EA039826B1 - Pyrrolobenzodiazepine-antibody conjugates - Google Patents

Abstract

A conjugate of formula (I): Ab-(DL)p, wherein Ab is an antibody that binds to AXL; DL is formula (A).

Description

Some pyrrolobenzodiazepines (PBBs) have the ability to recognize and bind to specific DNA sequences; the preferred sequence is PuGPu. The first PBB-based antitumor antibiotic, anthramycin, was discovered in 1965 (Leimgruber et al., J. Am. Chem. Soc, 87, 5793-5795 (1965); Leimgruber et al., J. Am. Chem. Soc , 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBBs have been reported and more than 10 synthetic routes to various analogs have been developed (Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. and Thurston D.E., Chem. Rev. 2011 111 ( 4), 2815-2864). Family members include abbeimycin (Hochlowski et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 ( Japanese patent 58180 487; Thurston et al., Chem. Brit., 26, 767-772 (1990); Bose et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto et al., J. Antibiotics, 33, 665-667 (1980)), neotramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porotramycin (Tsunakawa et al., J. Antibiotics, 41, 1366-1373 (1988)), protracarcinol (Shimizu et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomycin ( DC-102) (Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber et al., J. Am. Chem. Soc, 110, 2992-2993 (1988)) and tomycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs have a common structure:

About ³

They differ in the number, type, and position of substituents on both their aromatic A rings and pyrrole C rings, as well as in the degree of saturation of the C ring. Ring B contains either an imine (N=C) or carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center responsible for DNA alkylation. All of the known natural products have the ^-configuration at the chiral position C11a, which results in a right-handed twist conformation when viewed from the C ring to the A ring. This gives them a suitable three-dimensional shape for the B-form DNA minor groove isocoiling, resulting in a snug fit. at the binding site (Kohn, in Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove allows them to interfere with DNA processing, so they are used as antitumor agents.

One Pyrrolobenzodiazepine compound is described by Gregson et al. (Chem. Commun. 1999, 797-798) as Compound 1 and Gregson et al. (J. Med. Chem. 2001, 44, 1161-1174) as Compound 4a. This connection, also known as SG2000, is shown below:

oh oh

SG2000

WO 2007/085930 describes the preparation of dimeric PBB compounds containing linker groups for association with a cell-binding agent such as an antibody. The linker is present in the bridge connecting the monomer blocks of the PBD dimer.

Dimeric PBB compounds containing linker groups for connection to a cell-binding agent such as an antibody have been described in WO 2011/130613 and WO 2011/130616. The linker in these compounds is attached to the central part of the PBB at position C2 and is usually cleaved when the linker group is exposed to the enzyme. In WO 2011/130598 the linker in these compounds is attached to one of the available N10 positions on the central part of the PBB and is usually cleaved by the action of the enzyme on the linker group.

Antibody drug conjugates

Antibody therapy has been developed for the targeted treatment of patients with cancer, immunological and angiogenic disorders (Carter, P. (2006) Nature Reviews Immunology 6:343357). The use of antibody-drug conjugates (ADCs), i.e. immunoconjugates, for local delivery of cytotoxic or cytostatic agents, i.e. drugs for the destruction or inhibition of tumor cells in the treatment of cancer, targets the delivery of a drug fragment to tumors and intracellular accumulation in them, while systemic administration

- 1 039826 of these drugs in unconjugated form can lead to unacceptable levels of toxicity to normal cells (Xie et al. (2006) Expert. Opin. Biol. Ther. 6(3):281-291; Kovtun et al. (2006) Cancer Res 66(6):3214-3121 Law et al (2006) Cancer Res 66(4):2328-2337 Wu et al (2005) Nature Biotech 23(9): 1137-1145 Lambert J (2005) Current Opin. in Pharmacol. 5:543-549 Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103 Payne G. (2003) Cancer Cell 3:207-212 Trail et al (2003) Cancer Immunol Immunother 52:328-337 Syrigos and Epenetos (1999) Anticancer Research 19:605-614).

Thus, maximum efficiency with minimum toxicity is required. Efforts to develop and improve ADCs have focused on monoclonal antibody (MAB) selectivity as well as drug mechanism of action, drug attachment, drug/antibody ratio (load), and drug release properties (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO 2009/052249; McDonagh (2006) Protein Eng. Design & Sel 19(7): 299-307 Doronina et al (2006) Bioconj Chem 17:114-124 Erickson et al (2006) Cancer Res 66(8): 1-8 Sanderson et al ( 2005) Clin. Cancer Res. 11:843-852; Jeffrey et al. (2005) J. Med. Chem. 48:1344-1358; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070) . Drug moieties may exert their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, proteasome and/or topoisomerase inhibition. Some cytotoxic drugs are generally inactive or less active when conjugated to large antibodies or receptor protein ligands.

The authors of the present invention have developed specific conjugates of dimeric PBB and antibodies.

The essence of the invention

According to a first aspect of the present invention, there is provided a conjugate of formula (I): Ab - (DL)p (I) wherein

Ab is an antibody that binds to AXL;

DL represents

where

X is selected from the group consisting of a single bond, -CH ₂ - and -C ₂ H ₄ -;

n is from 1 to 8;

m is 0 or 1;

R ⁷ is methyl or phenyl;

if there is a double bond between C2 and C3, R ² is selected from the group consisting of:

(ia) a _C5-10 aryl group optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, carboxy, ester, C _1-7 alkyl, C _{3 7} heterocyclyl, and bis-hydroxy- _C1-3 alkylene;

(ib) C1-5 saturated aliphatic alkyl;

(ic) C ₃ - ₆ saturated cycloalkyl;

R ²² /^^R ²³ (id) ^r21 , where each of R ²¹ , R ²² and R ²³ is independently selected from H, C1-3 saturated alkyl, C ₂ - ₃ alkenyl, C _{2 - 3 alkynyl} and cyclopropyl, while the total number of carbon atoms in the group R ¹² is not more than 5;

R ^25b (ie) wherein one of R ^25a and R ^25b is H and the other is selected from phenyl, said phenyl being optionally substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

- 2 039826

(if) where R ²⁴ is selected from H; C _1-3 saturated alkyl; C _2-3 alkenyls; C _2-3 alkynyl; cyclo propyl; phenyl, said phenyl being optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl;

Xy ^p2 “if a single bond is present between C ₂ and C ₃ , R ² is ^R26b where R ^26a and R ^26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, with alkyl and alkenyl groups optionally substituted with a group selected from C1-4 alkylamido and C1-4 alkyl ester; or if one of R ^26a and R ^26b is H, the other is selected from nitrile and C1-4 alkyl ester;

if there is a double bond between C2' and C3', R ¹² is selected from the group consisting of:

(ia) a C5-10 aryl group optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, carboxy, ester, C _1-7 alkyl, C _3-7 heterocyclyl, and bis-oxy -C _1-3 alkylene;

(ib) C _1-5 saturated aliphatic alkyl;

(ic) C _3-6 saturated cycloalkyl;

^R32

A^R ³³ (id) ^r31 where each of R ³¹ , R ³² and R ³³ is independently selected from H, C _1-3 saturated alkyl, C _2-3 alkenyl, C _2-3 alkynyl and cyclopropyl, with the total number carbon atoms in the group R ¹² is not more than 5;

⁽ⁱ e) wherein one of R ^35a and R ^35b is H and the other is selected from phenyl, said phenyl being optionally substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if) where R ²⁴ is selected from H; C _1-3 saturated alkyl; C _2-3 alkenyls; C _2-3 alkynyl; cyclopropyl; phenyl, said phenyl being optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl;

Dx^K ³⁶ ' if a single bond is present between C2' and C3', R ¹² is ^R36b where R ^36a and R ^36b are independently selected from H, F, C _1-4 saturated alkyl, C _2-3 alkenyl, and alkyl and the alkenyl groups are optionally substituted with a group selected from C _1-4 alkylamido and C _1-4 alkyl ester; or if one of R ^36a and R ^36b is H, the other is selected from nitrile and C1-4 alkyl ester;

and p is from 1 to 8.

These conjugates were found to exhibit good activity and surprising tolerability compared to similar conjugates not containing a sulfonamide moiety.

Brief description of the drawings

In FIG. 1 shows the binding of the conjugate to AXL;

In FIG. 2 shows the performance of the conjugate under in vivo conditions;

In FIG. 3 shows the performance of the conjugates under in vivo conditions;

In FIG. 4 shows the performance of the conjugates under in vivo conditions;

In FIG. 5 shows the performance of the conjugates in vivo on a patient-derived xenograft; and

In FIG. 6 shows the in vivo performance of the conjugates on another patient-derived xenograft.

Detailed description of the invention

The present invention provides a PBB dimer with a linker attached at the N10 position on one of the antibody-conjugated PBB fragments as defined below.

The present invention is suitable for use in delivering a PBB compound to a preferred site in a subject's body. The conjugate allows the release of the active PBB compound which does not retain any portion of the linker. There is no overhang that could affect the reactivity of the PBB compound. Thus, the conjugate of formula (I) will release the RelA compound:

- 3 039826

Said bond between the PBB dimer and the antibody of the present invention is preferably stable outside of cells. Prior to transport or delivery to a cell, the antibody drug conjugate (ADC) is preferably stable and remains intact, i. the antibody remains attached to the drug moiety. Linkers are stable outside the target cell and can be cleaved to some effective extent inside the cell. An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) provide intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e. uncleaved, until the conjugate is delivered or transported to its target site; and (iv) maintain the cytotoxic, cytolytic effect, or cytostatic effect of the PBB drug moiety. The stability of the ADC can be measured using standard analytical techniques such as mass spectroscopy, HPLC, and LC/MS separation/analysis techniques.

Delivery of compounds of formulas RelA is achieved at the target site of activation of the conjugate of formula (I) by the action of an enzyme, such as cathepsin, on the connecting group and, in particular, on the valine-alanine dipeptide fragment.

Definition Substituents

As used herein, the term "optionally substituted" refers to a parent group which may be unsubstituted or which may be substituted.

Unless otherwise indicated, in this application, the term substituted refers to a parent group that carries one or more substituents. The term substituent is used in this application in the conventional sense and refers to a chemical moiety that is covalently attached or, if necessary, fused to the parent group. A wide range of substituents is well known, and the methods for their formation and introduction into various starting groups are also well known.

Substituent examples are described in more detail below.

C _1-12 alkyl: in this application, the term C _1-12 alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound containing from 1 to 12 carbon atoms, which may be aliphatic or alicyclic and which may be saturated or unsaturated (eg, partially unsaturated, completely unsaturated). In this application, the term C _1-4 alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound containing from 1 to 4 carbon atoms, which may be aliphatic or alicyclic and which may be saturated or unsaturated (for example, partially unsaturated , completely unsaturated). Thus, the term alkyl includes the subclasses alkenyl, alkynyl, cycloalkyl, etc. discussed below.

Examples of saturated alkyl groups include, but are not limited to, methyl (C ₁ ), ethyl (C ₂ ), propyl (C ₃ ), butyl (C ₄ ), pentyl (C 5 ), hexyl (C ₆ ), and heptyl (C ₇ ).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C ₂ ), n-propyl (C ₃ ), n-butyl (C ₄ ), n-pentyl (amyl) (C5), n -hexyl (Ce) and n-heptyl (C ₇ ).

Examples of saturated branched alkyl groups include isopropyl (C3), isobutyl (C4), sec-butyl (C ₄ ), tert-butyl (C ₄ ), isopentyl (C ₅ ) and neopentyl (C ₅ ).

C _2-12 alkenyl: As used herein, the term C _2-12 alkenyl refers to an alkyl group containing one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH ₂ ), 1-propenyl (-CH=CH-CH ₃ ), 2-propenyl (allyl, -CH-CH=CH ₂ ), isopropenyl (1-methylvinyl, -C(CH ₃ )=CH ₂ ), butenyl (C ₄ ), pentenyl (C ₅ ) and hexenyl (C ₆ ).

C _2-12 alkynyl: As used herein, the term C _2-12 alkynyl refers to an alkyl group containing one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (C=CH) and 2-propynyl (propargyl, —CH ₂ —C=CH).

C _3-12 cycloalkyl: in the present application, the term C _3-12 cycloalkyl refers to an alkyl group which is also a cyclyl group; those. a monovalent fragment obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, the fragment having 3 to 7 carbon atoms including 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, groups derived from saturated monocyclic hydrocarbon compounds:

cyclopropane (С ₃ ), cyclobutane (С ₄ ), cyclopentane (C ₅ ), cyclohexane (С ₆ ), cycloheptane (С ₇ ), methylcyclopropane (С ₄ ), dimethylcyclopropane (C ₅ ), methylcyclobutane (C ₅ ), dimethylcyclobutane ( C ₆ ),

- 4 039826 methylcyclopentane (C ₆ ), dimethylcyclopentane (C ₇ ) and methylcyclohexane (C ₇ );

unsaturated monocyclic hydrocarbon compounds:

cyclopropene (С ₃ ), cyclobutene (С ₄ ), cyclopentene (C ₅ ), cyclohexene (С ₆ ), methylcyclopropene (С ₄ ), dimethylcyclopropene (C ₅ ), methylcyclobutene (C ₅ ), dimethylcyclobutene (С ₆ ), methylcyclopentene ( C ₆ ), dimethylcyclopentene (C ₇ ) and dimethylcyclohexene (C ₇ ) and saturated polycyclic hydrocarbon compounds: norcaran (C ₇ ), norpinane (C ₇ ), norbornane (C ₇ ).

C _3-20 heterocyclyl: In this application, the term C _3-20 heterocyclyl refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, the moiety having 3 to 20 ring atoms, of which 1 to 10 are ring heteroatoms . Preferably, each ring contains 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms.

In this case, the prefixes (eg, C ₃ _ ₂₀ , C ₃ _ ₇ , C _5-6 , etc.) indicate the number of ring atoms or the range of the number of ring atoms, whether carbon atoms or heteroatoms. For example, in this application, the term C _5-6 heterocyclyl refers to a heterocyclyl group containing 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from

N1: aziridine (C ₃ ), azetidine (C ₄ ), pyrrolidine (tetrahydropyrrole) (C ₅ ), pyrroline (e.g. 3pyrroline, 2,5-dihydropyrrole) (C ₅ ), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C ₅ ), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);

O1: oxirane (C ₃ ), oxetane (C ₄ ) oxolane (tetrahydrofuran) (C ₅ ), oxol (dihydrofuran) (C ₅ ), oxan (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);

S1: thiirane (C ₃ ), thietane (C ₄ ), thiolane (tetrahydrothiophene) (C ₅ ), thiana (tetrahydrothiopyran) (C ₆ ), thiepane (C7);

O ₂ : dioxolane (C ₅ ), dioxane (C ₆ ) and dioxepane (C ₇ );

O ₃ : trioxane (C ₆ );

N ₂ : imidazolidine (C ₅ ), pyrazolidine (diazolidine) (C ₅ ), imidazoline (C ₅ ), pyrazoline (dihydropyrazole) (C ₅ ), piperazine (C ₆ );

N1O1: tetrahydrooxazole (C ₅ ), dihydrooxazole (C ₅ ), tetrahydroisoxazole (C ₅ ), dihydroisoxazole (C ₅ ), morpholine (C ₆ ), tetrahydrooxazine (C ₆ ), dihydrooxazine (C ₆ ), oxazine (C ₆ );

N1S1: thiazoline (C ₅ ), thiazolidine (C ₅ ), thiomorpholine (C ₆ );

N2O1: oxadiazine (C ₆ );

O1S1: oxathiol (C ₅ ) and oxatian (thioxane) (C ₆ ); and

N1O1S1: oxathiazine (C ₆ ).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides in cyclic form, e.g. furanose (C5) such as arabinofuranose, lyxofuranose, ribofuranose and xylofuranose, and pyranose ( _C6 ) such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulpyranose , idopyranose, galactopyranose and talopyranose.

C ₅ _ ₂₀ aryl: As used herein, the term C ₅ _ ₂₀ aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, said moiety having 3 to 20 ring atoms. In this application, the term C _5-7 aryl refers to a monovalent fragment obtained by removing a hydrogen atom from an atom of the aromatic ring of an aromatic compound, and the specified fragment contains from 5 to 7 ring atoms, and in this application the term C _5-10 aryl refers to a monovalent fragment , obtained by removing a hydrogen atom from an atom of the aromatic ring of an aromatic compound, and the specified fragment contains from 5 to 10 ring atoms. Preferably, each ring contains 5 to 7 ring atoms.

In this case, the prefixes (eg, C _3-20 , C _5-7 , C _5-6 , C _5-10 , etc.) indicate the number of ring atoms or the range of the number of ring atoms, whether carbon atoms or heteroatoms. For example, in this application, the term C _5-6 aryl refers to an aryl group containing 5 or 6 ring atoms.

All ring atoms may be carbon atoms, as in carboaryl groups.

Examples of carboaryl groups include, but are not limited to, groups derived from benzene (i.e. phenyl) (C ₆ ), naphthalene (C ₁₀ ), azulene (C ₁₀ ), anthracene (C ₁₄ ), phenanthrene (C ₁₄ ) , naphthacene (С18) and pyrene (С1 ₆ ).

Examples of aryl groups that contain fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indan (e.g., 2,3-dihydro-1H-indene) (C ₉ ), indene (C ₉ ), isoindene (C ₉ ), tetralin (1,2,3,4-tetrahydronaphthalene (C ₁₀ ), acenaphthene (C ₁₂ ), fluorene (C ₁₃ ), phenalene (C ₁₃ ), acephenanthrene (C ₁₅ ) and aceanthrene (C ₁₆ ).

Alternatively, the ring atoms may include one or more heteroatoms, as in

- 5 039826 heteroaryl groups. Examples of monocyclic heteroaryl groups include, but are not limited to, groups derived from:

N1: pyrrole (azole) (C ₅ ), pyridine (azine) (C ₆ );

Oh furan (oxol) (C ₅ );

Sy of thiophene (thiol) (C ₅ );

NjOy oxazole (C ₅ ), isoxazole (C ₅ ), isoxazine (C ₆ );

N2O1: oxadiazole (furazan) (C ₅ );

N ₃ O1: oxatriazole (C ₅ );

N ₁ S ₁ : thiazole (C ₅ ), isothiazole (C ₅ );

N2: imidazole (1,3-diazole) (C ₅ ), pyrazole (1,2-diazole) (C ₅ ), pyridazine (1,2-diazine) (C ₆ ), pyrimidine (1,3-diazine) ( C ₆ ) (eg, cytosine, thymine, uracil), pyrazine (1,4-diazine) (C ₆ );

N ₃ : triazole (C ₅ ), triazine (C ₆ ) and

N4: tetrazole (C ₅ ).

Examples of a heteroaryl that contains fused rings include, but are not limited to:

C ₉ (with 2 fused rings) derived from benzofuran (O ₁ ), isobenzofuran (O ₁ ), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine ( N4) (e.g. adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxol (O ₂ ), benzofurazan (N2O1), benzotriazole (N ₃ ), benzothiofuran (S ₁ ), benzothiazole (N ₁ S ₁ ), benzothiadiazole (N2S);

C ₁₀ (with 2 fused rings) derived from chromene (O1), isochromene (O1), chromane (O1), isochromane (O1), benzodioxane (O2), quinoline (N ₁ ), isoquinoline (N1), quinolysine (N1 ), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);

C11 (with 2 fused rings) derived from benzodiazepine (N2);

C ₁₃ (with 3 fused rings) derived from carbazole (Ni), dibenzofuran (O ₁ ), dibenzothiophene (S ₁ ), carboline (N2), perimidine (N2), pyridoindole (N2); and,

C ₁₄ (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O ₂ ), phenoxathiine (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1 ), thiantrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).

The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.

Halogen: -F, -Cl, -Br and -I.

Hydroxy: -OH.

Ether: -OR where R is an ether substituent, for example a C1-7 alkyl group (also referred to as C1-7 alkoxy, which is discussed below), a C _{3 -20} heterocyclyl group (also referred to as C _{3 -20} heterocyclyloxy) or C _{5_20} aryl group (also called C _{5 .2o} aryloxy group), preferably C1.7 alkyl group.

Alkoxy: -OR where R is an alkyl group, for example a C1.7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (npropoxy), -O(iPr) (isopropoxy), -O(nBu) (n- butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy) and -O(tBu) (tert-butoxy).

Carboxy (carboxylic acid): -C(=O)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C(=O)OR, where R represents an ester substituent, for example, a C 1-7 alkyl group, a C ₃ _ ₂₀ heterocyclyl group or a C ₅ _ ₂₀ aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH3, -C(=O)OCH2CH3, -C(=O)OC(CH3)3 and -C(=O)OPh.

Amino: -NR ¹ R ² where R ¹ and R ² independently represent amino substituents, for example hydrogen, C1-7 alkyl group (also called C1.7 alkylamino or di-C ₁ _7 alkylamino), C ₃ _ ₂₀ heterocyclyl group or a C ₅ _ ₂₀ aryl group, preferably an H or C1.7 alkyl group, or, in the case of a cyclic amino group, R ¹ and R ² , together with the nitrogen atom to which they are attached, form a heterocyclic ring containing from 4 to 8 ring atoms. Amino groups may be primary (-NH2), secondary (-NHR ¹ ) or tertiary (-NHR1R ² ), and in cationic form may be quaternary (-+NR ¹ R2R ³ ). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, -NHC(CH3)2, -N(CH3)2, -N(CH2CH3)2 and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=O)NR1R ² where R ¹ and R ² are independently amino substituents as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH ₂ , -C(=O)NHCH ₃ , -C(=O)N(CH ₃ ) ₂ , -C(=O)NHCH ₂ CH ₃ and

- 6 039826

-C(=O)N(CH ₂ CH ₃ ) ₂ , as well as amido groups in which R ¹ and R ² , together with the nitrogen atom to which they are attached, form a heterocyclic structure, for example, as in piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl and piperazinocarbonyl.

Nitro: -NO2.

Azido: -N ₃ .

Cyano (nitrile, carbonitrile): -CN.

Antibody

In one aspect, the antibody is an antibody that binds to AXL.

1H12

In some embodiments, the antibody comprises a VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the VH domain further comprises a VH CDR2 with the amino acid sequence of SEQ ID NO: 6 and/or a VH CDR1 with the amino acid sequence the sequence of SEQ ID NO: 5. In some embodiments, the antibody comprises a VH domain comprising a VH CDR1 with the amino acid sequence of SEQ ID NO: 5, a VH CDR2 with the amino acid sequence of SEQ ID NO.6, and a VH CDR3 with the amino acid sequence of SEQ ID NO: 7. According to preferred embodiments of the present invention, the antibody comprises a VH domain containing the sequence according to SEQ ID NO: 1.

The antibody may also contain a VL domain. In some embodiments, the antibody comprises a VL domain comprising a VL CDR3 with the amino acid sequence of SEQ ID NO: 10. In some embodiments of the present invention, the VL domain further comprises a VL CDR2 with the amino acid sequence of SEQ ID NO: 9 and/or a VL CDR1 with the amino acid sequence the sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises a VL domain comprising a VL CDR1 with the amino acid sequence of SEQ ID NO: 8, a VL CDR2 with the amino acid sequence of SEQ ID NO: 9, and a VL CDR3 with the amino acid sequence of SEQ ID NO: 10. According to preferred embodiments of the present invention, the antibody contains a VL domain containing the sequence according to SEQ ID NO: 2.

According to preferred embodiments of the present invention, the antibody comprises a VH domain and a VL domain. Preferably the VH contains the sequence of SEQ ID NO: 1 and the VL domain contains the sequence of SEQ ID NO: 2.

The VH and VL domain(s) can pair to form an antibody antigen-binding site that binds AXL.

According to some embodiments of the present invention, the antibody is an intact antibody containing a VH domain paired with a VL domain, while the VH and VL domains contain the sequences of SEQ ID NO.1 paired with SEQ ID NO: 2.

According to some embodiments of the present invention, the antibody comprises a heavy chain containing the sequence of SEQ ID NO: 3 paired with a light chain containing the sequence of SEQ ID NO: 4. In some embodiments of the present invention, the antibody is an intact antibody containing two heavy chains containing the sequence of SEQ ID NO: 3, each of which is paired with a light chain containing the sequence of SEQ ID NO: 4.

According to some embodiments of the present invention, the antibody comprises a heavy chain containing the sequence of SEQ ID NO: 24 paired with a light chain containing the sequence of SEQ ID NO: 4. In some embodiments of the present invention, the antibody is an intact antibody containing two heavy chains containing the sequence of SEQ ID NO: 24, each of which is paired with a light chain containing the sequence of SEQ ID NO: 4.

In one aspect, the antibody is an antibody described herein that has been modified (or further modified) as described below. In some embodiments of the present invention, the antibody is a humanized variant, a deimmunized variant, or a sequence altered variant of an antibody disclosed herein.

5F11

In some embodiments, the antibody comprises a VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO: 15. In some embodiments, the VH domain further comprises a VH CDR2 with the amino acid sequence of SEQ ID NO: 14 and/or a VH CDR1 with the amino acid sequence the sequence of SEQ ID NO: 13. According to some embodiments of the present invention, the antibody comprises a VH domain containing a VH CDR1 with the amino acid sequence of SEQ ID NO: 13, a VH CDR2 with the amino acid sequence of SEQ ID NO.14, and a VH CDR3 with the amino acid sequence of SEQ ID NO: fifteen.

- 7 039826

According to some embodiments of the present invention, the antibody contains a VH domain containing the sequence according to SEQ ID NO: 11.

In some embodiments, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 20. In some embodiments, the present invention the antibody contains a VH domain containing the sequence according to SEQ ID NO: 21.

The antibody may also contain a VL domain. In some embodiments, the antibody comprises a VL domain comprising a VL CDR3 with the amino acid sequence of SEQ ID NO: 18. In some embodiments of the present invention, the VL domain further comprises a VL CDR2 with the amino acid sequence of SEQ ID NO: 17 and/or a VL CDR1 with the amino acid sequence the sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a VL domain comprising a VL CDR1 with the amino acid sequence of SEQ ID NO: 16, a VL CDR2 with the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 with the amino acid sequence of SEQ ID NO: eighteen.

According to some embodiments of the present invention, the antibody contains a VL domain containing the sequence according to SEQ ID NO: 22.

According to preferred embodiments of the present invention, the antibody comprises a VH domain and a VL domain. In some embodiments, the VH comprises a VH CDR1 with the amino acid sequence of SEQ ID NO: 13, a VH CDR2 with the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 with the amino acid sequence of SEQ ID NO: 15; and the VL domain contains a VL CDR1 with the amino acid sequence of SEQ ID NO: 16, a VL CDR2 with the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 with the amino acid sequence of SEQ ID NO: 18.

According to some embodiments of the present invention, the antibody comprises a VH domain containing the sequence of SEQ ID NO: 19, and a VL domain containing the sequence of SEQ ID NO: 22. In some embodiments of the present invention, the antibody contains a VH domain containing the sequence of SEQ ID NO: 20, and a VL domain containing the sequence of SEQ ID NO: 22. According to some embodiments of the present invention, the antibody comprises a VH domain containing the sequence of SEQ ID NO: 21 and a VL domain containing the sequence of SEQ ID NO: 22.

The VH and VL domain(s) can pair to form an antibody antigen-binding site that binds AXL.

According to some variants of implementation of the present invention, the antibody is an intact antibody containing a VH domain paired with a VL domain.

In one aspect, the antibody is an antibody described herein that has been modified (or further modified) as described below. According to some embodiments of the present invention, the antibody is a humanized variant, a deimmunized variant, or a resurfaced variant of an antibody disclosed herein.

Terminology

In this application, the term antibody is used in the broadest sense and includes, in particular, monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (for example, bispecific antibodies), intact antibodies and antibody fragments, provided that they exhibit the desired biological activity. , for example, the ability to bind AXL. Antibodies can be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein produced by the immune system that is able to recognize and bind to a specific antigen. (Janeway C., Travers P., Walport M., Shlomchik (2001) Immuno Biology, 5th ed., Garland Publishing, New York). The target antigen typically has a plurality of binding sites, also referred to as epitopes, recognized by CDRs on a plurality of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody comprises a full length immunoglobulin molecule or an immunologically active portion of a full length immunoglobulin molecule, i. a molecule that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest, or a portion thereof, such targets include, but are not limited to, a cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. An immunoglobulin can be of any type (eg, IgQ IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass or allotype (eg, G1m1, G1m2, G1m3, non-G1m1 human [i.e. any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2 and Km3) immunoglobulin molecules. Immunoglobulins can be from any species, including humans, mice, or rabbits.

- 8 039826

As used herein, the term "binds AXL" means that an antibody binds AXL with higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank accession number CAA76847, variant no. CAA76847.1 GL3336842, record update date: January 7 2011, 2:30 p.m.). In some embodiments, the antibody binds AXL with an association constant (Ka) that is at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 ⁴ . 105 or 106 times higher than the association constant of an antibody for BSA when measured under physiological conditions. The antibodies of the present invention can bind CD22 with high affinity. For example, in some embodiments of the present invention, an antibody can bind CD22 to a KD that is equal to or less than about 10 ^-6 M, such as 1 x 10 -6, 10 ^-7 , 10 ⁸ , 10 ^-9 , 10' ¹⁰ , 10 · ¹¹ , ^10-12 , 10' ¹³ or ^10-14 .

AXL is a member of the TAM family of human receptor tyrosine kinases. In some embodiments of the present invention, the AXL polypeptide corresponds to Genbank accession number AAH32229, variant number AAH32229.1 GL21619004, record update date: March 6, 2012, 13:18 (SEQ ID NO: 9). According to one embodiment of the present invention, the nucleic acid encoding the AXL polypeptide corresponds to Genbank accession number M76125, variant number M76125.1 GL292869, record update date: June 23, 2010, 8:53 am. In some embodiments of the present invention, the AXL polypeptide comprises the sequence of SEQ ID NO: 23.

Antibody fragments contain a portion of a full-length antibody, usually its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and scFv fragments; diabody; linear antibodies; fragments produced by the Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region) and epitope-binding fragments of any of the above, which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and polyspecific antibodies formed from antibody fragments.

As used herein, the term monoclonal antibody refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. the individual antibodies that make up the population are identical except for possible natural mutations, which may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. In addition, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The circumstance monoclonal indicates the characterization of an antibody as derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be obtained by the hybridoma method, first described by Kohler et al. (1975) Nature 256: 495, or can be obtained by recombinant DNA methods (see US 4816567). Monoclonal antibodies can also be isolated from antibody phage libraries using the techniques described in Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597, or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4): 450-459).

Monoclonal antibodies as used herein specifically include chimeric antibodies in which a portion of the heavy and/or light chains are identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain( s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 68516855). Chimeric antibodies include primatized antibodies comprising antigen-binding variable domain sequences derived from a non-human primate (eg, Old World monkey or great ape) and human constant region sequences.

In the present application, an intact antibody is an antibody containing the VL and VH domains, as well as the light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains can be native sequence constant domains (eg, human native sequence constant domains) or an amino acid sequence variant thereof. An intact antibody may have one or more effector functions that relate to those biological activities associated with the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; binding to the Fc receptor; antibody-dependent cellular cytotoxic

- 9 039826 availability (AZKTs); phagocytosis and suppression of cell surface receptors such as the B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

Antibody modification

The antibodies disclosed in this application may be modified. For example, in order to make them less immunogenic in a human subject. This can be accomplished using any of a number of techniques familiar to those skilled in the art. Some of these techniques are described in more detail below.

humanization

Techniques for reducing the immunogenicity of a non-human antibody or antibody fragment in vivo include techniques referred to as humanization.

A humanized antibody refers to a polypeptide comprising at least a portion of a modified variable region of a human antibody, wherein a portion of the variable region, preferably a portion substantially smaller than an intact human variable domain, has been replaced with a corresponding sequence from a non-human species, wherein the modified variable region the region is fused to at least another portion of another protein, preferably a constant region of a human antibody. The expression humanized antibodies includes human antibodies in which one or more amino acid residues of the complementarity determining region (CDR) and/or one or more amino acid residues of the framework region (FW or FR) are replaced by amino acid residues from analogous sites in antibodies of rodents or other species, different from human. The expression humanized antibody also includes a variant of the amino acid sequence of an immunoglobulin, or a fragment thereof, which contains an FR containing essentially the amino acid sequence of a human immunoglobulin and a CDR containing essentially the amino acid sequence of a non-human immunoglobulin.

Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. Or, alternatively, a humanized antibody is a human antibody that also contains selected non-human (eg, murine) antibody sequences instead of human sequences. A humanized antibody may include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and/or biological activity. Such antibodies are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulins.

There are a number of humanization techniques, including CDR grafting, guided selection, deimmunization, resurfacing (also known as surface residue recombination), compound antibodies, optimization of human sequence region content, and framework shuffling.

CDR inoculation

In this technique, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient antibody are replaced with CDR residues from a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat, or rabbit having targeted properties (essentially, non-human CDRs are grafted onto a human frame). In some instances, framework region (FR) residues of a human immunoglobulin are replaced with corresponding non-human residues (this may occur, for example, if a particular FR residue has a significant effect on antigen binding).

In addition, humanized antibodies may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications are made to further improve and maximize antibody performance. Thus, in general, a humanized antibody will contain all of at least one, and in one aspect two, variable domains, in which all or all of the hypervariable loops correspond to those in a non-human immunoglobulin, and all or substantially all of the FR regions. are human immunoglobulin sequences. The humanized antibody will optionally also contain at least a portion of an immunoglobulin constant region (Fc) or that of a human immunoglobulin.

Guided Selection

The method consists in combining the VH or V _L domain of a specific non-human antibody

- 10 039826 a human VH or V _L library specific for a particular epitope, and specific human V domains are selected against the antigen of interest. The selected human VH is then combined with the V _L library to create a fully human VH x V _L combination. The method is described in Nature Biotechnology (NY) 12, (1994) 899-903.

Composite antibodies

In this method, two or more amino acid sequence segments from a human antibody are combined within the finished antibody molecule. They are constructed by combining several segments of the human VH and V _L sequences in combinations that restrict or avoid human T cell epitopes in the V regions of the final composite antibody. If necessary, T cell epitopes are limited or eliminated by replacing segments of the V region contributing to or encoding the T cell epitope with alternative segments that avoid T cell epitopes. This method is described in US 2008/0206239 A1.

Deimmunization

The method involves removing human (or other second species) T-cell epitopes from the V regions of the therapeutic antibody (or other molecule). The sequence of the V region of therapeutic antibodies is analyzed for the presence of MHC class II binding motifs, for example by comparison with MHC binding motif databases (such as the motif database hosted at www.wehi.edu.au). In another embodiment, class II MHC-binding motifs can be identified using computational pull methods such as those developed by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in these methods, successive overlapping peptides from V-region sequences are tested to determine their binding energy to MHC class II proteins. This data can then be combined with information about other sequence features that are relevant to successfully presented peptides, such as amphipathicity, Rothbard motifs, and cleavage sites for cathepsin B and other processing enzymes.

Once potential T-cell epitopes of a second species (eg, human) have been identified, they are eliminated by altering one or more amino acids.

The modified amino acids are usually found within the T cell epitope itself, but may also be adjacent to the epitope in terms of the primary or secondary structure of the protein (and therefore may not be adjacent in the primary structure). Most often, the change occurs by substitution, but in some cases, the addition or removal of an amino acid may be more appropriate.

All changes can be made using recombinant DNA technology so that the finished molecule can be obtained by expression in a recombinant host using well-known methods such as site-directed mutagenesis. However, it is also possible to use protein chemistry or any other means of molecular modification.

Surface change

This method includes:

(a) determining the conformational structure of the non-human (eg, rodent) variable region of the antibody (or fragment thereof) by constructing a three-dimensional model of the non-human antibody variable region;

(b) obtaining sequence alignments using relative availability distributions from X-ray crystallographic patterns of a sufficient number of heavy and light chain variable regions of a non-human antibody and a human antibody to obtain a group of heavy and light chain backbone positions where the positions of the alignment are identical at 98 % sufficient non-human antibody heavy and light chains;

(c) determining, for the non-human antibody to be humanized, the group of amino acid residues exposed on the surface of the heavy and light chains using the group of framework positions obtained in step (b);

(d) identifying in the amino acid sequences of the human antibody a group of amino acid residues exposed on the surface of the heavy and light chains that is most identical to the group of amino acid residues exposed on the surface determined in step (c), wherein the heavy and light chains from the human antibody are paired, or not mated naturally;

(e) replacing the group of amino acid residues exposed on the surface of the heavy and light chains determined in step (c) in the amino acid sequence of the non-human antibody to be humanized with the group of amino acid residues exposed on the surface of the heavy and light chains identified on step (d);

(f) building a three-dimensional model of the variable region of the non-human antibody resulting from the substitution indicated in step (e);

(g) identifying, by comparing the 3D models built in steps (a) and (f), any amino acid residues from the groups identified in steps (c) or (d) that are within 5 Angstroms of any atom of any residue from the regions the complementarity-determining non-human antibody to be humanized; and (h) replacing any human residues identified in step (g) with the original amino acid residue

- 11 039826 com, non-human, to determine the humanizing group of amino acid residues exposed on the surface of the non-human antibody; provided that step (a) need not be carried out first, but must be carried out before step (g).

Superhumanization

The method allows comparison of a non-human sequence with a functional repertoire of human germline genes. Human genes are selected that encode canonical structures that are identical or closely related to non-human sequences. Selected human genes with the highest homology within the CDR are selected as FR donors. Finally, non-human CDRs are grafted onto these human FRs. This method is described in WO 2005/079479 A2.

Optimization of the composition of human sequence regions

This method allows a non-human (eg, murine) sequence to be compared to the human germline gene repertoire, and differences are evaluated as human sequence region content (HSC), which quantifies the sequence at the level of potential MHC/T cell epitopes. The target sequence is then humanized by maximizing its HSC instead of using a common identity measure to generate a wide variety of humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).

Rearrangement of wireframes

The CDRs of the non-human antibody are hybridized in-frame to cDNA pools spanning all known human germline heavy and light chain framework genes. The humanized antibodies are then selected, for example by panning an antibody phage display library. This is described in Methods 36, 43-60 (2005).

Modification of an antibody using azide

An antibody can be prepared for conjugation to a drug linker using a three-step process:

(1) Expression of an antibody (Ab) bearing a central N-glycan in a suitable expression system (eg, CHO cell line). The central N-glycan is typically conjugated to the heavy chain Asn297 according to the Kabat numbering system;

(2) Pruning of all glycan isoforms (complex, hybrid, high mannose) with endoglycosidase to release the central GlcNAc; and (3) Enzymatic transfer to the central GlcNAc of an N-acetylgalactose residue bearing an azide group for conjugation with a drug linker.

For an overview of the above method, see van Geel, R., et al., Bioconjugate Chemistry, 2015, 26, 2233-2242; DOI: 10.1021/acs.bioconjchem.5b00224. Alternatively, the one-pot method can be used - see examples.

Implementation options X

In some embodiments of the present invention, X is a single bond.

In other embodiments of the present invention, X is -CH ₂ -.

According to additional embodiments of the present invention, X is -C ₂ H ₄ -.

According to some embodiments of the present invention, n is from 1 to 4.

According to some of these embodiments of the present invention, n is 1.

According to other of these embodiments of the present invention, n is 2.

According to other of these embodiments of the present invention, n is 4.

^R7

According to one embodiment of the present invention, R ⁷ is methyl.

According to another embodiment of the present invention, R ⁷ is phenyl.

^R2

If there is a double bond between C2 and C3, R ² is selected from:

^{(a) C} 5-10 aryl group optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, ether, C _1-7 alkyl, C _3-7 heterocyclyl and bis-oxyC _1-3 alkylene ;

(b) C _1-5 saturated aliphatic alkyl;

(c) C _3-6 saturated cycloalkyl;

(d) ^r21 , where each of R ²¹ , R ²² and R ²³ is independently selected from H, C _1-3 saturated alkyl, C 2-3 alkenyl, C ₂ - ₃ alkynyl and cyclopropyl, and the total number of carbon atoms in the group R ² is no more than 5;

- 12 039826

R ²⁵ » (e) ^R where one of R ^25a and R ^25b is H and the other is selected from phenyl, phenyl being optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl and thiophenyl; and (f) where R ²⁴ is selected from H; C _1-3 saturated alkyl; C ₂ - ₃ alkenyls; C _2-3 alkynyl; cyclopropyl; phenyl, wherein the phenyl is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl.

If R ² is a C _5-10 aryl group, it may be a C ₅ -7 aryl group. The C ₅ -7 aryl group may be a phenyl group or a C ₅ -7 heteroaryl group such as furanyl, thiophenyl and pyridyl. According to some variants of implementation of the present invention, R ² preferably represents phenyl. According to other variants of implementation of the present invention, R ¹² preferably represents thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.

If R ² is C ₅ . ₁₀ aryl group, it may be C ₈ . ₁₀ aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be linked to the PBB core at any available ring position. For example, quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4-yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl, and quinolin-8-yl. Of these, quinolin-3-yl and quinolin-6-yl may be preferred. Isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these, isoquinolin-3-yl and isoquinolin-6-yl may be preferred.

If R ² is C ₅ . ₁₀ aryl group, it can carry any number of substituent groups. It preferably bears 1 to 3 substituent groups, with 1 and 2 being more preferred and singly substituted groups being most preferred. The substituents may be in any position.

When R ² is a C ₅ _ ₇ aryl group, the single substituent is preferably on a ring atom that is not bond-adjacent to the rest of the compound, i.e. preferably it is β or γ relative to the rest of the compound. Therefore, when the C ₅ _ ₇ aryl group is phenyl, the substituent is preferably in the meta or para positions, and more preferably is in the para position.

When R ² is a C _5-10 aryl group, such as quinolinyl or isoquinolinyl, it may carry any number of substituents at any position on the quinoline or isoquinoline rings. In some embodiments of the present invention, it bears one, two, or three substituents, and these may be on either the proximal and distal rings, or both (if more than one substituent is present).

Substituents R ² if R ² is a C5-10 aryl group. If the substituent on R ² when R ² is a C5-10 aryl group is halogen, it is preferably F or Q, more preferably Cl.

If the substituent on R ² when R ² is a C _5-10 aryl group is an ether, it may, according to some embodiments of the present invention, be an alkoxy group, for example a C 1-7 alkoxy group (for example, methoxy, ethoxy), or it may, in some embodiments of the present invention, be a C _{5 -7} aryloxy group (eg, phenoxy, pyridyloxy, furanyloxy). The alkoxy group itself may be further substituted, for example, with an amino group (eg, dimethylamino).

If the substituent is on R ² when R ² is C ₅ . ₁₀ aryl group is C1. 7 alkyl, it may preferably be a C _1-4 alkyl group (eg methyl, ethyl, propyl, butyl).

If a substituent on R ² when R ² is a C _5-10 aryl group is C ₃ _ ₇ heterocyclyl, it may, according to some embodiments of the present invention, be a C ₆ nitrogen-containing heterocyclyl group, for example, morpholino, thiomorpholino, piperidinil, piperazinil. These groups can be linked to the rest of the PBB moiety via a nitrogen atom. These groups may be further substituted with, for example, C _1-4 alkyl groups. If the C ₆ nitrogen-containing heterocyclyl group is piperazinyl, said additional substituent may be on the second ring nitrogen atom.

If the substituent on R ² when R ² is a C _5-10 aryl group is bis-oxy-C _1-3 alkylene, it is preferably bis-oxymethylene or bis-oxyethylene.

If the substituent on R ² when R ² is a C _5-10 aryl group is an ester, it is preferably a methyl ester or an ethyl ester.

Particularly preferred substituents are when R ² is C ₅ . ₁₀ aryl group, include methoxy, ethoxy, fluorine, chlorine, cyano, bis-oxymethylene, methylpiperazinyl, morpholino and methyl

- 13 039826 thiophenyl. Other particularly preferred substituents for R ² are dimethylaminopropyloxy and carboxy.

Particularly preferred substituted R ² groups when R ² is a C ₅ aryl group include, but are not limited to, 4-methoxyphenyl, 3-methoxyphenyl, 4-ethoxyphenyl, 3ethoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 3, 4-bisoxymethylenephenyl, 4-methylthiophenyl, 4-cyanophenyl, 4phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl and naphthyl. Another possible substituted R ² group is 4-nitrophenyl. R ² groups of particular interest include 4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylenephenyl.

If R ² is Ci. ₅ saturated aliphatic alkyl, it can be methyl, ethyl, propyl, butyl or pentyl. In some embodiments of the present invention, it may be methyl, ethyl, or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. According to other embodiments of the present invention, it may be butyl or pentyl, which may be linear or branched.

When R ² is C ₃ _ ₆ saturated cycloalkyl, it can be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments of the present invention, it may be cyclopropyl.

If R ² is

R ²¹ each of R ²¹ , R ²² and R ²³ is independently selected from H, C1.3 saturated alkyl, C ₂ _ ₃ alkenyl, C ₂ _ ₃ alkynyl and cyclopropyl, and the total number of carbon atoms in the group R ² is not more than 5 In some embodiments of the present invention, the total number of carbon atoms in the R ² group is no more than 4 or no more than 3.

According to some embodiments of the present invention, one of R ²¹ , R ²² and R ²³ is H, while the other two groups are selected from H, C 1-3 saturated alkyl, C ₂ - ₃ alkenyl, C ₂ - ₃ alkynyl and cyclopropyl.

According to other embodiments of the present invention, two of R ²¹ , R ²² and R ²³ are H, with the other group selected from H, _C1.3 saturated alkyl, C _{2_3} alkenyl, C _{2_3} alkynyl _, and cyclopropyl.

In some embodiments of the present invention, the groups other than H are selected from methyl and ethyl. In some of these embodiments, groups that are not H are methyl.

According to some embodiments of the present invention, R ²¹ is H.

According to some embodiments of the present invention, R ²² is H.

According to some embodiments of the present invention, R ²³ is H.

According to some embodiments of the present invention, R ²¹ and R ²² are H.

According to some embodiments of the present invention, R ²¹ and R ²³ are H.

According to some embodiments of the present invention, R ²² and R ²³ are H.

The R ² group of particular interest is

If R ² is

R ^25b /%^x. _R 25a one of R ^25a and R ^25b is H and the other is selected from phenyl, the phenyl being optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments of the present invention, the group that is not H is an optionally substituted phenyl. If the optional phenyl substituent is halogen, it is preferably fluorine. In some embodiments of the present invention, the phenyl group is unsubstituted.

If R ² is

R ²⁴ is selected from: H; C1-3 saturated alkyl; C ₂ - ₃ alkenyls; C ₂ - ₃ alkynyl; cyclopropyl; phenyl, wherein the phenyl is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl and thiophenyl. If the optional phenyl substituent is halogen, it is preferably

- 14 039826 is fluorine. In some embodiments of the present invention, the phenyl group is unsubstituted.

In some embodiments of the present invention, R ²⁴ is selected from H, methyl, ethyl, ethenyl, and ethynyl. In some of these embodiments, R ²⁴ is selected from H and methyl.

If a single bond is present between C ₂ and C ₃ , R ² is

where R ^26a and R ^26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, with the alkyl and alkenyl groups optionally substituted with a group selected from C1-4 alkylamido and C1-4 alkyl ester; or if one of R ^26a and R ^26b is H, the other is selected from nitrile and C1-4 alkyl ester.

According to some embodiments of the present invention, preferably R ^26a and R ^26b are both H.

According to other embodiments of the present invention, preferably R ^26a and R ^26b are both methyl.

According to other embodiments of the present invention, preferably one of R ^26a and R ^26b is H and the other is selected from C _1-4 saturated alkyl, C _2-3 alkenyl, with the alkyl and alkenyl groups optionally substituted. In these additional embodiments, even more preferably the group that is not H is selected from methyl and ethyl.

^R12

The above preferences for R ² apply equally to R ¹² .

According to one embodiment of the present invention, DL is

Drug loading

The drug load is the average number of PBB drugs per antibody, eg antibody.

The average number of drugs per antibody in ADC preparations from conjugation reactions can be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA, and electrophoresis. The quantitative distribution of the ADC with respect to p can also be determined. ELISA can determine the average p value in a particular ADC preparation (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p (drug) values is not distinguishable based on antibody-antigen binding and due to limitation of ELISA detection. In addition, ELISA analysis for the detection of antibody-drug conjugates does not determine where drug fragments are attached to the antibody, such as heavy chain or light chain fragments, or specific amino acid residues. In some instances, separation, purification, and characterization of a homogeneous ADC, where p is some of the ADCs with other drug loading measures, can be accomplished by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

For antibody-drug conjugates of the present invention, p is limited by the number of attachment sites on the antibody, i. the number of azide groups. For example, an antibody may have only one or two azide groups to which a drug linker may be attached.

Typically, fewer than the theoretical maximum number of drug fragments are conjugated to an antibody during a conjugation reaction. The loading (drug/antibody ratio) of the ADC can be controlled in several different ways, including: (i) limiting the molar excess of the drug-linker intermediate (D-L) or linker reagent relative to the antibody, and (ii) limiting the conjugation reaction time or temperature.

- 15 039826

If more than one nucleophilic or electrophilic group of the antibody reacts with the drug-linker intermediate or linker reagent, and then the drug fragment, then the resulting product is a mixture of ADC compounds with a distribution of drug fragments attached to the antibody, for example 1, 2, 3 etc. Liquid chromatography techniques such as reverse phase polymer (PLRP) and hydrophobic interaction (HIC) can separate compounds in a mixture based on drug loading. ADCs with a single drug load (p) can be isolated, however, these ADCs with a single load can still be heterogeneous mixtures because the drug moieties can be linked by a linker at different sites in the antibody.

Thus, the antibody drug conjugate compositions of the present invention comprise mixtures of antibody drug conjugate compounds wherein the antibody contains one or more PBB drug moieties and in which the drug moieties can be attached to the antibody at different amino acid residues.

In one embodiment of the present invention, the average number of dimeric pyrrolobenzodiazepine groups per antibody is in the range of 1 to 8. In some embodiments of the present invention, the range is selected from 1-4, 1-4, 2-4, and 1-3.

In some embodiments, the antibody has one or two dimeric pyrrolobenzodiazepine groups.

Including other forms

Unless otherwise indicated, the above includes well-known ionic, salt, solvate and protected forms of these substituents. For example, reference to a carboxylic acid (COOH) also includes the anionic (carboxylate) form (-COO-), its salt or solvate, as well as standard protected forms. Similarly, reference to an amino group includes the protonated form (N+HR1R ² ), a salt or solvate of the amino group, eg the hydrochloride salt, as well as conventional protected forms of the amino group. Similarly, reference to a hydroxyl group also includes the anionic form (-O-), its salt or solvate, as well as conventional protected forms.

salt

It may be convenient or desirable to prepare, purify and/or process an appropriate salt of the active compound, for example a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

For example, if the compound is anionic or contains a functional group that may be anionic (eg, -COOH may be -COO-), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K+, alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ . Examples of suitable organic cations include, but are not limited to, the ammonium ion (ie NH4+) and substituted ammonium ions (eg NH3R ⁺ , NH2R2+, NHR3 ⁺ , NR4+). Examples of some suitable substituted ammonium ions include those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine . An example of a common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

If the compound is cationic or contains a functional group which may be cationic (eg, -NH2 may be -NH ₃ ), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucinic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, stearic, sulfonic tartaric, toluenesulfonic, trifluoroacetic acid and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify and/or process the appropriate solvate of the active compound. In this application, the term solvate is used in the conventional sense to refer to a complex of a solute (for example, an active compound, a salt of an acid

- 16 039826 active compound) and a solvent. When the solvent is water, the solvate is conveniently referred to as a hydrate, such as monohydrate, dihydrate, trihydrate, etc.

The present invention includes compounds in which the solvent is attached via an imine bond of the PBB moiety, as illustrated below, where the solvent is water or alcohol (RAOH, where R ^A is C _1-4 alkyl):

These forms may be referred to as the carbinolamine form and the carbinolamine ether form of PBB (as described in the R ¹⁰ section above). The balance of these equilibria depends on the conditions under which the compounds are located, as well as on the structure of the fragment itself.

These particular compounds may be isolated in solid form, for example by lyophilization.

Isomers

Some compounds of the present invention may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis and transforms; E- and Z-forms; c-, t- and r-forms; endo- and exoforms; R-, S- and mesoforms; D- and L-forms; d- and l-forms; (+) and (-) shapes; keto, enol and enolate forms; syn- and anti-forms; synclinal and anticlinal forms; α- and β-forms; axial and equatorial forms; forms of bath, armchair, twist, envelope and semi-armchair; and combinations thereof, hereinafter collectively referred to as isomers (or isomeric forms).

The term chiral refers to molecules whose mirror images are not able to overlap each other, while the term achiral refers to molecules whose mirror images overlap.

The term stereoisomers refers to compounds that have an identical chemical structure but differ in the arrangement of atoms or groups in space.

A 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 electrophoresis and chromatography.

Enantiomers refer to two stereoisomers of a compound that are not superimposed mirror images of each other.

In this application, stereochemical definitions and rules generally follow S. P. 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. Compounds of the present invention may contain asymmetric or chiral centers and therefore exist in various stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, that is, they have the ability 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 about its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of a plane-polarized light connection, with (-) or l indicating that the connection is left-handed. A connection with a (+) or d prefix is dextrorotatory. For a particular 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 stereoselection or stereospecificity in a chemical reaction or process. The terms racemic mixture and racemate refer to an equimolar mixture of two enantiomeric molecules devoid of optical activity.

It should be noted that, except as discussed below for tautomeric forms intentionally excluded from the term isomers as used in this application, these are structural (or structurally related) isomers (i.e., isomers that differ in the bonds between atoms, and not just by the position of atoms in space). For example, a reference to a methoxy group, -OCH3, should not be taken as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Likewise, a reference to ortho-chlorophenyl should not be taken as a reference to its structural iso.

- 17 039826 mer, meta-chlorophenyl. However, reference to a class of structures may well include structural isomeric forms that fall within that class (for example, C _1-7 alkyl includes n-propyl and isopropyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho- , meta- and para-methoxyphenyl).

The above exception does not apply to tautomeric forms, e.g. keto, enol and enolate forms, for example, as in the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/iminoalcohol, amidine/amidine, nitroso/ oxime, thioketone/enethiol, N-nitroso/hydroxyazo and nitro/acinitro.

The term tautomer or tautomeric form refers to structural isomers with different energies that are capable of interconversion 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 include interconversions by rearranging some of the bonding electrons.

It should be noted that the term isomer specifically includes compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including H ¹ , H ² (D) and H ³ (T); C may be in any isotopic form, including C ¹² , C ¹³ and C ¹⁴ ; The O may be in any isotopic form, including O ¹⁶ and O ¹⁸ ; etc.

Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, H ² (deuterium, D), H ³ (tritium) , C ¹¹ , C ¹³ , C ¹⁴ , N ¹⁵ , F ¹⁸ , R ³¹ , R ³² , S ³⁵ , Cl ³⁶ and I ¹²⁵ . Various isotopically labeled compounds of the present invention are included, for example those incorporating radioactive isotopes such as H ³ , C ¹³ and C ¹⁴ . Such isotopically labeled compounds can be used in metabolic studies, reaction kinetics studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including analyzes of drug or substrate distribution in tissue, or in radioactive treatment of patients. The deuterium-labeled or substituted therapeutic compounds of the present invention may have improved DMPK (metabolism and drug pharmacokinetics) distribution, metabolism and excretion (ADME) properties. Substitution with heavier isotopes such as deuterium may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. The F ¹⁸ labeled compound can be used for PET or SPECT studies. The isotopically labeled compounds of the present invention and their prodrugs can generally be prepared by following the procedures disclosed in the Schemes or in the Examples and Preparations described below, replacing the non-isotopically labeled reagent with a readily available isotopically labeled reagent. In addition, substitution with heavier isotopes, in particular deuterium (i.e., H ² or D), may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements or improved therapeutic index. It should be understood that in this case, deuterium is considered as a substituent. The concentration of such a heavier isotope, in particular deuterium, can be determined by the isotopic enrichment factor. In the compounds of the present invention, any atom not intentionally designated as a particular isotope is intended to represent any stable isotope of that atom.

Unless otherwise indicated, reference to a specific compound includes all such isomeric forms, including (in whole or in part) racemic and other mixtures thereof.

Methods for obtaining (for example, asymmetric synthesis) and separation (for example, fractional crystallization and chromatographic means) of such isomeric forms are either known in the art, or can be easily obtained in a known manner by adapting the methods described in this application, or known methods.

Biological activity

Cell proliferation assays in vitro

Typically, the cytotoxic or cytostatic activity of an antibody-drug conjugate (ADC) is measured by: exposing mammalian cells having receptor proteins to antibodies from the ADC in a cell culture medium; culturing the cells for a period of from about 6 hours to about 5 days; and cell viability measurements. In vitro cell assays are used to measure the viability (proliferation), cytotoxicity, and apoptosis (caspase activation) induction of ADCs of the present invention.

The in vitro performance of antibody-drug conjugates can be measured with

- 18 039826 using cell proliferation assay. The CellTiterGlo® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, WI, USA) homogeneous assay method based on recombinant expression of Coleoptera luciferase (US Pat. Nos. 5,583,024; 5,674,713 and 5,700,670). This cell proliferation assay determines the number of viable cells in culture based on the quantification of ATP present, an indicator of metabolically active cells (Crouch et al (1993) J. Immunol. Meth. 160:81-88; US 6602677). The CellTiter-Glo® assay is run in a 96-well format, making it suitable for automated high throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs 6:398-404). The homogeneous assay procedure involves adding one reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, media removal, and multiple pipetting steps are not required. The system only detects 15 cells per well in a 384-well format 10 minutes after reagent addition and mixing. Cells can be processed continuously using the ADC, or they can be processed and separated from the ADC. Typically, cells treated briefly, ie. 3 h showed efficiency similar to that of cells that were treated continuously.

The homogeneous add-mix-measure format results in cell lysis and a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The CellTiter-Glo® assay generates a luciferase-type luminescent signal that typically has a half-life of more than five hours, depending on the cell type and media used. Viable cells reflect in relative luminous units (RLU). The substrate, beetle luciferin, is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to AMP and emission of photons.

The in vitro efficacy of antibody-drug conjugates can also be measured by cytotoxicity assay. Cultured adherent cells are washed with PBS, separated using trypsin, diluted in complete medium containing 10% FST, centrifuged, resuspended in fresh medium and counted with a hemocytometer. Suspension cultures are counted directly. Monodisperse cell suspensions suitable for enumeration may require agitation of the suspension by repeated aspiration to break up cell clumps.

The cell suspension is diluted to the target seeding density and dispensed (100 µl per well) into black 96-well plates. Plates with attached cell lines are incubated overnight to allow attachment. The cell culture suspension can be used on the day of seeding.

A stock solution (1 ml) of ADC (20 μg/ml) was prepared in an appropriate cell culture medium. Serial 10-fold dilutions of stock ADC are prepared in 15 ml centrifuge tubes by sequentially transferring 100 µl to 900 µl of cell culture medium.

Wells with four replicates of each dilution of ADC (100 µl) are placed in 96-well black plates pre-seeded with cell suspension (100 µl) to give a final volume of 200 µl. Control wells are filled with cell culture medium (100 µl).

If the doubling time of the cell line exceeds 30 hours, the ADC incubation lasts 5 days, otherwise spend a four-day incubation.

At the end of the incubation period, cell viability is assessed using the Alamar blue assay. AlamarBlue (Invitrogen) is dispensed throughout the plate (20 µl per well) and incubated for 4 hours. Alamar blue fluorescence is measured at 570 nm excitation and 585 nm emission on a Varioskan Flash plate reader. Percent cell survival is calculated based on the average fluorescence intensity in the ADC-treated wells compared to the average fluorescence intensity in the control wells.

Application

The conjugates of the present invention can be used to provide PBB connection at the target location.

The target location is preferably a population of proliferative cells. An antibody is an antibody against an antigen present in a population of proliferative cells.

According to one embodiment of the present invention, the antigen is absent or present at a reduced level in a population of non-proliferative cells compared to the amount of antigen present in a population of proliferative cells, for example, a population of tumor cells.

At the target location, the linker can be cleaved to release the RelA compound. Thus, the conjugate can be used to selectively provide a RelA compound at a target location.

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The linker can be cleaved with an enzyme present at the target location.

The target location may be in vitro, in vivo or ex vivo.

The antibody-drug conjugate (ADC) compounds of the present invention include compounds that can be used for anticancer activity. In particular, the compounds include an antibody conjugated, i. covalently attached via a linker to the PBB drug moiety, i. e. toxin. If the drug is not conjugated to an antibody, the PBB drug has a cytotoxic effect. Thus, the biological activity of a PBB drug fragment is modulated by conjugation with an antibody. The antibody-drug conjugates (ADCs) of the present invention selectively deliver an effective dose of a cytotoxic agent to tumor tissue, whereby higher selectivity can be achieved, i.e. lower effective dose.

Thus, in one aspect, the present invention provides a conjugate compound described herein for use in therapy.

In a further aspect, a conjugate compound described herein is also provided for use in the treatment of a proliferative disease. According to a second aspect of the present invention, the use of a conjugate compound in the manufacture of a medicament for the treatment of a proliferative disease is provided.

One skilled in the art can easily determine if a candidate conjugate has a curative effect on a proliferative condition for any particular cell type. For example, assays that can be conveniently used to evaluate the activity provided by a particular compound are described in the examples below.

The term proliferative disease refers to unfavorable or uncontrolled cell proliferation of excess or abnormal cells that is undesirable, such as neoplastic or hyperplastic proliferation, whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to, benign, premalignant, and malignant cell proliferation, including, but not limited to, neoplasms and tumors (eg, histiocytoma, glioma, astrocytoma, osteoma), various cancers (eg, lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, esophageal cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma , osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone disease, fibroproliferative disorders (eg, connective tissue), and atherosclerosis. Various cancers of particular interest include, but are not limited to, leukemias and various ovarian cancers.

Any cell type can be treated, including, but not limited to, lung, gastrointestinal tract (including, for example, intestines, colon), breast (breast), ovary, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain and skin.

Disorders of particular interest include, but are not limited to, various types of cancer, including various types of metastatic cancer and metastatic cancer cells such as circulating tumor cells that can be found circulating in body fluids such as blood or lymph. Various cancers of particular interest include breast cancer, lung cancer, stomach cancer, head and neck cancer, colorectal cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, endometrial cancer, and prostate cancer, as well as lymphomas (eg, non-Hodgkin's lymphoma, NHL) and leukemia (eg, acute myelogenous leukemia, AML).

Other disorders of interest include any condition in which AXL is overexpressed or in which AXL antagonism would provide clinical benefit. These include immune disorders, cardiovascular disorders, thrombosis, various types of diabetes, immunity checkpoint disorders, fibrotic disorders (fibrosis) or proliferative diseases such as cancer, in particular metastatic cancer. In addition, AXL is known to be involved in many cancers of epithelial origin.

Fibrotic disorders of interest include strabismus, scleroderma, keloid, nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, nonalcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis of the liver, fibrosis of the kidneys, cancer and atherosclerosis. In these diseases, the chronic development of fibrosis in the tissue leads to noticeable changes in the structure of the affected organs and subsequently causes dysfunction of the organ. As a result of this process of persistent organ wasting, many diseases that involve fibrosis are often progressive conditions and have a poor long-term prognosis (see Rockey D.C., Bell, P.D. and Hill, J.A. (2015), N. Engl. Med., vol. 372 , pp. 1138-1149).

It is contemplated that the antibody-drug conjugates (ADC) of this ISO

- 20 039826 shaving can be used to treat various diseases or disorders, for example, characterized by overexpression of the tumor antigen. Exemplary conditions or hyperproliferative disorders include benign or malignant tumors; leukemia, hematological and lymphoid malignancies. Other examples include neuronal, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastocele, inflammatory, angiogenic, and immunological, including autoimmune, disorders.

Typically, the disease or disorder being treated is a hyperproliferative disease such as cancer. Examples of cancers to be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, or gastric cancer, including cancer. gastrointestinal tract, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary carcinoma gland cancer, kidney or kidney cancer, prostate cancer, vaginal cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

Autoimmune diseases for which ADC compounds may be used include rheumatological disorders (such as, for example, rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis ), osteoarthritis, autoimmune disorders of the gastrointestinal tract and liver (such as, for example, inflammatory bowel disease (eg, ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease ), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteritis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opso-myoclonal syndrome, myasthenia gravis, optic neuromyelitis, Parkinson's disease , Alzheimer's disease and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatological disorders (such as, for example, psoriasis, urticaria, allergic rash, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematological disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, posttransfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing disorders (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplantation and autoimmune endocrine disorders (such as, for example, diabetes-related autoimmune diseases such as insulin dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (eg, Graves' disease and thyroiditis). More preferably, such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

Methods of treatment

The conjugates of the present invention can be used in a therapy. Also provided is a method of treatment comprising administering to a subject in need of treatment a therapeutically effective amount of a conjugate compound of the present invention. The term "therapeutically effective amount" is an amount sufficient to demonstrate benefit to the patient. Such a benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and timing of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, such as deciding on dosage, is the responsibility of general practitioners and other physicians.

The compound of the present invention may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated. Examples of treatments and methods of therapy include, but are not limited to, chemotherapy (administration of active agents, including, for example, drugs such as chemotherapeutic agents); surgical intervention; and radiation therapy.

A chemotherapeutic agent is a chemical compound that can be used to treat cancer, regardless of the mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, plant alkaloids that block the mitotic spindle, cytotoxic/antineoplastic antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in targeted therapy and conventional chemotherapy.

- 21 039826

Examples of chemotherapeutic agents include: erlotinib (Tartceva®, Genentech/OSI Pharm.), docetaxel (Taxotere®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (Gemzar® , Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(P), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (Taxol®, Bristol-Myers Squibb Oncology, Princeton, NJ, USA), trastuzumab (Herceptin®, Genentech), temozolomide (4-methyl-5oxo-2,3,4,6,8-pentazabicyclo-[4.3 .0]-nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, temodar®, temodal®, Schering Plough), tamoxifen ((Z)-2-[4-(1, 2-diphenylbum-1-enyl)phenoxy]-N,Ndimethylethanamine, Nolvadex®, Itubal®, Valodex®) and doxorubicin (Adriamycin®), Akti-1/2, HPPD and rapamycin.

Other examples of chemotherapy agents include: oxaliplatin (Eloxatin®, Sanofi), bortezomib (Velcade®, Millennium Pharm.), sutent (Sunitinib®, SU11248, Pfizer), letrozole (Femara®, Novartis), imatinib mesylate (Gleevec®, Novartis) , XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (Faslodex®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, rapamune®, Wyeth), lapatinib (Tykerb® GSK572016 , Glaxo Smith Kline), lonafarnib (Sarasar™, SCH 66336, Schering Plough), sorafenib (Nexavar®, BAY43-9006, Bayer Labs), gefitinib (Press®, AstraZeneca), irinotecan (Camptosar®, CPT-11 Pfizer), tipifarnib (Zarnestra™, Johnson&Johnson), abraxane™ (no cremophor), albumin-modified paclitaxel nanoparticulate formulations (American Pharmaceutical Partne rs, Schaumberg, II), vandetanib (rINN, ZD6474, Zactima®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (Torisel®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (Telkita®, Telik), thiotepa, and cyclosphosphamide (Cytoxan®, Neosar®); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylomelamin; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CB1-TM1); eleuterobine; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediine antibiotics (e.g. calicheamicin, calicheamycin gamma-II, calicheamycin omega II (Angew Chem. Intl. Ed. Engl. (1994) 33: 183-186); dynemycin, dynemycin A; bisphosphonates such as clodronate ; esperamycin; as well as the chromophore part of neocarcinostatin and related chromophores of chromoprotein enediin antibiotics), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-oxo-diazo L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, kelamycin , rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; adrenal depressants such as aminoglutethimide, mitotane, trilostane; a folic acid supplement such as frolinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfornitin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidanin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubicin; loxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, OR, USA); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anhidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside ("Ara-S"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (navelbin®); novantron; teniposide; edatrexate; daunomycin; aminopterin; capezi

- 22 039826 tabine (Xeloda®, Roche); ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Also included within the definition of "chemotherapeutic agent" are: (i) antihormonal agents whose function is to regulate or inhibit the action of a hormone on tumors, such as antiestrogen and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex®; tamoxifen citrate ), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone and fareston® (toremifin citrate); (ii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, Megace® (megestrol acetate), Aromasin® (exemestane; Pfizer), formestanium, fadrozole , Rivisor® (Vorozol), Femara® (Letrozole; Novartis), and Arimidex® (Anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxocitabine (1,3-dioxolane-nucleoside analogue of cytosine); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, eg PKC-alpha, Raf and H-Ras, such as oblimersen (genasens®, Genta Inc.); (vii) ribozymes such as inhibitors of VEGF expression (eg angiozyme) and inhibitors of HER2 expression; (viii) vaccines, such as gene therapy vaccines, eg allovectin®, leuvectin® and vaxid®; proleukin® rIL-2; topoisomerase 1 inhibitors such as lurtotecan®; abarelix® rmRH; (ix) anti-angiogenic agents such as bevacizumab (Avastin®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

The definition of a chemotherapeutic agent also includes therapeutic antibodies such as alemtuzumab (Campat), bevacizumab (Avastin®, Genentech); cetuximab (Erbitux®, Imclone); panitumumab (Vectibix®, Amgen), rituximab (Rituxan®, Genentech/Biogen Idec), ofatumumab (Arzerra®, GSK), pertuzumab (Purgeta™, Omnitarg™, 2C4, Genentech), trastuzumab (Herceptin®, Genentech), tositumumab ( Bexar, Corixia) and an antibody-drug conjugate, gemtuzumab ozogamicin (Mylotarg®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the present invention include: alemtuzumab, apolizumab, azelizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusizumab, cidakulizumab, cidulizumab эфализумаб, эпратузумаб, эрлизумаб, фелвизумаб, фонтолизумаб, гемтузумаб озогамицин, инотузумаб озогамицин, ипилимумаб, лабетузумаб, линтузумаб, матузумаб, меполизумаб, мотавизумаб, мотовизумаб, натализумаб, нимотузумаб, ноловизумаб, нумавизумаб, окрелизумаб, омализумаб, паливизумаб, пасколизумаб, пекфуситузумаб, пектузумаб, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivisumab, reslizumab, recivisumab, rovelizumab, ruplizumab, sibrotuzumab, ciplizumab, sontuzumab, takatuzumab tetraxetane, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tumoluzumab, tumolizumab zumab, umavizumab, urtoxazumab and visilizumab.

Pharmaceutical compositions in accordance with the present invention and for use in accordance with the present invention may contain, in addition to the active ingredient, i. conjugate compounds, pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, such as dermal, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. The tablet may contain a solid carrier or an adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. The capsule may contain a solid carrier such as gelatin.

For intravenous, dermal or subcutaneous or lesion injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is free of pyrogens and has suitable pH, isotonicity and stability. Those skilled in the art are able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's solution injection, lactose Ringer's solution injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives may be included.

Lineups

While the conjugate compound may be used (eg, administered) alone, it is often preferred to present it as a composition or formulation.

- 23 039826

According to one implementation variant of the present invention, the composition is a pharmaceutical composition (for example, composition, preparation, drug) containing the conjugate compound described in this application, and a pharmaceutically acceptable carrier, diluent or excipient.

According to one implementation variant of the present invention, the composition is a pharmaceutical composition containing at least one conjugate compound described in this application, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (eg wetting agents), masking agents, coloring agents, flavoring agents and sweetening agents.

According to one implementation variant of the present invention, the composition additionally contains other active agents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical guidelines. See, for example, Handbook of Pharmaceutical Additives, 2nd ed. (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th ed., pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

Another aspect of the present invention relates to methods for preparing a pharmaceutical composition, comprising mixing at least one [C11]-radio-labeled conjugate or conjugate-like compound defined in this application, together with one or more other pharmaceutically acceptable ingredients well known to specialists in this field techniques, e.g. carriers, diluents, excipients, etc. When manufactured as discrete units (eg tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.

As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., which, under sound medical judgment, are suitable for use in contact with the tissues of the subject (e.g., human) in question, without undue toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be acceptable in terms of compatibility with the other ingredients of the formulation.

The compositions can be obtained using any of the methods well known in the pharmaceutical field. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. Typically, formulations are prepared by bringing the active compound into uniform and intimate association with carriers (eg, liquid carriers, finely dispersed solid carriers, etc.) and then shaping the product, if necessary.

The formulation can be formulated to provide fast or slow release; immediate, delayed, time-controlled or extended release; or their combinations.

Formulations suitable for parenteral administration (eg, by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (eg, solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (eg, in a liposome or other microparticle). Such liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other appropriate body fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerin, vegetable oils, and the like. Examples of isotonic vehicles suitable for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/mL to about 10 µg/mL, for example, from about 10 ng/mL to about 1 µg/mL. Formulations may be presented in sealed single-dose or multiple-dose containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Injectable solutions and suspensions for immediate administration may be prepared from sterile powders, granules and tablets.

Dosage

One skilled in the art will appreciate that suitable dosages of the conjugate compound and compositions containing the conjugate compound may vary from patient to patient. Determining the optimal dosage will usually involve compensating for the level of therapeutic benefit with whatever

- 24 039826 risk or harmful side effects. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of elimination of the compound, the duration of treatment, other drugs, compounds and/or materials used in combination, the severity of the condition. , as well as the type, sex, age, weight, condition, general health and history of the patient. The amount of the compound and the route of administration will ultimately be left to the discretion of the physician, veterinarian or clinician, although typically the dosage will be chosen to achieve local concentrations at the site of action that achieve the desired effect without causing significant hazardous or harmful effects. side effects.

The introduction can be carried out in a single dose, continuously or intermittently (for example, in divided doses at appropriate intervals) during the course of treatment. Methods for determining the most effective agents and dosage for administration are well known to those skilled in the art and will vary depending on the formulation used for therapy, the goal of therapy, the target cell(s) being(s) being treated, and the subject who is being treated. Single or multiple administration may be performed using the dose level and regimen selected by the attending physician, veterinarian or clinician.

Typically, a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram of subject body weight per day. If the active compound is a salt, an ester, an amide, a prodrug or the like, the amount to be administered is calculated based on the parent compound, and thus the actual weight to be applied is proportionally increased.

According to one embodiment of the present invention, the active compound is administered to a human patient according to the following dosing schedule: about 100 mg, 3 times a day.

According to one embodiment of the present invention, the active compound is administered to a human patient according to the following dosing schedule: about 150 mg, 2 times a day.

According to one embodiment of the present invention, the active compound is administered to a human patient according to the following dosing schedule: about 200 mg, 2 times a day.

However, according to one embodiment of the present invention, the conjugate compound is administered to a human patient according to the following dosing schedule: about 50 or about 75 mg, 3 or 4 times a day.

In one embodiment of the present invention, the conjugate compound is administered to a human patient according to the following dosing schedule: about 100 or about 125 mg, twice daily.

The dosages described above may be applied to the conjugate (including the PBB moiety and the linker to the antibody) or to an effective amount of the PBB compound provided, eg, the amount of the compound that can be released after cleavage of the linker.

For the prevention or treatment of a disease, the appropriate dosage of the ADC of the present invention will depend on the type of disease being treated as defined above, the severity and course of the disease, whether the molecule is being administered prophylactically or therapeutically, prior therapy, the patient's clinical history and response. on the antibody, as well as at the discretion of the attending physician. The molecule is administered in a suitable manner to the patient at one time or over several treatments. Depending on the type and severity of the disease, from about 1 μg/kg to about 15 mg/kg (e.g., 0.1-20 mg/kg), the molecules constitute the initial intended dosage for administration to the patient, for example, whether by one or more separate injections or by continuous infusion. A typical daily dosage may vary from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of ADC for administration to a patient is within the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, treatment is continued until targeted suppression of disease symptoms occurs. An exemplary dosing regimen includes a course of administration of an initial loading dose of about 4 mg/kg followed by the administration of additional doses of ADC every week, every two weeks, or every three weeks. Other dosing regimens may be used. The progress of this therapy is easily monitored using conventional techniques and assays.

Treatment

As used herein, the term treatment, when referring to the treatment of a condition, generally refers to treatment and therapy, whether human or animal (e.g., in veterinary applications), that achieves some intended therapeutic effect, such as inhibiting the progression of the condition, and includes reducing progression rate, progression rate delay, condition regression, condition improvement, and condition cure. Also included is treatment as a prophylactic measure (i.e. prophylaxis, prevention).

In this application, the term therapeutically effective amount refers to that amount of the active compound or material, composition or dosage form containing the active

- 25 039826 a compound that is effective to achieve some target therapeutic effect, commensurate with a reasonable benefit / risk ratio, when administered in accordance with the target treatment regimen.

Similarly, in this application, the term prophylactically effective amount refers to that amount of an active compound or material, composition or dosage form containing the active compound that is effective to achieve some desired prophylactic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with with targeted treatment.

Preparation of drug conjugates

The antibody-drug conjugates of the present invention can be prepared by conjugating the following drug linker:

with an azide-containing antibody using the methods described, for example, in van Geel, R., et al., Bioconjugate Chemistry, 2015, 26, 2233-2242; DOI: 10.1021/acs.bioconjchem.5b00224. Suitable ones include, but are not limited to, conjugation without copper, for example under aqueous conditions, with a solid co-solvent selected from DMF, DMSO and DMAA.

The drug linker can be synthesized according to the examples, with the methods optionally modified, for example with reference to WO 2016/053107 for the synthesis of the linker, and to the following documents for the PBB dimer, for example: WO 2011/130598, WO 2013/055987, WO2014 /057074.

Subject/Patient

The subject/patient can be an animal, mammal, placental mammal, marsupial (e.g., kangaroo, wombat), monotreme (e.g., platypus), rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse ), hare-like (for example, a rabbit), feathered (for example, a bird), a canine (for example, a dog), a feline (for example, a cat), an equine (for example, a horse), a porcine (for example, a pig), a sheep ( e.g., sheep), bovine (e.g., cow), primate, simian (e.g., ape or ape), ape (e.g., marmoset, baboon), great ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

In addition, the subject/patient may have any of its developmental forms, such as a fetus. According to one preferred embodiment of the present invention, the subject/patient is a human.

Examples

Synthesis of intermediate 3

- 26 039826

A solution of BCN alcohol (0.384 g, 2.55 mmol) in MeCN (25 ml) under N ₂ was cooled to 0°C and chlorosulfonyl isocyanate (CSI) (0.255 ml, 415 mg, 2.93 mmol, 1, 15 eq.). After stirring for 15 minutes, Et ₃ N (1.42 ml, 1.03 g, 10.2 mmol, 4 eq.) was added dropwise and stirring was continued for another 10 minutes. Then a solution of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (1.0 g, 6.1 mmol, 2.4 eq.) in H ₂ O (5 ml) was added and the reaction mixture was stirred to room temperature in for 2 hours After this time was added CHCl ₃ (50 ml) and H ₂ O (100 ml) and separated the layers. CH ₂ Cl ₂ (100 ml) was added to the aqueous layer in a separating funnel and the pH was adjusted to 4 with 1 N HCl before layers were separated. The aqueous layer was extracted twice with CH ₂ Cl ₂ (2x100 ml), the organic layers were combined, dried (Na ₂ SO ₄ ), filtered and concentrated. The residue was purified by silica gel reservoir column chromatography, eluting with CH ₂ Cl to 20% MeOH in CH ₂ Cl ₂ . The yield was 0.42 g (1.0 mmol, 39%) 3 as a colorless sticky wax.

Drug Linker Synthesis

Compound 1 can be synthesized as described in WO 2014/057074 - see compound 22.

(a) Palladium tetrakistriphenylphosphine (Pd(PPh ₃ ) ₄ , 4.8 mg, 4.15 µmol) is weighed and placed under an inert atmosphere. A solution of pyrrolidine (5.0 μl, 4.3 mg, 60 μmol) in DCM (1 ml) is degassed by bubbling N ₂ through the solution. A solution of 1 (27 mg, 24 μmol) in DCM (6 ml) was degassed by bubbling N2 through the solution. While the solution is being sparged with N2, a degassed pyrrolidine solution is added. Weighed Pd(PPh ₃ ) ₄ is dissolved in DCM (1 ml) and 0.9 ml of this solution is added. After 50 min sparging with N ₂ , DCM (25 ml) was added and the mixture was washed with aqueous saturated NH ₄ Cl (25 ml). After separation, the aqueous layer was extracted with DCM (2x25 ml). The combined organic layers are dried (Na ₂ SO ₄ ) and concentrated. Residue is purified

- 27 039826 by RP-HPLC (30-90% MeCN (0.1% formic acid) in H2O (0.1% formic acid). The combined fractions are passed through SPE columns (HCO ₃ ^- ) and concentrated. After addition of MeCN ( 50 ml), the mixture was again concentrated The resulting residue 2 was used in the next step.

The conversion of the reaction can be monitored by LC/MS analysis. Column: XBridge column VEN C18 Intelligent Speed (IS), 130 A, 3.5 µm (4.6 mm x 20 mm).

Mobile phase A: water (0.1% formic acid), mobile phase B (0.1% formic acid). Detection using PDA and ESI+. Samples can be obtained by diluting the reaction mixture with MeCN.

(b) To a solution of the above residue 2 in CHCl ₃ (5 ml) was added a solution of 3 (15 mg, 36 μmol, mol. wt. 418 g/mol) in CHCl ₃ (0.8 ml). The resulting mixture was added to the solid EDC. HCl (4.7 mg, 25 µmol), CHCl ₃ (5 ml) was added and the mixture was stirred for 30 minutes. DCM (30 ml) was added and the resulting mixture was washed with water (30 ml). After separation, the aqueous phase is extracted with DCM (30 ml). The combined organic layers are dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by RP-HPLC (30-90% MeCN (no acid) in H ₂ O (0.01% formic acid). The post-HPLC elution tubes were filled with 5% aqueous (NH4)HCO3 before collection. The combined HPLC fractions were extracted using DCM (3x20 mL) The combined organic layers were dried (Na2SO4) and concentrated to give Product 4 as a light yellow/white oil (21 mg, 16 µmol, mol wt 1323 g/mol, 67% after two steps) .

The conversion of the reaction can be monitored by LC/MS analysis. Column: XBridge column VEN C18 Intelligent Speed (IS), 130 A, 3.5 µm (4.6 mm x 20 mm). Mobile phase A: water (0.1% formic acid), mobile phase B (0.1% formic acid). Detection using PDA and ESI+.

Antibody modification Reaction conditions

Reaction conditions for one-pot glycan remodeling:

mg/ml antibody (~0.1 mM)

0.15 mg/ml EndoSH (1% w/w) from Streptococcus pyogenes

1.13 mg/mL His-TnGalNAcT (7.5% w/w) galactose-N-acetyltransferase enzyme (GalNAcT)

2.5 mM 6-N3GalNAc-UDP (25 equiv. vs. IgG) mM MnCl ₂ mM Tris-HCl, pH 8.0

150 mM NaCl

Incubate 16 hours at 30°C

Performed on AXL and B12.

Procedure

This example was run on a 25 mg scale, which can be modified as needed. The individual components are added in order and mixed:

106.5 µl 25 mM Tris, pH=8.0, 150 mM NaCl (to give a final volume of 1667 µl) ml 25 mg/ml antibody in 25 mM Tris, pH=8.0, 150 mM NaCl

71.4 µl 3.5 mg/ml EndoSH in 25 mM Tris, pH=8.0

389 µl 4.82 mg/ml His-TnGalNAcT in 25 mM Tris, pH=8.0

16.7 µl 1M MnCl ₂ in MQ

83.4 µl 0.1 M 6-N ₃ GalNAc-UDP in MQ

Mix for about 16 hours at 30°C. The readiness of the modified galactose residue can be assessed by subjecting the sample to MS analysis. After protein A affinity purification, a small sample of the product can be recovered using DTT and subsequently subjected to MS analysis. A typical mass spectrum of a successful transfer reaction shows the formation of one major product (90% of the total heavy chain), resulting from the transfer of a modified galactose to the central part of the GlcNAc(Fuc)-substituted antibody, and a minor product (±10% of the total heavy chain) , resulting from the transfer of the modified galactose to the central part of the GlcNAc-substituted antibody (without fucose).

Cleaning procedure

Buffers

Binding/wash buffer (TBS, pH=7.5):

mM tris-HCl, pH=7.5

150 mM NaCl

Wash buffer for endotoxin removal (TBS, pH=7.5 + Triton-X100):

mM tris-HCl, pH=7.5

150 mM NaCl

0.2% triton X-100

- 28 039826

Elution Buffer:

0.1 M glycine, pH=2.7

CIP buffer:

0.5M NaOH

Procedure

1. Wash the MabSelectSure 5 ml column (5 ml/min) with the following buffers to clean the column before sample loading:

Wash the column with at least 5 column volumes (CV) of TBS pH 7.5

Wash the column with 15 CV 0.5 M NaOH

Wash column with 5 CV TBS pH 7.5

Wash the column with 5 CV glycine pH=2.7

Wash column with TBS pH=7.5 until natural pH is obtained.

2. Remove the precipitate from the reaction mixture by centrifugation (5 minutes at 4000g) or filtration (0.22 or 0.45 µm filter).

3. Load sample at 2 ml/min and perform the following steps at 5 ml/min:

Washed with at least 20 CV TBS = 0.2% Triton X-100

Washed with at least 20 CV TBS

Elute with 0.1 M glycine pH=2.7

4. Immediately neutralize the fractions by adding 1/5 volume of 1 M Tris-HCl, pH=8.0 and mixing.

5. The sample is dialyzed against 3x>50 volumes of PBS, pH=7.4, at 4°C (3x>1 h).

6. Concentrate the sample using centrifugal filtration devices to ~20 mg/mL.

Conjugation 4 with modified antibody to produce ConjA and ConjB

Reaction Conditions mg/mL Azide Modified Antibody (0.1 M IgG)

0.5 mM 4 (5 eq vs. IgG = 2.5 eq per azide)

10% DMF or 25% propylene glycol

FSB with pH=7.4

Procedure

1) Add 9 volumes of 16.67 mg/ml azido-modified antibodies in PBS pH=7.

2) Add 1 vol. 5 mM 4 in DMF and mix immediately.

3) Incubate overnight.

4) Measure the conversion using RP-HPLC and MS.

conjugate Antibody ConjA AXL ConjB AT 12

Cleaning ADC

Getting a Sample

The following requirements must be met before loading onto the column:

Organic solvent <5%

Total sample volume <3% of CV (< 720 µl for Superdex 200 10/300 GL and <10 ml for Superdex 200 HiLoad 26/600)

No precipitation

The above requirements can be met using the following procedure:

1) The sample is diluted with PBS, pH=7.4, to a final concentration of organic solvent <5%

2) If the volume exceeds 3% CV, the sample was concentrated using Amicon Ultra centrifugal filters (HOMM 10 kDa)

3) Potential precipitate is removed by centrifugation (10 min at 13,000 rpm in a benchtop centrifuge)

cleaning

Purification was performed using a Superdex 200 10/300 GL column (CV = 23 ml, GE healthcare) on an Akta Purifier-10. The following washing steps are performed at a flow rate of 0.5 ml/min:

The column is washed with 1 CV of water.

The column was washed with 1 CV of 0.5 M NaOH.

Balance the PBS column with pH=7.4 (Sigma, D8537) until a neutral pH is obtained.

The sample is injected with 0.5 ml/min PBS, pH=7.4, and 1 ml fractions are collected (total cycle=1.5 CV). Monomeric fractions are pooled and dialyzed at 4° C. against 3 x 1 L of formulation buffer (30 mM histidine, 200 mM sorbitol, 0.02% (w/v) Tween-20, pH=6.0). Samples are sterilized using a 0.22 µm filter, flash frozen using liquid nitrogen and stored.

- 29 039826 at -80°C.

Mass spectral analysis of the manufacturer's digested sample showed one major product (observed mass 25691 Da, approximately 90% of the total Fc/2 fragment) corresponding to the conjugated Fc/2 fragment. RP-HPLC analysis of the reconstituted sample showed an average DAR of 1.98.

Cytotoxicity in vitro

H1299 cells were obtained from AtCs (ATCC number CRL-5803). Medium H1299 was Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Gibco PBS. Cells were grown at 37°C, 5% CO ₂ in a humidified incubator. Cell suspensions were dispensed into 96 well flat bottom plates (104 cells per well). A set of 8 x 10-fold dilutions of stock ADC was prepared in cell culture medium. Each dilution of ADC (50 μl per well) was dispensed into 4-replicate wells of a 96-well plate containing the cell suspension. Control wells were prepared by adding only the same volume of cell culture medium. After incubation for 96 hours, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, catalog no. G5421) according to the manufacturer's instructions. Absorbance was measured at 490 nm. Cell survival (%) was calculated based on the mean absorbance of the 4 ADC-treated wells compared to the mean absorbance of the 4 control wells (100%). Dose-response curves were generated from the average of data from three replicate experiments, and EC50 values were determined by fitting the data to a sigmoidal dose-response curve with a variable slope using Prism (GraphPad, San Diego, CA, USA). Error bars indicate standard deviation (SD).

The EC ₅₀ for ConjA was found to be 0.0554 µg/ml.

Antigen binding study

Maxisorp plates were coated with human Axl antigen (50 ng/well; batch in PBS) at +4° C. overnight. Non-reactive sites were blocked with SuperBlock buffer (overnight at +4°C or at room temperature). A set of 8 x 3-fold or 5-fold dilutions of stock ADC was prepared in sample buffer/PBS/Tween-20. Each dilution of ADC (60 μl/well) was dispensed into 4-replicate wells of the coated plate. Control wells were prepared by adding a similar volume of sample buffer/PBS/Tween-20. An anti-human kappa-IgG horseradish peroxidase (HRP) conjugate was used as a secondary antibody (1:5000, 1 h at room temperature). HRP was detected using a 1-Step Ultra TMB-ELISA substrate solution (75 μl/well; 5 min at room temperature). The reaction with the substrate was stopped using 0.6 M HCl (75 μl/well). Optical density was measured at 450 nm on an Envision using a peroxidase program at 450 nm. Antigen binding curves were generated from the average of 3 replicate Prism experiments (GraphPad, San Diego, CA, USA). In FIG. 1 shows the results obtained, where ^k is ConjA. Error bars indicate standard error of the mean (SEM). ConjA binds with high affinity to the extracellular domain of AXL plated.

In Vivo Efficacy Study - MDA-MB-231

5x10 ⁶ MDA-MB-231 tumor cells were implanted subcutaneously in female athymic nude mice. Dosing of ADC with vehicle or test article was started when tumor volumes reached 88-172 mm ³ . ConjA was administered intravenously (IV) by tail vein injection once at a dose level of 1 mg/kg. The dosing volume was 10 ml/kg body weight and increased according to the body weight of each individual animal. Animals were euthanized if their tumor volume reached a final volume of 1500 mm ³ or at the end of the study, whichever came first. Animal weight, signs of any adverse effects associated with treatment, and clinical signs were monitored during the study period. The following rule was used to calculate the mean group tumor volume: when an animal withdrew from the study due to tumor size, the final tumor volume recorded for the animal was included in the data used to calculate the mean volume at subsequent time points. Tumor volume and body weight values were not used to calculate group mean tumor volumes/body weights when less than 50% of animals per group remained in the study. Prism (GraphPad, San Diego, CA, USA) was used for graphical data presentation and statistical analyses. In FIG. 2 shows the results obtained, where is ConjA, and O is only a carrier. Error bars indicate SEM.

A single dose of ConjA 1 mg/kg strongly inhibited tumor growth, with 10/10 mice being tumor-free 60 days post-dose.

Toxicological study in rats

Method

ConjA was evaluated in a single intravenous dose tolerability study in rats. Male Sprague-Dawley rats (n=3/group) were dosed with 3 and 6 mg/kg ConjA on day 1 and necropsied 21 days post-dose. Body weight and food intake were often controlled by selection

- 30,039,826 lifetime samples for clinical pathology (blood at days 8 and 21) and resampling for pharmacokinetics. At autopsy, macroscopic observations were made, while selected organs were weighed and stored for possible histopathological examination.

ConjA was clinically well tolerated at doses of 3 and 6 mg/kg. Weight gain was reduced by 11% and 21% in the 3 and 6mg/kg groups, respectively, consistent with a reduction in food intake. Several hematological parameters were reduced on day 8, mainly in the 6 mg/kg group (reticulocytes (-76%), hemoglobin (-29%), leukocytes (-66%) and platelets (-37%)), while some signs of recovery were observed by day 21. At autopsy, a decrease in thymus weight was observed in all animals. Therefore, the maximum tolerated dose (MTD) for ConjA was 6 mg/kg.

In Vivo Efficacy Study - SN12C

Female severe combined immunodeficiency mice (Fox Chase SCID®, CB17/IcrPrkdcscid/IcrIcoCrl, Charles River) were ten weeks old with a body weight (BW) range of 18.0 to 21.6 g on study day 1. On the day of tumor implantation, each test mouse received 5 x 106 SN12C cells (0.1 ml cell suspension in 50% Matrigel® (Corning®) in phosphate buffered saline) implanted subcutaneously in the right flank. SN12C is a human renal carcinoma-derived xenograft model with high AXL expression (~88,000 copies per cell).

Tumor growth was controlled when the mean size approached the target range of 100 to 150 mm ³ . Tumors were assessed in two dimensions with a caliper and the volume was calculated using the formula:

where w = width and l = length in mm of the tumor. The mass of the tumor can be estimated with the assumption that 1 mg is equivalent to 1 mm ³ tumor volume.

Twenty-five days after tumor implantation, referred to as study day 1, animals were sorted into five groups (n=8) with individual tumor volumes from 108 to 172 mm ³ and group mean tumor volumes from 120 to 123 mm ³ . All treatments were administered intravenously in the lateral tail vein as a single injection on day 1 of the study. The dosing volume was 0.2 ml per 20 g body weight (10 ml/kg) and scaled according to the body weight of each individual animal. Tumors were measured with calipers twice a week and each animal was euthanized when its tumor reached a final volume of 2000 mm ³ or at the end of the study, whichever came first. The study ended on day 60. At a dose of 1 mg/kg, ConjA resulted in 7/8 complete responses (CR) and 6/8 tumor-free survivors (TFS) at the end of the study on day 60. In FIG. 3 shows the results obtained, where O is the carrier only; O is ConjB administered at 1 mg/kg; □ is ConjA administered at 0.3 mg/kg; △ is ConjA administered at 0.6 mg/kg; V is ConjA administered at a dose of 1 mg/kg. Error bars indicate SEM.

In vivo efficacy study - Karpas299 (AXL-negative)

Female severe combined immunodeficiency mice (Fox Chase SCID®, CB17/Icr-Prkdcscid/IcrIcoCrl, Charles River) were nine weeks old with a body weight (BW) range of 17.0 to 22.5 g on day 1 of the study. On the day of tumor implantation, each test mouse received 1 x 10 ⁷ Karpas-299 cells (0.1 ml cell suspension in PBS) implanted subcutaneously in the right flank.

Tumor growth was controlled when the mean size approached the target range of 100 to 150 mm ³ . Tumors were assessed in two dimensions using a caliper and the volume was calculated using the formula:

where w = width and l = length in mm of the tumor. The mass of the tumor can be estimated with the assumption that 1 mg is equivalent to 1 mm ³ tumor volume.

Ten days after tumor implantation, referred to as study day 1, animals were sorted into four groups with individual tumor volumes from 108 to 126 mm ³ and group mean tumor volumes from 113 to 117 mm ³ . All treatments were administered intravenously in the lateral tail vein as a single injection on day 1 of the study. The dosing volume was 0.2 ml per 20 grams of body weight (10 ml/kg) and scaled according to the body weight of each individual animal. Tumors were measured with calipers twice a week and each animal was euthanized when its tumor reached a final volume of 2000 mm ³ or at the end of the study, whichever came first. The study ended on day 29.

In FIG. 4 shows the results obtained, where O is the carrier only; □ is ConjA administered at 1 mg/kg.

Error bars indicate SEM.

- 31 039826

In vivo efficacy study - PAXF 1657 xenograft model derived from a patient with pancreatic cancer (PDX)

Female nu/nu (NU-Foxnlnu) mice from Charles River were at least 8 weeks old with a body weight (BW) range of 22.0 to 30.0 g on day 0. On the day of implantation, tumor fragments were obtained from xenografts in nude mice. After extraction from donor mice, the tumors were cut into fragments (edge length 3–4 mm) and placed in PBS containing 10% penicillin/streptomycin. Recipient animals were anesthetized with isoflurane inhalation and unilateral or bilateral tumor implants were injected subcutaneously into the flank.

Animals and tumor implants were monitored when their implant volumes approached the target range of 50 to 250 mm ³ in a sufficient number of animals. Tumors were assessed in two dimensions with a caliper and the volume was calculated using the formula:

Tumor volume (mm ³ ) = w ² xl/2 where w = width and l = length in mm of the tumor.

The day of randomization was designated day 0 of the experiment. On day 1 of the experiment, female nu/nu mice bearing subcutaneous PAXF 1657 xenografts (group mean tumor volumes 109.0110.1 mm ³ ) were sorted into groups (n=8 per group) and dosing started. The dosing volume was 0.1 ml per 20 g body weight (5 ml/kg) and scaled according to the body weight of each individual animal. All treatments were administered intravenously (IV) as a single injection on day 1 (qdx1). Tumors were measured with calipers twice a week and each animal was euthanized when its tumor reached a final volume of 2000 mm ³ or at the end of the study, whichever came first. The study ended on day 42. Each single dose of ConjA (0.3, 0.6 and 1 mg/kg) resulted in complete elimination of tumors at the end of the study.

In FIG. 5 shows the results obtained, where O is the carrier only; O is ConjB administered at 1 mg/kg; □ is ConjA administered at 0.3 mg/kg; △ is ConjA administered at 0.6 mg/kg; V is ConjA administered at a dose of 1 mg/kg.

Error bars indicate SEM. The vertical dotted line indicates the start of dosing (day 1).

In Vivo Efficacy Study - Xenograft Model ES0195 Derived from a Patient with Esophageal Cancer (PDX)

Balb/c female nude mice from Beijing AniKeeper Bio-Technology Co. Ltd. were 5-6 weeks of age with a body weight range of 20.2-24.6 g at baseline. Tumor fragments (2-3 mm in diameter in ice-cold RPMI1640 media without serum) were inoculated subcutaneously in the right flank of 24 female Balb/c nude mice.

All animals were randomly assigned to 3 different study groups. Randomization was performed using a multitasking method in the Study Log software on day 0. The mean tumor volume (mm ³ ) for each group + SD at randomization was as follows: ~ 141 mm ³ ± 47 mm ³ .

All treatments were administered intravenously (IV) as a single injection on day 1 (qdx1). Body weight was measured twice a week throughout the study. Tumor volumes were measured twice a week. The study was terminated 51 days after the start of dosing.

In FIG. 6 shows the results obtained, where O is the carrier only; □ is ConjA administered at 1 mg/kg; △ is ConjB administered at 1 mg/kg;

Error bars indicate SEM. The vertical dotted line indicates the start of dosing (day 1).

Study of passive activity in vitro conditions

The in vitro cytotoxicity of ConjA and the ADC isotype control, ConjB, was compared in SN12C and Karpas-299 cell lines, Karpas299 being AXL negative. SN12C are AXL positive. Attached SN12C cells were trypsinized, resuspended in 1 ml of growth medium, and mixed gently before counting to determine cell density. The Karpas299 cell suspension was counted without any pretreatment. Cell density was determined in duplicate by trypan blue exclusion using a LUNA-II™ automatic cell counter. The SN12C cell suspension was diluted to 1 x 10 ⁴ cells/ml in cell-specific growth media and dispensed at 100 μl/well into sterile white 96-well flat bottom microplates and incubated overnight to allow cell attachment. Karpas299 cells were seeded on the same day that ADC was applied.

Serial dilutions of filter-sterilized ADCs were made at a ratio of 1:10 and repeated to obtain eight serial dilutions using an initial concentration of ADC of 20 μg/ml and diluted in cell-specific growth medium in sterile 96-well polypropylene plates. Dilutions of ADC (including stock solution) were distributed according to

- 32 039826 wells, 2 replicates, 100 µl/well, labeled white 96-well flat bottom plate containing 100 µl of seeded cell suspension. For media control wells, 100 µl of growth medium was dispensed into 2-replicate wells, and for cell line control wells, 100 µl of growth medium was dispensed per 100 µl of the cell suspension previously dispensed into 2-replicate wells. All plates were incubated for 5 days at 37° C. in a CO ₂ gas (5%) incubator. Analyzes were performed using the same seeding densities and incubation times for both cell lines; 1 x 10 ³ cells/well were incubated for 5 days for each line (these conditions were pre-optimized for this study).

After a 5-day incubation period, the plates were centrifuged at 600 g (20°C) for 5 min, then 100 µl/well were carefully transferred to freshly prepared white 96-well flat-bottomed plates containing 100 µl of seeded Karpas-299 cells (previously distributed). All plates were incubated for 5 days at 37° C. in a CO2 gas (5%) incubator. After the incubation period, the plates were centrifuged at 600 g (20° C.) for 5 min, then 100 μl/well were carefully removed and discarded, and cell viability was measured on the remaining medium in the wells using the CellTiter-Glo® assay. Plates were read on Envision using the luminescence protocol and data was analyzed using Graphpad Prism software.

Cytotoxicity in vitro in Karpas299 cells [nM] ConjA ConjB primary culture 2.94 2.53 Media Transfer 0.016 3.77

Sequences

SEQ W NO1 D1H12 VH, CDR underlined]

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISSGGSYT YYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHPIYYTYDDTMDYWGOG TTVTVSS

SEQ ID NO 2 G1H12 VL, CDR underlined]

EIVLTOSPGTLSLSPGERATLSCSASSSVSSGNFHWYOOKPGLAPRLLIYRTSNLASGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCOOWSGYPWTFGGGTKLEIK

SEQ ID NO3 [heavy chain 1H12]

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISSGGSYT YYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHPIYYTYDDTMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQ SSGLYSLS S VVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYWSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

- 33 039826

VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEQ ID NO.4 [light chain 1H121

EIVLTQSPGTLSLSPGERATLSC S AS S S VS SGNFHWYQQKPGLAPRLLIYRTSNLASGIP A RF SGSGSGTDFTLTIS SLEPEDF AVYYCQQWSGYPWTFGGGTKLEIKRT VAAPS VFIFPP S DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGQNSQESVTEQDSKDSTYVSLSSTKTLSSKVACEK

SEP ID NO.5 ZN12 VH CDR11

SYGMS

SEP ID NO.b TSN12 VH CDR21

TISSGGSYTYYPDSVKG

SEP ID NO.7 TSN12 VH CDR31

HPIYYTYDDTMDY

SEP ID NO.8 ZN12 VL CDR11

SASSSVSSGNFH

SEP ID NO.9 ZN12 VL CDR21

RTSNLAS

SEP ID NO. 10 G1H12 VL CDR31

QQWSGYPWT

SEQ ID NO.II [5F11 VH mouse, CDR underlined!

EVKLLESGGGLVOPGGSLKLSCAASGFDFSRYWMSWVROAPGKGLEWIGEINPDSSTIN YTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCASPYYYGPFAYWGOGTLVTVSS SEQ ID NO.I2 [5F I I VL mice, CDR underlined!

DIVLTOSPASLAVSLGORAIISCKASOSVSFAGTSLMHWYOQKPGOQPKLLIYRASNLEA GFPTRFSGSGSRTDFTLNIHPVEEEDAATYYCOOSREYPRTFGGGTKLEVK

SEQ IDNO I3 [5F11 VH CDR11

RYWMS

SEQ W NO. 14 [5F11 VH CDR21

EINPDSSTINYTPSLKD

SEQ W NO. 15 [5F11 VH CDR31

PYYYGPFAY

SEQ IDNO I6 [5F11 VLCDR11

KASQSVSFAGTSLMH

SEP W NO. 17 [5F11 VL CDR21

- 34 039826

RASNLEA

SEP ID NO. 18 [5F11 VLCDR31

QQSREYPRT

SEO W NO. 19 F5F11 RHA1

QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVAEINPDSSTI NYTPSLKDRFAISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYYGPFAYWGQGTLVTV S

SEQ ID NO.20 F5F11 RHB1

EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVAEINPDSSTI NYTPSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYYYGPFAYWGQGTLVTV S

SEQ ID NO.21 [5F11RHC1

EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVSEINPDSSTIN YTPSLKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYYGPFAYWGQGTLVTVS SEQ ID NO.22 F5F11 RKA1

EIVLTQSPLSLPVTPGEPASISCKASQSVSFAGTSLMHWYLQKPGQSPQLLIYRASNLEAG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSREYPRTFGQGTKVEIK SEQ ID NO.23 [Axl human!

MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQ LQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQ CLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQ DAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLV SRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQL RLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHW QEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTA AGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILA LFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRD VMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEA VCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLP TQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDY YRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDY LRRGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEIL YVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQP ADRGSPAAPGQEDGA

SEQ ID NO.24 [heavy chain 1H121

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISSGGSYT YYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHPIYYTYDDTMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQ SSGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

CLAIM

Claims (25)

CLAIM 1. AXL binding conjugate of formula (I) Ab-(DL) _P (I) in which Ab is an antibody that binds to AXL; DL represents - 35 039826 where X is selected from the group consisting of a single bond, -CH ₂ - and -C2H4-; n is from 1 to 8; m is 0 or 1; R ⁷ is methyl or phenyl; if there is a double bond between C2 and C3, R ² is selected from the group consisting of: (ia) C _5-10 aryl group, unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, C _1-7 alkoxy, C _3-20 heterocyclyloxy, C _5-20 aryloxy, carboxy, C _1-7 alkyl, C _3-7 heterocyclyl, bis-oxy-C _1-3 alkylene and -C(=O)OR, where R represents a C _1-7 alkyl group, C _3-20 heterocyclyl group or C _{5- 20} an aryl group; (ib) C _1-5 saturated aliphatic alkyl; (ic) C _3-6 saturated cycloalkyl; ^R22 X^^R ²³ (id) r ²¹ , where each of R ²¹ , R ²² and R ²³ is independently selected from H, C _1-3 saturated alkyl, C _2-3 alkenyl, C _2-3 alkynyl and cyclopropyl, and the total the number of carbon atoms in the group R ² is not more than 5; (ie) ^R where one of R ^25a and R ^25b is H and the other is selected from phenyl, phenyl being unsubstituted or substituted by a group selected from halogen, methyl, methoxy; pyridyl and thiophenyl; and (if) where R ²⁴ is selected from H; C _1-3 saturated alkyl; C _2-3 alkenyls; C _2-3 alkynyl; cyclo propyl; phenyl, wherein the phenyl is unsubstituted or substituted by a group selected from halogen, methyl, methoxy; pyridyl and thiophenyl; D^p ^26a if a single bond is present between C2 and C3, R ² is ^R26b where R ^26a and R ^26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, with the alkyl and alkenyl groups unsubstituted or substituted with a group selected from C1-4 alkylamido and C1-4 alkyl ester; or if one of R ^26a and R ^26b is H, the other is selected from nitrile and C1-4 alkyl ester; if a double bond is present between C2' and C3', R ¹² is selected from the group consisting of (iia) a C _5-10 aryl group, unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, C _{1 -7} alkoxy, C _3-20 heterocyclyloxy, C _5-20 aryloxy, carboxy, C _1-7 alkyl, C _3-7 heterocyclyl, bis-oxy-C _1-3 alkylene and -C(=O)OR, where R is a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group; (iib) C _1-5 saturated aliphatic alkyl; (iic) C _3-6 saturated cycloalkyl; R ³² /^^R ³³ (iid) ^r31 , where each of R ³¹ , R ³² and R ³³ is independently selected from H, C _1-3 saturated alkyl, C _2-3 alkenyl, C _2-3 alkynyl and cyclopropyl, and the total number of carbon atoms in the group R ¹² is not more than 5; R ^35b (iie) where one of R ^35a and R ^35b is H and the other is selected from phenyl, phenyl being unsubstituted or substituted by a group selected from halogen, methyl, methoxy; pyridyl and thiophenyl; and - 36 039826 (iif) ^rM , where R ²⁴ is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyls; C _2-3 alkynyl; cyclopropyl; phenyl, wherein the phenyl is unsubstituted or substituted by a group selected from halogen, methyl, methoxy; pyridyl and thiophenyl; D^k ^36a if a single bond is present between C2' and C3', R ¹² is ^R where R ^36a and R ^36b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, with alkyl and alkenyl groups unsubstituted or substituted with a group selected from C1-4 alkylamido and C1-4 alkyl ester; or if one of R ^36a and R ^36b is H, the other is selected from nitrile and C1-4 alkyl ester and p is from 1 to 8; wherein the C _3-20 heterocyclyl group contains 3 to 20 ring atoms, of which 1 to 10 are ring heteroatoms; and the C _3-7 heterocyclyl group contains 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms. 2. The conjugate according to claim 1, characterized in that X is -CH ₂ -. 3. Conjugate according to claim 1 or 2, characterized in that n is 1-4. 4. Conjugate according to claim 3, characterized in that n is equal to 2. 5. A conjugate according to any one of claims 1 to 4, characterized in that a double bond is present between C2 and C3 and R ² is a C _1-5 saturated aliphatic alkyl group. 6. The conjugate according to claim 5, characterized in that R ² is methyl, ethyl or propyl. 7. A conjugate according to any one of claims 1 to 4, characterized in that there is a double bond between C2 and C3 and R ² is: (a) phenyl which bears one to three substituent groups, said substituents being selected from methoxy, ethoxy, fluoro, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl; or (b) cyclopropyl; or (c) a group of formula R ²¹ while the total number of carbon atoms in the group R ² is not more than 3; or (d) a group or (e) a group of formula in which R ²⁴ is selected from H or methyl. 8. The conjugate according to any one of claims 1 to 4, characterized in that there is a single bond between C2 and C3, R ² is and (a) R ^26a and R ^26b are both H; or (b) R ^26a and R ^26b are both methyl; or (c) one of R ^26a and R ^26b is H and the other is selected from C _1-4 saturated alkyl, C _2-3 alkenyl, the alkyl and alkenyl groups being unsubstituted or substituted. 9. A conjugate according to any one of claims 1 to 8, characterized in that a double bond is present between C2' and C3' and R ¹² is a C _1-5 saturated aliphatic alkyl group. 10. The conjugate according to claim 9, characterized in that R ¹² is methyl, ethyl or propyl. 11. A conjugate according to any one of claims 1 to 8, characterized in that there is a double bond between C2' and C3' and R ¹² is: (a) phenyl which bears one to three substituent groups, said substituents being selected from methoxy, ethoxy, fluoro, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl; or - 37 039826 (b) cyclopropyl; or (c) a group of formula while the total number of carbon atoms in the group R ¹² is not more than 3; or (d) a group; or (e) a group of formula in which R ³⁴ is selected from H or methyl. 12. The conjugate according to any one of claims 1 to 8, characterized in that there is a single bond between C2' and C3', R ¹² is and (a) R ^36a and R ^36b are both H; or (b) R ^36a and R ^36b are both methyl; or (c) one of R ^36a and R ^36b is H and the other is selected from C _1-4 saturated alkyl, C _2-3 alkenyl, the alkyl and alkenyl groups being unsubstituted or substituted. 13. The conjugate according to any one of claims 1 to 12, characterized in that said antibody contains a VH domain containing a VH CDR3 with the amino acid sequence of SEQ ID NO: 7, a VH CDR2 with the amino acid sequence of SEQ ID NO: 6, and a VH CDR1 with the amino acid sequence SEQ ID NO: 5. 14. The conjugate according to any one of claims 1 to 13, characterized in that said antibody contains a VH domain containing the sequence of SEQ ID NO: 1. 15. The conjugate according to any one of claims 1 to 14, characterized in that said antibody contains a VL domain containing a VL CDR3 with the amino acid sequence of SEQ ID NO: 10, a VL CDR2 with the amino acid sequence of SEQ ID NO: 9, and a VL CDR1 with the amino acid sequence SEQ ID NO: 8. 16. A conjugate according to any one of claims 1 to 15, characterized in that said antibody contains a VH domain containing the sequence of SEQ ID NO: 2. 17. A conjugate according to any one of claims 1 to 16, characterized in that said antibody contains a heavy chain containing the sequence of SEQ ID NO: 3 or SEQ ID NO: 24. 18. A conjugate according to any one of claims 1 to 17, characterized in that said antibody contains a pair with a light chain containing the sequence of SEQ ID NO: 4. 19. A composition for treating a proliferative disease in a subject, comprising a mixture of antibody-drug conjugate compounds according to any one of claims 1-18, wherein the average drug load per antibody in the mixture of antibody-drug conjugate compounds is from 1 to 4. 20. The use of a conjugate according to any one of claims 1 to 18 for the treatment of a proliferative disease in a subject. 21. Use according to claim 20, characterized in that said disease is cancer. 22. The use according to any one of claims 20 or 21, characterized in that the disease or cancer is characterized by the presence of a neoplasm containing both AXL+ and AXL cells. 23. A method for treating a proliferative disease, said method comprising: (i) detecting whether the subject has a neoplasm containing both AXL+ and AXL- cells; (ii) administering to said subject said antibody-drug conjugate according to any one of claims 1-18. 24. A pharmaceutical composition for the treatment of a proliferative disease, comprising a conjugate according to any one of claims 1 to 18 and a pharmaceutically acceptable diluent, carrier or excipient. 25. A pharmaceutical composition according to claim 24, further comprising a therapeutically effective amount of a chemotherapeutic agent.