioesters, thioethers, and diazonium compounds.

The term " bifunctional crosslinker ", " bifunctional linker ", or " crosslinkers " refers to modifiers that have two reactive groups; one of which is able to react with the cell-binding agent, while the other reacts with the cytotoxic compound, binding the two parts together. Such bifunctional crosslinkers are well known in the art (see, for example, Isalm and Dent in Bioconjugation chapter 5, p218-363, Groves Dictionaries Inc. New York, 1999). For example, bifunctional crosslinkers that provide linkage via a thioether linkage include N-succinimidyl-4-( N -maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) for introducing maleimido groups or with N -succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) for the introduction of iodoacetyl groups. Other bifunctional crosslinking agents that introduce maleimide groups or haloacetyl groups into the cell binding agent are well known in the art (see U.S. Patent Applications 2008/0050310, 20050169933, available from Pierce Biotechnology Inc. PO Box 117, Rockland, IL 61105, USA) and include, but are not limited to, bis-maleimidopolyethylene glycol (BMPEO), BM(PEO) ₂ , BM(PEO) ₃ , N-(β-maleimidopropyloxy)succinimide ester (BMPS), γ-N-maleiminobutyric acid succinimidyl ester (GMBS), Nε-hydroxysuccinimide ester of maleimidocaproic acid (EMCS), 5-maleimidovaleric acid NHS, HBVS, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), which is "long chain » analogue of SMCC (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl-3-(bromoacetamido) propionate (SBAP), N-succinimidyl iodoacetate (SIA), Nκ-succinimidyl ester-maleimine idoundecanoic acid (KMUA), N-succinimidyl-4-(p-maleimidophenyl)-butyrate) (SMPB), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobismaleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N -maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(γ- maleimidobutyloxy) sulfosuccinimide ester (Sulfo-GMBS), N-(ε-maleimidocaproyloxy) sulfosuccinimide ester (Sulfo-EMCS), N-(κ-maleimidoundecanoyloxy) sulfosuccinimide ester (Sulfo-KMUS), and sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate (sulfo-SMPB).

Heterobifunctional crosslinkers are bifunctional crosslinkers having two different reactive groups. Heterobifunctional crosslinkers containing both an amino-reactive N -hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link the cytotoxic compounds described herein to a cell-binding agent ( e.g. , antibody). Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH), and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates bearing an acid-labile bond can also be prepared using the hydrazine-containing benzodiazepine derivative of the present invention. Examples of bifunctional crosslinkers that can be used include succinimidyl p-formyl benzoate (SFB) and succinimidyl p-formylphenoxyacetate (SFPA).

Bifunctional crosslinkers that allow the cell binding agent to bind to cytotoxic compounds via disulfide bonds are known in the art and include N -succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N -succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), N -succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N -succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) to introduce dithiopyridyl groups. Other difunctional crosslinkers that can be used to introduce disulfide groups are known in the art and are described in US Pat. Alternatively, crosslinkers such as 2-iminothiolane, homocysteinthiolactone, or S-acetylsuccinic anhydride that introduce thiol groups can also be used.

" Linker ", " linker moiety " or " linking group " as defined herein refers to a moiety that joins two groups, such as a cell-binding agent and a cytotoxic compound, together. As a rule, the linker is essentially inert under conditions in which the two groups that it connects are connected. The bifunctional crosslinker may contain two reactive groups, one at each end of the linker moiety, such that one reactive group may first react with the cytotoxic compound to form a compound containing the linker moiety, and the second reactive group may then react with the cell binding agent. Alternatively, one end of the bifunctional cross-linking agent may first be reacted with the cell-binding agent to form a cell-binding agent containing a linker moiety and a second reactive group, which may then be reacted with a cytotoxic compound. The binding moiety may contain a chemical bond that releases the cytotoxic moiety at a particular site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds (see, for example, US Pat. ; 7276499, 7368565; 7388026 and 7414073). Preferred are disulfide bonds, thioether and peptidase-labile bonds. Other linkers that can be used in the present invention include non-cleavable linkers such as those detailed in US Publication No. 20050169933 or charged linkers or hydrophilic linkers described in US 2009/0274713, US 2010/01293140 and WO 2009/134976, which are all expressly incorporated into this document by reference.

The term " amino acid " refers to naturally occurring amino acids or non-naturally occurring amino acids. In some embodiments, the amino acid is NH ₂ -C(R ^aa' R ^aa )-C(=O)OH, where R ^aa and R ^aa' are each independently H, optionally substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl having from 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl, or R ^aa and the N-terminal nitrogen atom may together form a heterocyclic ring (eg, as in proline). The term " amino acid residue " refers to the corresponding residue when one hydrogen atom is removed from the amino and/or carboxy terminus of an amino acid, such as -NH-C(R ^aa' R ^aa )-C(=O)O-.

The term " peptide " refers to short chains of amino acid monomers linked by peptide (amide) bonds. In some embodiments, the implementation of the peptides contain from 2 to 20 amino acid residues. In other embodiments, the implementation of the peptides contain from 2 to 10 amino acid residues. In yet other embodiments, the implementation of the peptides contain from 2 to 5 amino acid residues. As used herein, when a peptide is part of a cytotoxic agent or linker as described herein, represented by a particular amino acid sequence, the peptide can be linked to the rest of the cytotoxic agent or linker in both directions. For example, the dipeptide X ₁ -X ₂ includes X ₁ -X ₂ and X ₂ -X ₁ . Similarly, the tripeptide X ₁ -X ₂ -X ₃ includes X ₁ -X ₂ -X ₃ and X ₃ -X ₂ -X ₁ and the tetrapeptide X ₁ -X ₂ -X ₃ -X ₄ includes X ₁ -X ₂ -X ₃ -X ₄ and X ₄ -X ₂ -X ₃ -X ₁ . X ₁ , X ₂ , X ₃ and X ₄ is an amino acid residue.

The term "cation" refers to an ion with a positive charge. The cation can be monovalent ( eg Na ⁺ , K ⁺ etc. ), divalent ( eg Ca ²⁺ , Mg ²⁺ etc. ) or multivalent ( eg Al ³⁺ etc. ) . Preferably, the cation is monovalent.

The term " cysteine-engineered antibody " includes an antibody with at least one cysteine (Cys) that is not normally present in a given light chain or heavy chain residue of an antibody. Such Cys, which may also be referred to as "engineered Cys", can be introduced, for example, using standard recombinant technology ( eg , by replacing the coding sequence for a non-Cys residue in the target residue with the coding sequence for Cys). In certain embodiments, a Cys-engineered antibody of the invention has a Cys-engineered heavy chain. In certain embodiments, the engineered Cys is in or near the heavy chain CH ₃ domain. In certain embodiments, the engineered Cys is located at heavy chain residue 442 (EU/OU numbering).

For the purposes of this document, all amino acid residues of antibodies described herein are numbered according to the EU index, Kabat et al ., Sequences of Proteins of Immunological Interest , 5th ^Ed ., NIH publication No. 91-3242, 1991 (EU/OU numbering, all contents incorporated herein by reference). Common isotypes are called G1, G2, G4, etc.

The C442 residue can be conjugated to the cytotoxic drug/agent via the free thiol group of the C442 residue, for example, by reacting with the cytotoxic drug's thiol-reactive agent ( eg , a maleimido group).

The term " aqueous solution " as used herein refers to a solution where the solvent is water or a mixture of water and one or more organic solvents.

As used herein, the term " treatment " or " treatment " includes changing, reducing, or stopping the symptoms, clinical signs, and underlying pathology of the condition in such a manner as to improve or stabilize the condition of the subject. In the context of this document, and as is well known in the art, "treatment" is an approach to obtain beneficial or desirable results, including clinical results. Beneficial or desirable clinical results may include, but are not limited to, reducing, improving, or slowing the progression of one or more symptoms or conditions associated with the condition, such as cancer, reducing the severity of the disease, stabilizing (i.e., not worsening) the disease state, delaying or slowing down the progression of the disease, improvement or mitigation of the disease state and remission (partial or complete), detectable or not detectable. "Treatment" can also mean prolonging survival compared to expected survival if no treatment is given. Exemplary favorable clinical results are described here.

METHODS OF THE PRESENT INVENTION

The present invention relates to new methods for preparing a conjugate of a cell-binding agent and a cytotoxic agent containing an imine-containing cytotoxic agent bearing a maleimide group covalently linked to a cell-binding agent (CCA).

In some embodiments, methods for preparing a cell-binding agent-cytotoxic agent conjugate of the present invention comprise the steps of:

(a) the interaction of the imine fragment in the imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof with sulfur dioxide, a bisulfite salt, or a metabisulfite salt in an aqueous solution at pH 1.9 to 5.0 to form a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent with the cell-binding agent to form a cell-binding agent-cytotoxic agent conjugate. In some embodiments, the bisulfite salt is sodium bisulfite or potassium bisulfite. More specifically, the bisulfite salt is sodium bisulfite. In yet other embodiments, the metabisulfite salt is sodium metabisulfite or potassium metabisulfite. More specifically, the metabisulfite salt is sodium metabisulfite.

The present invention also relates to a method for producing a modified cytotoxic agent, comprising the step of reacting an imine moiety in an imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof with sulfur dioxide, a bisulfite salt, or a metabisulfite salt in an aqueous solution at pH 1.9 to 5.0 to form a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof. In some embodiments, the reaction can be carried out under the reaction conditions described in the 1st, 2nd, 3rd, 4th, 5th, 6th or 7th aspect below.

In the 1st aspect, for the method of the present invention described above, the reaction of step (a) is carried out at a pH of 1.9 to 5.0. More specifically, the pH is 2.5 to 4.9, 1.9 to 4.8, 2.0 to 4.8, 2.5 to 4.5, 2.9 to 4.5, 2.9 to 4.0, 2.9 to 3.7, 3.1 to 3.5, or 3.2 to 3.4. In another specific embodiment, the reaction of step (a) is carried out at pH 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2 .8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 , 4.1, 4.2, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0. In yet another specific embodiment, the reaction of step (a) is carried out at pH 3.3.

In the context of this document, a specific pH value means a specific value of ±0.05.

In some embodiments, the reaction of step (a) is carried out in the presence of a buffer solution. Any suitable buffer solution known in the art may be used in the methods of the present invention. Suitable buffer solutions include, for example, but not limited to, citrate buffer, acetate buffer, succinate buffer, phosphate buffer, glycine-containing buffer ( e.g. glycine-HCl-buffer), phthalate buffer ( e.g. buffer solution containing sodium or potassium hydrogen phthalate) and their combination. In some embodiments, the buffer solution is a succinate buffer. In some embodiments, the buffer solution is a phosphate buffer. In some embodiments, the buffer is a citrate-phosphate buffer. In some embodiments, the buffer is a citrate-phosphate buffer containing citric acid and Na ₂ HPO ₄ . In other embodiments, the buffer is a citrate-phosphate buffer containing citric acid and K ₂ HPO ₄ . In some embodiments, the concentration of the buffer solution described above may be in the range of 10 to 250 mM, 10 to 200 mM, 10 to 150 mM, 10 to 100 mM, 25 to 100 mM, 25 to 75 mM , 10 to 50 mm or 20 to 50 mm.

In the 2nd aspect, the reaction step (a) is carried out in the absence of a buffer solution ( for example , the buffers described in the 1st aspect). In some embodiments, the present method comprises the steps of: (a) reacting an imine moiety in an imine-containing cytotoxic agent represented by formula (A), or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt, or a metabisulfite salt in aqueous solution to form a modified cytotoxic agent comprising modified imine moiety represented by the following formula:

or its pharmaceutically acceptable salt, where the aqueous solution does not contain a buffer; and

(b) reacting the modified cytotoxic agent with the cell-binding agent to form a cell-binding agent-cytotoxic agent conjugate. In some embodiments, the reaction of step (a) is carried out in a mixture of an organic solvent and water. More specifically, the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water. In some embodiments, the mixture of DMA and water contains less than 60% DMA by volume. More specifically, the volume ratio of DMA to water is 1:1.

In the third aspect, for the methods described above or in the 1st or 2nd aspect, 0.5-5.0 equivalents of the bisulfite salt or 0.25 or 2.5 equivalents of the metabisulfite salt are used for each 1 equivalent of the imine-containing cytotoxic agent in the reaction stage (a). In some embodiments, 0.5 to 4.5, 0.5 to 4.0, 0.5 to 3.5, 0.5 to 4.0, 0.5 to 3.5, 0.5 to 3.0, 0.5 to 2.5, 0.8 to 2.0, 0.9 to 1.8, 1.0 to 1.7, 1.1 to 1 .6 or 1.2 to 1.5 equivalents of the bisulfite salt or 0.25 to 2.25, 0.25 to 2.0, 0.25 to 1.75, 0.25 to 2.0 , 0.25 to 1.75, 0.25 to 1.5, 0.25 to 1.25, 0.4 to 1.0, 0.45 to 0.9, 0.5 up to 0.85, from 0.55 to 0.8 or from 0.6 to 0.75 equivalents of the metabisulfite salt is used for every 1 equivalent of the imine-containing cytotoxic agent. In other embodiments, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 1.4, 1.5, 1.6 , 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2 .9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5 or 5.0 bisulfite salt equivalents or 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0, 95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 2.0, 2.25, or 2.5 equivalents of the metabisulfite salt are used for every 1 equivalent of the imine-containing cytotoxic agent. In yet other embodiments, 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt is used for every 1 equivalent of the imine-containing cytotoxic agent. In other embodiments, 1.2 equivalents of the bisulfite salt or 0.6 equivalents of the metabisulfite salt is used for every 1 equivalent of the imine-containing cytotoxic agent.

Used here, the specific equivalent means a specific value of ± 0.05.

In the 4th aspect, for the methods of the present invention, the reaction of step (a) is carried out at a pH of 2.9 to 3.7 and 1.0 to 1.8 equivalents of the bisulfite salt or 0.5 to 0.9 equivalents of the metabisulfite salt is given in reaction with 1 equivalent of an imine-containing cytotoxic agent. In some embodiments, the reaction of step (a) is carried out at a pH of 3.1 to 3.5 and 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent. In other embodiments, the reaction of step (a) is carried out at a pH of 3.2 to 3.4 and 1.3 to 1.5 equivalents of the bisulfite salt or 0.65 to 0.75 equivalents of the metabisulfite is reacted with 1 equivalent of the imine containing cytotoxic agent. In other embodiments, the reaction of step (a) is carried out at a pH of 3.3 and 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent. In still other embodiments, the reaction of step (a) is carried out at a pH of 3.3 and 1.4 equivalents of sodium bisulfite is reacted with 1 equivalent of an imine-containing cytotoxic agent.

In the 5th aspect for the methods of the present invention described herein or in the 1st, 2nd, 3rd or 4th aspect, the reaction of step (a) is carried out in a mixture of an organic solvent and water. Any suitable organic solvent may be used. Representative organic solvents include, but are not limited to, alcohols (eg, methanol, ethanol, propanol, etc.), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, acetone, methylene chloride, etc. In some embodiments, the organic solvent is miscible with water. In other embodiments, the organic solvent is not miscible with water, i. the reaction of step (a) is carried out in a biphasic solution. In some embodiments, the organic solvent is dimethylacetamide (DMA). An organic solvent ( e.g. DMA) may be present in an amount of 1-99%, 1-95%, 10-80%, 20-70%, 30-70%, 1-60%, 5-60% 10-60%, 20-60%, 30-60%, 40-60%, 45-55%, 10-50% or 20-40% by volume of the total water and organic solvent. In some embodiments, the implementation of the reaction of stage (a) is carried out in a mixture of DMA and water, where the volume ratio of DMA and water is 1:1.

In the 6th aspect for the methods of the present invention described herein or in the 1st, 2nd, 3rd, 4th or 5th aspect, the reaction of step (a) can be carried out at any suitable temperature. In some embodiments, the reaction is carried out at a temperature of 0°C to 50°C, 10°C to 50°C, 10°C to 40°C, or 10°C to 30°C. In other embodiments, the reaction is carried out at a temperature of 15°C to 30°C, 20°C to 30°C, 15°C to 25°C, 16°C to 24°C, 17°C to 23 °C, 18°C to 22°C or 19°C to 21°C. In still other embodiments, the reaction can be carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25 °C In some embodiments, the reaction can be carried out from 0°C to 15°C, from 0°C to 10°C, from 1°C to 10°C, from 5°C to 15°C, or from 5°C to 10° C.

In the 7th aspect for the methods of the present invention described in this document or in the 1st, 2nd, 3rd, 4th, 5th or 6th aspect, the reaction of stage (a) is carried out from 1 minute up to 48 hours, from 5 minutes to 36 hours, from 10 minutes to 24 hours, from 30 minutes to 24 hours, from 30 minutes to 20 hours, from 1 hour to 20 hours, from 1 hour to 15 hours, from 1 hour to 10 hours, 2 hours to 10 hours, 3 hours to 9 hours, 3 hours to 8 hours, 4 hours to 6 hours, or 1 hour to 4 hours. In some embodiments, the reaction is allowed to proceed for 4-6 hours. In other embodiments, the reaction is allowed to proceed for 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours , 11 o'clock, 12 o'clock, 13 o'clock, 14 o'clock, 15 o'clock, etc. In other embodiments, the reaction may proceed within 4 hours. In still other embodiments, the reaction is allowed to proceed for 2 hours.

In the 8th aspect, for the methods of the present invention described herein or in the 1st, 2nd, 3rd, 4th, 5th, 6th or 7th aspect, the reaction of step (b) carried out at a pH of 4 to 9. In some embodiments, the reaction of step (b) is carried out at a pH of 4.5 to 8.5, 5 to 8.5, 5 to 8, 5 to 7.5, 5 up to 7, from 5 to 6.5 or from 5.5 to 6.5. In other embodiments, the reaction of step (b) is carried out at pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5, 9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

In some embodiments, for the methods of the present invention described herein or in the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th aspect, the step reaction (b) carried out in an aqueous solution containing a mixture of water and an organic solvent. Any suitable organic solvent described above may be used. More specifically, the organic solvent is DMA. In some embodiments, the aqueous solution contains less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3 %, less than 2% or less than 1% organic solvent ( e.g. DMA) by volume.

In some embodiments, for the methods of the present invention described herein or in the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th aspect, the modified cytotoxic agent is not purified before reaction with the cell-binding agent in step (b). Alternatively, the modified cytotoxic agent is purified prior to reaction with the cell-binding agent in step (b). Any suitable methods described herein can be used to purify the modified cytotoxic agent. In certain embodiments, the present invention relates to an isolated modified cytotoxic agent obtained by the present methods. The isolated modified cytotoxic agent may be stored for some time prior to reaction with the cell-binding agent. Preferably, the isolated modified cytotoxic agent is stored under conditions that will prevent degradation of the modified cytotoxic agent, e.g., as a purified solid or as a frozen solution, or stored at a low temperature (e.g., less than 10°C or less than 5°C). FROM).

In some embodiments for the methods of the present invention described herein, or in any of the embodiments described above, the cell-binding agent-cytotoxic agent conjugate of step (b) undergoes a purification step. In this regard, the cell-binding agent-cytotoxic agent conjugate can be purified from other components of the mixture using tangential flow filtration (FTF), non-adsorption chromatography, adsorption chromatography, adsorption filtration, selective precipitation, or any other suitable purification process, as well as combinations thereof.

In some embodiments, the cell-binding agent-cytotoxic agent conjugate is purified using a single purification step ( eg , PTP). Preferably, the conjugate is purified and replaced with the appropriate formulation using a single purification step ( eg FTP). In other embodiments, the cell-binding agent-cytotoxic agent conjugate is purified using two successive purification steps. For example, the conjugate may be first purified by selective precipitation, adsorption filtration, absorption chromatography, or non-absorption chromatography, followed by purification with FTP. One skilled in the art will appreciate that purification of the cell-binding agent-cytotoxic agent conjugate allows the isolation of a stable conjugate containing the cell-binding agent chemically bonded to the cytotoxic agent.

Any suitable FTP system can be used for cleaning, including the Pellicon type system (Millipore, Billerica, Mass.), the Sartocon cassette system (Sartorius AG, Edgewood, N.Y.) and the Centrasette type system (Pall Corp., East Hills, N.Y.)

Any suitable adsorption chromatography resin can be used for purification. Preferred adsorption chromatography resins include hydroxyapatite chromatography, charge induction hydrophobic chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye-ligand chromatography , affinity chromatography, reverse phase chromatography, and combinations thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT type I and type II, Bio-Rad Laboratories, Hercules, Calif.), hydroxyapatite HA Ultrogel (Pall Corp., East Hills, NY), and ceramic fluorapatite (CFT type I and type II, Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable HCIC resin is MEP Hypercel resin (Pall Corp., East Hills, NY). Examples of suitable HIC resins include butylsepharose, hexylsepharose, phenylsepharose and octylsepharose (all from GE Healthcare, Piscataway, NJ), as well as methyl macropreparations and tert-butyl resin macropreparations (Biorad Laboratories, Hercules, Calif.). Examples of suitable ion exchange resins include SP-Sepharose, CM-Sepharose and Q-Sepharose (all from GE Healthcare, Piscataway, NJ) and Unosphere S resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable mixed mode ion exchangers include Bakerbond ABx resin (JT Baker, Phillipsburg NJ). Examples of suitable IMAC resins include chelating sepharose resin (GE Healthcare, Piscataway, NJ) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable dye ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, NJ) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable affinity resins include protein A Sepharose resin ( e.g. , MabSelect, GE Healthcare, Piscataway, NJ) when the cell-binding agent is an antibody, and lectin affinity resins, e.g. , Lentil Lectin Sepharose resin (GE Healthcare, Piscataway, NJ). ) when the cell-binding agent carries the appropriate lectin binding sites. Alternatively, an antibody specific for a cell-binding agent can be used. Such an antibody can be immobilized, for example, on Sepharose 4 Fast Flow resin (GE Healthcare, Piscataway, NJ). Examples of suitable reverse phase resins include C4, C8 and C18 resins (Grace Vydac, Hesperia, Calif.).

Any suitable non-adsorption chromatographic resin can be used for purification. Examples of suitable non-adsorption chromatography resins include, but are not limited to, SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™ resins (e.g. S-200 and S-300), SUPERDEX™ resins (e.g. SUPERDEX™ 75 and SUPERDEX™ 200), BIO-GEL® resins (eg P-6, P-10, P-30, P-60 and P-100), and others known to those skilled in the art.

In some embodiments, the purified cell-binding agent-cytotoxic agent conjugate is formulated in a suitable preparative buffer. In some embodiments, the formulation buffer contains a bisulfite salt, such as sodium bisulfite or potassium bisulfite. More specifically, the formulation buffer contains sodium bisulfite. In some embodiments, the formulation buffer comprises 5 to 200 µM, 10 to 200 µM, 10 to 150 µM, 20 to 100 µM, 30 to 90 µM, 40 to 80 µM, 50 to 70 µM, 40 to 60 µM, 45 to 55 µM, or 55- to 65 µM bisulfite salt ( e.g. sodium bisulfite). In other embodiments, the formulation buffer comprises 20, 30, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 70, 80, 90 or 100 µM bisulfite salt ( e.g. sodium bisulfite).

In other embodiments, the formulation buffer further comprises trehalose. Any suitable amount of trehalose may be used. In some embodiments, the formulation buffer further comprises 2 to 15%, 5 to 10%, 6 to 10%, 7 to 9%, or 6 to 8% trehalose by weight.

In another embodiment, the formulation buffer has a pH of 4 to 6, 4 to 5, or 4 to 4.5. In other embodiments, the formulation buffer pH is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 ,0.

In still other embodiments, the formulation buffer contains 10 mM sodium succinate, 50 μM sodium bisulfite, 8% trelose dihydrate, and 0.01% polysorbate 20 at pH 4.2.

In some embodiments, for the methods of the present invention described herein, the reaction of step (a) does not result in significant sulfonation of the maleimide group. In some embodiments, less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the maleimide group is sulfonated. The percentage of maleimide sulfonation is equal to the total amount of maleimide-sulfonated cytotoxic agent (a cytotoxic agent that has sulfonation on maleimide only) and disulphated cytotoxic agent (cytotoxic agent that has sulfonation on both the maleimide and imine moieties) divided by the initial amount of the imine-containing cytotoxic agent before its reaction with a bisulfite salt or a metabisulfite salt.

In a 9th aspect, the present invention relates to a method for producing a cell-binding agent-cytotoxic agent conjugate represented by the following formula:

;

;

or a pharmaceutically acceptable salt thereof, comprising the steps of:

(a) the interaction of the imine fragment in the imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof with sulfur dioxide, a bisulfite salt or a metabisulfite salt in an aqueous solution at a pH of 3.1 to 3.5 to form a modified cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent, or a pharmaceutically acceptable salt thereof, with the cell-binding agent Ab to form a cell-binding agent-cytotoxic agent conjugate, wherein:

Ab is an anti-CD123 antibody comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:25 and an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:26; and

w is 1 or 2.

In some embodiments, the implementation of the method according to the 9th aspect is carried out under the reaction conditions described in the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th or 8th aspect, and in any of the embodiments described therein.

In the 10th aspect, the reaction of step (a) in the method of the 9th aspect is carried out at a pH of 3.2 to 3.4. More specifically, the pH is 3.3.

In some embodiments, the implementation of the reaction of stage (a) is carried out in the presence of a buffer solution. Typical buffer solutions include, but are not limited to, citrate buffer, acetate buffer, succinate buffer, or phosphate buffer. More specifically, the buffer is a succinate buffer.

In the 11th aspect, for the reaction of step (a) in the method of the 9th aspect, 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent. The remaining reaction conditions are as described above in the 10th aspect and any embodiments described therein. In some embodiments, 1.3 to 1.5 equivalents of the bisulfite salt or 0.65 to 0.75 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent. More specifically, 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt are reacted with 1 equivalent of the imine containing cytotoxic agent.

In the 12th aspect, the reaction of step (a) in the process of the 9th aspect is carried out at a pH of 3.2 to 3.4 and 1.3 to 1.5 equivalents of sodium bisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent. In some embodiments, the reaction is carried out at pH 3.3 and 1.4 equivalents of sodium bisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent.

In some embodiments, the reaction of step (a) in the method of the 9th, 10th, 11th or 12th aspect is carried out in a suitable solvent or mixture of solvents. In some embodiments, the reaction of step (a) is carried out in an aqueous solution containing a mixture of water and an organic solvent. Any suitable organic solvent described above may be used. More specifically, the organic solvent is DMA. In some embodiments, the aqueous solution contains less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3 %, less than 2% or less than 1% organic solvent ( e.g. DMA) by volume.

In some embodiments, the reaction of step (a) is carried out at a suitable temperature, for example, at room temperature or at a temperature of 15 to 25°C, for a sufficient period of time, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 24 hours, 48 hours, etc.

In some embodiments, the modified cytotoxic agent obtained in the reaction of step (a) is not purified prior to reaction with the cell-binding agent in step (b).

In some embodiments, the modified cytotoxic agent obtained in the reaction of step (a) is purified prior to reaction with the cell-binding agent in step (b). Any suitable purification methods described herein may be used.

In the 13th aspect, the reaction of step (b) in the method of the 9th aspect is carried out at a pH of 5.5 to 6.5; and the remaining reaction conditions are as described in the 9th, 10th, 11th or 12th aspect. In some embodiments, the pH is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the pH is 6.0.

In some embodiments, the implementation of the conjugate obtained by the methods of the 9th, 10th, 11th, 12th or 13th aspect is purified by a suitable method described herein. In one embodiment, the conjugate is purified by tangential flow filtration to obtain a purified conjugate.

In some embodiments, the purified conjugate is prepared in a formulation buffer containing 40 to 80 μM sodium bisulfite having a pH of 4 to 5. In some embodiments, the formulation buffer contains 50 μM sodium bisulfite having a pH of 4.2. In some embodiments, the formulation buffer contains 10 mM sodium succinate, 50 μM sodium bisulfite, 8% trehalose dihydrate, and 0.01% polysorbate 20 at pH 4.2.

In some embodiments, for the methods of the present invention described herein (for example, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th aspect and any embodiments described therein), the cell-binding agent is not an anti-CD123 antibody.

In some embodiments, for the methods of the present invention described herein (for example, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th aspect and any embodiments described therein), the cell-binding agent is not an anti-CD123 antibody comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO: 25 and an immunoglobulin light chain having the amino acid sequence of SEQ ID NO: 26.

In some embodiments, a cell-binding agent-cytotoxic agent conjugate prepared by the methods of the present invention described herein (e.g., 1st, 2nd, 3rd, 4th, 5th, 6th, 7- th or 8th aspect and any embodiments described therein) is not a conjugate represented by the following structure:

;

;

or a pharmaceutically acceptable salt thereof, wherein Ab is an anti-CD123 antibody comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:25 and an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:26; and

w is 1 or 2.

IMIN-CONTAINING CYTOTOXIC AGENTS And CONJUGATES OF THE PRESENT INVENTIONS

In some embodiments, for the methods of the present invention described herein, the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof, and

the cell-binding agent-cytotoxic agent conjugate is represented by the following formula:

where D ^M is a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

представляет собой связывающий клетки агент, связанный с цитотоксическим агентом посредством тиольной группы; и w является целым числом от 1 до 10.or a pharmaceutically acceptable salt thereof; L is a linker;

is a cell-binding agent linked to a cytotoxic agent via a thiol group; and w is an integer from 1 to 10.

In some embodiments, CCA is an antibody, and the antibody is linked to a cytotoxic agent via one or more cysteine thiol groups. In some embodiments, the free thiol group is located on the engineered Cys residue in the CH3 region of the heavy chain of the antibody, at position 442 of that heavy chain (or C442 for short) under EU/OU numbering. More specifically, the cysteine residue at position 442 is recombinantly introduced into the antibody.

In other embodiments, w is 1 or 2. More specifically, w is 2.

The following describes certain embodiments and specific embodiments for the methods of the present invention described herein (for example, methods 1st, 2nd, 3rd, 4th, 5th, 6th, 7th or 8th aspect described above and the embodiments described therein).

In a 1st specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is represented by the following structural formula:

or their pharmaceutically acceptable salt, where:

one of L', L'' and L''' is represented by the following formula:

and the other two are each independently selected from -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , halogen, guanidinium [ -NH(C=NH)NH ₂ ], -OR, -NR'R'', -NO ₂ , -NR'COR'', -SR, -SOR', -SO ₂ R', -SO ₃ H, -OSO ₃ H, -SO ₂ NR'R'', cyano, azido, -COR', -OCOR' and -OCONR'R';

one of Z ₁ and Z ₂ is -C(=O)- and the other is -NR ₅ -;

P ₁ is an amino acid residue or a peptide containing 2 to 20 amino acid residues;

R _x1 is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

R ^e represents -H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or -(CH ₂ -CH ₂ -O) _n -R ^k , where R ^k represents -H, linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (eg -NHR ¹⁰¹ ) or tertiary amino (-NR ¹⁰¹ R ¹⁰² ) group, or a 5- or 6-membered nitrogen-containing heterocycle, such as piperidine or morpholine, where R ¹⁰¹ and R ¹⁰² are each independently linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

R is at each occurrence independently selected from the group consisting of -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted 3-18 membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N and P;

R' and R'' are each independently selected from -H, -OH, -OR, -NHR, -NR ₂ , -COR, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol link -(CH ₂ CH ₂ O) _n -R ^c and optionally substituted 3-18-membered heterocyclic ring having from 1 to 6 heteroatoms independently selected from O, S, N and P;

R ^c represents -H or optionally substituted linear or branched alkyl having from 1 to 4 carbon atoms;

n is an integer from 1 to 24;

X ₁ ' is selected from -H, an amino protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , an optionally substituted aryl , having from 6 to 18 carbon atoms, an optionally substituted 5-18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3-18-membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N and P;

Y ₁ ' is selected from -H, oxo (i.e., Y ₁ ' together with the carbon atom to which it is attached forms a -C(=O)-group), optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl , having from 1 to 10 carbon atoms, optionally substituted 6-18 membered aryl, optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and

sulfur, an optionally substituted 3-18 membered heterocyclic ring having 1 to 6 heteroatoms;

R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' are each independently selected from the group consisting of -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl , having from 1 to 10 carbon atoms, a polyethylene glycol link - (CH ₂ CH ₂ O) _n -R ^c , halogen, guanidinium [-NH (C \u003d NH) NH ₂ ], -OR, -NR'R'', - NO ₂ , -NCO, -NR'COR'', -SR, -SOR', -SO ₂ R', -SO ₃ ^- H, -OSO ₃ H, -SO ₂ NR'R'', cyano, azido, -COR', -OCOR' and -OCONR'R'';

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo;

G is -CH- or -N-;

A and A' are the same or different and are independently selected from -O-, oxo(-C(=O)-), -CRR'O-, -CRR'-, -S-, -CRR'S-, -NR ₅ and -CRR'N(R ₅ )-; and

R ₅ at each occurrence is independently -H or optionally substituted linear or branched alkyl having from 1 to 10 carbon atoms.

In a more specific embodiment, D is represented by the formula (IGN1') or (IGN1).

In another more specific embodiment, for formulas (IGN1')-(IGN4') and (IGN1)-(IGN4), one of L', L" and L'" is represented by formula (A') or (D') and the others are -H, linear or branched alkyl having from 1 to 6 carbon atoms, halogen, -OH, (C ₁ -C ₆ )alkoxy or -NO ₂ .

In another more specific embodiment, for formulas (IGN1')-(IGN4') and (IGN1)-(IGN4), L' is represented by formula (A'); and L” and L'” are both -H.

In another more specific embodiment, for formulas (IGN1')-(IGN4') and (IGN1)-(IGN4), L' is represented by formula (D'); and L” and L'” are both -H.

In another more specific embodiment, for formulas (IGN1')-(IGN4') and (IGN1)-(IGN4), R _x1 is linear, branched or cyclic alkyl having 1 to 6 carbon atoms, optionally substituted with halogen, -OH , -SO ₃ H, (C ₁ -C ₃ )alkyl, (C ₁ -C ₃ )alkoxy, halo(C ₁ -C ₃ )alkyl, or a charged substituent or an ionizable group Q.

In the 2nd specific aspect, for the formulas (IGN1')-(IGN4') and (IGN1)-(IGN4), L' is represented by the following formula:

where:

each of R _a and R _b at each occurrence independently represents -H, (C ₁ -C ₃ )alkyl or a charged substituent or an ionizable group Q;

s is an integer from 1 to 6;

s1' is 0 or an integer from 1 to 6; and

Cy is cyclic alkyl with 5 or 6 ring carbon atoms, optionally substituted with halogen, -OH, (C ₁ -C ₃ )alkyl, (C ₁ -C ₃ )alkoxy, or halo(C ₁ -C ₃ )alkyl;

and the remaining variables are as described above in the 1st specific aspect or any specific or more specific embodiments described therein.

In a more specific embodiment, R _a and R _b are both H; Cy in formulas (B2') and (C2') is cyclohexane; and R ₅ is H or Me. More specifically, s1' is 0 or 1.

In the 3rd specific aspect for the formulas (IGN1')-(IGN4') and (IGN1)-(IGN4) P ₁ is a peptide containing from 2 to 10 amino acid residues; and the remaining variables are as described in the 1st or 2nd specific aspect or any specific or more specific embodiments described therein.

In specific embodiments, P ₁ is a peptide containing 2 to 5 amino acid residues.

In other specific embodiments, P ₁ is Gly-Gly-Gly, Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile- Cit, Trp, Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu- Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly- Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys , D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D -Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe and Gln-Ala. More specifically, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In the 4th specific aspect, for the formulas (IGN1')-(IGN4') and (IGN1)-(IGN4) X ₁ ' is selected from the group consisting of -H, -OH, optionally substituted linear, branched or cyclic alkenyl alkyl or alkynyl having 1 to 10 carbon atoms and phenyl; Y ₁ ' is selected from the group consisting of -H, oxo, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; and the remaining variables are as described above in the 1st, 2nd or 3rd specific aspect, or in any specific or more specific embodiments described therein. More specifically, X ₁ ' represents -H, -OH, (C ₁ -C ₃ )alkyl, halo(C ₁ -C ₃ )alkyl or phenyl; and Y ₁ ' is -H, oxo, (C ₁ -C ₃ ) alkyl, or halo(C ₁ -C ₃ ) alkyl. In another more specific embodiment, X ₁ ' represents -H, -OH or -Me; and Y ₁ ' represents -H or oxo. More specifically, X ₁ ' represents -H; and Y ₁ ' represents -H.

In the 5th specific aspect for the formulas (IGN1')-(IGN4') and (IGN1)-(IGN4) A and A' are the same or different and represent -O-, -S-, -NR ₅ - or oxo -(C=O)-; and the remaining variables are as described above in the 1st, 2nd, 3rd or 4th specific aspect, or in any specific or more specific embodiments described therein. More specifically, A and A' are the same or different and represent -O- or -S-. More specifically, A and A' are -O-.

In the 6th specific aspect for the formulas (IGN1')-(IGN4') and (IGN1)-(IGN4) R ₆ represents -OR where R represents an alkyl group having from 1 to 6 carbon atoms; and the remaining variables are as described above in the 1st, 2nd, 3rd, 4th, or 5th specific aspect, or any specific or more specific embodiments described therein. More specifically, R ₆ is -OMe.

In the 7th specific aspect for the formulas (IGN1)-(IGN4) R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' each independently represents -H, halogen , -NO ₂ , -OH, (C ₁ -C ₃ )alkyl, halo(C ₁ -C ₃ )alkyl or (C ₁ -C ₃ ) alkoxy; and the remaining variables are as described above in the 1st, 2nd, 3rd, 4th, 5th, or 6th specific aspect, or any specific or more specific embodiments described therein. More specifically, R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' represent -H.

In the 8th specific aspect, for the formulas (IGN1')-(IGN4') and (IGN1)-(IGN4), R, R', R" and R ₅ are each independently -H or (C ₁ -C ₃ )alkyl ; and the remaining variables are as described above in the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th specific aspect, or any specific or more specific embodiments described in them.

In the 9th specific aspect, for the formulas (IGN1')-(IGN4') and (IGN1)-(IGN4):

R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' are all -H;

R ₆ is -OMe;

X ₁ ' and Y ₁ ' are both -H; and

A and A' are -O-;

and the remaining variables are as described above in the 1st, 2nd or 3rd specific aspect, or in any specific or more specific embodiments described therein. More specifically, R, R', R" and R ₅ are each independently -H or ((C ₁ -C ₃ )alkyl. More specifically, R, R', R" and R ₅ are -H.

In a 10th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is represented by the following structural formula:

;

;

;

;

;

;

;

;

;

;

;

;

;

;

or a pharmaceutically acceptable salt thereof; and the remaining variables are described for formula (A) or (B) described above.

In an 11th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by the following structural formula:

where:

s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group;

R ₂₃ and R ₂₄ are each independently H or optionally substituted alkyl;

m' is an integer from 0 to 10; and

R ^h ' represents H or optionally substituted alkyl;

and the remaining variables are as described above for formula (A) or formula (B), or the 1st to 10th specific aspect, or any specific or more specific embodiments described therein.

In a specific embodiment, m' is an integer from 1 to 6. More specifically, m' is an integer from 1 to 3.

In another specific embodiment, R ₂₃ and R ₂₄ are each independently H or (C ₁ -C ₃ ) alkyl. More specifically, _R23 and _R24 are both H.

In another specific embodiment, R ^h ' represents H or (C ₁ -C ₃ ) alkyl. In particular, R ^h ' represents H.

In another specific embodiment, R ₂₃ and R ₂₄ are both H; m' is an integer from 1 to 6. More specifically, m' is an integer from 1 to 3.

In a 12th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by the following structural formula:

;

;

where s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group; and the remaining variables are as described above for formula (A) or formula (B), or the 1st to 10th specific aspect, or any specific or more specific embodiments described therein.

In a 13th specific aspect, the imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, and the modified cytotoxic agent is represented by the following formula:

,

,

or their pharmaceutically acceptable salt. More specifically, the modified cytotoxic agent is represented by the following formula:

,

,

or their sodium or potassium salt. Even more specifically, the modified cytotoxic agent is represented by the following formula:

.

.

In some embodiments, the imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, and the modified cytotoxic agent is represented by the following formula:

,

,

or their pharmaceutically acceptable salt. More specifically, the modified cytotoxic agent is represented by the following formula:

,

,

or their sodium or potassium salt. Even more specifically, the modified cytotoxic agent is represented by the following formula:

.

.

In a 14th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is represented by the following structural formula:

or their pharmaceutically acceptable salt, where:

X' is selected from the group consisting of -H, -OH, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl and an amino protecting group;

Y' is selected from the group consisting of -H, oxo, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

A and A' are selected from -O- and -S-;

W' is absent or selected from -O-, -N(R ^e )-, -N(R ^e )-C(=O)-, -N(C(=O)R ^e )-, -S- or - CH ₂ -S-, -CH ₂ NR ^e -;

R ^x is absent or selected from linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;

R ^e represents -H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or -(CH ₂ -CH ₂ -O) _n -R ^k where R ^k represents -H, linear branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (for example, -NHR ¹⁰¹ ) or tertiary amino (-NR ¹⁰¹ R ¹⁰² ) group or a 5- or 6-membered nitrogen-containing heterocycle, where R ¹⁰¹ and R ¹⁰² is each independently linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

n is an integer from 1 to 24;

G is selected from -CH- or -N-;

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo; and

R is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or a PEG group -(CH ₂ CH ₂ O) _n -R ^c , where n is an integer from 1 to 24, and R ^c represents a linear or branched alkyl with 1-4 carbon atoms;

R' and R' are each independently selected from -H, -OH, -OR, -NRR ^g ', -COR, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted aryl , having from 6 to 18 carbon atoms, optionally substituted 3-18-membered heterocyclic ring, having from 1 to 6 heteroatoms selected from O, S, N and P, group PEG -(CH ₂ CH ₂ O) _n -R ^c , and R ^g ' represents -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or a PEG group -(CH ₂ CH ₂ O) _n -R ^c .

In a more specific embodiment, D is represented by formula (IGN5') or (IGN5) above, or a pharmaceutically acceptable salt thereof.

In another specific embodiment, for formulas (IGN5')-(IGN8') and (IGN5)-(IGN8):

X' and Y' are both -H;

A and A' are both -O-;

R ₆ is -OMe;

W' represents -N(R ^e )- or -N(R ^e )-C(=O)-;

R ^e represents -H, linear or branched alkyl with 1-4 carbon atoms or -(CH ₂ -CH ₂ -O) _n -R ^k , where R ^k represents -H, linear or branched alkyl with 1-4 atoms carbon;

n is an integer from 2 to 6; and

R ^x is straight or branched alkyl having 1 to 6 carbon atoms.

In a 15th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is represented by the following structural formula:

or its pharmaceutically acceptable salt, where the remaining variables are as described above for formula (A) or (B).

In a 16th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by the following structural formula:

where:

s1 is a site covalently linked to D and s2 is a site covalently linked to a maleimide group;

E is -(CR ₁₀ R ₁₁ ) _q -, cycloalkyl or cycloalkylalkyl;

Z is absent, is -SO ₂ NR ₉ -, -NR ₉ SO ₂ -, -C(=O)-NR ₉ -, -NR ₉ -C(=O)-, -C(=O)-O- , -OC(=O)-, -C(=O)-NR ₉ -(CH ₂ CH ₂ O) _p -, -NR ₉ -C(=O)-(CH ₂ CH ₂ O) _p -, - (OCH ₂ CH ₂ ) _p -C(=O)NR ₉ - or -(OCH ₂ CH ₂ ) _p -NR ₉ -C(=O)-;

p is an integer from 1 to 24;

Q is H, a charged substituent, or an ionizable group;

R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are each independently H or optionally substituted alkyl; and

q and r are in each case independently integers from 0 to 10; and the remaining variables are as described above for formula (A) or formula (B), or in the 14th or 15th specific aspect, or in any specific or more specific embodiments described therein.

In a more specific embodiment, q and r are independently integers from 1 to 6, more specifically from 1 to 3.

In another more specific embodiment, R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are each independently H or C _1-3 alkyl. More specifically, R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are all -H.

In another more specific embodiment, p is an integer from 2 to 14. More specifically, p is an integer from 2 to 8, 2 to 6, or 2 to 4.

In a more specific embodiment, E is -(CR ₁₀ R ₁₁ ) _q -; and the remaining variables in the formula (L2) are as described above in the 16th specific aspect.

; а остальные переменные в формуле (L2) являются такими, как описаны выше в 16-ом конкретном аспекте.In another more specific embodiment, E is

; and the remaining variables in the formula (L2) are as described above in the 16th specific aspect.

In yet another specific embodiment, Z is -C(=O)-NR ₉ - or -NR ₉ -C(=O)-; and the remaining variables in formula (L2) are as described above in the 16th specific aspect or any specific or more specific embodiments described above. More specifically, R ₉ is H or Me. Alternatively, R ₉ is H.

In another more specific embodiment, Q is i) H; ii) -SO ₃ H, -Z'-SO ₃ H, -OPO ₃ H ₂ , -Z'-OPO ₃ H ₂ , -PO ₃ H ₂ , -Z'-PO ₃ H ₂ , -CO ₂ H, -Z'-CO ₂ H, -NR ₁₄ R ₁₅ or -Z'-NR ₁₄ R ₁₅ , or a pharmaceutically acceptable salt thereof; or iii) -N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- or -Z'-N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- ; Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted phenylene; each R ₁₄ -R ₁₆ is independently optionally substituted alkyl; and X ^- is a pharmaceutically acceptable anion; and the remaining variables in formula (L2) are as described above in the 16th embodiment or any of the more specific embodiments described above. In some embodiments, Z' is an optionally substituted alkylene. In yet other embodiments, Z' is alkylene (eg, -CH ₂ ); and each R ₁₄ -R ₁₆ is independently C _1-4 alkyl.

In yet another specific embodiment, Q is H, or —SO ₃ H, or a pharmaceutically acceptable cation (eg, sodium or potassium salt); and the remaining variables in formula (L2) are as described above in the 16th specific aspect or any specific or more specific embodiments described above.

In another more specific embodiment, for formula (L2):

each of R ₁₂ and R ₁₃ , at each occurrence independently represents H or (C ₁ -C ₃ )alkyl;

Q is H or -SO ₃ H or a pharmaceutically acceptable salt thereof;

Z is -C(=O)-NR ₉ - or -NR ₉ -C(=O)-;

R ₉ is H or (C ₁ -C ₃ )alkyl;

E is -(CR ₁₀ R ₁₁ ) _q -.

R ₁₀ and R ₁₁ are each independently H or (C ₁ -C ₃ ) alkyl; and

q and r are integers from 1 to 5.

In a 17th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by any one of the following structural formulas:

or a pharmaceutically acceptable salt thereof (eg sodium or potassium salt), where s1 is a site covalently linked to D and s2 is a site covalently linked to a maleimide group;

q and r are each independently an integer from 1 to 6; and the remaining variables are as described above for formula (A) or formula (B), or in the 14th or 15th specific aspect, or in any specific or more specific embodiments described therein. More specifically, q and r are each independently an integer from 1 to 3.

In an 18th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by any one of the following structural formulas:

, и

, and

,

,

or a pharmaceutically acceptable salt thereof (eg sodium or potassium salt), where s1 is a site covalently linked to D and s2 is a site covalently linked to a maleimide group;

and the remaining variables are as described above for formula (A) or formula (B), or in the 14th or 15th specific aspect, or in any specific or more specific embodiments described therein.

In a 19th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by the following structural formula:

where:

s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group;

R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ are each independently H or optionally substituted alkyl;

m and n are each independently an integer from 0 to 10;

R ^h is H or optionally substituted alkyl;

P _L is an optionally substituted alkylene, -(CH ₂ -CH ₂ -O) _j - (wherein the oxygen atom is linked to a -(C=O)-group linked to P), an amino acid residue or a peptide containing from 2 to 20 amino acid residues; and

j is an integer from 1 to 24; and the remaining variables are as described above for formula (A) or formula (B), or the 14th or 15th specific aspect, or any specific or more specific embodiments described therein.

In the 20th specific aspect of formula (L3), R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ each represents H or (C ₁ -C ₃ )alkyl; m and n are each independently an integer from 1 to 6; and the remaining variables are as described above in the 19th specific embodiment or any specific or more specific embodiment described therein. More specifically, R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ are all H. Even more specifically, R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ are all H; and m and n are each independently an integer from 1 to 3.

In the 21st specific aspect for the formula (L3) P _L is an amino acid residue or a peptide containing from 2 to 10 amino acid residues; and the remaining variables are as described above in the 19th or 20th specific aspect, or any specific or more specific embodiment described therein. More specifically, P _L is a peptide containing 2 to 5 amino acid residues.

In some embodiments, each amino acid residue is an amino acid residue independently selected from: a naturally occurring amino acid, a synthetic amino acid, an amino acid analog, and an amino acid mimetic that functions similarly to a naturally occurring amino acid.

In other embodiments, each amino acid residue is an amino acid residue independently selected from the group consisting of: histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, N-methyl-histidine, N-methyl-alanine, N-methyl-isoleucine, N-methyl-arginine, N-methyl-leucine, N-methyl-asparagine, N- methyl-lysine, N-methyl-aspartic acid, N-methyl-methionine, N-methyl-cysteine, N-methyl-phenylalanine, N-methyl-glutamic acid, N-methyl-threonine, N-methyl-glutamine, N- methyl-tryptophan, N-methyl-glycine, N-methyl-valine, N-methyl-proline, N-methyl-serine, N-methyl-tyrosine, hydroxyproline, γ-carboxyglutamate, selinocysteine, O-phosphoserine, homoserine, norleucine, methionine sulfoxide, methylsulfonium methionine, citrulline, ornithine, cysteine sulfonic acid, cysteine sulfonic acid, 3-aminoalanine, 3-dimethylamine oalanine, 2-amino-4-(dimethylamino)butanoic acid, 2,4-diaminobutanoic acid, 2-amino-6-(dimethylamino)hexanoic acid, 2-amino-5-(dimethylamino)pentanoic acid and β-alanine, each independently as the L or D isomer. More specifically, each amino acid residue is an independently selected glycine or alanine residue.

In other embodiments, P _L is a protease-cleavable peptide. More specifically, P _L is a peptide cleaved by a protease expressed in tumor tissue. In an alternative embodiment, P _L is a lysosomal protease cleavable peptide.

In still other embodiments, P _L is selected from the group consisting of: Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala- Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe- Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D -Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala -Ala, D-Ala-D-Ala., Ala-Met, Met-Ala, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, Gly-Gly-Gly, Ala-Ala-Ala, D- Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Val-Cit, Ala-Val-Ala and β-Ala-Gly-Gly-Gly. More specifically, P _L is Gly-Gly-Gly, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Val-Ala or β-Ala-Gly-Gly-Gly .

In a 22nd specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by the following structural formula:

,

,

or a pharmaceutically acceptable salt thereof, where s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group; and the remaining variables are as described above for formula (A) or formula (B), or in the 14th or 15th specific aspect.

In the 23rd specific aspect, the imine-containing cytotoxic agent is represented by the following formula:

; или

; or

,

,

or a pharmaceutically acceptable salt thereof, and the modified cytotoxic agent is represented by the following formula:

or their pharmaceutically acceptable salt.

In a more specific embodiment, the modified cytotoxic agent is represented by the following formula:

or their sodium or potassium salt. Even more specifically, the modified cytotoxic agent is represented by the following formula:

In some embodiments, the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof, and the modified cytotoxic agent is represented by the following formula:

; или

; or

,

,

or their pharmaceutically acceptable salt.

In the 24th specific aspect, the conjugate that can be obtained by the methods of the present invention is represented by the following structural formula:

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

; или

; or

,

,

or a pharmaceutically acceptable salt thereof (eg sodium or potassium salt).

In one aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is an imine-containing PBB compound.

In a 25th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is represented by the following formula:

(ПБД1');

(PBD1');

(ПБД1);

(PBD1);

(ПБД2');

(PBD2');

(ПБД2);

(PBD2);

(ПБД3');

(PBD3');

(ПБД3);

(PBD3);

(ПБД4'); или

(PBD4'); or

(ПБД4),

(PBD4),

or their pharmaceutically acceptable salt, where:

W is selected from C=O, C=S, CH ₂ , BH, SO and SO ₂ ;

X ₁ ' is selected from -H, an amino protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , an optionally substituted aryl , having from 6 to 18 carbon atoms, an optionally substituted 5-18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3-18-membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N and P;

Y ₁ ' is selected from -H, oxo, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted 6-18 membered aryl, optionally substituted 5-18 membered heteroaryl ring, containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, optionally substituted 3-18-membered heterocyclic ring having from 1 to 6 heteroatoms;

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo;

A and A' are the same or different and are independently selected from -O-, oxo (-C(=O)-), -CRR'O-, -CRR'-, -S-, -CRR'S-, -NR ₅ and -CRR'N(R ₅ )-,

R is at each occurrence independently selected from the group consisting of -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted 3-18 membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N and P;

R' and R'' are each independently selected from -H, -OH, -OR, -NHR, -NR ₂ , -COR, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol link -(CH ₂ CH ₂ O) _n -R ^c and optionally substituted 3-18-membered heterocyclic ring having from 1 to 6 heteroatoms independently selected from O, S, N and P;

R ^c represents -H or a substituted or unsubstituted linear or branched alkyl having from 1 to 4 carbon atoms, or a linking group with a reactive group associated with it;

n is an integer from 1 to 24;

R ₅ at each occurrence is independently —H or optionally substituted linear or branched alkyl having from 1 to 10 carbon atoms;

D ₀ and D ₀ ' are the same or different and are independently absent or selected from the group consisting of an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an amino acid, a peptide containing from 2 to 6 amino acids, and polyethylene glycol link (-OCH ₂ CH ₂ ) _n -;

L is absent, is a linker, a polyethylene glycol unit (-OCH ₂ CH ₂ ) _n -, optionally substituted linear, branched or cyclic alkyl or alkenyl having from 1 to 10 carbon atoms, an optionally substituted phenyl group, an optionally substituted 3-18-membered a heterocyclic ring or a 5-18 membered heteroaryl ring having 1 to 6 heteroatoms independently selected from O, S, N and P;

R _a , R _a ', R _b and R _b ' are the same or different and independently selected from the group consisting of -H, halide or optionally substituted branched, linear or cyclic alkyl having from 1 to 10 carbon atoms; or R _a and R _a ' and/or R _b and R _b ' together form a double bond containing the group =B and =B', respectively;

═B and ═B' are the same or different and are independently selected from an optionally substituted branched or linear alkenyl or carbonyl group;

Q is Q ₁ -Ar-Q ₂ ;

Q' is Q ₁ '-Ar'-Q ₂ ';

Q ₁ and Q ₁ ' each independently absent, is a linear, branched or cyclic alkyl with 1-6 carbon atoms or-CH=CH link;

Ar and Ar' are each independently absent or represent an aryl group;

Q ₂ and Q ₂ ' are each independently selected from -H, linker, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -R ^c '-(OCH ₂ CH ₂ ) _n -R ^c or a substituent selected from halogen, guanidinium [-NH(C=NH)NH ₂ ], -R, -OR, -NR'R'', -NO ₂ , -NCO, -NR'COR'' , NR'(C=O)OR'' -SR, sulfoxide represented by -SOR', sulfone represented by -SO ₂ R', sulfonate represented by -SO ₃ M, sulfate -OSO ₃ M, sulfonamide represented by SO ₂ NR'R '', cyano, azido, -COR', -OCOR' or -OCONR'R''; and

R ^c ' is absent or selected from linear or branched alkyl, alkenyl or alkynyl having from 1 to 5 carbon atoms.

In a 26th specific aspect, for the imine-containing cytotoxic agent of formula (A) or conjugate of formula (B), D is selected from one of the following:

(ПБД5')

(PBD5')

(ПБД5),

(PBD5),

(ПБД6'),

(PBD6'),

(ПБД6),

(PBD6),

(ПБД7'),

(PBD7'),

(ПБД7),

(PBD7),

(ПБД8'),

(PBD8'),

(ПБД8),

(PBD8),

(ПБД9'),

(PBD9'),

(ПБД9),

(PBD9),

(ПБД10') и

(PBD10') and

(ПБД10),

(PBD10),

or their pharmaceutically acceptable salt, where:

one of L', L'' and L''' in the formula (PBD5'), (PBD6'), (PBD7'), (PBD8'), (PBD5), (PBD6), (PBD7) or (PBD8) represented by the following formula:

and the other two are each independently selected from -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , halogen, guanidinium [ -NH(C=NH)NH ₂ ], -OR, -NR'R'', -NO ₂ , -NR'COR'', -SR, -SOR', -SO ₂ R', -SO ₃ H, -OSO ₃ H, -SO ₂ NR'R'', cyano, azido, -COR', -OCOR' and -OCONR'R';

one of Q ₂ and Q ₂ ' in the formula (PBD9'), (PBD10'), (PBD9) or (PBD10) is represented by the following formula:

and the other is selected from -H, -R, -OR, -NR'R'', -NO2, -NR'(C=O)OR'', -SR or -NO ₂ ;

one of Z ₁ and Z ₂ is -C(=O)- and the other is -NR ₅ -;

P ₁ is an amino acid residue or a peptide containing 2 to 20 amino acid residues;

R _x1 is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

R ^e represents -H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or -(CH ₂ -CH ₂ -O) _n -R ^k where R ^k represents -H, linear branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (for example, -NHR ¹⁰¹ ) or tertiary amino (-NR ¹⁰¹ R ¹⁰² ) group or a 5- or 6-membered nitrogen-containing heterocycle, where R ¹⁰¹ and R ¹⁰² is each independently linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

R ^c represents -H or optionally substituted linear or branched alkyl having from 1 to 4 carbon atoms;

R ₅ at each occurrence is independently -H or optionally substituted linear or branched alkyl having from 1 to 10 carbon atoms.

R _a '' and R _b '' are the same or different and are selected from -H and -Me; and

n' is selected from 0, 1, 2 and 3; and the remaining variables are as described in the 25th specific aspect.

In the 27th specific aspect, one of L', L" and L"' in the formula (PBD5'), (PBD6'), (PBD7'), (PBD8'), (PBD5), (PBD6), (PBD7) or (PBD8) is represented by formula (A') or (D') and the other two are -H, linear or branched alkyl having 1 to 6 carbon atoms, halogen, -OH, (C ₁ -C ₆ )alkoxy or -NO ₂ ; or one of Q ₂ and Q ₂ ' in the formula (PBD9'), (PBD10'), (PBD9) or (PBD10) is represented by formula (A'), (D') or (E'); and the other is -H, linear or branched alkyl having from 1 to 6 carbon atoms, halogen, -OH, (C ₁ -C ₆ )alkoxy or -NO ₂ ; and the remaining variables are as described in the 26th specific aspect.

In the 28th specific aspect, L” and L”' are both -H; and L' in the formula (PBD5'), (PBD6'), (PBD7'), (PBD8'), (PBD5), (PBD6), (PBD7) or (PBD8) is represented by the following formula:

one of Q ₂ and Q ₂ ' in the formula (PBD9'), (PBD10'), (PBD9) or (PBD10) is represented by the following formula:

where:

each of R _a and R _b at each occurrence independently represents -H, (C ₁ -C ₃ )alkyl or a charged substituent or an ionizable group Q;

s is an integer from 1 to 6;

s1' is 0 or an integer from 1 to 6; and

Cy is cyclic alkyl with 5 or 6 ring carbon atoms, optionally substituted with halogen, -OH, (C ₁ -C ₃ )alkyl, (C ₁ -C ₃ )alkoxy, or halo(C ₁ -C ₃ )alkyl; and the remaining variables are as described in the 27th specific aspect. In some embodiments, R _a and R _b are both H; Cy in formulas (B2') and (C2') is cyclohexane; R ₅ is H or Me; and s1' represents 0 or 1.

In the 29th specific aspect, P ₁ is a peptide containing 2 to 10 amino acid residues; and the remaining variables are as described in the 28th specific aspect. In some embodiments, P ₁ is a peptide containing 2 to 5 amino acid residues. In some embodiments, P ₁ is Gly-Gly-Gly, Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala- Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe- Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D -Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala -Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe and Gln-Ala. In some embodiments, P ₁ is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In the 30th specific aspect, for the methods described in any of the 25th, 26th, 27th, 28th or 29th specific aspect and embodiments described therein, R ₆ is -OMe; X ₁ ' and Y ₁ ' are both -H; and A and A' are -O-.

In a 31st specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is represented by the following structural formula:

;

;

;

;

;

;

;

;

; или

; or

,

,

or their pharmaceutically acceptable salt.

In the 32nd specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), -L- is represented by the following structural formula:

,

,

or their pharmaceutically acceptable salt, where:

s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group;

R ₂₃ and R ₂₄ are each independently H or optionally substituted alkyl;

m' is an integer from 0 to 10; and

R ^h' is H or optionally substituted alkyl; and the remaining variables are as described above in the 25th, 26th, 27th, 28th, 29th, 30th, 31st or 32nd specific aspect.

In some embodiments, R ₂₃ and R ₂₄ are both H; and m' is an integer from 1 to 6.

In some embodiments, R ^h' is H.

In some embodiments, L is represented by the following structural formula:

.

.

In the 34th specific aspect, the imine-containing cytotoxic agent is represented by the following formula:

or their pharmaceutically acceptable salt.

In some embodiments, the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof; the modified imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof; and the conjugate is represented by the following formula:

or a pharmaceutically acceptable salt thereof.

In a 35th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), D is represented by the following formula:

(ПБД11'),

(PBD11'),

(ПБД11),

(PBD11),

(ПБД12'),

(PBD12'),

(ПБД12),

(PBD12),

(ПБД13'),

(PBD13'),

(ПБД13),

(PBD13),

(ПБД14'),

(PBD14'),

(ПБД14),

(PBD14),

(ПБД15'),

(PBD15'),

(ПБД15),

(PBD15),

(ПБД16') и

(PBD16') and

(ПБД16),

(PBD16),

or their pharmaceutically acceptable salt, where:

X ₁ ' is selected from the group consisting of -H, -OH, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl and an amine protecting group;

Y ₁ ' is selected from the group consisting of -H, oxo, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

one of Q ₂ and Q ₂ ' in the formula (PBD15) or (PBD16) is -W'-R ^x -S-; and the other is selected from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , halogen, guanidinium [-NH( C=NH)NH ₂ ], -OR, -NR'R'', -NO ₂ , -NR'COR'', -SR, -SOR', -SO ₂ R', -SO ₃ H, -OSO ₃ H, -SO ₂ NR'R'', cyano, azido, -COR', -OCOR' and -OCONR'R'';

W' is absent or selected from -O-, -N(R ^e )-, -N(R ^e )-C(=O)-, -N(C(=O)R ^e )-, -S- or - CH ₂ -S-, -CH ₂ NR ^e -;

R ^x is absent or selected from linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;

R ^e represents -H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or -(CH ₂ -CH ₂ -O) _n -R ^k where R ^k represents -H, linear branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (for example, -NHR ¹⁰¹ ) or tertiary amino (-NR ¹⁰¹ R ¹⁰² ) group or a 5- or 6-membered nitrogen-containing heterocycle, where R ¹⁰¹ and R ¹⁰² is each independently linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

n is an integer from 1 to 24;

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo; and

R is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or a PEG group -(CH ₂ CH ₂ O) _n -R ^c , where n is an integer from 1 to 24, and R ^c represents a linear or branched alkyl with 1-4 carbon atoms;

R' and R" are each independently selected from -H, -OH, -OR, -NRR ^g' , -COR, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted aryl , having from 6 to 18 carbon atoms, optionally substituted 3-18-membered heterocyclic ring, having from 1 to 6 heteroatoms selected from O, S, N and P, group PEG -(CH ₂ CH ₂ O) _n -R ^c , and R ^g' represents -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or a PEG group -(CH ₂ CH ₂ O) _n -R ^c .

In the 36th specific aspect, for the formula (PBD11'), (PBD12'), (PBD13'), (PBD14'), (PBD15'), (PBD16'), (PBD11), (PBD12), (PBD13) , (PBD14), (PBD15) or (PBD16) X ₁ ' and Y ₁ ' are both -H;

R ₆ is -OMe;

W' represents -N(R ^e )- or -N(R ^e )-C(=O)-;

R ^e represents -H, linear or branched alkyl with 1-4 carbon atoms or -(CH ₂ -CH ₂ -O) _n -R ^k , where R ^k represents -H, linear or branched alkyl with 1-4 atoms carbon;

n is an integer from 2 to 6; and

R ^x is straight or branched alkyl having 1 to 6 carbon atoms; and the remaining variables correspond to those described in the 35th specific aspect.

In a 37th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), L is represented by the following structural formula:

,

,

or their pharmaceutically acceptable salt, where:

s1 is a site covalently linked to D and s2 is a site covalently linked to a maleimide group;

E is -(CR ₁₀ R ₁₁ ) _q -, cycloalkyl or cycloalkylalkyl;

Z is absent, is -SO ₂ NR ₉ -, -NR ₉ SO ₂ -, -C(=O)-NR ₉ -, -NR ₉ -C(=O)-, -C(=O)-O- , -OC(=O)-, -C(=O)-NR ₉ -(CH ₂ CH ₂ O) _p -, -NR ₉ -C(=O)-(CH ₂ CH ₂ O) _p -, - (OCH ₂ CH ₂ ) _p -C(=O)NR ₉ - or -(OCH ₂ CH ₂ ) _p -NR ₉ -C(=O)-;

p is an integer from 1 to 24;

Q is H, a charged substituent, or an ionizable group;

R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are each independently H or optionally substituted alkyl; and

q and r are in each case independently integers from 0 to 10; and the remaining variables are as described in the 35th or 36th specific aspect.

In some embodiments, E is -(CR ₁₀ R ₁₁ ) _q -. In some embodiments, Z is -C(=O)-NR ₉ - or -NR ₉ -C(=O)-. In some embodiments, R ₉ is -H. In some embodiments, R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are H; and q and r are each independently an integer from 1 to 6.

In some embodiments, for -L- described in the 37th specific aspect:

R ₁₂ and R ₁₃ are each independently H or (C ₁ -C ₃ )alkyl;

Q is H or -SO ₃ H or a pharmaceutically acceptable salt thereof

Z is -C(=O)-NR ₉ - or -NR ₉ -C(=O)-;

R ₉ is H or (C ₁ -C ₃ )alkyl;

E is -(CR ₁₀ R ₁₁ ) _q -.

R ₁₀ and R ₁₁ are each independently H or (C ₁ -C ₃ ) alkyl; and

q and r are integers from 1 to 5.

In a 38th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), L is represented by any one of the following structural formulas:

, и

, and

,

,

or their pharmaceutically acceptable salt, and the remaining variables are as described in the 35th or 36th specific aspect.

In a 39th specific aspect, for an imine-containing cytotoxic agent of formula (A) or a conjugate of formula (B), L is represented by the following structural formula:

,

,

or their pharmaceutically acceptable salt, where:

s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group;

R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ are each independently H or optionally substituted alkyl;

m and n are each independently an integer from 0 to 10;

R ^h is H or optionally substituted alkyl;

P _L is an optionally substituted alkylene, -(CH ₂ -CH ₂ -O) _j - (wherein the oxygen atom is linked to a -(C=O)-group linked to P), an amino acid residue or a peptide containing from 2 to 20 amino acid residues; and

j is an integer from 1 to 24; and the remaining variables are as described in the 35th or 36th specific aspect.

In some embodiments, R ₁₉ , R ₂₀ , R ₂₁ , and R ₂₂ are each H; and m and n are each independently an integer from 1 to 6.

In some embodiments, P _L is a peptide containing 2 to 10 amino acid residues. More specifically, P _L is a peptide containing 2 to 5 amino acid residues. More specifically, P _L is selected from the group consisting of: Ala-Val, Val-Ala, Val-Cit, Cit-Val. Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ - nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu ( SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D -Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D -Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala., Ala-Met, Met-Ala, Gln- Val, Asn-Ala, Gln-Phe, Gln-Ala, Gly-Gly-Gly, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Val-Cit, Ala-Val-Ala and β-Ala-Gly-Gly-Gly.

In some embodiments, L is represented by the following structural formula:

,

,

or their pharmaceutically acceptable salt.

In the 40th specific embodiment, for the methods described herein, the imine-containing cytotoxic agent is represented by the following formula:

(ПБД17');

(PBD17');

(ПБД17);

(PBD17);

(ПБД18') или

(PBD18') or

(ПБД18),

(PBD18),

or their pharmaceutically acceptable salt, where:

dotted lines indicate the optional presence of a double bond;

R ^3'' represents a C _3-12 alkylene group,

each X' is at each occurrence independently -O-, -S- or -N(H)-;

each R ² is independently selected from -H, -OH, -CN, -R ^1' , -OR ^1' , -O-SO ₂ -R ^1' , -CO ₂ R ^1' , -COR ^1' or halogen, or both R ² taken together are ═O, ═CH ₂ , ═CH-R ^a or ═C(R ^a ) ₂ ;

each R ^2' is independently selected from -H, -OH, -CN, -R ^1' , -OR ^1' , -O-SO ₂ -R ^1' , -CO ₂ R ^1' , -COR ^1' or halogen;

R ^4b is a leaving group selected from -OR ⁶ ', -OCOR ⁴ ', -OCOOR ⁴ ', -OCONR ^4' R ^5' , -NR ^4' R ^5' , -NR ^4' COR ^5' , -NR ^4' NR ^4' R ⁵ , an optionally substituted 5- or 6-membered nitrogen-containing heterocycle (e.g. piperidine, tetrahydropyrrole, pyrazole, morpholine), guanidinium represented by -NR ^4' (C=NH)NR ^4' R ^5' , amino acids or a peptide represented by -NR ^6' COP', where P' is an amino acid or a polypeptide containing from 2 to 20 amino acid units, -SR ^6' , -SOR ^4' , -SO ₂ M, -SO ₃ M, -OSO ₃ M, halo, cyano and azido;

^RL is a linker carrying a maleimide moiety that can form a covalent bond with a cell-binding agent (CCA);

R ⁶ and R ⁹ are independently selected from -H, -R ^1' , -OH, -OR ^1' , -SH, -SR ^1' , -NH ₂ , -NHR ^1' , -NR ^1' R ^3' , - NO ₂ , Me ₃ Sn and halogen; and

R ^1' and R ^3' are each independently selected from optionally substituted C _1-12 alkyl, C _3-20 heterocyclyl, or C _5-20 aryl groups, and optionally in connection with the group -NR ^1' R ^3' , R ^1' and R ^3' together with nitrogen, the atom to which they are attached forms an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring; or any pair of adjacent groups from R ⁶ to R ⁹ together form a group -O-(CH ₂ ) _p -O-, where p is 1 or 2;

R ^4' and R ^5' are each independently selected from -H, -OH, -OR ^6' , -NHR ^6' , -NR ^6' ₂ , -COR ⁶ , optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl, having 1 to 10 carbon atoms, a polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^b or an optionally substituted 3-18 membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N or P ;

R ^6' at each occurrence is independently selected from the group consisting of -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, -(CH ₂ CH ₂ O) _n -R ^b , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen or sulfur, and an optionally substituted 3-18 membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N, or P;

R ^a is independently selected from -R ^1' , -CO ₂ R ^1' , -COR ^1' , -CHO, -CO ₂ H or halogen;

R ^b is -H or substituted or unsubstituted linear or branched alkyl having from 1 to 4 carbon atoms;

M is H or a pharmaceutically acceptable cation; and

n is an integer from 1 to 24.

In the 41st specific aspect for the methods described herein, the imine-containing cytotoxic agent is represented by the following formula:

(ПБД19');

(PBD19');

(ПБД19);

(PBD19);

(ПБД20') или

(PBD20') or

(ПБД20),

(PBD20),

or its pharmaceutically acceptable salt, where R ^3'' represents C _3-5 alkylene and the remaining variables as described above in the 40th specific aspect.

, где L¹ представляет собой расщепляемый линкер, A представляет собой связывающую группу, несущую малеимид, способный связывать L¹ со связывающим клетки агентом, L² представляет собой ковалентную связь или вместе с -OC(=O)- образует саморасщепляющийся линкер; а остальные переменные являются такими, как описаны выше в 40-ом или 41-ом конкретном аспекте.In the 42nd specific aspect, for the formula (PBD17'), (PBD18'), (PBD19'), (PBD20'), (PBD17), (PBD18), (PBD19) or (PBD20), R ^L is

, where L ¹ is a cleavable linker, A is a linking group bearing a maleimide capable of linking L ¹ with a cell-binding agent, L ² is a covalent bond or together with -OC(=O)- forms a self-cleaving linker; and the remaining variables are as described above in the 40th or 41st specific aspect.

In the 43rd specific aspect for R ^L described in the 42nd specific aspect, L ¹ is a peptide containing from 2 to 10 amino acid residues; and the remaining variables are as described above in the 42nd specific aspect. More specifically, L ¹ is a peptide containing 2 to 5 amino acid residues. Even more specifically, L ¹ is selected from the group consisting of Phe-Lys, Val-Ala, Val-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, Trp- Cit, Lys-Lys, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D- Arg-D-Arg, Gly-Gly-Gly, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO:1), β-Ala-Leu-Ala-Leu (SEQ ID NO:2) and Gly-Phe-Leu-Gly (SEQ ID NO:3).

In the 44th specific aspect for R ^L described in the 42nd specific aspect, -C(=O)O- and L ² together form the group:

where the asterisk indicates the point of attachment to position N10, the wavy line indicates the point of attachment to the L ¹ linker, Y' represents -NH-, -O-, -C(=O)NH- or -C(=O)O-, and n is from 0 to 3; and the remaining variables correspond to those described in the 42nd or 43rd specific aspect.

In the 45th specific aspect for R ^L described in the 42nd specific aspect, -C(=O)O- and L ² together form the group:

,

,

and the remaining variables correspond to those described in the 42nd or 43rd specific aspect.

In the 46th specific aspect for R ^L described in the 42nd specific aspect, L ¹ and L ² together with -OC(=O)- constitute a group selected from:

,

,

where the asterisk indicates the point of attachment to position N10, and the wavy line indicates the point of attachment to the remainder of the linker L ¹ or the point of attachment to A.

In the 47th specific aspect for R ^L described in the 42nd specific aspect, A is represented by one of the following:

, где звездочка обозначает точку присоединения к L¹, а p равен от 1 до 6; или(i)

, where the asterisk denotes the point of attachment to L ¹ and p is from 1 to 6; or

, где звездочка обозначает точку присоединения к L¹, r равен 0 или 1, а s равен от 0 до 30;(ii)

, where the asterisk denotes the point of attachment to L ¹ , r is 0 or 1, and s is from 0 to 30;

and the remaining variables are as described in the 42nd, 43rd, 44th, 45th or 46th specific aspect.

In some embodiments, p is from 4 to 6; r is 1; and s is between 1 and 10.

In the 48th specific aspect for the methods described in the present invention, the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof, the modified imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, and the conjugate is represented by the following formula:

where s is from 2 to 8; w is 1 or 2; and SKA is a cell-binding agent described here. In some embodiments, s is 7.

In some embodiments, for the methods described in the present invention, the imine-containing cytotoxic agent is represented by the following formula:

;

;

or a pharmaceutically acceptable salt thereof, the modified imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, and the conjugate is represented by the following formula:

where s is from 2 to 8; w is 1 or 2; and SKA is a cell-binding agent described here. In some embodiments, s is 7.

In the 49th specific aspect for the methods described in the present invention, the imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, the modified imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, and the conjugate is represented by the following formula:

or its pharmaceutically acceptable salt, where s is from 2 to 8; w is 1 or 2; and SKA is a cell-binding agent described here. In some embodiments, s is 7.

In some embodiments, for the methods described in the present invention, the imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, the modified imine-containing cytotoxic agent is represented by the following formula:

,

,

or a pharmaceutically acceptable salt thereof, and the conjugate is represented by the following formula:

or its pharmaceutically acceptable salt, where s is from 2 to 8; w is 1 or 2; and SKA is a cell-binding agent described here. In some embodiments, s is 7.

In some embodiments, the charged substituent or ionizable group Q described in any of the above embodiments is i) -SO ₃ H, -Z'-SO ₃ H, -OPO ₃ H ₂ , -Z'-OPO ₃ H ₂ , -PO ₃ H ₂ , -Z'-PO ₃ H ₂ , -CO ₂ H, -Z'-CO ₂ H, -NR ₁₁ R ₁₂ or -Z'-NR ₁₁ R ₁₂ , or a pharmaceutically acceptable salt thereof; or ii) -N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- or -Z'-N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- ; Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted phenylene; each R ₁₄ to R ₁₆ is independently optionally substituted alkyl; and X ^- is a pharmaceutically acceptable anion. More specifically, Q is -SO ₃ H or a pharmaceutically acceptable salt thereof. More specifically, Q is -SO ₃ Na.

BINDERS CELLS AGENTS

In some embodiments, the cell-binding agent may be used in the present methods as an antibody having an engineered cysteine residue (eg, at position 442 of the heavy chain(s) according to EU/OU numbering). The engineered Cys residue may be located on one or both of the heavy chains of an antibody, or on one or both of the light chains of an antibody, or an antigen-binding portion thereof, or a combination thereof. In some cases, this means that the Cys residue is at position 442 of the heavy chain(s) of the antibody according to the EU/OU numbering. In some embodiments, the antibody is a cysteine engineered antibody as described herein.

In some embodiments, an antibody of the present invention is a monoclonal antibody, a chimeric antibody, a humanized antibody, a resurfaced antibody, or a human antibody.

In other embodiments, the cell-binding agent is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a fragment (or "antigen-binding portion") of a monoclonal antibody that specifically binds to the target cell , a chimeric antibody, a fragment (or "antigen-binding portion") of a chimeric antibody that specifically binds to a target cell, a domain-specific antibody (eg, sdAb), or a fragment of a domain-specific antibody that specifically binds to a target cell.

In still other embodiments, the cell-binding agent is a humanized antibody, a humanized single chain antibody, or a fragment (or "antigen-binding portion") of a humanized antibody. In a specific embodiment, the humanized antibody is huMy9-6 or another related antibody as described in US Patent Nos. 7,342,110 and 7,557,189. No. 8,557,966. In still other embodiments, the humanized antibody is an anti-CD123 antibody described in U.S. Application No. 15/195401, filed June 28, 2016, titled "ANTI-CD123 ANTIBODIES AND CONJUGATES AND DERIVATIVES THEREOF" ("ANTI-CD123 ANTIBODIES AND CONJUGATES AND THEIR DERIVATIVES). The contents of all of these applications are incorporated herein by reference in their entirety.

In yet other embodiments, the cell-binding agent is a surface-altered antibody, a surface-altered single chain antibody, an antibody fragment (or "antigen-binding portion"), or a bispecific antibody.

In another aspect of the invention, a recombinant antibody is provided comprising a mature truncated heavy chain, light chain, or antigen-binding portion thereof derived from the heavy chain (HC), light chain (LC) of the recombinant antibody in question, or an antigen-binding portion thereof, as described herein.

For example, the recombinant antibody may be or may comprise a scFv-Fc, Fcab, mAb2, small modular immunopharmaceutical (SMIP), Genmab/unibody or duotelo, minibody, IgGΔCH2, DVD-Ig, protelo, intrabody, or multispecific antibody.

^DuoBody® is a heterodimeric, bispecific, modified IgG1 antibody based on the IgG1 hinge region, which typically contains (i) a stable hinge region that contains the CPPC sequence and does not allow Fab fragment exchange in vivo , and (ii) an IgG4-like CH3 domain, modified to contain the residues F405L and K409R, which allows the exchange of Fab fragments in vivo (See, for example, WO2008119353 and WO2011131746).

In some embodiments, a recombinant antibody may comprise 1, 2, 3, or 4 mature truncated heavy chain, light chain, or antigen-binding portions thereof, each derived from either the heavy chain (HC), light chain (LC) of the recombinant antibody in question, or their antigen-binding portion described herein.

In other embodiments, the recombinant antibody may be a heterodimeric antibody comprising a first heavy chain polypeptide and a second heavy chain polypeptide, wherein the Fc region of the first heavy chain polypeptide and the Fc region of the second heavy chain polypeptide are in contact at a contact surface, and the contact surface of the Fc region the second heavy chain polypeptide contains a bulge with the possibility of being located in the depression on the contact surface of the Fc region of the first heavy chain polypeptide. In some embodiments, the tang-to-trough technique for promoting specific heavy chain pairing in a bispecific antibody can be further improved based on, for example, Genentech/Roche's CrossMab technology, e.g., by shuffling the CH1 and kappa constant regions to further reduce or eliminate mismatch of light chains.

Alternatively, similar results can also be achieved using LC heterodimers, such as the Zymeworks Azymetric™ IgG1 heterodimer light chain platform technology, which fully complements many other biological approaches, including conventional light chain, domain antibody, and single chain formats, when developed completely bispecific antibodies.

In some embodiments, the Fc region of the second heavy chain polypeptide has been changed from the template/parent polypeptide to encode the bump, or the Fc region of the first heavy chain polypeptide has been changed from the template/parent polypeptide to encode the recess, or both.

In other embodiments, the bulge and the recess contain a naturally occurring amino acid residue.

In other embodiments, the Fc region of the second heavy chain polypeptide containing the bulge is obtained by replacing the original residue at the template/parent polypeptide interface with an insertion residue having a larger side chain volume than the original residue.

In yet other embodiments, the Fc region of a second heavy chain polypeptide containing a bulge is obtained by a method comprising the step of replacing nucleic acids encoding an initial residue from the contact surface of said polypeptide with nucleic acids encoding an insertion residue having a larger side chain volume, than the original.

In some embodiments, the antibody comprises bispecific, multispecific, and monospecific antibody variants that include antigen-binding regions and a heavy chain constant domain, wherein the heavy chain constant domain is modified to include Cys at position 442 according to EU/OU numbering.

In other embodiments, the antibody can bind to a ligand on a target cell, such as a ligand on a cell surface, including cell surface receptors.

Specific exemplary antigens or ligands may include renin; growth hormone (eg, human growth hormone and bovine growth hormone); growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1 antitrypsin; A-chain of insulin; B-chain of insulin; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factor (eg, vmc factor, factor IX, tissue factor, and von Willebrand factor); an anti-clotting factor (eg, protein C); atrial natriuretic factor; lung surfactant; a plasminogen activator (eg, urokinase, human or tissue-type urinary plasminogen activator); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor alpha and beta; enkephalinase; RANTES (i.e. regulator of normal T cell expression and secretion activation); inflammatory protein-1-alpha human macrophages; serum albumin (human serum albumin); a Mueller inhibitor; A-chain of relaxin; B-chain of relaxin; prorelaxin; mouse gonadotropin-associated peptide; microbial protein (beta-lactamase); DNases; IgE; cytotoxic antigen associated with T-lymphocytes (for example, CTLA-4); inhibin; activin; vascular endothelial growth factor; hormone or growth factor receptor; protein A or D; rheumatoid factor; neurotrophic factor (eg, bone-derived neurotrophic factor, neurotrophin-3, -4, -5 or -6), nerve growth factor (eg, NGF-β); platelet growth factor; fibroblast growth factor (eg, aFGF and bFGF); fibroblast growth factor receptor 2; epidermal growth factor; transforming growth factor (eg, TGF-alpha, TGF-β1, TGFβ2, TGFβ3, TGFβ4 and TGFβ5); insulin-like growth factor-I and -II; des(1-3)-IGF-I (IGF-I brain); a protein that binds insulin-like growth factor; melanotransferrin; Epcam; GD3; FLT3; PSMA; PSCA; MUC1; MUC16; STEAP; CEA; TENB2; EpA receptor; EphB receptor; folate receptor; FOLR1; mesothelin; crypto; alpha _v beta ₆ ; integrins; VEGF; VEGFR; EGFR; transferrin receptor; IRTA1; IRTA2; IRTA3; IRTA4; IRTA5; CD proteins (eg, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52 , CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD123, CD134, CD137, CD138, and CD152), one or more tumor-associated antigens or cell surface receptors (see U.S. Publication No. 2008/0171040 or U.S. Publication US No. 2008/0305044, incorporated by reference in its entirety); erythropoietin; osteoinductive factor; immunotoxin; bone morphogenetic protein; interferon (eg, interferon-alpha, -beta and -gamma); colony stimulating factor (eg M-CSF, GM-CSF and G-CSF); interleukins (eg, IL-1 to IL-10); superoxide dismutase; T-cell receptor; surface membrane protein; decay acceleration factor; viral antigen (for example, part of the envelope of HIV); transport protein, homing receptor; addressin; regulatory protein; integrin (eg CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM;) tumor associated antigen (eg HER2, HER3 and HER4 receptors); endoglin; c-Met; c-kit; 1GF1R; PSGR; NGEP; PSMA; PSCA; TMEFF2; LGR5; B7H4; and fragments of any of the above polypeptides.

In some embodiments, the cell-binding agent is an anti-folate receptor antibody. More specifically, an anti-folate receptor antibody is a humanized antibody that specifically binds human folate receptor 1, wherein the antibody contains: (a) a heavy chain CDR1 containing GYFMN (SEQ ID NO: 4); a heavy chain CDR2 containing RIHPYDGDTFYNQXaa ₁ FXaa ₂ Xaa ₃ (SEQ ID NO: 5); and a heavy chain CDR3 containing YDGSRAMDY (SEQ ID NO: 6); and (b) a light chain CDR1 containing KASQSVSFAGTSLMH (SEQ ID NO: 7); Light chain CDR2 containing RASNLEA (SEQ ID NO: 8); and a light chain CDR3 containing QQSREYPYT (SEQ ID NO: 9); where Xaa ₁ is selected from K, Q, H and R; Xaa ₂ is selected from Q, H, N and R; and Xaa ₃ is selected from G, E, T, S, A, and V. Preferably, the heavy chain CDR2 sequence contains RIHPYDGDTFYNQKFQG (SEQ ID NO: 10).

В других вариантах осуществления антитело против фолатного рецептора представляет собой гуманизированное антитело или его антигенсвязывающий фрагмент, которое специфически связывает фолатный рецептор 1 человека, содержащее тяжелую цепь, имеющую аминокислотную последовательность QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQ KFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLCLSPG (SEQ ID NO: 11).

В других вариантах осуществления антитело против фолатного рецептора представляет собой гуманизированное антитело или его антигенсвязывающий фрагмент, которое специфически связывает фолатный рецептор 1 человека, содержащее легкую цепь, имеющую аминокислотную последовательность DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD RFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12); или DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD RFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13).

In other embodiments, the anti-folate receptor antibody is a humanized antibody, or an antigen-binding fragment thereof, that specifically binds the human folate receptor 1, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. Preferably, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 13 (huMov19).

In other embodiments, the anti-folate receptor antibody is a humanized antibody, or an antigen-binding fragment thereof, containing an engineered Cys residue (e.g., C442) and a heavy chain variable domain at least about 90%, 95%, 99%, or 100% identical to QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQ KFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSS (SEQ ID NO: 14), и вариабельный домен легкой цепи, по меньшей мере на около 90%, 95%, 99% или 100% идентичный DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD RFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR (SEQ ID NO: 15); or DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPD RFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR (SEQ ID NO: 16).

In a specific embodiment, the humanized antibody is huMy9-6 or another related antibody as described in U.S. Patent Nos. 7,342,110 and 7,557,189. described in U.S. Patent No. 8,577,966. In some embodiments, the humanized antibody is an anti-CD37 (e.g., anti-CD37-3) antibody described in U.S. Patent No. 8,765,917. In some embodiments, the humanized antibody is an anti-EGFR antibody described in U.S. Pat. No. 8790649. In other embodiments, the implementation of the antibody is an antibody against EGFR. In some embodiments, the anti-EGFR antibody is a non-antagonist antibody, including, for example, the antibodies described in WO2012058592, incorporated herein by reference. In other embodiments, the anti-EGFR antibody is a non-functional antibody, such as humanized ML66. More specifically, the anti-EGFR antibody is huML66.

In some embodiments, the antibody is an anti-CD123 antibody, such as the humanized huCD123 antibody described in U.S. Application No. 15/195401, filed Jun. CD123 ANTIBODIES AND CONJUGATES AND THEIR DERIVATIVES") (full content, including all sequences and graphics, is incorporated herein). In a specific embodiment, an anti-CD123 antibody or antigen-binding fragment thereof comprises: a) at least one heavy chain variable region or fragment thereof containing three consecutive complementarity determining regions (CDRs) CDR1, CDR2 and CDR3, respectively, wherein CDR1 has the sequence SSIMH ( SEQ ID NO: 17), CDR2 has the sequence YIKPYNDGTKYNEKFKG (SEQ ID NO: 18) and CDR3 has the sequence EGGNDYYDTMDY (SEQ ID NO: 19); and b) at least one light chain variable region or fragment thereof comprising three consecutive complementarity determining regions (CDRs) CDR1, CDR2 and CDR3, respectively, wherein CDR1 has the sequence RASQDINSYLS (SEQ ID NO: 20), CDR2 has the sequence RVNRLVD ( SEQ ID NO: 21) and CDR3 has the sequence LQYDAFPYT (SEQ ID NO: 22).

In other specific embodiments, the CD123 antibody, or antigen-binding fragment thereof, contains an engineered Cys residue (eg, C442); an immunoglobulin heavy chain variable domain at least about 90%, 95%, 99%, or 100% identical to QXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNE KFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSS (SEQ ID NO:23); and an immunoglobulin light chain variable region having an amino acid sequence at least about 90%, 95%, 99%, or 100% identical to DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSG SGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKR (SEQ ID NO:24). In certain embodiments, Xaa, the second residue from the N-terminus of SEQ ID NO: 23, is Phe. In other embodiments, Xaa is Val.

В некоторых вариантах осуществления антитело против CD123 или его антигенсвязывающий фрагмент могут содержать: область тяжелой цепи иммуноглобулина, имеющую аминокислотную последовательность QVQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNE KFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLCLSPG (SEQ ID NO:25);

и область легкой цепи иммуноглобулина, имеющую аминокислотную последовательность DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSG SGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:26).

COMPOSITIONS AND METHODS OF APPLICATION

The present invention also includes cell-binding agent-cytotoxic agent conjugates prepared by any of the methods of the present invention, a composition (e.g., a pharmaceutical composition) comprising cell-binding agent-cytotoxic agent conjugates prepared by any of the methods of the present invention, and a carrier (pharmaceutically acceptable carrier) . The present conjugates and compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (eg, human). The present invention includes a method for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human), comprising administering to said mammal a therapeutically effective amount of the conjugates obtained by the methods of the present invention described above, or a composition thereof, alone or in combination with a second therapeutic agent.

In some embodiments, the proliferative disorder is cancer. The cancer may include hematologic cancer or a solid tumor. More specifically, the cancer is a leukemia (e.g., acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) such as acute B-lymphoblastic leukemia (B-ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL) ) or lymphoma, melanoma, lung cancer (eg, non-small cell lung cancer), ovarian cancer, endometrial cancer, peritoneal cancer, pancreatic cancer, breast cancer, prostate cancer, and cervical cancer.

The present invention also provides methods of treatment comprising administering to a subject in need of treatment an effective amount of any of the conjugates described above.

Similarly, the present invention provides a method of inducing cell death in selected cell populations, comprising contacting target cells or tissue containing target cells with an effective amount of a cytotoxic agent containing any of the conjugates described above. Target cells are cells to which a cell-binding agent can bind.

If desired, other active agents, such as other anticancer agents, can be administered along with the conjugate.

Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and may be determined by those skilled in the art depending on the clinical situation. Examples of suitable carriers, diluents and/or excipients include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, with or without about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0, 9% saline (0.9% w/v NaCl) and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.

EXAMPLES

Example 1 Preparation of imine-containing cytotoxic agents

The following solvents, reagents, protecting groups, moieties, and other designations may be abbreviated in parentheses:

Me methyl Et ethyl Pr propyl i-Pr isopropyl Bu butyl t-Bu tert-butyl Ph phenyl AC acetyl AcOH/HOAc acetic acid Ala alanine aq. water ACN / CH ₃ CN acetonitrile Divoda deionized water DCM / CH ₂ Cl ₂ dichloromethane boc/boc tert-butoxycarbonyl G gram DMA N,N-dimethylacetamide DIEA or DIPEA N,N-diisopropylethylamine h hour DMF N,N-dimethylformamide EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide min minutes EtOAc ethyl acetate IER or ER electrospray ionization mg milligram LC liquid chromatography HPLC high performance liquid chromatography ml milliliters mmol mmol HCMS liquid chromatography - mass spectrometry mcg microgram µmol micromol MS mass spectrometry µl microliters MeOH methanol NHS N-hydroxysuccinamide us. or sat. saturated CT or CT room temperature (ambient temperature approx. 25 °C) NMR nuclear magnetic resonance spectroscopy THF tetrahydrofuran TEA triethylamine (Et ₃ N) RP-HPLC or RP-HPLC reverse phase high performance liquid chromatography

Synthesis N1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-N6-((S)-1-(((S)-1-((3- ((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indo-9- yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5.6][1.4]diazepino[1.2-a ]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)adipamide, compound D1

NHS ester 1a (8.2 mg, 7.6 µmol) (obtained according to the procedures described in US-2016/0082114, incorporated herein by reference) and 1-(2-aminoethyl)-1H-pyrrole hydrochloride -2,5-dione (2.2 mg 0.011 mmol) was dissolved in anhydrous dichloromethane (305 μl) at room temperature. DIPEA (2.66 μl, 0.015 mmol) was added and the reaction mixture was stirred for 3.5 hours. The reaction mixture was concentrated and purified by RP-HPLC (C18 column, CH ₃ CN/H ₂ O gradient, 35% to 55%). Fractions of the desired product were frozen and lyophilized to give maleimide compound D1 as a white solid powder (5.3 mg, 58% yield). LCMS = 5.11 min (8 min method). MS (m/z): 1100.6 (M + 1) ⁺ .

B. Synthesis of N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-11-(3-((((S)-8-methoxy-6- oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-(((( S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl) -13,13-dimethyl-2,5,8-trioxa-14,15-dithia-11-azanonedecane-19-amide, compound D2

Anhydrous diisopropylethylamine (0.015 ml, 0.085 mmol) and stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated ammonium chloride and diluted with dichloromethane. The resulting mixture was separated in a separating funnel. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and distilled off under reduced pressure. The residue was purified by semi-preparative reverse phase HPLC (column C18, CH ₃ CN/H ₂ O). Fractions that contained pure product were pooled, frozen and lyophilized to give the desired NHS ester, 2b (29.7 mg, 60% yield). LCMS = 9.1 min (15 min method). MS (m/z): 1157.3 (M + 1) ⁺ .

DIPEA (0.0022 ml , 0.013 mmol). The mixture was stirred at room temperature for 3 hours, then it was distilled off under reduced pressure. The residue was purified by semi-preparative reverse phase HPLC (column C18, CH ₃ CN/H ₂ O). Fractions that contained pure product were pooled, frozen and lyophilized to give the desired maleimide, Compound D2 (10 mg, 80% yield). LCMS = 8.3 min (15 min method). MS (m/z): 1181.8 (M + 1) ⁺ .

C. Synthesis 1-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-((5-((3-((((S) -8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl) -5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl )oxy)methyl)phenyl)amino)-2-methyl-5-oxopentan-2-yl)disulfanyl)-1-oxobutane-2-sulfonic acid, compound D3

To a suspension of free thiol 3а (88 mg, 0.105 mmol) and 1-((2,5-dioxopyrrolidin-1-yl)oxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butan-2-sulfonic acid ( 64.0 mg, 0.158 mmol) in anhydrous dichloromethane (2.10 ml) was added DIPEA (55.0 μl, 0.315 mmol) under nitrogen at room temperature. The mixture was stirred for 16 hours and then 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (55.6 mg, 0.315 mmol), anhydrous dichloromethane (1.0 ml) and DIPEA (0.055 ml) were added. , 0.315 mmol). The mixture was stirred for an additional 5 hours at room temperature, after which the reaction mixture was concentrated in vacuo. The resulting residue was purified by RP-HPLC (C18, CH ₃ CN/H ₂ O). Fractions containing the desired product were frozen and lyophilized to give maleimide compound D3 (20 mg, 16% yield) as a white solid. LCMS = 4.92 min (8 min method). MS (m/z): 1158.6 (M + 1) ⁺ .

D. Synthesis N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-11-(3-((((S)-8-methoxy-6-oxo -11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S )-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5.6][1.4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)- 2,5,8-trioxa-11-azapentadecan-15-amide, compound D4

To a solution of NHS ester 4a (5 mg, 4.82 µmol) and 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1.7 mg, 9.64 µmol) in anhydrous dichloromethane (200 µl) DIPEA (1.512 µl, 8.68 µmol) was added under nitrogen atmosphere. The mixture was stirred at room temperature for 4 hours and then concentrated in vacuo. The resulting residue was purified by RP-HPLC (C18, CH ₃ CN/H ₂ O). Fractions containing the desired product were frozen and lyophilized to give maleimide, Compound D4 (3.5 mg, 68% yield). LCMS = 4.61 min (15 min method). MS (m/z): 1062.8 (M + 1) ⁺ .

Example 2. Selective sulfonation imine-containing cytotoxic agent, containing maleimide group

To a mixture of 50 mM sodium succinate pH 3.3 (116.5 ml) and DMA (98.5 ml) was added D1 (263.6 mg) dissolved in 21.4 ml DMA. Then 3.4 ml of a 100 mM solution of sodium bisulfite (1.4 equivalents) in water containing 1 v/v% DMA was introduced into the reaction. The homogeneous mixture was allowed to react for 2 h at 25°C, after which the completeness of the reaction was analyzed using SELC-MS. The reaction mixture is suitable for conjugation without further purification. As shown in FIG. 1, analysis of the reaction mixture by SELC-MS shows 92.5% imine-sulfo D1, 1.9% unreacted D1, 0.8% maleimide-sulfo D1 and 4.8% disulfo-D1. Mode of determination of negative ions ESI-MS [MH] - calculated for imine-sulfo D1 (C ₆₀ H ₆₂ N ₉ O ₁₅ S ^- ): 1180.41; found: 1180.03.

Example 3. Influence bisulfite sodium on the selective sulfonation

As indicated in Table 1, the following reagents were added to the required volume of 50 mM sodium succinate pH 3.3 buffer in the following order: DMA (38.8 μl), required volume of 20 or 40 mM aqueous sodium bisulfite solution containing 1 v/v % DMA, and 8.9 mM D1 in DMA (11.2 µl). The resulting reaction mixture containing 50% DMA by volume was allowed to react for 20 hours at 25°C. The reaction products were analyzed using SELC-MS. The relative content of the observed products disulfo D1, maleimide-sulfo D1, imine-sulfo D1 and unsulfonated products D1 are shown in Table 1 .

Table one

Sodium succinate buffer (µl) Initial mixture of sodium bisulfite Reaction products, D1 (%) Reaction Total Equivalents Initial mixture concentration (mM) Volume added (µl) disulfo Maleimide-sulfo imine-sulfo Nonsulfonated Total Reactive Maleimide Remaining one 46 0.8 twenty four 0.6 1.4 35.9 62.1 98.0 2 45 1.0 twenty 5 1.1 1.4 53.4 44.2 97.6 3 44 1.2 twenty 6 2.0 1.2 69.6 27.2 96.8 four 43 1.4 twenty 7 2.6 1.1 79.1 17.2 96.3 5 42 1.6 twenty eight 5.0 1.1 85.7 8.2 93.9 6 41 1.8 twenty 9 8.3 1.2 84.6 5.9 90.5 7 45 2.0 40 5 8.7 0.8 88.2 2.3 90.5 eight 44 2.4 40 6 13.0 0.9 83.9 2.2 86.1 9 43 2.8 40 7 16.4 1.1 79.8 2.7 82.5 ten 42 3.2 40 eight 19.8 1.5 75.8 2.9 78.7 eleven 41 3.6 40 9 22.9 1.8 71.9 3.4 75.3

Example four. Influence pH on the selective sulfonation

As detailed in Table 2, to 44.0 µl of 50 mM sodium succinate buffer at the indicated pH, the following reagents were added in the following order: 20 mM aqueous sodium bisulfite (6.0 µl) containing 1 v/v % DMA, DMA (38.8 µl) and 8.9 mM D1 in DMA (11.2 µl). The resulting reaction mixture containing 50% DMA by volume was allowed to react for 4 hours at 25°C. The reaction products were analyzed using SELC-MS. The relative content of the observed products disulfo, maleimide-sulfo, imine-sulfo and unsulfonated D1 is shown in Table 2 .

Table 2.

Sodium succinate buffer pH Sodium bisulfite (total equivalents) Reaction products, DGN549-C (%) Reaction disulfo Maleimide-sulfo imine-sulfo Nonsulfonated Total Reactive Maleimide Remaining one 2.9 1.2 0.7 1.0 68.5 29.8 98.3 2 3.1 1.2 0.8 1.3 65.0 33.0 97.9 3 3.3 1.2 1.1 1.7 66.9 30.3 97.1 four 3.4 1.2 1.3 1.4 66.5 30.7 97.3 5 3.7 1.2 2.3 2.7 65.6 29.4 94.9

Example 5. Selective sulfonation With buffer or without

As detailed in Table 3 , to 21.6 µl of DMA was added 22.0 µl of water or 50 mM sodium succinate buffer at pH indicated, 20 mM aqueous sodium bisulfite (3.0 µl) containing 1 v/v % DMA , and 14.5 mM D1 in DMA (3.4 μl). The resulting reaction mixture containing 50% DMA by volume was allowed to react for 6 hours at 25°C. The reaction products were analyzed using SELC-MS. The relative content of the observed products disulfo, maleimide-sulfo, imine-sulfo and unsulfonated D1 is shown in Table 3 .

Table 3.

Sodium succinate buffer pH Sodium bisulfite (total equivalents) Reaction products, D1 (%) Reaction disulfo Maleimide-sulfo imine-sulfo Nonsulfonated Total Reactive Maleimide Remaining one 3.5 1.2 0.8 0.5 86.9 11.8 98.7 2 4.0 1.2 4.8 2.9 70.0 22.8 92.8 3 only water 1.2 2.8 1.8 75.8 19.6 95.4

Similarly, as detailed in Table 4 , to 47.6 µl of DMA was added 55.6 µl of water or 50 mM sodium succinate buffer pH 4.75, 20 mM aqueous sodium bisulfite (6.9 µl) containing 1 v/v % DMA, and 8.4 mM D1 in DMA (14.9 μl). The resulting reaction mixture, containing 50% DMA by volume, was allowed to react for 24 h at 25°C. The reaction products were analyzed by SALC-MS (see Fig. 2). The relative content of the observed products of disulfo, maleimide-sulfo, imine-sulfo and non-sulfonated DGN549-C is indicated in table 4 .

Table four.

Sodium succinate buffer pH Sodium bisulfite (total equivalents) Reaction products, D1 (%) Reaction disulfo Maleimide-sulfo imine-sulfo Nonsulfonated Total Reactive Maleimide Remaining one 4.75 1.1 9.8 40.7 13.4 36.1 49.5 3 only water 1.1 1.5 0.9 73.8 23.8 97.6

In another experiment, as detailed in Table 5 , the indicated volume of water, the indicated volume of 20 mM aqueous sodium bisulfite solution, was added to 24.6 μl of DMA. The pH of these solutions was as indicated in Table 5 . To these mixtures was added 11.2 mm D1 in DMA (5.4 μl). The resulting reaction mixtures containing 50% DMA by volume were left to react for 1-2 h at 25°C. The reaction products were analyzed using SELC-MS. The relative content of the observed products of disulfo, maleimide-sulfo, imine-sulfo and unsulfonated D1 is indicated in table 5 .

Table 5

Water (µl) Initial mixture of sodium bisulfite Reaction products, DGN549-C (%) Reaction Total Equivalents Initial mixture concentration (mM) Volume added (µl) Observed reaction pH disulfo Maleimide-sulfo imine-sulfo Nonsulfonated Total Reactive Maleimide Remaining one 21.0 3.0 twenty 9.0 4.2 6.9 - 90.9 2.2 93.1 2 15.0 5.0 twenty 15.0 4.2 20.5 - 78.0 1.5 79.5

"-" means not observed by HPLC.

Example 6. Cooking conjugates antibody-cytotoxic agent

The sulfonation reaction mixture (240 ml, 3.5 eq.) prepared according to Example 2 was then introduced into a 50 mM potassium phosphate pH 6.0 solution containing 10 g of anti-CD123 antibody with reduced engineered cysteine residues C442. At a final antibody concentration of 2 mg/ml and 15 v/v % DMA, the conjugation reaction was allowed to proceed for 18 hours at 25°C. SEC analysis of the reaction product gives ADC with a DAR (drug to antibody ratio) of 1.9 and % HMW (percentage of high molecular weight particles) of 4.4% compared to 3.7% before conjugation.

Conjugates with two other humanized C442 engineered cysteine monoclonal antibodies were also prepared according to the same procedures described above.

Example 7. Selective sulfonation imine-containing dimers PBB, containing maleimide group

To 21.2 µl of 50 mM sodium succinate buffer, pH 3.3, was added in the following order: 20 µl of DMA, 3.8 µl of 20 mM sodium bisulfite aqueous solution, and 5.0 µl of 10.0 mM thalirin in DMA. This corresponds to 1.5 equivalents of bisulfite with respect to talerin. The resulting reaction mixture containing 50% DMA by volume was allowed to react for 4 hours at 25°C. The reaction products were analyzed using SELC-MS. The relative abundances of the observed imindisulfo, total iminmonosulfo, nonsulfonated and total maleimidesulfo products were determined as indicated in Table 6 . IER-MS calculated. for talirin (C ₆₀ H ₆₅ N ₈ O ₁₂ ⁺ ) [M+H] ⁺ 1089.4716, found 1089.4716; calculated for imine-monosulfated talerin (C ₆₀ H ₆₅ N ₈ O ₁₅ S ^- ) [MH] ^- 1169.4296, found 1169.4345; calculated for imine-disulfonated thalyrin (C ₆₀ H ₆₇ N ₈ O ₁₈ S ₂ ^- ) [MH] ^- 1251.4020, found 1251.4053. A typical chromatogram (absorbance at 330 nm) of the final reaction mixture is shown in FIG. 3. Structures of identified reaction products

Table 6.

Sodium succinate buffer (µl) Initial mixture of sodium bisulfite Reaction products, thalirin (%) Reaction Total Equivalents
(compared to load) Initial mixture concentration (mM) Volume added (µl) Imindisulfo Total imindisulfo Total kinds of maleimide sulfo Nonsulfonated Total Reactive Maleimide Remaining talirin 21.2 1.5 twenty 3.8 57.3 39.0 <1 3.7 >99.0

talirin

Imine monosulfothalirin (isomer 1)

Imine monosulfothalirin (isomer 2)

Imine di-sulfo-thalyrin

Example 8. Selective sulfonation imine-containing dimers IGN, containing maleimide group

As indicated in Table 7 , to the required volume of 50 mM sodium succinate buffer, pH 3.3, was added in the following order: DMA (21.2 μl), required volume of 20 mM aqueous sodium bisulfite and 13.2 mM D5 in DMA (3.8 µl). The resulting reaction mixture containing 50% DMA by volume was allowed to react for 4 hours at 25°C. The reaction products were analyzed using SELC-MS. The relative content of the observed imindisulfo, iminmonosulfo, nonsulfonated and total maleimidesulfonated products was determined as indicated in Table 7 . IER-MS calculated. for D5 (C ₆₀ H ₆₀ N ₉ O ₁₂ ⁺ ) [M+H] ⁺ 1098.4356, found 1098.4351; calculated for monosulfonated imine D5 (C ₆₀ H ₆₀ N ₉ O ₁₅ S ^- ) [MH] ^- 1178.3935, found 1178.4006; calculated for dimethylsulfonated imine D5 (C ₆₀ H ₆₂ N ₉ O ₁₈ S ₂ ^- ) [MH] ^- 1260.3660, found 1260.3704; calculated for iminsulfansylated maleimidesulfonated D5 (C ₆₀ H ₆₄ N ₉ O ₂₁ S ₃ ^- ) [M-2H] ² z=2670.6653, found 670.6693. Representative chromatograms (absorbance at 330 nm) of the final reaction mixture with 2.0 and 2.5 equivalents of bisulfite are shown in FIG. 4A and 4B. The structures of the identified reaction products are shown below.

Table 7

Sodium succinate buffer (µl) Initial mixture of sodium bisulfite Reaction products, IGN148-mal (%) Reaction Total Equivalents
(compared to load) Initial mixture concentration (mM) Volume added (µl) Imindisulfo Iminmonosulfo Total kinds of maleimide sulfo Nonsulfonated Total Reactive Maleimide Remaining one 21.2 1.5 twenty 3.8 41.9 46.9 1.4 9.7 98.6 2 20.0 2.0 twenty 5.0 76.0 20.3 1.9 1.8 98.1 3 18.7 2.5 twenty 6.3 83.3 6.7 10.0 - 90.0

D5

Imine monosulfo D5

Imine di-sulfo D5

Imine di-sulfo maleimide D5

--->

SEQUENCE LIST

<110> IMMUNOGEN, INC.

<120> SELECTIVE SULPHATION OF BENZODIAZEPINE DERIVATIVES

<130> 128037-03820

<140> PCT/US2017/062989

<141> 11/22/2017

<150> 62/425.761

<151> 11/23/2016

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 4

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Ala Leu Ala Leu

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<221> source

<223> /note="Artificial sequence description: synthetic

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<220>

<221> MOD_OST

<222> (1)..(1)

<223> Beta-Ala

<400> 2

Xaa Leu Ala Leu

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<211> 4

<212> Protein

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<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 3

Gly Phe Leu Gly

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<210> 4

<211> 5

<212> Protein

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<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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Gly Tyr Phe Met Asn

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<210> 5

<211> 23

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<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<220>

<221> OPTION

<222> (14)..(14)

<223> Gln, His or Arg

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<221> OPTION

<222> (18)..(18)

<223> His, Asn or Arg

<220>

<221> OPTION

<222> (21)..(21)

<223> Glu, Thr, Ser, Ala or Val

<220>

<221> OTHER_INDICATIONS

<222> (1)..(23)

<223> /note="Variant residues given in sequence,

are not preferred over those given in the footnotes

for variant positions"

<400> 5

Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Ala Ala

1 5 10 15

Phe Gln Ala Ala Gly Ala Ala

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<210> 6

<211> 9

<212> Protein

<213> Artificial sequence

<220>

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<223> /note="Artificial sequence description: synthetic

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<400> 6

Tyr Asp Gly Ser Arg Ala Met Asp Tyr

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<210> 7

<211> 15

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<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 7

Lys Ala Ser Gln Ser Val Ser Phe Ala Gly Thr Ser Leu Met His

1 5 10 15

<210> 8

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 8

Arg Ala Ser Asn Leu Glu Ala

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<210> 9

<211> 9

<212> Protein

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<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 9

Gln Gln Ser Arg Glu Tyr Pro Tyr Thr

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<210> 10

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 11

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

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Leu His Asn His Tyr Thr Gln Lys Ser Leu Cys Leu Ser Pro Gly

435 440 445

<210> 12

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 12

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala

20 25 30

Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser

65 70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

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Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 13

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 13

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

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Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala

20 25 30

Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser

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Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

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Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

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Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 14

<211> 118

<212> Protein

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<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

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Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys

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Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 15

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 15

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

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Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala

20 25 30

Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser

65 70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 16

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 16

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala

20 25 30

Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 17

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 17

Ser Ser Ile Met His

fifteen

<210> 18

<211> 17

<212> Protein

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<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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Tyr Ile Lys Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

<210> 19

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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Glu Gly Gly Asn Asp Tyr Tyr Asp Thr Met Asp Tyr

1 5 10

<210> 20

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 20

Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser

1 5 10

<210> 21

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 21

Arg Val Asn Arg Leu Val Asp

fifteen

<210> 22

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<400> 22

Leu Gln Tyr Asp Ala Phe Pro Tyr Thr

fifteen

<210> 23

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

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<220>

<221> MOD_OST

<222> (2)..(2)

<223> Any amino acid

<400> 23

Gln Xaa Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Ser

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Lys Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ser Asp Arg Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Asn Asp Tyr Tyr Asp Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Val Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ala Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 25

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Ser

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Lys Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ser Asp Arg Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Asn Asp Tyr Tyr Asp Thr Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Cys Leu Ser

435 440 445

ProGly

450

<210> 26

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Val Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ala Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 27

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<220>

<221> MOD_OST

<222> (1)..(1)

<223> Beta-Ala

<400> 27

Xaa Gly Gly Gly

one

<---

Claims (164)

1. A method for producing a cell-binding agent-cytotoxic agent conjugate, comprising the steps: (a) interaction of an imine moiety in an imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt, or a metabisulfite salt in an aqueous solution at a pH of 2.9 to 4.0 to form a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and (b) reacting the modified cytotoxic agent with a cell-binding agent to form a cell-binding agent-cytotoxic agent conjugate, where the cell-binding agent is an antibody or antibody fragment; D is an imine-containing benzodiazepine compound; and L is a linker, and D is represented by the following formula:

or a pharmaceutically acceptable salt thereof, where: R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' are all H; R ₆ are -OMe; X ₁ ' and Y ₁ ' are both -H; A and A' are -O-; L'' and L''' are both H; L' is represented by the following formula: -NR ₅ -P ₁ -C(=O)-(CR _a R _b ) _s -C(=O)- (B1'); -NR ₅ -P ₁ -C(=O)-Cy-(CR _a R _b ) _s1' -C(=O)- (B2'); -C(=O)-P ₁ -NR ₅ -(CR _a R _b ) _s -C(=O)- (C1'), or -C(=O)-P ₁ -NR ₅ -Cy-(CR _a R _b ) _s1' -C(=O)- (C2'), where: each of R _a and R _b at each occurrence independently represents -H, (C ₁ -C ₃ )alkyl or a charged substituent or an ionizable group Q; s is an integer from 1 to 6; s1' is 0 or an integer from 1 to 6; Cy is cyclic alkyl with 5 or 6 ring carbon atoms optionally substituted with halogen, —OH, (C ₁ -C ₃ )alkyl, (C ₁ -C ₃ )alkoxy, or halo(C ₁ -C ₃ )alkyl; P ₁ is an amino acid residue or a peptide containing 2 to 20 amino acid residues; G is -CH- or -N-; R ₅ at each occurrence is independently —H or optionally substituted linear or branched alkyl having from 1 to 10 carbon atoms; L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where: s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group; R ₂₃ and R ₂₄ are each independently H or optionally substituted alkyl; m' is an integer from 0 to 10; and R ^h ' represents H or optionally substituted alkyl. 2. The method of claim 1, wherein the reaction in step (a) is carried out at a pH of 2.9 to 3.7. 3. Process according to claim 1, wherein the reaction in step (a) is carried out at a pH of 3.1 to 3.5. 4. The method according to p. 1, where the reaction in stage (a) is carried out at a pH of from 3.2 to 3.4. 5. The method according to p. 1, where the reaction in stage (a) is carried out at pH 3.3. 6. The method according to any one of paragraphs. 1-5, where the reaction in step (a) is carried out in the presence of a buffer solution. 7. The method of claim 6 wherein the buffer solution is citrate buffer, acetate buffer, succinate buffer and phosphate buffer. 8. The method of claim 6 wherein the buffer solution is a succinate buffer. 9. A method for producing a cell-binding agent-cytotoxic agent conjugate, comprising the steps: (a) interaction of an imine moiety in an imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt or a metabisulfite salt in an aqueous solution in the absence of a buffer for the formation of a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and (b) reacting the modified cytotoxic agent with a cell-binding agent to form a cell-binding agent-cytotoxic agent conjugate, where the cell-binding agent is an antibody or antibody fragment; D is an imine-containing benzodiazepine compound; and L is a linker, and D is represented by the following formula:

or a pharmaceutically acceptable salt thereof, where: R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' are all H; R ₆ are -OMe; X ₁ ' and Y ₁ ' are both -H; A and A' are -O-; L'' and L''' are both -H; L' is represented by the following formula: -NR ₅ -P ₁ -C(=O)-(CR _a R _b ) _s -C(=O)- (B1'); -NR ₅ -P ₁ -C(=O)-Cy-(CR _a R _b ) _s1' -C(=O)- (B2'); -C(=O)-P ₁ -NR ₅ -(CR _a R _b ) _s -C(=O)- (C1') or -C(=O)-P ₁ -NR ₅ -Cy-(CR _a R _b ) _s1' -C(=O)- (C2'), where: each of R _a and R _b at each occurrence independently represents -H, (C ₁ -C ₃ )alkyl or a charged substituent or an ionizable group Q; s is an integer from 1 to 6; s1' is 0 or an integer from 1 to 6; and Cy is cyclic alkyl with 5 or 6 ring carbon atoms optionally substituted with halogen, —OH, (C ₁ -C ₃ )alkyl, (C ₁ -C ₃ )alkoxy, or halo(C ₁ -C ₃ )alkyl; P ₁ is an amino acid residue or a peptide containing 2 to 20 amino acid residues; G is -CH- or -N-; R ₅ at each occurrence is independently —H or optionally substituted linear or branched alkyl having from 1 to 10 carbon atoms; L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where: s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group; R ₂₃ and R ₂₄ are each independently H or optionally substituted alkyl; m' is an integer from 0 to 10; and R ^h ' represents H or optionally substituted alkyl. 10. The method according to any one of paragraphs. 1-9, wherein 0.5 to 5 equivalents of the bisulfite salt or 0.25 to 2.5 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent. 11. The method of claim 10 wherein 0.8 to 2.0 equivalents of the bisulfite salt or 0.4 to 1.0 equivalents of the metabisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent. 12. The method of claim 10 wherein 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent. 13. The method of claim 10, wherein 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent. 14. The process of claim 1, wherein the reaction of step (a) is carried out at a pH of 2.9 to 3.7 and 1.0 to 1.8 equivalents of the bisulfite salt or 0.5 to 0.9 equivalents of the metabisulfite salt is given in reaction with 1 equivalent of an imine-containing cytotoxic agent. 15. The process of claim 1, wherein the reaction of step (a) is carried out at a pH of 3.1 to 3.5 and 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt is given in reaction with 1 equivalent of an imine-containing cytotoxic agent. 16. The method of claim 1 wherein the reaction of step (a) is carried out at pH 3.3 and 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent. 17. The method according to any one of paragraphs. 1-16, where the reaction of step (a) is carried out in a mixture of an organic solvent and water. 18. The method of claim 17 wherein the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water. 19. The method according to claim 17, wherein the reaction of step (a) is carried out in a mixture of DMA and water, where the volume ratio of DMA to water is 1:1. 20. The method according to any one of paragraphs. 1-19, where in step (a) an imine-containing cytotoxic agent is reacted with sodium bisulfite. 21. The method according to any one of paragraphs. 1-19, where in step (a) an imine-containing cytotoxic agent is reacted with sodium metabisulphite. 22. The method according to any one of paragraphs. 1-21, where the modified cytotoxic agent is not purified prior to reaction with the cell-binding agent in step (b). 23. The method according to any one of paragraphs. 1-21, where the reaction in step (b) is carried out at pH 4 to 9. 24. The method of claim 23, wherein the reaction in step (b) is carried out at a pH of 5 to 8.5. 25. The method of claim 23, wherein the reaction in step (b) is carried out at a pH of 5.5 to 6.5. 26. The method according to any one of paragraphs. 1-25, where the conjugate is purified by tangential flow filtration to obtain a purified conjugate. 27. The method of claim 26, wherein the purified conjugate is prepared in formulation buffer containing a bisulfite salt. 28. The method of claim 27, wherein the formulation buffer contains 10 to 200 µM sodium bisulfite. 29. The method of claim 27 wherein the formulation buffer contains 40 to 80 µM sodium bisulfite. 30. The method of claim 27 wherein the formulation buffer contains 50 µM sodium bisulfite. 31. The method according to any one of paragraphs. 27-30, wherein the formulation buffer has a pH of 4 to 5. 32. The method of claim 31 wherein the formulation buffer has a pH of 4.2. 33. The method according to any one of paragraphs. 1-32, where R _a and R _b are both H; Cy in formulas (B2') and (C2') is cyclohexane; and R ₅ is H or Me. 34. The method of claim 33 wherein s1' is 0 or 1. 35. The method according to any one of paragraphs. 1-34, where the charged substituent or ionizable group Q is i) -SO ₃ H, -Z'-SO ₃ H, -OPO ₃ H ₂ , -Z'-OPO ₃ H ₂ , -PO ₃ H ₂ , - Z'-PO ₃ H ₂ , -CO ₂ H, -Z'-CO ₂ H, -NR ₁₁ R ₁₂ or -Z'-NR ₁₁ R ₁₂ or a pharmaceutically acceptable salt thereof; or ii) -N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- or -Z'-N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- ; Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted phenylene; each R ₁₄ -R ₁₆ is independently optionally substituted alkyl; and X ^- is a pharmaceutically acceptable anion. 36. The method of claim 35 wherein Q is -SO ₃ H or a pharmaceutically acceptable salt thereof. 37. The method according to any one of paragraphs. 1-36, where P ₁ is a peptide containing from 2 to 10 amino acid residues. 38. The method according to p. 37, where P ₁ is a peptide containing from 2 to 5 amino acid residues. 39. The method according to p. 38, where P ₁ is Gly-Gly-Gly, Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit , Ile-Cit, Trp-Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu -Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly -Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val- Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe and Gin-Ala. 40. The method of claim 39 wherein P ₁ is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala. 41. The method according to any one of paragraphs. 1-40, where R ₅ represents -H or (C ₁ -C ₃ )alkyl. 42. The method according to any one of paragraphs. 1-32, where D is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof. 43. The method according to any one of paragraphs. 1-42, where R ₂₃ and R ₂₄ are both H; and m' is an integer from 1 to 6. 44. The method according to any one of paragraphs. 1-43, where R ^h ' represents H. 45. The method according to any one of paragraphs. 1-42, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof. 46. The method according to any one of paragraphs. 1-32, where the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof, and the modified cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof. 47. The method according to p. 46, where the modified cytotoxic agent is represented by the following formula:

or their sodium or potassium salt. 48. The method according to p. 47, where the modified cytotoxic agent is represented by the following formula:

49. The method according to any one of paragraphs. 1-48, where the cell-binding agent is an antibody having an engineered cysteine residue. 50. The method of claim 49 wherein the engineered cysteine residue is at position 442 according to the EU/OU numbering of one or both of the antibody heavy chains. 51. The method of claim 50 wherein the engineered cysteine residue is in both heavy chains. 52. The method of claim 50 wherein the engineered cysteine residue is in one of the heavy chains. 53. The method according to any one of paragraphs. 1-52, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a resurfaced antibody, or a human antibody. 54. The method of claim 53 wherein the antibody is an anti-CD123 antibody. 55. The method of claim 54, wherein the anti-CD123 antibody comprises an immunoglobulin heavy chain region having the amino acid sequence of SEQ ID NO:25 and an immunoglobulin light chain region having the amino acid sequence of SEQ ID NO:26. 56. The method according to any one of paragraphs. 1-32, characterized in that it is intended to obtain a cell-binding agent-cytotoxic agent conjugate represented by the following formula:

or a pharmaceutically acceptable salt thereof, the method comprising the steps of: (a) interaction of an imine moiety in an imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt or a metabisulfite salt in an aqueous solution at a pH of 3.1 to 3.5 to form a modified cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof; and (b) reacting the modified cytotoxic agent, or a pharmaceutically acceptable salt thereof, with the cell-binding agent Ab to form a cell-binding agent-cytotoxic agent conjugate, wherein: Ab is an anti-CD123 antibody comprising an immunoglobulin heavy chain region having the amino acid sequence of SEQ ID NO:25 and an immunoglobulin light chain region having the amino acid sequence of SEQ ID NO:26; and w is 1 or 2. 57. The method according to p. 56, where the reaction in stage (a) is carried out at a pH of from 3.2 to 3.4. 58. The method according to p. 56, where the reaction in stage (a) is carried out at pH 3.3. 59. The method according to any one of paragraphs. 56-58, where the reaction in step (a) is carried out in the presence of a buffer solution. 60. The method of claim 59, wherein the buffer solution is a citrate buffer, an acetate buffer, a succinate buffer, or a phosphate buffer. 61. The method of claim 60 wherein the buffer is a succinate buffer. 62. The method according to any one of paragraphs. 56-61, wherein 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent. 63. The method of claim 62, wherein 1.3 to 1.5 equivalents of the bisulfite salt or 0.65 to 0.75 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent. 64. The method of claim 62, wherein 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent. 65. The method according to any one of paragraphs. 56-64, wherein the reaction of step (a) is carried out at a pH of 3.2 to 3.4 and 1.3 to 1.5 equivalents of sodium bisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent. 66. The method according to any one of paragraphs. 56-65, where the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water. 67. The method according to any one of paragraphs. 56-66, where the reaction in step (a) is carried out at room temperature. 68. The method according to any one of paragraphs. 56-67, where the modified cytotoxic agent is not purified prior to reaction with the cell-binding agent in step (b). 69. The method according to any one of paragraphs. 56-68, where the reaction in step (b) is carried out at a pH of 5.5 to 6.5. 70. The method according to any one of paragraphs. 56-69, where the conjugate is purified by tangential flow filtration to obtain a purified conjugate. 71. The method of claim 70 wherein the purified conjugate is prepared in formulation buffer containing 40 to 80 µM sodium bisulfite having a pH of 4 to 5. 72. The method of claim 71 wherein the formulation buffer contains 50 µM sodium bisulfite having a pH of 4.2.

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