EA040749B1 - PYRROLOBENZODIAZEPINE-ANTIBODY CONJUGATES - Google Patents

Description

Cross-reference to related applications

This application claims priority under GB 1702031.4 filed on February 8, 2017, GB 1719391.3 filed on November 22, 2017, GB 1719398.8 filed on November 22, 2017, and gB 1719393.9 filed on November 22, 2017.

Technical field

The present invention relates to pyrrolobenzodiazepines (PBD) containing a labile protecting group in the form of an antibody linker.

State of the art

Pyrrolobenzodiazepines

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 have been developed for various analogs (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, 9197 (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:

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 isospiral, 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:

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 the C2 position and is usually cleaved by the action of the enzyme on the linker group. 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.

- 1 040749

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 to kill or inhibit tumor cells in cancer treatment, targets delivery of the drug moiety to and intracellular accumulation in tumors, while systemic administration of these drugs in the 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; WO2009/052249; 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, a conjugate of formula (I) is provided:

Ab-(DL) _P (I) in which

Ab is an antibody that binds to DLK1;

DL represents

Where

X is selected from the group consisting of a single bond, -CH2- 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) 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-C _1-3 alkylene;

(ib) C1. ₅ saturated aliphatic alkyl;

- 2 040749 (ic) C _3-6 saturated cycloalkyl;

(id) where each of R ²¹ , R ²² and R ²³ is independently selected from H, C _1-3 saturated alkyl,

C _2-3 α-alkenyl, C _2-3 α-kynyl and cyclopropyl, wherein the total number of carbon atoms in the R ¹² group is not more than 5;

⁽ ie) wherein one of R ^25a and R ^25b is H and the other is selected from: phenyl, wherein said phenyl is 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;

^ ^R2Sa if there is a single bond between C2 and C3, R ² is R ^26b where R ^26a and R ^26b are independently selected from H, F, C _1-4 saturated alkyl, C _2-3 alkenyl, alkyl and alkenyl the groups are optionally substituted with a group selected from C _1-4 alkylamido and C _1-4 alkyl ester; or if one of R ^26a and R ^26b is H, the other is selected from nitrile and C _1-4 alkyl ester;

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

(ia) a C _5-10 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;

R ³³ (id) ^R , 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 αkynyl and cyclopropyl, with the total number of carbon atoms in group R ¹² composition-

lays no more than 5;

(ie) wherein one of R ^35a and R ^35b is H and the other is selected from: phenyl, wherein said phenyl is optionally substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if) R ³⁴ where R ²⁴ is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyls; C _2-3 αkynyl; cyclopropyl; phenyl, said phenyl being optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl;

Lu ^p36E if a single bond is present between C2' and C3', R ¹² is R ^36b where R ^36a and R ^36b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, with the alkyl and alkenyl groups optionally substituted 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.

These conjugates were found to exhibit good activity and surprising tolerance compared to similar conjugates without a sulfonamide group.

Brief description of the drawings

In FIG. 1 shows the cytotoxicity of a conjugate according to the first aspect of this ISO.

- 3 040749 shaving in vitro.

In FIG. 2 shows the in vivo performance of the conjugate according to the first aspect of the present invention.

In FIG. 3 shows the cytotoxicity of a conjugate according to the first aspect of the present invention in A204 and Hep3B cells in 2D and 3D in vitro cell cultures.

In FIG. 4 shows the in vitro cytotoxicity of the conjugate according to the second aspect of the present invention.

In FIG. 5 shows the in vivo performance of the conjugate according to the second aspect of the present invention.

In FIG. 6 shows the antitumor activity of a conjugate according to the second aspect of the present invention in an in vivo SN12C xenograft model.

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 providing 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:

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 with some degree of efficiency 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

Deputies.

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

Unless otherwise indicated, as used herein, the term substituted refers to a parent group that carries one or more substituents. The term substituent is used herein in its 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: As used herein, 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). As used herein, 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 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g., 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 (C2), propyl (C ₃ ), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C ₇ ).

Examples of saturated linear alkyl groups include, but are not limited to, methyl

- 4 040749 (C1), ethyl (C2), n-propyl (C ₃ ), n-butyl (C ₄ ), n-pentyl(amyl) (C ₅ ), n-hexyl (C ₆ ) and n-heptyl (C ₇ ).

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

C2.12 alkenyl: As used herein, the term C2.12 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=CH2), 1-propenyl (—CH=CH— _CH3 ), 2-propenyl (allyl, —CH—CH= _CH2 ), 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: As used herein, 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 (C ₃ ), cyclobutane (C ₄ ), cyclopentane (C ₅ ), cyclohexane (C ₆ ), cycloheptane (C ₇ ), methylcyclopropane (C ₄ ), dimethylcyclopropane (C ₅ ), methylcyclobutane (C ₅ ), dimethylcyclobutane ( C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7);

unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C3), cyclobutene (C ₄ ), cyclopentene (C ₅ ), cyclohexene (C6), methylcyclopropene (C ₄ ), dimethylcyclopropene (C ₅ ), methylcyclobutene (C ₅ ), dimethylcyclobutene (C ₆ ), methylcyclopentene (C6) , dimethylcyclopentene (C7) and dimethylcyclohexene (C7);

saturated polycyclic hydrocarbon compounds:

norcaran (C ₇ ), norpinan (C ₇ ), norbornane (C ₇ ).

C ₃ _ ₂₀ heterocyclyl: As used herein, 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 , _{C5-6 ,} etc.) indicate the number of ring atoms or the range of the number of ring atoms, whether carbon atoms or heteroatoms. For example, as used herein, 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, groups 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 (C ₆ ), dihydropyridine (C ₆ ), tetrahydropyridine (C ₆ ), azepine (C ₇ );

O1: oxirane (C3), oxetane (C ₄ ), oxolane (tetrahydrofuran) (C ₅ ), oxol (dihydrofuran) (C ₅ ), oxan (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);

S1: thiirane (C3), thietane (C ₄ ), thiolane (tetrahydrothiophene) (C ₅ ), thiana (tetrahydrothiopyran) (C ₆ ), thiepane (C7);

O2: dioxolane (C5), dioxane (C6) and dioxepane (C7);

O ₃ : trioxane (C ₆ );

N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), 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 );

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: in this document, the term C ₅ _ ₂₀ aryl refers to a monovalent fragment, according to

- 5 040749 radiated by removing a hydrogen atom from an atom of the aromatic ring of an aromatic compound, and the specified fragment contains from 3 to 20 ring atoms. As used herein, the term C _5-7 aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, said moiety having 5 to 7 ring atoms, and as used herein, the term C _5-10 aryl refers to a monovalent moiety , 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, as used herein, 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 (C18) and pyrene (C16).

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

Np pyrrole (azole) (C ₅ ), pyridine (azine) (C ₆ );

O ₁ : furan (oxol) (C ₅ );

S1: thiophene (thiol) (C ₅ );

N ₁ O ₁ : oxazole (C ₅ ), isoxazole (C ₅ ), isoxazine (C ₆ );

N ₂ Of 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 );

N3: triazole (C5), triazine (C6);

N4: tetrazole (C5).

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 (N ₁ ), isoindole (N ₁ ), indolizine (N ₁ ), indoline (N ₁ ), isoindoline (N ₁ ), purine (N ₄ ) (e.g. adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N ₁ O ₁ ), benzisoxazole (N ₁ O ₁ ), benzodioxol (O2), benzofurazan (N ₂ O ₁ ), benzotriazole (N ₃ ), benzothiofuran (S ₁ ), benzothiazole (N ₁ S ₁ ), benzothiadiazole (N ₂ S);

C ₁₀ (with 2 fused rings) derived from chromene (O ₁ ), isochromene (O ₁ ), chromane (O ₁ ), isochromane (O ₁ ), benzodioxane (O ₂ ), quinoline (N ₁ ), isoquinoline (N ₁ ), quinolysine (N ₁ ), benzoxazine (N ₁ O ₁ ), 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 (N ₁ ), dibenzofuran (O ₁ ), dibenzothiophene (S ₁ ), carboline (N2), perimidine (N2), pyridoindole (N2);

C ₁₄ (with 3 fused rings), derived from acridine (N ₁ ), xanthene (O ₁ ), thioxanthene (S ₁ ), oxantrene (O ₂ ), phenoxathiine (O ₁ S ₁ ), phenazine (N2), phenoxazine ( N ₁ O ₁ ), phenothiazine (N ₁ S ₁ ), thiantrene (S ₂ ), phenanthridine (N ₁ ), 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, e.g. C _1-7 alkyl group (also called C _1-7 alkoxy group, which is discussed below), C _3-20 heterocyclyl group (also called C 3-20 heterocyclyloxy group) or a C5-20 aryl group (also referred to as a _C5-20 aryloxy group), preferably a _C1-7 alkyl group.

Alkoxy: -OR where R is an alkyl group, for example a C _1-7 alkyl group.

- 6 040749

Examples of C ₁ - ₇ alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), O(nPr) (n-propoxy), -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 is an ester substituent, e.g. a C _{1 -7} alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group , preferably a C ₁ - ₇ alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH ₃ , -C(=O)OCH ₂ CH ₃ , -C(=O)OC(CH ₃ ) ₃ and -C(=O)OPh .

Amino: -NR ¹ R ² where R ¹ and R ² independently represent amino substituents, for example, hydrogen, C ₁ - ₇ alkyl group (also called C ₁ - ₇ alkylamino or di-C _{1 .7} αkylαmino), C _{3 -20} heterocyclyl group or C ₅ - ₂₀ aryl group, preferably H or C ₁ - 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 4 to 8 ring atoms. Amino groups may be primary (-NH2), secondary (-NHR ¹ ) or tertiary (-NHR ¹ R 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)NR ¹ R 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)NH2, -C(=O)NHCH ₃ , -C(=O)N(CH ₃ ) ₂ , -C(=O)NHCH2CH ₃ and -C (=O)N(CH2CH3)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: -NO ₂ .

Azido: -N ₃ .

Cyano (nitrile, carbonitrile): -CN.

Antibody.

Antibody to DLK1.

In one aspect, the antibody is an antibody that binds to DLK1.

Ligand-homologous delta protein 1 (DLK-1) is an EGF-like membrane-bound protein consisting of six tandem EGF-like repeats, a peri-membrane region with a TACE-mediated cleavage site (ADAM17), a transmembrane domain, and a short intracellular tail. DLK-1 is highly expressed during fetal development, but its expression is reduced and severely restricted in adults. In contrast, DLK-1 is re-expressed in several tumors such as neuroblastoma, hepatocellular carcinoma (HCC), rhabdomyosarcoma, small cell lung cancer, myelodysplastic syndrome, and acute myeloid leukemia. Interestingly, in HCC, DLK1 has been shown to be a marker of cancer stem cells, a subset of cells responsible for tumor initiation, growth, metastasis, and recurrence.

Overall, DLK-1 is a promising target for an antibody-drug conjugate (ADC) approach due to its selective expression in a wide range of malignancies and limited expression in healthy organs, as well as its expression on HCC cancer stem cells.

HuBa-1-3d.

In some embodiments, the antibody comprises a VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO. 7. According to some embodiments of the present invention, the VH domain further comprises a VH CDR2 with the amino acid sequence of SEQ ID NO. 6 and/or CDR1 VH with the amino acid 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, CDR2 VH with the amino acid sequence of SEQ ID NO. 6 and CDR3 VH 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. According to 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 CDR1 VL with the amino acid sequence of SEQ ID NO. 8. According to some embodiments of the present invention, the antibody comprises a VL domain containing a VL CDR1 with the amino acid sequence of SEQ ID NO. 8, CDR2 VL with the amino acid sequence of SEQ ID NO. 9 and CDR3 VL with the amino acid sequence of SEQ ID NO. 10. According to the preferred variants of real

- 7 040749 lysis of the present invention, the antibody contains a VL domain containing a sequence in accordance with 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 SEQ ID NO. 2.

The VH and VL domain(s) can pair to form an antibody antigen-binding site that binds DLK1.

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, while the VH and VL domains contain the sequences of SEQ ID NO. 1 paired with SEQ ID NO. 2.

According to some variants of implementation of the present invention, the antibody contains a heavy chain containing the sequence of SEQ ID NO. 3 paired with a light chain containing the sequence of SEQ ID NO. 4. According to 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 variants of implementation of the present invention, the antibody contains a heavy chain containing the sequence of SEQ ID NO. 11 paired with a light chain containing the sequence of SEQ ID NO. 4. According to some embodiments of the present invention, the antibody is an intact antibody containing two heavy chains containing the sequence of SEQ ID NO. 11, each of which is paired with a light chain containing the sequence of SEQ ID NO. 4.

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

Terminology.

As used herein, the term antibody is used in its broadest sense and includes, in particular, monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), intact antibodies, and antibody fragments, provided they exhibit the intended biological activity. , for example, the ability to bind DLK1. 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 may be of any type (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass or allotype (eg, G1m1, G1m2, G1m3, non- Human G1m1 (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 derived from any species including human, mouse or rabbit.

As used herein, the term binds DLK1 means that an antibody binds DLK1 with a higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank accession number CAA76847, variant no. CAA76847.1 GI:3336842, record update date: January 7 2011, 14:30). In some embodiments of the present invention, an antibody binds DLK1 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 the antibody for BSA, when measured under physiological conditions. The antibodies of the present invention can bind DLK1 with high affinity. For example, in some embodiments of the present invention ^, an antibody may bind DLK1 to a KD that is equal to or less than about 10'6 M, such as 1x10'6, ^10'7 , 10'8, ^10'9 , ^10'10 , ^10'11 , 10' ¹² , 10' ¹³ or 10' ¹⁴ .

DLK1 is a member of the EGF-like family of homeotic proteins. In some embodiments of the present invention, the DLK1 polypeptide corresponds to Genbank Accession Number CAA78163, Variant No. CAA78163.1, Record Update Date: February 2, 2011, 10:34 AM (SEQ ID NO. 12). According to one embodiment of the present invention, a nucleic acid encoding

- 8 040749 DLK1 polypeptide, corresponding to Genbank accession number Z12172, version no. Z12172.1, entry date updated: February 2, 2011, 10:34 am. According to some embodiments of the present invention, DLK1 contains the sequence of SEQ ID NO. 13.

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 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 containing antigen-binding variable domain sequences derived from a non-human primate (eg, Old World monkey or great ape) and human constant region sequences.

As used herein, an intact antibody is an antibody containing the VL and VH domains, as well as the light chain constant domain (CL) and the 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 cytotoxicity (ADCC); phagocytosis; and downregulation 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 herein may be modified. For example, in order to make them less immunogenic in a human subject. This can be done using any of a number of techniques known 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

- 9 040749 in vivo include techniques called 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 selective 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 the content of human sequence regions, and framework shuffling.

CDR graft.

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 cases, framework region (FR) residues of a human immunoglobulin are replaced with corresponding non-human residues (this may be the case, 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, wherein 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 particular non-human antibody specific for a particular epitope with a human VH or V _L library, and the particular human V domains are selected against the antigen of interest. The selected human VH is then combined with the VL library to create a fully human VHxVL combination. The method is described in Nature Biotechnology (NY) 12, (1994) 899-903.

Compound 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 multiple segments of human VH and VL sequences in combinations that limit or avoid human T cell epitopes in the V regions of the final composite antibody. Where 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). Sequence V-region therapy

- 10 040749 cal antibodies are 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 structures 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;

(h) replacing any human residues identified in step (g) with the original non-human amino acid residue to determine the humanizing group of surface-exposed amino acid residues 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 sections of the human sequence.

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

- 11 040749 by maximizing its HSC instead of applying a common identity measure to produce a wide variety of humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).

Rearrangement of frame sections.

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 an 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.

(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) a C5-10 aryl group optionally substituted with one or more substituents selected from the group consisting of halo, nitro, cyano, ether, C _1-7 alkyl, C ₃ _ ₇ heterocyclyl, and bis-oxyC1-3 alkylene;

(b) C _1-5 saturated aliphatic alkyl;

(c) C3-6 saturated cycloalkyl;

R ²² /^i^R ²³ (d) R ²¹ 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, the total number of carbon atoms in the R ² group being not more than 5;

R ^25b (e) where 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;

(f) ^R where R ²⁴ is selected from H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 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.

- 12 040749 group, for example, 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 a C _5-10 aryl group, it may be a C _8-10 aryl group, such as 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 a C ₅ - ₁₀ aryl group, it may carry any number of substituent groups. It preferably bears from 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 _8-10 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 C _5-10 aryl group.

If the substituent on R ² when R ² is a C _5-10 aryl group is halogen, it is preferably F or Cl, more preferably Cl.

If the substituent is on R ² when R ² is C ₅ . ₁₀ aryl group is an ether, it may, according to some variants of implementation of the present invention, be an alkoxy group, for example, C ₁ . ₇ alkoxy (eg, methoxy, ethoxy), or it may, according to 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 a substituent on R ² when R ² is a C _5-10 aryl group is C _1-7 alkyl, it may preferably be a C _1-4 alkyl group (eg methyl, ethyl, propyl, butyl).

If the substituent is on R ² when R ² is C ₅ . ₁₀ aryl group is C ₃ _ ₇ heterocyclyl, it may, according to some embodiments of the present invention, be a C6 nitrogen-containing heterocyclyl group, for example, morpholino, thiomorpholino, piperidinyl, piperazinyl. 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 C6 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 when R ² is a C _5-10 aryl group include methoxy, ethoxy, fluorine, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl. Other particularly preferred substituents for R ² are dimethylaminopropyloxy and carboxy.

Particularly preferred substituted R ² groups when R ² is a C _5-10 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 3,4bisoxymethylenephenyl.

When R ² is C _1-5 saturated aliphatic alkyl, it can be methyl, ethyl, propyl, butyl or pentyl. According to some embodiments of the present invention

- 13 040749 tenia it can 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 may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments of the present invention, it may be cyclopropyl.

When R ² is , each of R ²¹ , R ²² and R ²³ is independently selected from H, Ci-3 on saturated alkyl, C _2-3 alkenyl, C ₂ - ₃ alkynyl and cyclopropyl, with the total number of carbon atoms in the group R ² is no more than 5. According to 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, C _1-3 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: ·

^R2Sb

When R ² is , 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 halo, 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; 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 the optional phenyl substituent is halogen, it is preferably 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.

X^R ^26a

If a single bond is present between C2 and C3, R ² is R ^26b 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 chosen from 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

- 14 040749

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.

If more than one nucleophilic or electrophilic group of an antibody reacts with a drug-linker intermediate or linker reagent followed by a drug fragment, then the resulting product is a mixture of antibody-drug conjugates with a distribution of drug fragments attached to the antibody, e.g., 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 loading (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 on the antibody.

Thus, the antibody-drug conjugate compositions of the present invention comprise mixtures of antibody-drug conjugates in which the antibody contains one or more PBB drug moieties and in which the drug moieties

- 15 040749 means can be attached to the antibody at various amino acid residues.

According to one embodiment of the present invention, the average number of dimeric pyrrolobenzodiazepine groups per antibody ranges from 1 to 8.

According to some embodiments of the present invention, the range is selected from 1-4, 1-4, 24, 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, ammonium ion (ie NH4 ⁺ ) and substituted ammonium ions (eg NH3R ⁺ , NH2R ₂ ⁺ , 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 document, the term solvate is used in the conventional sense to mean a complex of a solute (eg, active compound, salt of the 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 in which

- 16 040749 of which the compounds are located, as well as from 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 trans forms; E- and Z-forms; c-, t- and r-forms; endo- and exoforms; R-, S- and mesoforms; D and L-shapes; 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; bath, armchair, twist, envelope and semi-armchair forms; 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 document, 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, i.e. 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 indicate 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 herein, these are structural (or structural) 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, -OCH ₃ , 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 isomer, 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/acine-nitro.

- 17 040749 ? ,O h he H ⁺ _h O' -cc ^z - c=c' - c=c ^z

I ^{4 z 4} H ⁺ / \ keto enol enolate

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 involve 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 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) associated distribution, metabolism and excretion (ADME) properties. Substitution with heavier isotopes, such as deuterium, may provide certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. 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 (eg, asymmetric synthesis) and separation (eg, 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 herein or known methods.

Biological activity

In vitro cell proliferation assays.

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 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 potency of antibody-drug conjugates can be measured by cell proliferation assay. The CellTiter-Glo® 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). Analysis

- 18 040749

CellTiter-Glo® 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) were dispensed into 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.

In 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, such as 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.

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 (conjugates) 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. So

- 19 040749 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, there is also provided a conjugate compound described herein 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 cellular proliferation of excess or pathological cells that is undesirable, such as neoplastic or hyperplastic proliferation, whether in vitro or in vivo.

Conjugates directed against DLK-1.

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, colon cancer, colon cancer, breast carcinoma, ovarian carcinoma, 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. Cancers of particular interest include: hepatocellular carcinoma, hepatoblastoma, non-small cell lung cancer, small cell lung cancer, colon cancer, breast cancer, gastric cancer, pancreatic cancer, neuroblastoma, adrenal cancer, pheochromocytoma, paraganglioma, medullary thyroid carcinoma glands, skeletal muscle cancer, liposarcoma, glioma, Wilms tumor, neuroendocrine tumors, acute myeloid leukemia and myelodysplastic syndrome.

Other disorders of interest include any condition in which DLK1 is overexpressed or in which DLK1 antagonism would provide clinical benefit. These include immune disorders, cardiovascular disorders, thrombosis, diabetes, immune response checkpoint disorders, fibrotic disorders (fibrosis), or proliferative diseases such as cancer, in particular metastatic cancer.

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. Cancers of particular interest include mesothelioma, lung cancer, ovarian cancer and pancreatic cancer.

Other disorders of interest include any condition in which mesothelin is overexpressed or in which mesothelin antagonism would provide clinical benefit. These include immune disorders, cardiovascular disorders, thrombosis, diabetes, immune response checkpoint disorders, fibrotic disorders (fibrosis) or proliferative diseases such as cancer, in particular metastatic cancer.

It is contemplated that the antibody-drug conjugates (ADCs) of the present invention may be used to treat various diseases or disorders, for example those characterized by overexpression of a tumor antigen. Exemplary conditions or hyperproliferative disorders include benign or malignant tumors; leukemia, hematological and lymphoid malignancies. Other examples include neuronal, glial,

- 20 040749 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, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, 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), Behçet'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.

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),

- 21 040749

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-diphenylbut-1-enyl)phenoxu]-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 (Iressa®, AstraZeneca), irinotecan (Camptosar®, CPT-11 Pfizer), tipifarnib (Zarnestra™, Johnson&Johnson), abraxane™ (no cremophor), paclitaxel albumin-modified nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, Zaktima®, 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. calicheamycin, calicheamycin gamma-I, calicheamycin omega-I (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemycin, dynemycin A; bisphosphonates such as clodronate; esperamycin; as well as the chromophore part of neocarzinostatin and related chromophores of chromoprotein enediin antibiotics), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-oxo-diazo-5-oxo-diazo- -L-norleucine, morpholinodoxorubicin, 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-C); 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; capecitabine (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.

- 22 040749

Also included within the definition of a 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, 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 compound, 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.

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 herein, and a pharmaceutically acceptable carrier, times

- 23 040749 additive or excipient.

In one embodiment of the present invention, the composition is a pharmaceutical composition comprising at least one conjugate compound described herein, 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 as defined herein with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art. 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 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, non-pyrogenic, 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 multi-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 any risk or deleterious side effects. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, route of administration, time of administration, rate of elimination of the compound, duration of treatment, other drug

- 24 040749 means, 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 its 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, in 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.

According to 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, 2 times a day.

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 upon 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 15 mg/kg (e.g., 0.1-20 mg/kg) 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).

As used herein, the term "therapeutically 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 therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with the intended regimen. treatment.

- 25 040749

Similarly, as used herein, 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 according to 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 methods include, but are not limited to, conjugation without copper, for example under aqueous conditions, with an optional co-solvent selected from DMF, DMSO and DMAA.

The drug linker can be synthesized according to the examples, with appropriate modifications, 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, WO 2014 /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 ), lagomorph (eg, rabbit), feathered (eg, bird), canine (eg, dog), feline (eg, cat), equine (eg, horse), porcine (eg, pig), ovine ( 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.

Statement of the Invention

The following numbered embodiments describe some specific contemplated combinations of the present invention. Conjugates directed against DLK1 1. Conjugate of formula (I):

Ab-(DL) _P (I) in which

Ab is an antibody that binds to DLK1;

DL represents

- 26 040749

Where

X is selected from the group consisting of: single bond, -CH2- and -C2H 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 C _5-10 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 6is -oxy-C _1-3 alkylene;

(ib) C _1-5 saturated aliphatic alkyl;

(ic) C _3-6 saturated cycloalkyl;

(id) 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, with said total number of carbon atoms in the R ² group not exceeding 5:

_R2 5 _b (ie) where one of R ^25a and R ^25b is H and the other is selected from:

phenyl, wherein the phenyl is optionally substituted with 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; cyclopropyl; phenyl, wherein the phenyl is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl;

D^ ^26a if a single bond is present between C2 and C3, R ² is R ^26b where R ^26a and R ^26b are independently selected from H, F, C1-4 saturated alkyl, C1-3 alkenyl, with the alkyl and alkenyl groups optionally substituted 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:

(iia) a C _5-10 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 6is -oxy-C _1-3 alkylene;

(iib) C _1-5 saturated aliphatic alkyl;

(iic) C _3-6 saturated cycloalkyl;

- 27 040749 (iid) R ³¹ , 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, said total number of carbon atoms in the R ¹² group being not more than 5;

(iie) wherein one of R ^35a and R ^35b is H and the other is selected from: phenyl, wherein phenyl is optionally substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (iif) ^R 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;

,R ^36a if a single bond is present between C2' and C3', R ¹² is R ^36b where R ^36a and R ^36b 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 ^36a and R ^36b is H, the other is selected from nitrile and C1-4 alkyl ester; and p is from 1 to 8.

2. The conjugate of claim 1 wherein X is a single bond.

3. The conjugate of claim 1 wherein X is -CH ₂ -.

4. The conjugate of claim 1 wherein X is -C ₂ H ₄ -.

5. A conjugate according to any one of claims 1-4, wherein n is 1-4.

6. Conjugate according to statement 5, in which n is equal to 1.

7. Conjugate according to statement 5, in which n is equal to 2.

8. Conjugate according to statement 5, in which n is 4.

9. A compound according to any one of claims 1-8, wherein a double bond is present between C2 and C3 and R ² is a C _5-7 aryl group.

10. The compound of claim 9 wherein R ² is phenyl.

11. A compound according to any one of claims 1-8, wherein a double bond is present between C2 and C3 and R ² is a C _8-10 aryl group.

12. A compound according to any one of claims 9-11, wherein R ² carries one to three substituent groups.

13. A compound according to any one of claims 9 to 12, wherein said substituents are selected from methoxy, ethoxy, fluorine, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl.

14. A compound according to any one of claims 1-8, wherein a double bond is present between C2 and C3 and ^R2 is a _C1-5 saturated aliphatic alkyl group.

15. The compound of claim 14 wherein R ² is methyl, ethyl or propyl.

16. A compound according to any one of claims 1-8, wherein a double bond is present between C2 and C3 and ^R2 is a _C3-6 saturated cycloalkyl group.

17. The compound of claim 16 wherein R ² is cyclopropyl.

18. A compound according to any one of claims 1 to 8, wherein a double bond is present between C2 and C3 and R ² is a group of the formula:

19. The compound according to claim 18, in which the specified total number of carbon atoms in the group R ² is not more than 4.

20. The compound according to claim 19, in which the specified total number of carbon atoms in the group R ² is not more than 3.

21. The compound according to any one of claims 18-20, in which 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.

22. The compound according to any one of claims 18-20, wherein two of R ²¹ , R ²² and R ²³ are H, while the other group is selected from H, C _1-3 saturated alkyl, C _2-3 alkenyl, C _2-3 alkynyl and cyclopropyl.

- 28 040749

23. A compound according to any one of claims 1 to 8, wherein a double bond is present between C2 and C3 and R ²

25. A compound according to any one of claims 1 to 8, wherein a double bond is present between C2 and C3 and R ² is a group of the formula:

^R24

26. The compound of claim 25 wherein R ²⁴ is selected from H, methyl, ethyl, ethenyl and ethynyl.

27. The compound of claim 26 wherein R ²⁴ is selected from H and methyl.

28. The compound according to any one of claims 1 to 8, in which a single bond is present between C2 and C3,

R ² is

, and R ^26a and R ^26b are both H.

29. The compound according to any one of claims 1 to 8, in which a single bond is present between C2 and C3,

R ² is

, and R ^26a and R ^26b are both methyl.

30. A compound according to any one of claims 1 to 8, wherein a single bond is present between C2 and C3,

R ² is

, 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 optionally substituted.

31. A compound according to any one of claims 1-30, wherein a double bond is present between C2' and C3' and R ¹ is a C _5-7 aryl group.

32. The compound of claim 31 wherein R ¹² is phenyl.

33. A compound according to any one of claims 1-30, wherein a double bond is present between C2' and C3' and R ¹ is a C _8-10 aryl group.

34. A compound according to any one of claims 31-33, wherein R ¹² carries one to three substituent groups.

35. A compound according to any one of claims 31-34, wherein said substituents are selected from methoxy, ethoxy, fluorine, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl.

36. A compound according to any one of claims 1-30, wherein a double bond is present between C2' and C3' and is a C _1-5 saturated aliphatic alkyl group.

37. The compound of claim 36 wherein R ¹² is methyl, ethyl or propyl.

38. The compound according to any one of claims 1-30, wherein a double bond is present between C2' and C3' and is a _C3-6 saturated cycloalkyl group.

39. The compound of claim 38 wherein R ¹² is cyclopropyl.

40. The compound according to any one of claims 1 to 30, in which a double bond is present between C2' and C3' and is a group of the formula:

_R 12 _R 12

^R12

41. The compound according to claim 40, in which the specified total number of carbon atoms in the group R is not more than 4.

42. The compound according to claim 41, in which the specified total number of carbon atoms in the group R is not more than 3.

43. The compound according to any one of claims 40-42, in which one of R ³¹ , R ³² and R ³³ 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.

44. The compound according to any one of claims 40-42, in which two of R ³¹ , R ³² and R ³³ are H, when

- 29 040749 this other group is selected from H, C 1-3 saturated alkyl, C _2-3 alkenyl, C _2-3 alkynyl and cyclopropyl.

45. A compound according to any one of claims 1 to 30, wherein a double bond is present between C2' and C3' and R ¹² is a group of the formula:

46. Connection according to the statement 45, in which R ¹² represents a group:

47. A compound according to any one of claims 1 to 30, wherein a double bond is present between C2' and C3' and R ¹² is a group of the formula:

48. The compound of claim 47 wherein R ³⁴ is selected from H, methyl, ethyl, ethenyl and ethynyl.

49. The compound of claim 48 wherein R ³⁴ is selected from H and methyl.

50. A compound according to any one of claims 1 to 30, wherein a single bond is present between C2' and C3', R ¹² is ^R36b and R ^36a and R ^36b are both H.

51. A compound according to any one of claims 1 to 30, wherein a single bond is present between C2' and C3', R ¹² is ^R36b and R ^36a and R ^36b are both methyl.

52. The compound according to any one of claims 1-30, in which a single bond is present between C2' and C3', R ¹²

Al ^p36a is ^R36b , one of ^R36a and ^R36b is H and the other is selected from C _1-4 saturated alkyl, C _2-3 alkenyl, the alkyl and alkenyl groups being optionally substituted.

53. The conjugate according to claim 1, in which DL is:

54. A conjugate according to any one of claims 1 to 53, wherein said antibody contains a VH domain containing a VH CDR3 with the amino acid sequence of SEQ ID NO. 7.

55. A conjugate according to any one of claims 1 to 54, wherein said antibody contains a VH domain containing a VH CDR2 with the amino acid sequence of SEQ ID NO. 6 and/or CDR1 VH with the amino acid sequence of SEQ ID NO. 5.

56. A conjugate according to any one of claims 1 to 55, wherein said antibody contains a VH domain containing a VH CDR3 with the amino acid sequence of SEQ ID NO. 7, CDR2 VH with the amino acid sequence of SEQ ID NO. 6 and CDR1 VH with the amino acid sequence of SEQ ID NO. 5.

57. The conjugate according to any one of claims 1-56, in which the specified antibody contains a VH domain, containing

- 30 040749 the general sequence of SEQ ID NO. 1.

58. A conjugate according to any one of claims 1 to 57, wherein said antibody contains a VL domain containing a VL CDR3 with the amino acid sequence of SEQ ID NO. 10.

59. A conjugate according to any one of claims 1 to 58, wherein said antibody contains a VL domain containing a VL CDR2 with the amino acid sequence of SEQ ID NO. 9 and/or CDR1 VL with the amino acid sequence of SEQ ID NO. 8.

60. A conjugate according to any one of claims 1 to 59, wherein said antibody contains a VL domain containing a VL CDR3 with the amino acid sequence of SEQ ID NO. 10, CDR2 VL with the amino acid sequence of SEQ ID NO. 9 and CDR1 VL with the amino acid sequence of SEQ ID NO. 8.

61. A conjugate according to any one of claims 1-60, wherein said antibody contains a VL domain containing the sequence of SEQ ID NO. 2.

62. A conjugate according to any one of claims 1-61, wherein said antibody is an intact antibody.

63. A conjugate according to any one of claims 1-62, wherein said antibody contains a heavy chain containing the sequence of SEQ ID NO. 3, or a heavy chain containing the sequence of SEQ ID NO. eleven.

64. A conjugate according to any one of claims 1-63, wherein said antibody contains a light chain containing the sequence of SEQ ID NO. 4.

65. The conjugate of any one of claims 1-64, wherein said antibody is a humanized, deimmunized, or surface-altered antibody.

66. A conjugate according to any one of claims 1 to 65, wherein said antibody lacks unconjugated azide groups.

67. A conjugate according to any one of claims 1-66, wherein p is 1, 2, 3, or 4.

68. A composition comprising a mixture of antibody-drug conjugates according to any one of claims 1-67, wherein said average drug load per antibody in said mixture of antibody-drug conjugates is from about 1 to about 4.

69. A conjugate according to any one of claims 1-67 for use in therapy.

70. A conjugate according to any one of claims 1-67 for use in the treatment of a proliferative disease in a subject.

71. A conjugate of claim 70 wherein said disease is cancer.

72. The conjugate of claim 71, wherein said cancer is a cancer selected from the group consisting of: hepatocellular carcinoma, hepatoblastoma, non-small cell lung cancer, small cell lung cancer, colon cancer, breast cancer, gastric cancer, pancreatic cancer, neuroblastoma , adrenal cancer, pheochromocytoma, paraganglioma, medullary thyroid carcinoma, skeletal muscle cancer, liposarcoma, glioma, Wilms tumor, neuroendocrine tumors, acute myeloid leukemia, and myelodysplastic syndrome.

73. A pharmaceutical composition containing a conjugate according to any one of claims 1 to 67 and a pharmaceutically acceptable diluent, carrier or excipient.

74. The pharmaceutical composition of claim 73, further comprising a therapeutically effective amount of a chemotherapeutic agent.

75. The use of a conjugate according to any one of claims 1 to 67 in the preparation of a medicament for use in the treatment of a proliferative disease in a subject.

76. A method of treating cancer, comprising administering to a patient a pharmaceutical composition according to claim 74.

77. The method of claim 76 wherein said patient is administered a chemotherapeutic agent in combination with said conjugate.

Examples

Review.

The ADCs disclosed herein are synthesized in a two-step process. The first step is glycan remodeling, in which the native N-linked glycan is trimmed to a central GlcNAc and then azido-modified GalNAc is added to form an antibody-GalNAc-N ₃ intermediate. This compound is purified by protein A chromatography and then conjugated to a drug linker in a second step. These steps are described below in the following description of the drug-linker synthesis.

- 31 040749

Synthesis of intermediate compound 3.

A solution of BCN alcohol (0.384 g, 2.55 mmol) in MeCN (25 ml) under N2 was cooled to 0°C and chlorosulfonyl isocyanate (CSI) (0.255 ml, 415 mg, 2.93 mmol, 1.15 equiv.). 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. A solution of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (1.0 g, 6.1 mmol, 2.4 eq.) in H2O (5 ml) was then added and the reaction mixture was stirred to room temperature for 2 h. After this time, CHCl ₃ (50 ml) and H2O (100 ml) were added and the layers were separated. CH ₂ Cl ₂ (100 ml) was added to the aqueous layer in a separating funnel and the pH was adjusted to 4 with 1N HCl before separating the layers. The aqueous layer was extracted twice using 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 ₂ to 20% MeOH in CH ₂ Cl ₂ . The yield was 0.42 g (1.0 mmol, 39%) 3 as a colorless sticky wax.

- 32 040749

Synthesis of a drug linker.

oh oh

- 33 040749

ABOUT

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) was degassed by bubbling N2 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 N2, DCM (25 ml) was added and the mixture was washed with aqueous saturated NH4Cl (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. The residue was purified by RP-HPLC (30-90% MeCN (0.1% formic acid) in H2O (0.1% formic acid)). The combined fractions are passed through SPE columns (HCO3 ^- ) and concentrated. After MeCN (50 ml) was added, the mixture was concentrated again. The resulting residue 2 is used in the next step.

The conversion of the reaction can be monitored by LC/MS analysis. Column: XBridge BEN C18 Intelligent Speed (IS), 130 Λ, 3.5 µm (4.6x20 mm) column.

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 CHCl3 (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.HC1 (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)). Before collection, the HPLC elution tubes are filled with 5% aqueous (NH4)HCO ₃ . The combined HPLC fractions were extracted using DCM (3x20 ml). The combined organic layers are dried (Na ₂ SO ₄ ) and concentrated. Product 4 is obtained 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 BEN C18 Intelligent Speed (IS), 130 Λ, 3.5 µm (4.6x20 mm) column. Mobile phase A: water (0.1% formic acid), mobile phase B (0.1% formic acid). Detection using PDA and ESI+.

Glycan remodeling (ADC directed against DLK1, ConjA1)

Obtaining an antibody.

Buffer of approximately 60 mg of anti-DLK1 antibody was replaced with 25 mM Tris/Cl, 150 mM NaCl, pH=8.0 using a G25 desalting column; 4x2.5ml at 6mg/ml loaded onto 4x obes columns

- 34 040749 salting PD10 (GE 17085101). The buffer exchanged antibody was then concentrated to at least 25 mg/mL using a Vivaspin 20 centrifugal concentrator (Sigma Z614637). By UV analysis at A280-320 nm, using an extinction coefficient of 1.5, the protein concentration was confirmed to be 28.4 mg/mL.

remodeling reaction.

Glycan remodeling was performed in a one pot reaction overnight (16 h) at room temperature. The following reaction mixture was prepared with the addition of solutions/reagents in the order detailed below.

Solution Volume (µl) Final concentration 25mMtris/C1, 150MMNaCl, pH=8.0 663.8 No data Antibody to DLK1 in 25 mM Tris/C1, 150 mM NaCl, pH=8.0 2000 15 mg/ml 3.5 mg/ml EndoSH in 25 mM Tris/C1, pH=8.0 165.7 1% w/w regarding MAT 4.82 mg/ml His-TnGalNAcT in 25 mM Tris/C1, pH=8.0 902.5 7.5% w/w, relative to MAT 1 M MgCE in water MQ 38.7 10 mM 0.1 M 6-N ₃ GalNAc-UDP in water MQ 96 25 eq. relatively MAT

Purification procedure on protein A.

Protein A binding and elution was performed on a HiTrap MabSelect Sure 5 ml column (GE 110034-94). All chromatographic steps were performed at a flow rate of 240 cm/h using the AKTA Prime plus system. The column was prepared and used as follows.

Solution CV Notes 0.1 MNaOH 3 Disinfection 25 mM Tris/C1, 25 mM NaCl, pH=8.0 10 Balancing DLK1 antibody sample As needed Loading 0.4 M potassium phosphate, 0.2% triton 100, pH=7.0 20 Flushing/Endotoxin Removal Step 25 mM Tris/C1, 25 mM NaCl, pH=8.0 20 Washing/removing the newt 0.15 M acetic acid As Picking a peak from 0.1 to need 0.1 opt. units

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 an antibody with a substituted central GlcNAc(Fuc), and a minor product (±10% of the total heavy chain), resulting from the transfer of a modified galactose to an antibody with a substituted central GlcNAc (no fucose).

- 35 040749

Buffer replacement after purification on protein A.

Next, the buffer in the remodeled and purified GalNAc-N ₃ anti-DLK1 antibody was replaced with phosphate buffered saline (PBS) and concentrated to approximately 16.6 mg/ml using a Vivaspin 20 centrifugal concentrator (Sigma Z614637). The eluate after purification on protein A was diluted with PBS in a ratio of 1:1, then re-concentrated to the original volume and repeated 6 times. Finally, the volume was reduced to the target value of 16-17 mg/ml and the sample was removed from the device. By UV analysis at A280-320 nm, using an extinction coefficient of 1.5, the protein concentration was confirmed to be 16.4 mg/ml, and a total of 2.7 ml was isolated.

Conjugation 4 with modified antibody to generate ConjA1

reaction conditions.

2.7 ml 16.4 mg/ml azido-modified anti-DLK1 antibody (in this example HuBa-1-3d described herein) 0.3 ml 10 mM 4 in dimethylacetamide.

The reaction mixture was thoroughly stirred and allowed to conjugate overnight (16 h) at room temperature. The conjugation mixture was filtered through a 0.2 μm PVDF filter (Millipore SLGV033RS) prior to final purification and formulation.

Purification of ConjA1.

The filtered conjugation mixture was purified using a Vivaspin 20 centrifugal concentrator (Sigma Z614637). The conjugation mixture was diluted with 30 mm histidine HCl, 200 mm sorbitol, pH=6.0 in a ratio of 1:1 and re-concentrated to the original volume. This was repeated 12 times before removing the purified ADC mass from the centrifuge.

Protein concentration was determined by quantitative analysis by gel permeation chromatography (SEC) using an antibody calibration curve and conjugate diluted to approximately 5 mg/ml with additional 30 mM histidine HCl, 200 mM sorbitol, pH=6.0. Tween 20 was added to 0.02% w/v. from a 1% stock solution in 30 mM histidine HCl, 200 mM sorbitol, pH=6.0, and the concentration was retested by SEC quantitation. A sample was taken for testing, and the residue was divided into 1 ml aliquots and frozen at -80°C.

Product analysis showed the following properties:

transparent, colorless, without particles

5.17 [protein] SEC mean DAR 1.87 (reverse phase chromatography)

0.09 EU/mg endotoxin <4.5% free drug-linker pH=6.06

Glycan remodeling (ADC directed against KAAG1, ConjA2)

Obtaining an antibody.

Buffer of approximately 150 mg of anti-KAAG1 antibody (approximately 25 ml at 6.13 mg/ml in PBS, pH=7.4) was replaced with 25 mM Tris/Cl, 150 mM NaCl, pH=8.0 and concentrated to >25 mg /ml using Vivaspin 20 centrifugal concentrators (Sigma Z614637). The antibody was first concentrated to 12 ml, then diluted with 25 mM Tris/Cl, 150 mM NaCl, pH 8.0 in a ratio of 1:1, then re-concentrated to 12 ml, and this method was repeated 6 times. Finally, the buffer exchanged stock solution was further concentrated to 6 ml. Protein concentration was determined by UV analysis at A280-320 nm using an extinction coefficient of 1.5 and then diluted to 25 mg/ml using 25 mM Tris/Cl, 150 mM NaCl, pH=8.0.

remodeling reaction.

Glycan remodeling was performed in a one pot reaction overnight (16 h) at room temperature. The following reaction mixture was prepared by adding solutions/reagents in the order detailed below.

Solution Volume (µl) Final concentration 25 mM Tris/Cl, 150 mM NaCl, pH=8.0 893 No data MAT to KAAG1 in 25 mM Tris/C1, 150 mM NaCl, pH=8.0 6000 15 mg/ml

- 36 040749

3.5 mg/ml EndoSH in 25 mM Tris/C1, pH=8.0 428.4 1% w/w regarding MAT 4.82 mg/ml His-TnGalNAcT in 25 mM Tris/C1, pH=8.0 2330 7.5% w/w regarding MAT 1 M MgCE in water MQ 100.2 10 mM 0.1 M 6-N ₃ GalNAc-UDP in water MQ 250 25 eq. regarding MAT

Purification procedure on protein A.

Protein A binding and elution was performed on a HiScreen MabSelect Sure 4.7 ml column (GE 28-9269-77). All chromatographic steps were performed at a flow rate of 240 cm/h using the AKTA Prime plus system. The column was prepared and used as follows.

Solution CV Notes 0.1 MNaOH 3 Disinfection 25 mM Tris/C1, 25 mM NaCl, pH=8.0 10 Balancing Antibody sample As needed Loading 0.4 M potassium phosphate, 0.2% triton 100, pH=7.0 20 Wash/remove step endotoxins 25 mM Tris/C1, 25 mM NaCl, pH=8.0 20 Washing/removing the newt 0.15 M acetic acid As needed Picking a peak from 0.1 to 0.1 opt. units

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 an antibody with a substituted central GlcNAc(Fuc) and a minor product (±10% of the total heavy chain), resulting from the transfer of a modified galactose to an antibody with a substituted central GlcNAc (no fucose).

Buffer replacement after purification on protein A.

The protein A eluate containing remodeled/purified Ab-GalNAc-N ₃ was pH adjusted by adding 1.5 M Tris base at 3.2% v/v, then the buffer was replaced with PBS and concentrated to ≈17 mg/mL using centrifugal concentrators Vivaspin 20 (Sigma Z614637). The combined pH-adjusted product was first diluted 1:1 with PBS, then re-concentrated to the original volume and this process was repeated 6 times. Finally, the buffer exchanged stock solution was further concentrated to a target value of ≈17 mg/mL. By UV analysis at A280-320 nm, using an extinction coefficient of 1.5, the protein concentration was confirmed to be 16.5 mg/mL; a total of 7.9 ml was isolated with a yield of 88%.

Conjugation 4 with modified antibody to produce ConjA2

To 7.9 ml of 16.5 mg/ml Ab-GalNAc-N ₃ (Ab=3A4 described herein) was added 0.788 ml of 10 mM PL1601 in DMF (10% final v/v DMF). The reaction mixture was thoroughly stirred and allowed to conjugate overnight (16 h) at room temperature. The conjugation mixture was filtered through a 0.22 µm PES filter (Millipore SLGV033RS) prior to final purification and formulation.

Purification of ConjA2.

The filtered conjugation mixture was purified by constant volume diafiltration using a Pellicon 3 30 kDa membrane with membrane area ≥50 g/m ² , cross flow 5.0 ± 0.25 L/min/m ² , TMP 1.0 ± 0.2 bar and a total of 12 diavolumes of exchange buffer for PBS pH=7.4. The diafiltered pooled product was recovered from the UFDF and filtered through a 0.22 µm PES membrane filter (Millipore SLGV033RS) into sterile tubes manufactured

- 37 040749

Eppendorf. The protein concentration was determined by UV analysis at A280-320 nm using an extinction coefficient of 1.5 and was 4.9 mg/mL. A sample was taken for testing and the residue was stored at 4°C.

Product analysis showed the following properties:

transparent, colorless, without particles

4.9 mg/mL [protein] spectroscopy at A280/330 nm average DAR 1.9 (reverse phase chromatography)

0.07 EU/mg endotoxin

3% free drug-linker compound

98.3% monomer by gel permeation chromatography

Glycan remodeling (ADC directed against mesothelin, ConjA3)

Obtaining an antibody.

Buffer of approximately 60 mg of anti-mesothelin antibody is replaced with 25 mM Tris/Cl, 150 mM NaCl, pH=8.0 using a G25 desalting column; 4x2.5ml at 6mg/ml loaded onto 4x PD10 desalting columns (GE 17085101). The buffer exchanged antibody is then concentrated to at least 25 mg/ml using a Vivaspin 20 centrifugal concentrator (Sigma Z614637). The protein concentration is confirmed by UV analysis at A280-320 nm using an extinction coefficient of 1.5.

remodeling reaction.

Glycan remodeling is performed in a one-pot reaction overnight (16 hours) at room temperature. The following reaction mixture is prepared by adding solutions/reagents in the order detailed below.

Solution Volume (µl) Final concentration 25 mM Tris/Cl, 150 mM NaCl, pH=8.0 663.8 No data Antibody to mesothelin in 25 mM Tris/C1, 150 mM NaCl, pH=8.0 2000 15 mg/ml 3.5 mg/ml EndoSH in 25 mM Tris/Cl, pH=8.0 165.7 1% w/w regarding MAT 4.82 mg/ml His-TnGalNAcT in 25 mM Tris/C1, pH=8.0 902.5 7.5% w/w, relative to MAT 1 M MgCH in water MQ 38.7 10 mM 0.1 M 6-N ₃ GalNAc-UDP in water MQ 96 25 eq. relatively MAT

Purification procedure on protein A.

Protein A binding and elution was performed on a 5 ml HiTrap MabSelect Sure column (GE 11-0034-94). All chromatographic steps are performed at a flow rate of 240 cm/h using the AKTA Prime plus system. The column is prepared and used as follows.

- 38 040749

Solution CV Notes 0.1 MNaOH 3 Disinfection 25 mM Tris/C1, 25 mM NaCl, pH=8.0 10 Balancing Mesothelin antibody sample As needed Loading 0.4 M potassium phosphate, 0.2% triton 100, pH=7.0 20 Flushing/Endotoxin Removal Step 25 mM Tris/C1, 25 mM NaCl, pH=8.0 20 Washing/removing the newt 0.15 M acetic acid As needed Picking a peak from 0.1 to 0.1 opt. units

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 an antibody with a substituted central GlcNAc(Fuc) and a minor product (±10% of the total heavy chain), resulting from the transfer of a modified galactose to an antibody with a substituted central GlcNAc (no fucose).

Buffer replacement after purification on protein A.

Next, the buffer in the remodeled and purified GalNAc-N3 mesothelin antibody was replaced with phosphate buffered saline (PBS) and concentrated to approximately 16.6 mg/mL using a Vivaspin 20 centrifugal concentrator (Sigma Z614637). The protein A eluate is diluted 1:1 with PBS, then re-concentrated to the original volume and this is repeated 6 times. Finally, the volume is reduced to the target value of 16-17 mg/ml and the sample is removed from the device. The protein concentration is confirmed by UV analysis at A280-320 nm using an extinction coefficient of 1.5.

Conjugation 4 with modified antibody to produce ConjA3

reaction conditions.

2.7 ml ~15 mg/ml azido-modified mesothelin antibody.

0.3 ml 10 mM 4 in dimethylacetamide.

The reaction mixture is thoroughly stirred and allowed to conjugate overnight (16 hours) at room temperature. The conjugation mixture is filtered through a 0.2 μm PVDF filter (Millipore SLGV033RS) prior to final purification and formulation.

Purification of ConjA3.

The filtered conjugation mixture is purified using a Vivaspin 20 centrifugal concentrator (Sigma Z614637). The conjugation mixture is diluted with 30 mm histidine HCl, 200 mm sorbitol, pH=6.0 in a ratio of 1:1, and then re-concentrate to the original volume. This is repeated 12 times before removing the cleaned mass of ADC from the centrifuge device.

Protein concentration is determined by SEC quantitation using a standard curve of antibody and conjugate diluted to approximately 5 mg/mL with additional 30 mM histidine HCl, 200 mM sorbitol, pH=6.0. Tween 20 is added to 0.02% w/v. from a 1% stock solution in 30 mM histidine HCl, 200 mM sorbitol, pH=6.0, and the concentration is retested by quantitative SEC analysis. A sample is taken for testing and the remainder is aliquoted into 1 ml aliquots and frozen at -80°C.

Cytotoxicity of ConjA1 in vitro.

Flasks with Lu135 cells or SK-N-FI cells were trypsinized and the released cells were washed and resuspended in fresh medium. Cell density was determined by mixing with trypan blue (0.4% (w/v) Sigma TB154) at a ratio of 1:1 and counting clear/blue (live/dead) cells using a Luna II automatic cell counter (Logos Biosystems) . The cell suspension was diluted to the desired seeding density (20 x 104/ml), dispensed into white flat-bottomed 96-well microplates (50 µl/well) and incubated overnight.

A stock solution (1 ml) of ConjA1 (20 µg/ml) was prepared by diluting ConjA1, sterilized

- 39 040749 by filtration, in the same cell culture medium. A set of 8 x 10-fold dilutions of ConjA1 stock solution was prepared in a sterile 24-well plate by sequentially transferring 100 μl to 900 μl of cell culture medium. Each dilution of ConjA1 was dispensed, 50 μl/well, into 4-replicate wells of a 96-well cell suspension plate. Control wells were filled with the same volume of culture medium only.

After a period of exposure to ConjA1, cell viability was measured with Promega CellTiter-Glo by adding 100 µl/well, mixing for 2 min and reading the signal on Envision using the luminescence protocol. Data was analyzed using Graphpad Prism software.

The EC ₅₀ of ConjA1 against Lu-135 cells was found to be 0.01765 μg/ml. The EC ₅₀ of the ADC control was 0.5326 μg/ml (see FIG. 1A*).

ConjA1 EC ₅₀ against SK-N-FI cells was found to be 0.1565 µg/ml. The EC ₅₀ of the ADC control was 5x105 μg/ml (see FIG. 1B*).

* In both Figs. 1A * 1B ▼ is ConjA1 and • is an otherwise identical ADC control containing B12 antibody non-specific for DLK1.

In vivo efficacy studies using ConjA1.

Antitumor activity in the LI1097 xenograft model derived from a liver cancer patient (PDX) in vivo.

LI1097 seed tumors were engrafted subcutaneously in NOD/SCID mice and maintained subcutaneously in BALB/c nude mice prior to implantation. When tumor volumes reached 700-1500 mm ³ , the tumors were harvested and cut into pieces with a diameter of approximately 2-3 mm ³ . Tumors or tumor pieces were washed with ice-cold RPMI1640 media (no serum) and then placed in ice-cold media for use.

Prior to tumor implantation, the skin of five to six week old female BALB/c nude mice was disinfected with iodophor on the right flank. Each mouse was inoculated without anesthesia, subcutaneously, in the upper part of the right flank with one fragment of a primary human liver tumor LI1097 for tumor development.

After tumor inoculation, the morbidity and mortality of the animals were monitored daily. Tumor size was measured with a caliper twice a week in two dimensions. Tumor volume was expressed in mm ³ using the formula: TV=0.5axb2 where a and b are the long and short tumor diameters, respectively.

On study day 12, mice were randomized into 5 groups of 8 mice each; the mean tumor volume was ~170 mm ³ in the cohort. Mice were dosed with test agents on study day 13 (day 1, indicated by the vertical dotted line in the graph). Test mice in this study received a single dose of their assigned test article and dose level on day 1, after which tumor growth was monitored until day 51.

The results are shown in FIG. 2A, on which:

o=vehicle, qdx1 ◊ = ADC control using B12 antibody, non-specific for DLK1, 1 mg/kg, qdx1 □ = ConjA1, 0.1 mg/kg, qdx1

Δ = ConjA1, 0.3 mg/kg, qdx1 ▼ = ConjA1, 1.0 mg/kg, qdx1

As follows from FIG. 2A, ConjA1 at 1.0 mg/kg resulted in the greatest reduction in tumor growth, followed by ConjA1 at 0.3 mg/kg. In addition, at the highest dose tested, ConjA1 resulted in 3/8 partial responses (PR) and 2/8 complete responses (CR), while none of the mice treated with vehicle or ADC for isotype control (1 mg/kg, single dose) dose), had no PR, CR, or did not survive tumor free (TFS).

Antitumor Activity in the SK-N-FI Neuroblastoma Xenograft Model in Vivo

Female NOD-SCID mice were six weeks old on the day of implantation. SK-N-FI cells were harvested during the log expansion phase and resuspended in PBS with 50% Matrigel. 100 μl (3x106 cells) of the cell suspension mixture was injected subcutaneously with a 26 G syringe into the right flank of each mouse. Animals were examined twice a week to assess body weight and tumor size. Tumor size was measured using digital calipers and calculated according to the following expression:

Tumor volume (mm ³ ) = (minor axis)2* (major axis)* π/6

Eighteen days after transplantation of cancer cells, 50 mice, whose tumor volume ranged from 99.0 mm ³ to 155.2 mm ³ (average 116.2 mm ³ ), were divided into 5 groups (N=10 in each group). On the day of dosing, the test subjects were administered by intravenous injection into the tail vein. The endpoint of the study was set as each tumor ^reaching the 1000 mm

- 40 040749 PS), whichever comes first.

The results are shown in FIG. 2B, on which:

◊ = vehicle, qdxl o = ADC control with B12 antibody, non-specific for DLK1, 0.5 mg/kg, qdxl □ = ADC control with B12 antibody, non-specific for DLK1, 1 mg/kg, qdx1

Δ = ConjAl, 0.5 mg/kg, qdxl ▼ = ConjAll, 1.0 mg/kg, qdxl

As follows from FIG. 2B, ConjAl at 1.0 mg/kg resulted in the greatest tumor growth retardation, followed by ConjAl at 0.5 mg/kg.

In a xenograft model of SK-N-FI derived from human neuroblastoma, a single dose of ConjAl at 0.5 or 1 mg/kg showed dose-dependent antitumor activity compared to mice treated with vehicle and ADC for isotype control.

At the highest dose tested, ConjAl resulted in 1/9 partial response (PR) and 4/9 complete response (CR) animals, one of which survived tumor free (TFS) at the end of the study at day 60 (one animal from l0 the original animals in this group were excluded from the final figures for reasons unrelated to treatment).

Toxicological study in rats

Toxicology study in rats (off-target toxicity assessment, ADC tested does not bind rat Dlk-l).

Method.

ConjAl was evaluated in a single intravenous dose tolerability study in rats. Male Sprague-Dawley rats (u=3/group) were dosed at 5 mg/kg on day 1 and necropsied 21 days post-dose. Body weight and food intake were often monitored with lifetime sampling for clinical pathology (blood at days 8 and 21) and repeated sampling for pharmacokinetics. Gross observations were made at autopsy, with selected organs weighed and stored for possible histopathological examination.

Results.

ConjAl was clinically well tolerated at 5 mg/kg without any significant adverse clinical signs. Body weight gain was reduced, with the animals being about 15% lighter than the control group at the end of the study. The white blood cell count decreased by day 8 (neutrophil count decreased by about 95% compared to the simultaneous control), while there were signs of recovery by day 22.

General conclusion.

ConjAl had good stability, was well tolerated and showed a favorable pharmacokinetic profile in rats with a half-life of 9 days at 5 mg/kg. This indicates that the MTD in rats is at least 5 mg/kg or higher.

Cytotoxicity in A204 and Hep3B cells in 2D and 3D cell cultures in vitro

Materials and methods.

Cells were seeded in onco media (RPMI, 5% FBS, 2 mM L-alanyl-L-glutamine, l mM sodium pyruvate, and 1% penicillin/streptomycin) in 384-well Elplasia plates pre-coated with pHEMA.

Compounds ADC, ConjAl, and B12-1601 were added 24 hours after cell seeding, at an initial concentration of 10 mg/ml, 10-fold serial dilution for 9 concentrations with four replicates. Incubation periods with ADC compounds were 5 days in 2D and 7 days in 3D cultures, with media changed every 3 days for a total of l4 days.

At the end of the incubation period, cells were lysed and analyzed to determine cell viability. Cell proliferation endpoint was analyzed as percentage of control (ROS) using the following formula:

ROS=relative cell count (compound wells)/relative cell count (vehicle control wells) x!00%

Data was analyzed using Graphpad Prism software.

Results.

In FIG. 3 shows cytotoxicity data in A204 and Hep3B cells in 3D in vitro cell culture.

In FIG. 3A shows data for A204 cells [IC ₅₀ 3D ADC=0.001460 μg/ml, IC ₅₀ 3D B12PL1601=0.7662 μg/ml, IC ₅₀ 2D ADC=0.006399 μg/ml, IC ₅₀ 3D B12-PL1601= 0.4059 µg/m];

In FIG. 3B shows data for Hep3B cells [IC ₅₀ 3D ADC=0.2271 μg/ml, IC ₅₀ 3D B12PL1601=~432.6 μg/ml, IC ₅₀ 2D ADC=~59.29 μg/ml, IC ₅₀ 2D B12- PL1601=~3.957 µg/mL].

- 4l 040749

Cytotoxicity of ConjA2 in vitro

Flasks with SN12C cells or MDA-MB-231FI cells were trypsinized and the released cells were washed and resuspended in fresh medium. Cell density was determined by mixing with trypan blue (0.4% (w/v) Sigma TB154) at a ratio of 1:1 and counting clear/blue (live/dead) cells using a Luna II automatic cell counter (Logos Biosystems) . The cell suspension was diluted to the desired seeding density (20x10 ⁴ /ml), dispensed into white 96-well flat bottom microplates (50 μl/well) and incubated overnight.

A stock solution (1 ml) of ConjA2 (20 μg/ml) was prepared by diluting filter-sterilized ConjA2 in the same cell culture medium. A set of 8 x 10-fold dilutions of ConjA2 stock solution was prepared in a sterile 24-well plate by sequentially transferring 100 μl to 900 μl of cell culture medium. Each dilution of ConjA2 was dispensed, 50 μl/well, into 4-replicate wells of a 96-well cell suspension plate. Control wells were filled with the same volume of culture medium only.

After a period of exposure to ConjA2, cell viability was measured with Promega CellTiter-Glo by adding 100 μl/well, mixing for 2 min and reading the signal on Envision using the luminescence protocol. Data was analyzed using Graphpad Prism software.

ConjA2 EC ₅₀ against SN12C cells was found to be 0.0663 µg/ml. The ADC control EC ₅₀ was undetectable (see FIG. 4A*).

ConjA2 EC ₅₀ against MDA-MB-231 cells was found to be 0.226 μg/ml. The ADC control EC ₅₀ was undetectable (see FIG. 4B*).

* In both Figures 4A and 4B, ♦ is ConjA2 and • is an otherwise identical ADC control containing B12 antibody non-specific for KAAG1.

ConjA2 efficacy study in vivo

Female nude mice (Crl:NU(Ncr)-Foxnlnu, Charles River) were eight weeks old with a body weight (BW) range of 20.7 to 31.2 g on day 1 of the study.

On the day of implantation, MDA-MB-231 tumor cells used for implantation were harvested in logarithmic expansion phase and resuspended in phosphate buffered saline (PBS) at 5 x 10 ⁷ cells/ml. Each mouse was injected subcutaneously (sc) in the right flank with 5×10 ⁶ cells (0.1 ml of cell suspension) and tumors were controlled when their volumes approached the target range of 100 to 150 mm ³ . Tumors were measured with calipers in two dimensions 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 mass of the tumor can be estimated with the assumption that 1 mg is equivalent to 1 mm ³ tumor volume.

Sixteen days after tumor implantation, referred to as study day 1, animals were sorted into groups, each consisting of 8 mice with individual tumor volumes from 108 to 144 mm ³ and group mean tumor volumes of 112.5-123.8 mm ³ . On Study Day 1, all treatments were administered intravenously (IV) as a single injection (qdx1) by injection into the tail vein at a dosing volume of 0.2 ml per 20 g of body weight (10 ml/kg), recalculated according to weight body of each individual animal. Tumors were measured with calipers twice a week and each animal was euthanized when its tumor volume reached the end point of 1500 mm ³ or at the end of the study, whichever came first. The study ended on day 59.

The results are shown in FIG. 5, on which:

o = vehicle, qdx 1 (top line) □ = ADC control using B12 antibody, non-specific for KAAG1, 0.6 mg/kg, qdx1 o = ConjA2, 0.6 mg/kg, qdx1 (bottom line)

As follows from FIG. 5, ConjA2 at 0.6 mg/kg resulted in a significant slowdown in tumor growth.

Antitumor activity of ConjA2 in SN12C xenograft model in vivo

Female severe combined immunodeficiency mice (Fox Chase SCID®, CB17/IcrPrkdcscid/IcrlcoCrl, Charles River) were nine weeks old with a body weight (BW) range of 15.4 to 22.2 g on day 1 of the study.

On the day of tumor implantation, each test mouse received 5x10 ⁶ SN12C cells (0.1 ml cell suspension in 50% Matrigel® (Corning®) matrix in phosphate buffered saline) 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 measured with calipers in two dimensions and the volume was calculated using the formula:

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Tumor volume (mm ³ ) = w ² xl/2 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-three days after tumor implantation, referred to as study day 1, animals were sorted into nine groups (n=8) with individual tumor volumes ranging from 108 to 172 mm ³ and group mean tumor volumes of 129 mm ³ .

On study day 1, all treatments were administered intravenously (IV) as a single injection (qdx 1) by injection into the tail vein at a dosing volume of 0.2 ml per 20 g body weight (10 ml/kg), recalculated according to body weight of each individual animal. Tumors were measured with calipers twice a week, and each animal was euthanized when its tumor volume reached the end point of 1000 mm ³ or at the end of the study, whichever came first. The study ended on day 60.

The data is shown in FIG. 6, which shows that administration of ADC (ConjA2) reduced tumor growth in a dose dependent manner.

Cytotoxicity in vitro

Flasks with OVCAR3, CAPAN-2 or HPAC cells are trypsinized and the released cells are washed and resuspended in fresh medium. Cell density is determined by mixing with trypan blue (0.4% (w/v) Sigma TB154) at a ratio of 1:1 and counting clear/blue (live/dead) cells using a Luna II automatic cell counter (Logos Biosystems) . The cell suspension is diluted to the desired seeding density (20x104/ml), dispensed into flat-bottom white 96-well microplates (50 µl/well) and incubated overnight.

A stock solution (1 ml) of ConjA3 (20 μg/ml) was prepared by diluting filter-sterilized ConjA3 in the same cell culture medium. A set of 8 x 10-fold dilutions of ConjA3 stock solution is prepared in a sterile 24-well plate by sequentially transferring 100 μl to 900 μl of cell culture medium. Each dilution of ConjA3 was dispensed, 50 μl/well, into 4-replicate wells of a 96-well cell suspension plate. Control wells are filled with the same volume of culture medium only.

After a period of exposure to ConjA3, cell viability is measured with a Promega CellTiter-Glo by adding 100 µl/well, mixing for 2 minutes, and reading the signal on Envision using a luminescence protocol. Data is analyzed using Graphpad Prism software.

ConjA3 efficacy study in vivo

Antitumor activity in the OVCAR3 in vivo model.

Seed tumors are engrafted subcutaneously in NOD/SCID mice and maintained subcutaneously in BALB/c nude mice prior to implantation. When tumor volumes have reached 700-1500 mm ³ , the tumors are harvested and cut into pieces with a diameter of approximately 2-3 mm ³ .

Tumors or tumor pieces are washed with ice-cold RPMI1640 media (no serum) and then placed in ice-cold media for use.

Prior to tumor implantation, the skin of five to six week old female BALB/c nude mice is disinfected with iodophor on the right flank. Each mouse is inoculated without anesthesia, subcutaneously, in the upper part of the right flank with one tumor fragment for tumor development.

After inoculation of the tumor daily check the morbidity and mortality of animals. Tumor size is measured with a caliper twice a week in two dimensions. Tumor volume is expressed in mm ³ using the formula: TV=0.5axb2 where a and b are the long and short tumor diameters, respectively.

On study day 12, mice are randomized into 5 groups of 8 mice each; the mean tumor volume is ~170 mm ³ in the cohort. Mice are dosed with test agents on day 13 of the study. Test mice in this study receive a single dose of their assigned test article and dose level on day 1, after which tumor growth is monitored for up to day 51.

- 43 040749

Sequences.

SEQID NO. 1 VCO HuBa-l-3d, CDR underlined]

QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGN TNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQ GTMVTVSS

SEQID NO. 2 [VL HuBa-l-3d, CDR underlined]

DIVMTPSPDSLAVSLGERATINCKSSPSLLNSSNPKNYLAWYPPKPGPPPKLLVYFAST RESGVPDRF SGSGSGTDFTLTIS SLOAEDVAVYYCOQHYSTPPTFGOGTKLEIK

SEQID NO. 3 [heavy chain HuBa-l-3d]

QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGN TNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQID NO. 4 [light chain HuBa-l-3d]

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSSNQKNYLAWYQQKPGQPPKLLVYFAST RESGVPDRF SGSGSGTDFTLTIS SLQAEDVAVYYCQQHYSTPPTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKTHSPEKVTKVTKVGLNKTHSPACEVACE

SEQID NO. 5 [CDR1 VHHuBa-l-3d]

DYAMH

SEQID NO. 6 [CDR2 VH HuBa-l-3d]

VISTYYGNTNYNPKFKG

SEQID NO. 7 [CDR3 VH HuBa-l-3d]

GGLREYYYAMDY

SEQID NO. 8 [CDR1 VL HuBa-l-3d]

KSSPSLLNSSNPKNYLA

SEQID NO. 9 [CDR2 VL HuBa-l-3d]

FASTRES

SEPID NP. 10 [CDR3 VLHuBa-l-3d]

PPHYSTPPPT

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SEQID NO. 11 [heavy chain HuBa-l-3d, terminal K1

QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGN TNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEQID NO. 12 [DLK1 human, variant 11

MTATEALLRVLLLLLAFGHSTYGAECFPACNPQNGFCEDDNVCRCHVGWQGPLCDQC VTSPGCLHGLCGEPGQCICTDGWDGELCDRDVRACSSAPCANNGTCVSLDGGLYECSC APGYSGKDCQKKDGPCVINGSPCQHGGTCVDDEGRASHASCLCPPGFSGNFCEIVANSC TPNPCENDGVCTDIGGDFRCRCP AGFIDKTC SRP VTNC AS SPCQNGGTCLQHTQ VS YECL CKPEFTGLTCVKKRALSPQQVTRLPSGYGLAYRLTPGVHELPVQQPEHRILKVSMKELN KKTPLLTEGQAICFTILGVLTSLVVLGTVGIVFLNKCETWVSNLRYNHMLRKKKNLLLQ YNSGEDLAVNIIFPEKIDMTTFSKEAGDEEI

SEQID NO. 13 [DLK1 human, variant 21

MTATEALLRVLLLLLAFGHSTYGAECFPACNPQNGFCEDDNVCRCQPGWQGPLCDQCV TSPGCLHGLCGEPGQCICTDGWDGELCDRDVRACSSAPCANNRTCVSLDDGLYECSCA PGYSGKDCQKKDGPCVINGSPCQHGGTCVDDEGRASHASCLCPPGFSGNFCEIVANSCT PNPCENDGVCTDIGGDFRCRCPAGFIDKTCSRPVTNCASSPCQNGGTCLQHTQVSYECL CKPEFTGLTCVKKRALSPQQVTRLPSGYGLAYRLTPGVHELPVQQPEHRILKVSMKELN KKTPLLTEGQAICFTILGVLTSLVVLGTVGIVFLNKCETWVSNLRYNHMLRKKKNLLLQ YNSGEDLAVNIIFPEKIDMTTFSKEAGDEEI

SEQID NO. 101 [VH 3 A4, CDR underlined!

OIOLVOSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGOGLEWIGDINPYNGD TNYNOKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGOGTLVTV ss

SEO ID NO. 102 [VL 3A4, CDR underlined!

DIVMTOTPLSLPVTPGEPASISCRSSOSLLHSNGNTYLEWYLQKPGOSPOLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGOGTKLEIK

SEQ W NO. 103 [heavy chain 3A41

- 45 040749

QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGD TNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQID NO. 104 [light chain 3A41

DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVF IFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSGSTYSLVEKHK

SEQID NO. 105 [CDR1 VH3A41

GYTFTDDYMS

SEQID NO. 106[CDR2 VH3A41

DINPYNGDTN

SEQID NO. 107 [CDR3 VH3A41

DPGAMDY

SEQID NO. 108 [CDR1 VL3A41

RSSPSLLHSNGNTYLE

SEQID NO. 109[CDR2 VL3A41

TVSNRFS

SEQID NO. 110 [CDR3 VL3 A41

FPGSHVPLT

SEQID NO. Ill [heavy chain 3 A4, terminal K1

PIPLVPSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKPAPGPGLEWIGDINPYNGD TNYNPKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGPGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLP SSGLYSLSSVVTVPSSSLGTPTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYN*STYRVVSVLTVLHPDWLNGKEYKCKVSNKALPAPIEKTISKAKGPPREPPVYTLP PSRDELTKNPVSLTCLVKGFYPSDIAVEWESNGPPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWPPGNVFSCSVMHEALHNHYTPKSLSLSPGK

- 46 040749

N* stands for Asn297

SEP ID NO. 112 [KAAG1 human!

MDDDAAPRVEGVPVAVHKHALHDGLRQVAGPGAAAAHLPRWPPPQLAASRREAPPLS QRPHRTQGAGSPPETNEKLTNPQVKEK

SEP ID NO. 113 [VL 3A4-L2, CDR underlined!

DVVMTOTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLOKPGOSPKLLIYTVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGOGTKLEIK

SEQID NO. 114 [light chain 3 A4-L21

DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSV FIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSPVKHSFK

SEQID NO. 115 [VL 3A4-K4, CDR underlined!

DIVMTOSPDSLAVSLGERATINCRSSOSLLHSNGNTYLEWYOQKPGOPPKLLIYTVSNRF SGVPDRF SGSGSGTDFTLTIS SLOAEDVAVYYCFQGSHVPLTFGOGTKVEIK

SEQID NO. 116 [light chain 3A4-K41

DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLEWYQQKPGQPPKLLIYTVSNRF SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPLTFGQGTKVEIKRTVAAPSV FIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSKDSTYSFPVTLVTEQDSKDSKADYEKHKHK

SEQID NO. 201 [UN XA4, CDR underlined!

OVHLVESGGGVVOPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSH EYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGOG TLVTVSS

SEQID NO. 202 [VL XA4, CDR underlined!

EIVLTOSPATLSLSPGERATLSCRASOSVSSYLAWYOOKPGOAPRLLIYDASNRATGIPA RF SGSGSGTDFTLTIS SLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

SEQID NO. 203 [heavy chain XA41

QVHLVESGGGVVQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSH EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQ SSGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT I<PREEQYN2STYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TISI<AI<GQPREPQ

- 47 040749

VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQID NO. 204 [light chain XA41

EIVLTQSPATLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RF SGSGSGTDFTLTIS SLEPEDF AVYYCQQRSNWPLTFGGGTKVEIKRT VAAPS VFIFPP S DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYRGSLSSTL TLSKADYTKGLQPVYACE

SEQID NO. 205 [CDR1 VH XA41

IYGMH

SEQID NO. 206 [CDR2 VH XA41

VLWYDGSHEYYADSVKG

SEQID NO. 207 [CDR3 VH XA41

DGDYYDSGSPLDY

SEQID NO. 208 [CDR1 VL XA41

RASPSVSSYLA

SEQID NO. 209 [CDR2 VL XA41

DASNRAT

SEQID NO. 210 [CDR3 VL XA41

PPRSNWPLT

SEQID NO. 211 [heavy chain XA4, terminal K1

PVHLVESGGGVVPPGRSLRLSCVASGITFRIYGMHWVRPAPGKGLEWVAVLWYDGSH EYYADSVKGRFTISRDNSKNTLYLPMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGPG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLP SSGLYSLS S VVTVPS S SLGTpTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEPYN^STYRVVSVLTVLHPDWLNGKEYKCKVSNKALPAPIEKTISKAKGPPREPP VYTLPPSREEMTKNPVSLTCLVKGFYPSDIAVEWESNGPPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWPPGNVFSCSVMHEALHNHYTPKSLSLSPGK

N* stands for Asn297

SEQID NO. 212 [VH XFT, CDR underlined!

OVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVROAPGKGLEWMGIIDPGDSRTRY SPSFOGOVTISADKSISTAYLOWSSLKASDTAMYYCARGQLYGGTYMDGWGOGTLVT vss

SEO ID NO. 213 [VL XFT, CDR underlined!

- 48 040749

DIALTOPASVSGSPGOSITISCTGTSSDIGGYNSVSWYOQHPGKAPKLMIYGVNNRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIK

SEQ W NO. 214 [XFT1 heavy chain

QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRY SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEP ID NO. 215 [XFT1 light chain

SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIKRTVAAPSVFI

SEQID NO. 216 [XFT CDR1 VH1

GYSFTSYWIG

SEQID NO. 217 [CDR2 VH XFT1 WMGIIDPGDSRTRYSPSFQG SEQ ID NO. 218 [CDR3 VH XFT1 GQLYGGTYMDG

SEQID NO. 219 [CDR1 VL XFT1

TGTSSDIGGYNSVS

SEQID NO. 220 [CDR2 VL XFT1

LMIYGVNNRPS

SEQID NO. 221 [CDR3 VL XFT1

SSYDIESATP

SEQ W NO. 222 [XFT heavy chain, terminal K1

PVELVPSGAVKKPGESLKISCKGSGYSFTSYWIGWVRPAPGKGLEWMGIIDPGDSRTRY SPSFPGPVTISADKSISTAYLPWSSLKASDTAMYYCARGPLYGGTYMDGWGPGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL PSSGLYSLSSVVTVPSSSLGTPTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

- 49 040749

EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEP ID NO. 223 VCO X09, CDR underlined]

OVOLOQSGPELEKPGASVKISCKASGYSFTGYTMNWVKOSHGKSLEWIGLITPYNGASS YNOKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT vss

SEO ID NO. 224 [VL X09, CDR underlined]

DIELTOSPAIMSASPGEKVTMTCSASSSVSYMHWYOOKSGTSPKRWIYDTSKLASGVPG RFSGSGSGNSYSLTISSVEAEDDATYYCOQWSKHPLTFGSGTKVEIK

SEO ID NO. 225 [heavy chain X091

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQID NO. 226 [light chain X09]

DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPG RFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTL TLSKADYEKHKSSYTKTL TLSKADYEKHKSSYTKNSVTHQNSYACEVQ

SEQID NO. 227 CDR1 VH[X09]

GYSFT

SEQID NO. 228 [CDR2 UN X09]

LITPYNGAS S YNQKFRG

SEQID NO. 229 [CDR3 UN X09]

GGYDGRGFDY

SEQID NO. 230 [CDR1 VL X09]

SASSSVSYMH

SEQID NO. 231 [CDR2 VL X09]

- 50 040749

DTSKLAS

SEQID NO. 232 [CDR3 VL X091

QQWSKHPLT

SEQID NO. 233 [heavy chain X09, terminal K1

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEQID NO. 234 VCO X09.2, CDR underlined!

OVOLOQSGPELEKPGASVKISCKASGYSFTGYTMNWVKOSHGKSLEWIGLITPYNGASS YNOKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGOGTTVT vss

SEO ID NO. 235 [VL X09.2, CDR underlined!

DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYOOKSGTSPKRWIYDTSKLASGVPG RF SGSGSGNS YSLTIS SVEAEDDATYYCOQWSGYPLTFGAGTKLEIK

SEQID NO. 236 [heavy chain X09.21

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQID NO. 237 [light chain X09.21

DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPG RFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTL TLSKADYEKHKSSYTKTL TLSKADYEKHKSSYTKNSVTHQNSYACEVQ

- 51 040749

SEO ID NO. 238 [CDR1 UN X09.21

GYTMN

SEP ID NO. 239 [CDR2 CR X09 21

LITPYNGAS S YNQKFRG

SEP ID NO. 240 [CDR3 UN X09 21

GGYDGRGFDY

SEP ID NO. 241 [CDR1 VL X09.21

SASSSVSYMH

SEP ID NO. 242 [CDR2 VL X09.21

DTSKLAS

SEP ID NO. 243 [CDR3 VL X09.21

QQWSGYPLT

SEQID NO. 244 [heavy chain X09.2, terminal K1

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEQID NO. 245 [human mesothelium, variant 11

MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPL DLLLFLNPD AF SGPQ ACTRFF SRITKANVDLLPRGAPERQRLLP AALACWGVRGSLLSEA DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPST WSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDEL YPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQ VATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDP RQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRT DAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGY LVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA

SEQID NO. 246 [human mesothelium, variant 21

- 52 040749

MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPL DLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPST WSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDEL YPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRF VKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYP KARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTV AEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQ EALSGTPCLLGPGPVLTVLALLLASTLA

- 53 040749

Sequence listing.

<110> ADC Therapeutics SA Medlmmune Limited <120> PYRROLOBENZODIAZEPINE-ANTIBODY CONJUGATES <130> RKA/LP338742 <150> GB 1719391.3 <151> 2017-11-22 <150> GB 1719398.8 <151> 2017 <-11-22 GB 1719393.9 <151> 2017-11-22 <150> GB 1702031.4 <151> 2017-02-08 <160> 246 <170> Patentin version 3.5 <210> 1 <211> 121 <212> PROTEIN <213> Artificial sequence <220>

< 223> VH HuBa-l-3d <400>1

Gin Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala 15 1015

Ser Vai Lys Vai Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 2530

Ala Met His Trp Vai Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp lie 35 4045

Gly Vai lie Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gin Lys Phe 50 5560

Lys Gly Lys Ala Thr Met Thr Vai Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly

100 105110

Gin Gly Thr Met Vai Thr Vai Ser Ser

- 54 040749

115 120 <210> 2 <211> 113 <212> PROTEIN <213> Artificial sequence <220>

< 223> VL HuBa-l-3d < 400> 2

Asp lie Vai Met Thr Gin Ser Pro 1 5

Asp Ser Leu Ala Vai Ser Leu Gly

15

Glu Arg Ala Thr lie Asn Cys Lys 20

Ser Ser Gin Ser Leu Leu Asn Ser

thirty

Ser Asn Gin Lys Asn Tyr Leu Ala

40

Trp Tyr Gin Gin Lys Pro Gly Gin 45

Pro Pro Lys Leu Leu Vai Tyr Phe 50 55

Ala Ser Thr Arg Glu Ser Gly Vai 60

Pro Asp Arg Phe Ser Gly Ser Gly 65 70

Ser Gly Thr Asp Phe Thr Leu Thr

80 lie Ser Ser Leu Gin Ala Glu Asp 85

Vai Ala Vai Tyr Tyr Cys Gin Gin

95

His Tyr Ser Thr Pro Pro Thr Phe

100

Gly Gin Gly Thr Lys Leu Glu lie

105 110

Lys <210> 3 <211> 450 <212> PROTEIN <213> Artificial sequence <220>

<223> Heavy Chain HuBa-l-3d <220>

< 221> MOD_RES < 222> (301) . . (301) <223> Xaa is Asn297 <400> 3

Gin Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala

10 15

- 55 040749

Ser Vai Lys Vai Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 2530

Ala Met His Trp Vai Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp lie 35 4045

Gly Vai lie Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gin Lys Phe 50 5560

Lys Gly Lys Ala Thr Met Thr Vai Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105110

Gin Gly Thr Met Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120125

Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135140

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

145 150 155160

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

165 170175

Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai 180 185190

Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Vai Asn His

195 200205

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

210 215220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235240

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

245 250255 lie Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His 260 265270

- 56 040749

Glu asp Pro 275 Glu Vai Lys Phe Asn 280 trp Tyr Vai asp Gly 285 Vai Glu Vai His Asn 290 Ala Lys Thr Lys Pro 295 Arg Glu Glu gin Tyr 300 xaa Ser Thr Tyr Arg 305 Vai Vai Ser Vai Leu 310 Thr Vai Leu His gin 315 asp trp Leu Asn Gly 320 Lys Glu Tyr Lys Cys 325 Lys Vai Ser Asn Lys 330 Ala Leu Pro Ala Pro 335 He Glu Lys Thr lie 340 Ser Lys Ala Lys Gly 345 gin Pro Arg Glu Pro 350 gin Vai Tyr Thr Leu 355 Pro Pro Ser Arg Glu 360 Glu Met Thr Lys Asn 365 gin Vai Ser Leu Thr 370 Cys Leu Vai Lys Gly 375 Phe Tyr Pro Ser asp 380 lie Ala Vai Glu trp 385 Glu Ser Asn gly gin 390 Pro Glu Asn Asn Tyr 395 Lys Thr Thr Pro Pro 400 Vai Leu asp Ser Asp 405 gly Ser Phe Phe Leu 410 Tyr Ser Lys Leu Thr 415 Vai asp Lys Ser Arg 420 trp gin gin gly Asn 425 Vai Phe Ser Cys Ser 430 Vai Met His Glu Ala 435 Leu His Asn His Tyr 440 Thr gin Lys Ser Leu 445 Ser Leu Ser Pro Gly 450 <210> <211> <212> <213> : 4 220 PROTEIN Artificial sequence <220> <223> . Light chain I luba- -l-3c i <400> 4 asp 1 lie Vai Met Thr 5 Gln Ser Pro asp Ser 10 Leu Ala Vai Ser Leu 15 gly Glu Arg Ala Thr lie Asn Cys Lys Ser Ser gin Ser Leu Leu Asn Ser

- 57 040749

2530

Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 4045

Pro Pro Lys Leu Leu Vai Tyr Phe Ala Ser Thr Arg Glu Ser Gly Vai 50 5560

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 7580 lie Ser Ser Leu Gin Ala Glu Asp Vai Ala Vai Tyr Tyr Cys Gin Gin 85 9095

His Tyr Ser Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Leu Glu He 100 105110

Lys Arg Thr Vai Ala Ala Pro Ser Vai Phe He Phe Pro Pro Ser Asp

115 120125

Glu Gin Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn

130 135140

Phe Tyr Pro Arg Glu Ala Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu

145 150 155160

Gin Ser Gly Asn Ser Gin Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp

165 170175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185190

Glu Lys His Lys Vai Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser 195 200205

Ser Pro Vai Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215220 <210> 5 <2H> 5 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR1 VH HuBa-l-3d < 400> 5

Asp Tyr Ala Met His

5

- 58 040749 <210> 6 <211> 17 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR2 VH HuBa-l-3d <400>6

Vai lie Ser Thr Tug Tug Gly Asn Thr Asn Tyr Asn Gin Lys Phe Lys

1015

Gly <210> 7 <211>12 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR3 VH HuBa-l-3d <400> 7

Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr

1510 <210>8 <211>17 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR1 VL HuBa-l-3d <400>8

Lys Ser Ser Gin Ser Leu Leu Asn Ser Ser Asn Gin Lys Asn Tyr Leu

1015

Ala <210>9 <211> 7 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR2 VL HuBa-l-3d <400> 9

Phe Ala Ser Thr Arg Glu Ser

5

- 59 040749

<210> 10 <211> 9 <212> PROTEIN <213> Artificial sequence <220> <223> CDR3 VL HuBa-l-3d <400> 10 Gin Gin l His Ser Thr Pro Pro Thr 1 5

<210> 11 <211> 451 <212> PROTEIN <213> Artificial sequence <220>

<223> Heavy chain HuBa-l-3d, terminal K <220>

< 221> MOD_RES < 222> (301) . . (301) <223> Xaa is Asn297 <400> 11

Gin 1 Vai gin Leu Wai 5 gin Ser gly Ala Glu 10 Vai Lys Lys Pro Gly 15 Ala Ser Vai Lys Vai 20 Ser Cys Lys gly Ser 25 gly Tyr Thr Phe Thr 30 asp Tyr Ala Met His 35 trp Vai Arg gin Ala 40 Pro gly gin gly Leu 45 Glu trp lie gly Vai 50 lie Ser Thr Tyr Tyr 55 gly Asn Thr Asn Tyr 60 Asn gin Lys Phe Lys 65 gly Lys Ala Thr Met 70 Thr Vai asp Lys Ser 75 Thr Ser Thr Ala Tyr 80 Met Glu Leu Arg Ser 85 Leu Arg Ser asp Asp 90 Thr Ala Vai Tyr Tyr 95 Cys Ala Arg gly Gly 100 Leu Arg Glu Tyr Tyr 105 Tyr Ala Met asp Tyr 110 trp gly gin gly Thr 115 Met Vai Thr Vai Ser 120 Ser Ala Ser Thr Lys 125 gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser gly gly Thr Ala

- 60 040749

130 135140

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

145 150 155160

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

165 170175

Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai

180 185190

Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His

195 200205

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

210 215220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235240

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

245 250255

He Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His

260 265270

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

275 280285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Xaa Ser Thr Tyr

290 295300

Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly

305 310 315320

Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro He

325 330335

Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai

340 345350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Vai Ser 355 360365

Leu Thr Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp He Ala Vai Glu

370 375380

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

- 61 040749

385 390 395400

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

405 410415

Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met

420 425430

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

435 440445

Pro Gly Lys 450 <210>12 <211> 383 <212> PROTEIN <213> Homo sapiens <400>12

Met Thr Ala Thr Glu Ala Leu Leu Arg Vai Leu Leu Leu Leu Leu Ala

1015

Phe Gly His Ser Thr Tyr Gly Ala Glu Cys Phe Pro Ala Cys Asn Pro 20 2530

Gin Asn Gly Phe Cys Glu Asp Asp Asn Vai Cys Arg Cys His Vai Gly 35 4045

Trp Gin Gly Pro Leu Cys Asp Gin Cys Vai Thr Ser Pro Gly Cys Leu 50 5560

His Gly Leu Cys Gly Glu Pro Gly Gin Cys lie Cys Thr Asp Gly Trp 65 70 7580

Asp Gly Glu Leu Cys Asp Arg Asp Vai Arg Ala Cys Ser Ser Ala Pro 85 9095

Cys Ala Asn Asn Gly Thr Cys Vai Ser Leu Asp Gly Gly Leu Tyr Glu

100 105110

Cys Ser Cys Ala Pro Gly Tyr Ser Gly Lys Asp Cys Gin Lys Lys Asp

115 120125

Gly Pro Cys Vai lie Asn Gly Ser Pro Cys Gin His Gly Gly Thr Cys 130 135140

Vai Asp Asp Glu Gly Arg Ala Ser His Ala Ser Cys Leu Cys Pro Pro

145 150 155160

- 62 040749

Gly Phe Ser Gly Asn Phe Cys Glu 165

He Vai Ala Asn Ser Cys Thr Pro

170 175

Asn Pro Cys Glu Asn Asp Gly Vai 180

Cys Thr Asp He Gly Gly Asp Phe 185 190

Arg Cys Arg Cys Pro Ala Gly Phe

195 200

He Asp Lys Thr Cys Ser Arg Pro 205

Vai Thr Asn Cys Ala Ser Ser Pro

210 215

Cys Gin Asn Gly Gly Thr Cys Leu

220

Gin His Thr Gin Vai Ser Tyr Glu 225 230

Cys Leu Cys Lys Pro Glu Phe Thr

235 240

Gly Leu Thr Cys Vai Lys Lys Arg 245

Ala Leu Ser Pro Gin Gin Vai Thr

250 255

Arg Leu Pro Ser Gly Tyr Gly Leu

260

Ala Tyr Arg Leu Thr Pro Gly Vai

265 270

His Glu Leu Pro Vai Gin Gin Pro

275 280

Glu His Arg He Leu Lys Vai Ser 285

Met Lys Glu Leu Asn Lys Lys Thr

290 295

Pro Leu Leu Thr Glu Gly Gin Ala 300

He Cys Phe Thr He Leu Gly Vai 305 310

Leu Thr Ser Leu Vai Vai Leu Gly

315 320

Thr Vai Gly He Vai Phe Leu Asn 325

Lys Cys Glu Thr Trp Vai Ser Asn

330 335

Leu Arg Tyr Asn His Met Leu Arg 340

Lys Lys Lys Asn Leu Leu Leu Gin 345 350

Tyr Asn Ser Gly Glu Asp Leu Ala

355 360

Vai Asn He He Phe Pro Glu Lys

365

He Asp Met Thr Thr Phe Ser Lys

370 375

Glu Ala Gly Asp Glu Glu He 380 <210> 13 <2H> 383 < 212> PROTEIN <213> Homo sapiens <400> 13

- 63 040749

Met Thr Ala Thr Glu Ala Leu Leu Arg Vai Leu Leu Leu Leu Leu Ala 15 1015

Phe Gly His Ser Thr Tyr Gly Ala Glu Cys Phe Pro Ala Cys Asn Pro 20 2530

Gin Asn Gly Phe Cys Glu Asp Asp Asn Vai Cys Arg Cys Gin Pro Gly 35 4045

Trp Gin Gly Pro Leu Cys Asp Gin Cys Vai Thr Ser Pro Gly Cys Leu 50 5560

His Gly Leu Cys Gly Glu Pro Gly Gin Cys lie Cys Thr Asp Gly Trp 65 70 7580

Asp Gly Glu Leu Cys Asp Arg Asp Vai Arg Ala Cys Ser Ser Ala Pro 85 9095

Cys Ala Asn Asn Arg Thr Cys Vai Ser Leu Asp Asp Gly Leu Tyr Glu

100 105110

Cys Ser Cys Ala Pro Gly Tyr Ser Gly Lys Asp Cys Gin Lys Lys Asp 115 120125

Gly Pro Cys Vai lie Asn Gly Ser Pro Cys Gin His Gly Gly Thr Cys 130 135140

Vai Asp Asp Glu Gly Arg Ala Ser His Ala Ser Cys Leu Cys Pro Pro

145 150 155160

Gly Phe Ser Gly Asn Phe Cys Glu lie Vai Ala Asn Ser Cys Thr Pro

165 170175

Asn Pro Cys Glu Asn Asp Gly Vai Cys Thr Asp lie Gly Gly Asp Phe 180 185190

Arg Cys Arg Cys Pro Ala Gly Phe lie Asp Lys Thr Cys Ser Arg Pro

195 200205

Vai Thr Asn Cys Ala Ser Ser Pro Cys Gin Asn Gly Gly Thr Cys Leu

210 215220

Gin His Thr Gin Vai Ser Tyr Glu Cys Leu Cys Lys Pro Glu Phe Thr

225 230 235240

Gly Leu Thr Cys Vai Lys Lys Arg Ala Leu Ser Pro Gin Gin Vai Thr

245 250255

- 64 040749

Arg Leu Pro Ser Gly Tyr Gly Leu 260

Ala Tyr Arg Leu Thr Pro Gly Vai 265 270

His Glu Leu Pro Vai Gin Gin Pro

275 280

Glu His Arg lie Leu Lys Vai Ser 285

Met Lys Glu Leu Asn Lys Lys Thr

290 295

Pro Leu Leu Thr Glu Gly Gin Ala

300 lie Cys Phe Thr lie Leu Gly Vai

305 310

Leu Thr Ser Leu Vai Vai Leu Gly

315 320

Thr Vai Gly lie Vai Phe Leu Asn 325

Lys Cys Glu Thr Trp Vai Ser Asn

330 335

Leu Arg Tyr Asn His Met Leu Arg 340

Lys Lys Lys Asn Leu Leu Leu Gin 345 350

Tyr Asn Ser Gly Glu Asp Leu Ala

355 360

Vai Asn lie lie Phe Pro Glu Lys 365 lie Asp Met Thr Thr Phe Ser Lys

370 375

Glu Ala Gly Asp Glu Glu lie 380 <210>14 <400>14

000 <210>15 <400>15

000 <210>16 <400>16

000 <210>17 <400>17

000 <210>18 <400>18

000 <210>19 <400>19

000 <210> 20

- 65 040749 <400> 20 LLC <210> 21 <400> 21 LLC <210> 22 <400> 22 LLC <210> 23 <400> 23 LLC <210> 24 <400> 24 LLC <210> 25 <400 > 25 LLC <210> 26 <400> 26 LLC <210> 27 <400> 27 LLC <210> 28 <400> 28 LLC <210> 29 <400> 29 LLC <210> 30 <400> 30 LLC <210 > 31 <400> 31 OOO <210> 32 <400> 32 OOO

- 66 040749 <210>33 <400>33

000 <210>34 <400>34

000 <210>35 <400>35

000 <210>36 <400>36

000 <210>37 <400>37

000 <210>38 <400>38

000 <210>39 <400>39

000 <210>40 <400>40

000 <210>41 <400>41

000 <210>42 <400>42

000 <210>43 <400>43

000 <210>44 <400>44

000 <210>45 <400>45

000

- 67 040749 <210> 46 <400> 46,000 <210> 47 <400> 47,000 <210> 48 <400> 48

000 <210> 49 <400> 49,000 <210> 50 <400> 50,000 <210> 51 <400> 51,000 <210> 52 <400> 52,000 <210> 53 <400> 53,000 <210> 54 <400> 54,000 <210> 55 <400> 55,000 <210> 56 <400> 56,000 <210> 57 <400> 57,000 <210> 58 <400> 58

- 68 040749

OOO <210> 59 <400> OOO 59 <210> 60 <400> OOO 60 <210> 61 <400> OOO 61 <210> 62 <400> OOO 62 <210> 63 <400> OOO 63 <210> 64 <400> OOO 64 <210> 65 <400> OOO 65 <210> 66 <400> OOO 66 <210> 67 <400> OOO 67 <210> 68 <400> OOO 68 <210> 69 <400> OOO 69 <210> 70 <400> OOO 70 <210> 71

- 69 040749 <400> 71 LLC <210> 72 <400> 72 LLC <210> 73 <400> 73 LLC <210> 74 <400> 74 LLC <210> 75 <400> 75 LLC <210> 76 <400 > 76

LLC <210> 77 <400> 77 LLC <210> 78 <400> 78 LLC <210>79 <400> 79 LLC <210>80 <400>80

LLC <210>81 <400>81

LLC <210>82 <400>82

LLC <210>83 <400>83

OOO <210>84

- 70 040749 <400>84

LLC <210>85 <400>85

LLC <210>86 <400>86

LLC <210>87 <400>87

LLC <210>88 <400>88

LLC <210>89 <400>89

OOO <210>90 <400>90

LLC <210>91 <400>91

LLC <210>92 <400>92

LLC <210>93 <400>93

LLC <210>94 <400>94

LLC <210>95 <400>95

OOO <210>96 <4 Ο 0>9 6

OOO

- 71 040749 <210>97 <400>97

000 <210>98 <400>98

000 <210>99 <400>99

000 <210>100 <400>100

000 <210>101 <211>116 <212> PROTEIN <213> Artificial sequence <220>

< 223> VH 3Α4 < 400> 101

Gin lie Gin Leu Vai Gin Ser Gly 1 5

Ala Glu Vai Lys Lys Pro Gly Ala 10 15

Ser Vai Lys Vai Ser Cys Lys Ala 20

Ser Gly Tyr Thr Phe Thr Asp Asp 25 30

Tyr Met Ser Trp Vai Lys Gin Ala

40

Pro Gly Gin Gly Leu Glu Trp lie 45

Gly Asp lie Asn Pro Tyr Asn Gly

55

Asp Thr Asn Tyr Asn Gin Lys Phe 60

Lys Gly Lys Ala Thr Leu Thr Vai 65 70

Asp Lys Ser Thr Ser Thr Ala Tyr

80

Met Glu Leu Ser Ser Leu Arg Ser 85

Glu Asp Thr Ala Vai Tyr Tyr Cys

95

Ala Arg Asp Pro Gly Ala Met Asp 100

Tyr Trp Gly Gin Gly Thr Leu Vai 105 110

Thr Vai Ser Ser

115 <210> 102 <211> 112

- 72 040749 <212> PROTEIN <213> Artificial sequence <220>

< 223> VL 3A4 <400>102

Asp lie Vai Met Thr Gin Thr Pro Leu Ser Leu Pro Vai Thr Pro Gly

1015

Glu Pro Ala Ser He Ser Cys Arg Ser Ser Gin Ser Leu Leu His Ser 20 2530

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 4045

Pro Gin Leu Leu He Tyr Thr Vai Ser Asn Arg Phe Ser Gly Vai Pro 50 5560

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 7580

Ser Arg Vai Glu Ala Glu Asp Vai Gly Vai Tyr Tyr Cys Phe Gin Gly 85 9095

Ser His Vai Pro Leu Thr Phe Gly Gin Gly Thr Lys Leu Glu He Lys 100 105110 <210>103 <2H>445 <212> PROTEIN <213> Artificial sequence <220>

<223> Heavy Chain 3A4 <220>

<221> MOD_RES <222> (296)..(296) <223> Haa is Asn297 <400>103

Gin He Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala

1015

Ser Vai Lys Vai Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp 20 2530

Tyr Met Ser Trp Vai Lys Gin Ala Pro Gly Gin Gly Leu Glu Trp lie 35 4045

- 73 040749

Gly Asp lie Asn Pro Tyr Asn Gly Asp Thr Asn Tyr Asn Gin Lys Phe 50 5560

Lys Gly Lys Ala Thr Leu Thr Vai Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Asp Pro Gly Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu Vai

100 105110

Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala 115 120125

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

130 135140

Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly

145 150 155160

Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser

165 170175

Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu 180 185190

Gly Thr Gin Thr Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr

195 200205

Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Vai Phe

225 230 235240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro

245 250255

Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai

260 265270

Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr

275 280285

Lys Pro Arg Glu Glu Gin Tyr Xaa Ser Thr Tyr Arg Vai Vai Ser Vai

290 295300

- 74 040749

Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315320

Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser

325 330335

Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro

340 345350

Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr Cys Leu Vai

355 360365

Lys Gly Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly

370 375380

Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp

385 390 395400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp

405 410415

Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His 420 425430

Asn His Tyr Thr Gin Lys Ser Leu

435440

Ser Leu Ser Pro Gly

445 <210>104 <2H> 219 <212> PROTEIN <213> Artificial sequence <220>

<223> Light chain 3A4 <400>104

Asp He Vai Met Thr Gin Thr Pro 15

Leu Ser Leu Pro Vai Thr Pro Gly 1015

Glu Pro Ala Ser He Ser Cys Arg 20

Ser Ser Gin Ser Leu Leu His Ser

thirty

Asn Gly Asn Thr Tyr Leu Glu Trp

40

Tyr Leu Gin Lys Pro Gly Gin Ser 45

Pro Gin Leu Leu He Tyr Thr Vai

55

Ser Asn Arg Phe Ser Gly Vai Pro 60

Asp Arg Phe Ser Gly Ser Gly Ser 65 70

Gly Thr Asp Phe Thr Leu Lys lie

80

- 75 040749

Ser Arg Vai Glu Ala Glu Asp Vai Gly Vai 8590

Tyr Tyr Cys

Phe Gin Gly 95

Ser His Vai Pro Leu Thr Phe Gly Gin Gly Thr Lys Leu Glu He Lys 100 105110

Arg Thr Vai Ala Ala Pro Ser Vai Phe He Phe Pro Pro Ser Asp Glu

115 120125

Gin Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe

130 135140

Tyr Pro Arg Glu Ala Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin

145 150 155160

Ser Gly Asn Ser Gin Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser

165 170175

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

180 185190

Lys His Lys Vai Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser 195 200205

Pro Vai Thr Lys Ser Phe Asn Arg Gly Glu Cys

210215 <210>105 <2H>10 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR1 VH 3A4 < 400>105

Gly Tyr Thr Phe Thr Asp Asp Tyr Met Ser 1510 < 210>106 < 2H>10 < 212> PROTEIN < 213> Artificial sequence <220>

<223> CDR2 VH 3A4 <400> 106

Asp He Asn Pro Tyr Asn Gly Asp Thr Asn 15 10

- 76 040749 <210> 107 <211> 7 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR3 VH 3Α4 < 400>107

Asp Pro Gly Ala Met Asp Tyr <210>108 <211>16 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR1 VL 3A4 <400>108

Arg Ser Ser Gin Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Glu

1015 <210>109 <211> 7 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR2 VL 3A4 <400>109

Thr Vai Ser Asn Arg Phe Ser <210>110 <211>9 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR3 VL 3A4 <400>110

Phe Gin Gly Ser His Vai Pro Leu Thr 15 <210>111 <211> 446 <212> PROTEIN <213> Artificial sequence <220>

- 77 040749 <223> Heavy chain ZA4, terminal K <220>

<221> MOD_RES <222> (296)..(296) <223> Haa is Asn297 <400>111

Gin Not Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Ala 15 1015

Ser Vai Lys Vai Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asp 20 2530

Tyr Met Ser Trp Vai Lys Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 4045

Gly Asp He Asn Pro Tyr Asn Gly Asp Thr Asn Tyr Asn Gin Lys Phe 50 5560

Lys Gly Lys Ala Thr Leu Thr Vai Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Vai Tyr Tyr Cys 85 9095

Ala Arg Asp Pro Gly Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu Vai

100 105110

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

115 120125

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

130 135140

Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly 145 150 155160

Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser

165 170175

Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu 180 185190

Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His Lys Pro Ser Asn Thr 195 200205

Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215220

- 78 040749

Cys Pro Pro Cys Pro Ala Pro Glu 225 230

Leu Leu Gly Gly Pro Ser Vai Phe

235 240

Leu Phe Pro Pro Lys Pro Lys Asp 245

Thr Leu Met lie Ser Arg Thr Pro

250 255

Glu Vai Thr Cys Vai Vai Vai Asp 260

Vai Ser His Glu Asp Pro Glu Vai

265 270

Lys Phe Asn Trp Tyr Vai Asp Gly

275 280

Vai Glu Vai His Asn Ala Lys Thr

285

Lys Pro Arg Glu Glu Gin Tyr Xaa

290 295

Ser Thr Tyr Arg Vai Vai Ser Vai 300

Leu Thr Vai Leu His Gin Asp Trp

305 310

Leu Asn Gly Lys Glu Tyr Lys Cys

315 320

Lys Vai Ser Asn Lys Ala Leu Pro

325

Ala Pro lie Glu Lys Thr lie Ser

330 335

Lys Ala Lys Gly Gin Pro Arg Glu 340

Pro Gin Vai Tyr Thr Leu Pro Pro 345 350

Ser Arg Asp Glu Leu Thr Lys Asn

355 360

Gin Vai Ser Leu Thr Cys Leu Vai 365

Lys Gly Phe Tyr Pro Ser Asp lie

370 375

Ala Vai Glu Trp Glu Ser Asn Gly 380

Gin Pro Glu Asn Asn Tyr Lys Thr 385 390

Thr Pro Pro Vai Leu Asp Ser Asp

395 400

Gly Ser Phe Phe Leu Tyr Ser Lys 405

Leu Thr Vai Asp Lys Ser Arg Trp

410 415

Gin Gin Gly Asn Vai Phe Ser Cys 420

Ser Vai Met His Glu Ala Leu His

425 430

Asn His Tyr Thr Gin Lys Ser Leu

435440

Ser Leu Ser Pro Gly Lys

445 <210>112 <211>84 <212> PROTEIN <213> Homo sapiens <400>112

- 79 040749

Met 1 asp asp asp Ala 5 Ala Pro Arg Vai Glu 10 gly Vai Pro Vai Ala 15 Vai His Lys His Ala 20 Leu His asp gly Leu 25 Arg gin Vai Ala Gly 30 Pro gly Ala Ala Ala 35 Ala His Leu Pro Arg 40 trp Pro Pro Pro Gin 45 Leu Ala Ala Ser Arg 50 Arg Glu Ala Pro Pro 55 Leu Ser gin Arg Pro 60 His Arg Thr gin Gly Ala 65 Wai Lys <210> <211> <212> <213> <220> <223> <400> Gly Ser Pro Pro Glu Thr Asn Glu 70 Glu Lys 113 112 PROTEIN Artificial sequence VL 3A4-L2 113 Lys 75 Leu Thr Asn Pro Gin 80 asp 1 Vai Vai Met Thr 5 gin Thr Pro Leu Ser 10 Leu Pro Vai Thr Pro 15 gly Glu Pro Ala Ser 20 lie Ser Cys Arg Ser 25 Ser gin Ser Leu Leu 30 His Ser Asn gly Asn 35 Thr Tyr Leu Glu TRP 40 Tyr Leu gin Lys Pro 45 gly gin Ser Pro Lys 50 Leu Leu lie Tyr Thr 55 Vai Ser Asn Arg Ph60 Ser gly Vai Pro Asp 65 Arg Phe Ser gly Ser 70 gly Ser gly Thr Asp 75 Phe Thr Leu Lys lie 80 Ser Arg Vai Glu Ala 85 Glu asp Vai gly Wai 90 Tyr Tyr Cys Phe Gin 95 gly Ser His Vai Pro 100 Leu Thr Phe gly gin 105 gly Thr Lys Leu Glu 110 lie Lys

<210> 114

- 80 040749 <211> 219 <212> PROTEIN <213> Artificial sequence <220>

<223> Light chain 3A4-L2 <400>114

Asp Vai Vai Met Thr Gin Thr Pro Leu Ser Leu Pro Vai Thr Pro Gly

1015

Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Ser 20 2530

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 4045

Pro Lys Leu Leu lie Tyr Thr Vai Ser Asn Arg Phe Ser Gly Vai Pro 50 5560

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 7580

Ser Arg Vai Glu Ala Glu Asp Vai Gly Vai Tyr Tyr Cys Phe Gin Gly 85 9095

Ser His Vai Pro Leu Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105110

Arg Thr Vai Ala Ala Pro Ser Vai Phe lie Phe Pro Pro Ser Asp Glu

115 120125

Gin Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe

130 135140

Tyr Pro Arg Glu Ala Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin

145 150 155160

Ser Gly Asn Ser Gin Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser

165 170175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185190

Lys His Lys Vai Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser

195 200205

Pro Vai Thr Lys Ser Phe Asn Arg Gly Glu Cys

210215

- 81 040749 <210> 115 <2Η> 112 <212> PROTEIN <213> Artificial sequence <220>

<223> VL 3Α4-Κ4 <400>115

Asp He Vai Met Thr Gin Ser Pro Asp Ser Leu Ala Vai Ser Leu Gly 15 1015

Glu Arg Ala Thr He Asn Cys Arg Ser Ser Gin Ser Leu Leu His Ser 20 2530

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gin Gin Lys Pro Gly Gin Pro 35 4045

Pro Lys Leu Leu He Tyr Thr Vai Ser Asn Arg Phe Ser Gly Vai Pro 50 5560

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He 65 70 7580

Ser Ser Leu Gin Ala Glu Asp Vai Ala Vai Tyr Tyr Cys Phe Gin Gly 85 9095

Ser His Vai Pro Leu Thr Phe Gly Gin Gly Thr Lys Vai Glu He Lys 100 105110 <210>116 <2H> 219 <212> PROTEIN <213> Artificial sequence <220>

< 223> Light chain 3A4-K4 < 400> 116

Asp He Vai Met Thr Gin Ser Pro 1 5

Asp Ser Leu Ala Vai Ser Leu Gly 10 15

Glu Arg Ala Thr He Asn Cys Arg 20

Ser Ser Gin Ser Leu Leu His Ser

thirty

Asn Gly Asn Thr Tyr Leu Glu Trp

40

Tyr Gin Gin Lys Pro Gly Gin Pro 45

Pro Lys Leu Leu He Tyr Thr Vai 50 55

Ser Asn Arg Phe Ser Gly Vai Pro 60

- 82 040749

Asp Arg Phe Ser Gly Ser Gly Ser 65 70

Gly Thr Asp Phe Thr Leu Thr He

80

Ser Ser Leu Gin Ala Glu Asp Vai 85

Ala Vai Tyr Tyr Cys Phe Gin Gly 90 95

Ser His Vai Pro Leu Thr Phe Gly 100

Gin Gly Thr Lys Vai Glu He Lys 105 110

Arg Thr Vai Ala Ala Pro Ser Vai

115 120

Phe He Phe Pro Ser Asp Glu 125

Gin Leu Lys Ser Gly Thr Ala Ser

130 135

Vai Vai Cys Leu Leu Asn Asn Phe 140

Tyr Pro Arg Glu Ala Lys Vai Gin 145 150

Trp Lys Vai Asp Asn Ala Leu Gin

155 160

Ser Gly Asn Ser Gin Glu Ser Vai 165

Thr Glu Gin Asp Ser Lys Asp Ser

170 175

Thr Tyr Ser Leu Ser Ser Thr Leu 180

Thr Leu Ser Lys Ala Asp Tyr Glu 185 190

Lys His Lys Vai Tyr Ala Cys Glu

195 200

Vai Thr His Gin Gly Leu Ser Ser 205

Pro Vai Thr Lys Ser Phe Asn Arg

210215

Gly Glu Cys <210>117 <400>117

000 <210>118 <400>118

000 <210>119 <400>119

000 <210>120 <400> 120,000 <210>121 <400>121

- 83 040749

LLC <210>122 <400>122

LLC <210>123 <400>123

LLC <210>124 <400>124

OOO <210>125 <400>125

LLC <210>126 <400>126

LLC <210>127 <400>127

LLC <210>128 <400>128

LLC <210>129 <400>129

OOO <210>130 <400>130

LLC <210>131 <400>131

LLC <210>132 <400>132

LLC <210>133 <400>133

OOO <210>134

- 84 040749 <400>134

LLC <210>135 <400>135

LLC <210>136 <400>136

LLC <210>137 <400>137

LLC <210>138 <400>138

LLC <210>139 <400>139

OOO <210>140 <400>140

LLC <210>141 <400>141

LLC <210>142 <400>142

LLC <210>143 <400>143

LLC <210>144 <400>144

OOO <210>145 <400>145

LLC <210>146 <400>146

OOO <210>147

- 85 040749 <400>147

LLC <210>148 <400>148

LLC <210>149 <400>149

OOO <210>150 <400>150

LLC <210>151 <400>151

LLC <210>152 <400>152

LLC <210>153 <400>153

LLC <210>154 <400>154

LLC <210>155 <400>155

LLC <210>156 <400>156

LLC <210>157 <400>157

LLC <210>158 <400>158

LLC <210>159 <400>159

OOO

- 86 040749 <210>160 <400>160

000 <210>161 <400>161

000 <210>162 <400>162

000 <210>163 <400>163

000 <210>164 <400>164

000 <210>165 <400>165

000 <210>166 <400>166

000 <210>167 <400>167

000 <210>168 <400>168

000 <210>169 <400>169

000 <210>170 <400>170

000 <210>171 <400>171

000 <210>172 <400>172

000

- 87 040749

<210> 173 <400> 000 173 <210> 174 <400> 000 174 <210> 175 <400> 000 175 <210> 176 <400> 000 176 <210> 177 <400> 000 177 <210> 178 <400> 000 178 <210> 179 <400> 000 179 <210> 180 <400> 000 180 <210> 181 <400> 000 181 <210> 182 <400> 000 182 <210> 183 <400> 000 183 <210> 184 <400> 000 184 <210> 185 <400> 185

- 88 040749

OOO <210> 186 <400> OOO 186 <210> 187 <400> OOO 187 <210> 188 <400> OOO 188 <210> 189 <400> OOO 189 <210> 190 <400> OOO 190 <210> 191 <400> OOO 191 <210> 192 <400> OOO 192 <210> 193 <400> OOO 193 <210> 194 <400> OOO 194 <210> 195 <400> OOO 195 <210> 196 <400> OOO 196 <210> 197 <400> OOO 197 <210> 198

- 89 040749 <400>198

LLC <210>199 <400>199

OOO <210> 200 <400> 200 OOO <210> 201 <211> 122 <212> PROTEIN <213> Artificial sequence <220>

<223> VH ΧΑ4 <400> 201

Gin Vai His Leu Vai Glu Ser Gly Gly Gly Vai Vai Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Vai Ala Ser Gly lie Thr Phe Arg lie Tyr 20 2530

Gly Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai 35 4045

Ala Vai Leu Trp Tyr Asp Gly Ser His Glu Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala lie Tyr Tyr Cys 85 9095

Ala Arg Asp Gly Asp Tyr Tyr Asp Ser Gly Ser Pro Leu Asp Tyr Trp

100 105110

Gly Gin Gly Thr Leu Vai Thr Vai Ser Ser

115120 <210> 202 <211>107 <212> PROTEIN <213> Artificial sequence <220>

<223> VL XA4 <400> 202

- 90 040749

Glu He Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr 20 2530

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu He 35 4045

Tyr Asp Ala Ser Asn Arg Ala Thr Gly He Pro Ala Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Glu Pro 65 70 7580

Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu 85 9095

Thr Phe Gly Gly Gly Thr Lys Vai Glu He Lys

100105 <210> 203 <2H> 451 <212> PROTEIN <213> Artificial sequence <220>

< 223> Heavy chain XA4 <220>

< 221> MOD_RES < 222> (302) . . (302) <223> Xaa is Asn297 <400> 203

Gin Vai His Leu Vai Glu Ser Gly 1 5

Gly Gly Vai Vai Gin Pro Gly Arg 10 15

Ser Leu Arg Leu Ser Cys Vai Ala 20

Ser Gly He Thr Phe Arg He Tyr 25 30

Gly Met His Trp Vai Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Vai 45

Ala Vai Leu Trp Tyr Asp Gly Ser

55

His Glu Tyr Tyr Ala Asp Ser Vai 60

Lys Gly Arg Phe Thr He Ser Arg 65 70

Asp Asn Ser Lys Asn Thr Leu Tyr

80

- 91 040749

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala He Tyr Tyr Cys 85 9095

Ala Arg Asp Gly Asp Tyr Tyr Asp Ser Gly Ser Pro Leu Asp Tyr Trp 100 105110

Gly Gin Gly Thr Leu Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro

115 120125

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

130 135140

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

145 150 155160

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

165 170175

Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr

180 185190

Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn 195 200205

His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser

210 215220

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

225 230 235240

Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250255

Met He Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser 260 265270

His Glu Asp Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu

275 280285

Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Xaa Ser Thr

290 295300

Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn

305 310 315320

Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro

325 330335

- 92 040749 lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 340 345350

Vai Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Vai

355 360365

Ser Leu Thr Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp lie Ala Vai

370 375380

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

385 390 395400

Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410415

Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai 420 425430

Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu

435 440445

Ser Pro Gly 450 <210> 204 <2H> 214 <212> PROTEIN <213> Artificial sequence <220>

<223> Light chain XA4 <400> 204

Glu lie Vai Leu Thr Gin Ser Pro 1 5

Ala Thr Leu Ser Leu Ser Pro Gly 10 15

Glu Arg Ala Thr Leu Ser Cys Arg 20

Ala Ser Gin Ser Vai Ser Ser Tyr 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro

40

Gly Gin Ala Pro Arg Leu Leu He 45

Tyr Asp Ala Ser Asn Arg Ala Thr 50 55

Gly He Pro Ala Arg Phe Ser Gly 60

Ser Gly Ser Gly Thr Asp Phe Thr 65 70

Leu Thr He Ser Ser Leu Glu Pro

80

Glu Asp Phe Ala Vai Tyr Tyr Cys

Gin Gin Arg Ser Asn Trp Pro Leu

- 93 040749

9095

Thr Phe Gly Gly Gly Thr Lys Vai Glu He Lys Arg Thr Vai Ala Ala 100 105110

Pro Ser Vai Phe He Phe Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120125

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

130 135140

Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

145 150 155160

Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr 180 185190

Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser 195 200205

Phe Asn Arg Gly Glu Cys 210 <210> 205 <2H> 5 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR1 VH XA4 < 400> 205

He Tyr Gly Met His

5 <210> 206 <2H> 17 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR2 VH XA4 < 400> 206

Vai Leu Trp Tyr Asp Gly Ser His Glu Tyr Tyr Ala Asp 15 10

Ser Vai Lys 15

gly

- 94 040749 <210> 207 <211> 13 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR3 VH ΧΑ4 <400> 207

Asp Gly Asp Tyr Tyr Asp Ser Gly Ser Pro Leu Asp Tyr 15 10 <210> 208 <211> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR1 VL XA4 <400> 208

Arg Ala Ser Gin Ser Vai Ser Ser Tyr Leu Ala

10 <210> 209 <211> 7 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR2 VL XA4 < 400> 209

Asp Ala Ser Asn Arg Ala Thr

5 <210> 210 <211> 9 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR3 VL XA4 < 400> 210

Gin Gin Arg Ser Asn Trp Pro Leu Thr

5 <210> 211 <211> 452 <212> PROTEIN <213> Artificial sequence

- 95 040749 <220>

< 223> Heavy chain ΧΑ4 , terminal K <220>

< 221> MOD_RES < 222> (302) .. (302) < 223> Haa is Asn297 < 400> 211

Gin Vai His Leu Vai Glu Ser Gly Gly Gly Vai Vai Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Vai Ala Ser Gly lie Thr Phe Arg lie Tyr 20 2530

Gly Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai 35 4045

Ala Vai Leu Trp Tyr Asp Gly Ser His Glu Tyr Tyr Ala Asp Ser Vai 50 5560

Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala lie Tyr Tyr Cys 85 9095

Ala Arg Asp Gly Asp Tyr Tyr Asp Ser Gly Ser Pro Leu Asp Tyr Trp

100 105110

Gly Gin Gly Thr Leu Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro

115 120125

Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135140

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

145 150 155160

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

165 170175

Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr 180 185190

Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Vai Asn

195 200205

His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser

- 96 040749

210 215

220

Cys Asp Lys Thr His Thr Cys Pro

225 230

Pro Cys Pro Ala Pro Glu Leu Leu

235 240

Gly Gly Pro Ser Vai Phe Leu Phe 245

Pro Pro Lys Pro Lys Asp Thr Leu

250 255

Met He Ser Arg Thr Pro Glu Vai 260

Thr Cys Vai Vai Vai Asp Vai Ser 265 270

His Glu Asp Pro Glu Vai Lys Phe

275 280

Asn Trp Tyr Vai Asp Gly Vai Glu 285

Vai His Asn Ala Lys Thr Lys Pro

290 295

Arg Glu Glu Gin Tyr Xaa Ser Thr 300

Tyr Arg Vai Vai Ser Vai Leu Thr 305 310

Vai Leu His Gin Asp Trp Leu Asn

315 320

Gly Lys Glu Tyr Lys Cys Lys Vai 325

Ser Asn Lys Ala Leu Pro Ala Pro

330 335

He Glu Lys Thr He Ser Lys Ala 340

Lys Gly Gin Pro Arg Glu Pro Gin 345 350

Vai Tyr Thr Leu Pro Pro Ser Arg

355 360

Glu Glu Met Thr Lys Asn Gin Vai 365

Ser Leu Thr Cys Leu Vai Lys Gly

370 375

Phe Tyr Pro Ser Asp He Ala Vai 380

Glu Trp Glu Ser Asn Gly Gin Pro 385 390

Glu Asn Asn Tyr Lys Thr Thr Pro

395 400

Pro Vai Leu Asp Ser Asp Gly Ser

405

Phe Phe Leu Tyr Ser Lys Leu Thr

410 415

Vai Asp Lys Ser Arg Trp Gin Gin 420

Gly Asn Vai Phe Ser Cys Ser Vai 425 430

Met His Glu Ala Leu His Asn His

435 440

Tyr Thr Gin Lys Ser Leu Ser Leu 445

Ser Pro Gly Lys 450 <210> 212

- 97 040749 <2H> 119 <212> PROTEIN <213> Artificial sequence <220>

< 223> VH XFT <400> 212

Gin Vai Glu Leu Vai Gin Ser Gly Ala Vai Lys Lys Pro Gly Glu Ser

1015

Leu Lys He Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp 20 2530

He Gly Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Met Gly 35 4045

He He Asp Pro Gly Asp Ser Arg Thr Arg Tyr Ser Pro Ser Phe Gin 50 5560

Gly Gin Vai Thr He Ser Ala Asp Lys Ser He Ser Thr Ala Tyr Leu 65 70 7580

Gin Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala 85 9095

Arg Gly Gin Leu Tyr Gly Gly Thr Tyr Met Asp Gly Trp Gly Gin Gly 100 105110

Thr Leu Vai Thr Vai Ser Ser

115 <210> 213 <2H>111 <212> PROTEIN <213> Artificial sequence <220>

<223> VL XFT <400> 213

Asp He Ala Leu Thr Gin Pro Ala Ser Vai Ser Gly Ser Pro Gly Gin

1015

Ser He Thr He Ser Cys Thr Gly Thr Ser Ser Asp He Gly Gly Tyr 20 2530

Asn Ser Vai Ser Trp Tyr Gin Gin His Pro Gly Lys Ala Pro Lys Leu 35 4045

Met He Tyr Gly Vai Asn Asn Arg Pro Ser Gly Vai Ser Asn Arg Phe

- 98 040749

5560

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr lie Ser Gly Leu 65 70 7580

Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Asp lie Glu 85 9095

Ser Ala Thr Pro Vai Phe Gly Gly Gly Thr Lys Leu Glu lie Lys

100 105110 <210> 214 <211> 448 <212> PROTEIN <213> Artificial sequence <220>

<223> XET heavy chain <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Haa is Asn297 < 400> 214

Gin Vai Glu Leu Vai Gin Ser Gly 1 5

Ala Vai Lys Lys Pro Gly Glu Ser 10 15

Leu Lys Lie Ser Cys Lys Gly Ser 20

Gly Tyr Ser Phe Thr Ser Tyr Trp 25 30 lie Gly Trp Vai Arg Gin Ala Pro 35 40

Gly Lys Gly Leu Glu Trp Met Gly 45 lie lie Asp Pro Gly Asp Ser Arg 50 55

Thr Arg Tyr Ser Pro Ser Phe Gin 60

Gly Gin Vai Thr lie Ser Ala Asp 65 70

Lys Ser lie Ser Thr Ala Tyr Leu

80

Gin Trp Ser Ser Leu Lys Ala Ser 85

Asp Thr Ala Met Tyr Tyr Cys Ala

95

Arg Gly Gin Leu Tyr Gly Gly Thr 100

Tyr Met Asp Gly Trp Gly Gin Gly 105 110

Thr Leu Vai Thr Vai Ser Ser Ala

115 120

Ser Thr Lys Gly Pro Ser Vai Phe 125

Pro Leu Ala Pro Ser Ser Lys Ser

Thr Ser Gly Gly Thr Ala Ala Leu

- 99 040749

130

135

140

Gly 145

Cys

Leu

Vai

Lys

Asp 150

Tyr

Phe

Pro

Glu

Pro

155

Vai

Thr

Vai

TRP 160

Asn

gly

Ala

Leu

165

Thr

gly

Vai

His

170

Thr

Phe

Pro

Ala

Vai

175

Leu

gin

Gly 180

Leu

Tyr

Leu

Vai

Vai

Thr

185

Vai

190

Pro

Leu

195

gly

Thr

gin

Thr

Tyr 200

Cys

Asn

Vai

Asn

205

His

Lys

Pro

Asn

210

Thr

Lys

Vai

asp

Lys 215

Lys

Vai

Glu

Pro

Lys 220

Cys

asp

Lys

Thr

225

His

Thr

Cys

Pro

Pro

230

Cys

Pro

Ala

Pro

Glu

235

Leu

Leu

gly

gly

Pro

240

Vai

Phe

Leu

Phe

245

Pro

Pro

Lys

Pro

Lys

250

asp

Thr

Leu

Met

255

Arg

Thr

Pro

Glu

260

Vai

Thr

Cys

Vai

Vai

265

Vai

asp

Vai

His

270

Glu

asp

Pro

Glu

Vai

275

Lys

Phe

Asn

trp

Tyr 280

Vai

asp

gly

Vai

Glu

285

Vai

His

Asn

Ala Lys Thr Lys Pro Arg Glu Glu 290 295

Gin Tyr Xaa Ser Thr Tyr Arg Vai 300

Vai Ser Vai Leu Thr Vai Leu His

305 310

Gin Asp Trp Leu Asn Gly Lys Glu

315 320

Tyr Lys Cys Lys Vai Ser Asn Lys 325

Ala Leu Pro Ala Pro lie Glu Lys

330 335

Thr lie Ser Lys Ala Lys Gly Gin 340

Pro Arg Glu Pro Gin Vai Tyr Thr

345 350

Leu Pro Pro Ser Arg Glu Glu Met

355 360

Thr Lys Asn Gin Vai Ser Leu Thr 365

Cys Leu Vai Lys Gly Phe Tyr Pro

370 375

Ser Asp lie Ala Vai Glu Trp Glu 380

Ser Asn Gly Gin Pro Glu Asn Asn

Tyr Lys Thr Thr Pro Pro Vai Leu

- 100 040749

385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu 405 Tyr Ser Lys Leu Thr Vai Asp 410 415 Lys Ser Arg Trp Gin 420 Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His 425 430 Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 215 <2H> 218 <212> PROTEIN <213> Artificial sequence <220> <223> Light chain XET <400> 215 gly Asp He Ala Leu 1 Thr Gin Pro Ala 5 Ser Vai Ser Gly Ser Pro Gly 10 15 gin Ser He Thr He 20 Ser Cys Thr Gly Thr Ser Ser Asp He Gly Gly 25 30 Tyr Asn Ser Vai Ser 35 Trp Tyr Gin Gin 40 His Pro Gly Lys Ala Pro Lys 45 Leu Met He Tyr Gly 50 Vai Asn Asn Arg 55 Pro Ser Gly Vai Ser Asn Arg 60 Phe Ser Gly Ser Lys 65 Ser Gly Asn Thr 70 Ala Ser Leu Thr He Ser Gly 75 Leu 80 Gin Ala Glu Asp Glu Ala Asp Tyr 85 Tyr Cys Ser Ser Tyr Asp He 90 95 Glu Ser Ala Thr Pro 100 Vai Phe Gly Gly Gly Thr Lys Leu Glu He Lys 105 110 Arg Thr Vai Ala Ala 115 Pro Ser Vai Phe 120 He Phe Pro Ser Asp Glu 125 gin Leu Lys Ser Gly 130 Thr Ala Ser Vai 135 Vai Cys Leu Leu Asn Asn Phe 140 Tyr Pro Arg Glu Ala 145 Lys Vai Gin Trp 150 Lys Vai Asp Asn Ala Leu Gin 155 Ser 160

- 101 040749

Gly Asn Ser Gin Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr

165 170175

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

180 185190

His Lys Vai Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro

195 200205

Vai Thr Lys Ser Phe Asn Arg Gly Glu Cys

210215 <210> 216 <2H>10 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR1 VH XFT <400> 216

Gly Tyr Ser Phe Thr Ser Tyr Trp He Gly

10 <210> 217 <2H> 20 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR2 VH XFT <400> 217

Trp Met Gly He He Asp Pro Gly Asp Ser Arg Thr Arg Tyr Ser Pro

10 15

Ser Phe Gin Gly 20 <210> 218 <2H> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR3 VH XFT <400> 218

Gly Gin Leu Tyr Gly Gly Thr Tyr Met Asp Gly

10 <210> 219

- 102 040749 <211> 14 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR1 VL XFT <400> 219

Thr Gly Thr Ser Ser Asp lie Gly Gly Tyr Asn Ser Vai Ser 15 10 <210> 220 <211> 11 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR2 VL XFT <400> 220

Leu Met lie Tyr Gly Vai Asn Asn Arg Pro Ser

10 <210> 221 <211> 10 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR3 VL XFT <400> 221

Ser Ser Tyr Asp lie Glu Ser Ala Thr Pro 15 10 <210> 222 <211> 449 <212> PROTEIN <213> Artificial sequence <220>

<223> XFT heavy chain terminal K <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Haa is Asn297

<400> 222 Gin 1 Vai Glu Leu Wai 5 gin Ser gly Ala Wai 10 Lys Lys Pro gly Glu 15 Ser Leu Lys lie Ser 20 Cys Lys gly Ser Gly 25 Tyr Ser Phe Thr Ser thirty Tyr trp

- 103 040749

He Gly Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Met Gly 35 4045

He He Asp Pro Gly Asp Ser Arg Thr Arg Tyr Ser Pro Ser Phe Gin 50 5560

Gly Gin Vai Thr He Ser Ala Asp Lys Ser He Ser Thr Ala Tyr Leu 65 70 7580

Gin Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala 85 9095

Arg Gly Gin Leu Tyr Gly Gly Thr Tyr Met Asp Gly Trp Gly Gin Gly

100 105110

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

115 120125

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

130 135140

Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp

145 150 155160

Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu

165 170175

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser

180 185190

Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His Lys Pro

195 200205

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

210 215220

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

225 230 235240

Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser

245 250255

Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp

260 265270

Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

275 280285

- 104 040749

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Xaa Ser Thr Tyr Arg Vai 290 295300

Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

305 310 315320

Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys

325 330335

Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

340 345350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Vai Ser Leu Thr

355 360365

Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp He Ala Vai Glu Trp Glu 370 375380

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu

385 390 395400

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

405 410415

Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu 420 425430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

435 440445

Lys <210> 223 <2H>119 <212> PROTEIN <213> Artificial sequence <220>

<223> VH X09 <400> 223

Gin Vai Gin Leu Gin Gin Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

10 15

Ser Vai Lys lie Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30

Thr Met Asn Trp Vai Lys Gin Ser His Gly Lys Ser Leu Glu Trp He

- 105 040749

Gly Leu 50

Arg Gly 65

Met Asp

Ala Arg

He Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn 5560

Lys Ala Thr Leu Thr Vai Asp Lys Ser Ser Ser 7075

Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Vai 8590

Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp 100105

Gin Lys Phe

Thr Ala Tyr 80

Tyr Phe Cys 95

Gly Ser Gly 110

Thr Pro Vai Thr Vai Ser Ser

115 <210> 224 <2H> 106 <212> PROTEIN <213> Artificial sequence <220>

< 223> VL X09 < 400> 224

Asp He Glu Leu Thr Gin Ser Pro 1 5

Ala He Met Ser Ala Ser Pro Gly 10 15

Glu Lys Vai Thr Met Thr Cys Ser 20

Ala Ser Ser Ser Vai Ser Tyr Met 25 30

His Trp Tyr Gin Gin Lys Ser Gly 35 40

Thr Ser Pro Lys Arg Trp He Tyr 45

Asp Thr Ser Lys Leu Ala Ser Gly 50 55

Vai Pro Gly Arg Phe Ser Gly Ser 60

Gly Ser Gly Asn Ser Tyr Ser Leu 65 70

Thr He Ser Ser Vai Glu Ala Glu

80

Asp Asp Ala Thr Tyr Tyr Cys Gin 85

Gin Trp Ser Lys His Pro Leu Thr

95

Phe Gly Ser Gly Thr Lys Vai Glu 100

He Lys 105 <210> 225 <2H> 448 <212> PROTEIN

- 106 040749 <213> Artificial sequence <220>

<223> Heavy Chain X09 <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Haa is Asn297 < 400> 225

Gin Vai Gin Leu Gin Gin Ser Gly 1 5

Pro Glu Leu Glu Lys Pro Gly Ala 10 15

Ser Vai Lys He Ser Cys Lys Ala 20

Ser Gly Tyr Ser Phe Thr Gly Tyr 25 30

Thr Met Asn Trp Vai Lys Gin Ser

40

His Gly Lys Ser Leu Glu Trp He 45

Gly Leu He Thr Pro Tyr Asn Gly 50 55

Ala Ser Ser Tyr Asn Gin Lys Phe 60

Arg Gly Lys Ala Thr Leu Thr Vai 65 70

Asp Lys Ser Ser Ser Thr Ala Tyr 75 80

Met Asp Leu Leu Ser Leu Thr Ser 85

Glu Asp Ser Ala Vai Tyr Phe Cys 90 95

Ala Arg Gly Gly Tyr Asp Gly Arg

100

Gly Phe Asp Tyr Trp Gly Ser Gly

105 110

Thr Pro Vai Thr Vai Ser Ser Ala

115 120

Ser Thr Lys Gly Pro Ser Vai Phe 125

Pro Leu Ala Pro Ser Ser Lys Ser

130 135

Thr Ser Gly Gly Thr Ala Ala Leu 140

Gly Cys Leu Vai Lys Asp Tyr Phe 145 150

Pro Glu Pro Vai Thr Vai Ser Trp

155 160

Asn Ser Gly Ala Leu Thr Ser Gly 165

Vai His Thr Phe Pro Ala Vai Leu

170 175

Gin Ser Ser Gly Leu Tyr Ser Leu 180

Ser Ser Vai Vai Thr Vai Pro Ser

185 190

Ser Ser Leu Gly Thr Gin Thr Tyr

195 200

He Cys Asn Vai Asn His Lys Pro 205

- 107 040749

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

210 215 220

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

225 230 235 240

Ser Vai Phe Leu Phe Pro Pro Lys 245

Pro Lys Asp Thr Leu Met He Ser

250 255

Arg Thr ProGlu

260

Vai Thr

Cys Vai Vai Vai Asp Vai

265

Ser His Glu Asp

270

Pro Glu Vai Lys

275

Phe Asn

Trp Tyr Vai Asp Gly Vai

280

Glu Vai His Asn 285

Ala Lys Thr Lys 290

Pro Arg

Glu Glu Gin Tyr Xaa Ser

295 300

Thr Tyr Arg Vai

Vai Ser Vai Leu Thr Vai Leu His

305 310

Gin Asp Trp Leu Asn Gly Lys Glu

315 320

Tyr Lys

Cys Lys Vai 325

Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys

330 335

Thr He

Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

340 345 350

Leu Pro

Cys Leu

370

Ser Asn

385

Asp Ser

Pro 355

Vai

gly

asp

Ser Arg Asp Glu Leu

360

Lys Gly Phe Tyr Pro 375

Gin Pro Glu Asn Asn

390

Gly Ser Phe Phe Leu 405

Thr Lys Asn Gin Vai Ser Leu Thr 365

Ser Asp He Ala Vai Glu Trp Glu 380

Tyr Lys Thr Thr Pro Pro Vai Leu

395 400

Tyr Ser Lys Leu Thr Vai Asp Lys

410 415

Ser Arg Trp Gin Gin Gly Asn Vai 420

Phe Ser Cys Ser Vai Met His Glu 425 430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445 <210> 226 <2H> 213 <212> PROTEIN

- 108 040749 <213> Artificial sequence <220>

<223> Light chain X09 <400> 226

Asp lie Glu Leu Thr Gin Ser Pro 1 5

Ala He Met Ser Ala Ser Pro Gly

15

Glu Lys Vai Thr Met Thr Cys Ser 20

Ala Ser Ser Ser Vai Ser Tyr Met 25 30

His Trp Tyr Gin Gin Lys Ser Gly 35 40

Thr Ser Pro Lys Arg Trp He Tyr 45

Asp Thr Ser Lys Leu Ala Ser Gly 50 55

Vai Pro Gly Arg Phe Ser Gly Ser 60

Gly Ser Gly Asn Ser Tyr Ser Leu 65 70

Thr He Ser Ser Vai Glu Ala Glu

80

Asp Asp Ala Thr Tyr Tyr Cys Gin 85

Gin Trp Ser Lys His Pro Leu Thr 90 95

Phe Gly Ser Gly Thr Lys Vai Glu 100

He Lys Arg Thr Vai Ala Ala Pro 105 110

Ser Vai Phe He Phe Pro Pro Ser

115 120

Asp Glu Gin Leu Lys Ser Gly Thr 125

Ala Ser Vai Vai Cys Leu Leu Asn

130 135

Asn Phe Tyr Pro Arg Glu Ala Lys 140

Vai Gin Trp Lys Vai Asp Asn Ala 145 150

Leu Gin Ser Gly Asn Ser Gin Glu

155 160

Ser Vai Thr Glu Gin Asp Ser Lys 165

Asp Ser Thr Tyr Ser Leu Ser Ser

170 175

Thr Leu Thr Leu Ser Lys Ala Asp 180

Tyr Glu Lys His Lys Vai Tyr Ala 185 190

Cys Glu Vai Thr His Gin Gly Leu

195 200

Ser Ser Pro Vai Thr Lys Ser Phe 205

Asn Arg Gly Glu Cys 210 <210> 227

- 109 040749 <211> 5 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR1 VH X09 < 400> 227

Gly Tyr Ser Phe Thr

5 <210> 228 <211> 17 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR2 VH X09 <400> 228

Leu lie Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gin Lys Phe Arg

10 15

gly

<210> <211> <212> <213> 229 10 PROTEIN Artificial sequence

<220> <223> CDR3 VH X09 <400> 229

Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr

1 5 10 <210> <211> <212> <213> 230 10 PROTEIN Artificial sequence

<220> <223> CDR1 VL X09 <400> 230

Ser Ala Ser Ser Ser Vai Ser Tyr Met His

1 5 10 <210> <211> <212> <213> 231 7 PROTEIN Artificial sequence

- 110 040749 <220>

<223> CDR2 VL Χ09 <400> 231

Asp Thr Ser Lys Leu Ala Ser

5 <210> 232 <2H> 9 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR3 VL X09 < 400> 232

Gin Gin Trp Ser Lys His Pro Leu Thr

5 <210> 233 <2H> 449 <212> PROTEIN <213> Artificial sequence <220>

<223> Heavy chain X09, terminal K <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Haa is Asn297 < 400> 233

Gin Vai Gin Leu Gin Gin Ser Gly 1 5

Pro Glu Leu Glu Lys Pro Gly Ala 10 15

Ser Vai Lys He Ser Cys Lys Ala 20

Ser Gly Tyr Ser Phe Thr Gly Tyr 25 30

Thr Met Asn Trp Vai Lys Gin Ser

40

His Gly Lys Ser Leu Glu Trp He 45

Gly Leu He Thr Pro Tyr Asn Gly

55

Ala Ser Ser Tyr Asn Gin Lys Phe 60

Arg Gly Lys Ala Thr Leu Thr Vai 65 70

Asp Lys Ser Ser Ser Thr Ala Tyr

80

Met Asp Leu Leu Ser Leu Thr Ser 85

Glu Asp Ser Ala Vai Tyr Phe Cys

95

Ala Arg Gly Gly Tyr Asp Gly Arg

Gly Phe Asp Tyr Trp Gly Ser Gly

- 111 040749

100

105

BUT

Thr Pro Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe 115 120125

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

130 135140

Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp

145 150 155160

Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu

165 170175

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser 180 185190

Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His Lys Pro 195 200205

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

210 215220

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

225 230 235240

Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser

245 250255

Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp 260 265270

Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

275 280285

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Xaa Ser Thr Tyr Arg Vai

290 295300

Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

305 310 315320

Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys

325 330335

Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

340 345350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr

- 112 040749

355 360365

Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu

370 375380

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu

385 390 395400

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

405 410415

Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu 420 425430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440445

Lys <210> 234 <2H>119 <212> PROTEIN <213> Artificial sequence <220>

<223> VH X09.2 <400> 234

Gin Vai Gin Leu Gin Gin Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

1015

Ser Vai Lys He Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 2530

Thr Met Asn Trp Vai Lys Gin Ser His Gly Lys Ser Leu Glu Trp He 35 4045

Gly Leu He Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gin Lys Phe 50 5560

Arg Gly Lys Ala Thr Leu Thr Vai Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 7580

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Vai Tyr Phe Cys 85 9095

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gin Gly 100 105110

- 113 040749

Thr Thr Vai Thr Vai Ser Ser

115 <210> 235 <2H> 106 <212> PROTEIN <213> Artificial sequence <220>

<223> VL X09.2 <400>235

Asp He Glu Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly

1015

Glu Lys Vai Thr Met Thr Cys Ser Ala Ser Ser Ser Vai Ser Tyr Met 20 2530

His Trp Tyr Gin Gin Lys Ser Gly Thr Ser Pro Lys Arg Trp He Tyr 35 4045

Asp Thr Ser Lys Leu Ala Ser Gly Vai Pro Gly Arg Phe Ser Gly Ser 50 5560

Gly Ser Gly Asn Ser Tyr Ser Leu Thr He Ser Ser Vai Glu Ala Glu 65 70 7580

Asp Asp Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Gly Tyr Pro Leu Thr 85 9095

Phe Gly Ala Gly Thr Lys Leu Glu He Lys 100105 <210> 236 <2H>448 <212> PROTEIN <213> Artificial sequence <220>

<223> Heavy chain X09.2 <220>

<221> MOD_RES <222> (299)..(299) <223> Haa is Asn297 <400> 236

Gin Vai Gin Leu Gin Gin Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

10 15

Ser Vai Lys He Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

- 114 040749

2530

Thr Met Asn Trp Vai Lys Gin Ser His Gly Lys Ser Leu Glu Trp He 35 4045

Gly Leu He Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gin Lys Phe 50 5560

Arg Gly Lys Ala Thr Leu Thr Vai Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 7580

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Vai Tyr Phe Cys 85 9095

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Ser Gly 100 105110

Thr Pro Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe 115 120125

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

130 135140

Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp

145 150 155160

Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu

165 170175

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser 180 185190

Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His Lys Pro 195 200205

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

210 215220

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

225 230 235240

Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser

245 250255

Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp 260 265270

Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

- 115 040749

275 280285

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Xaa Ser Thr Tyr Arg Vai 290 295300

Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

305 310 315320

Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys

325 330335

Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr 340 345350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr

355 360365

Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp He Ala Vai Glu Trp Glu

370 375380

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu

385 390 395400

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

405 410415

Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu 420 425430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440445 <210> 237 <2H> 213 <212> PROTEIN <213> Artificial sequence <220>

<223> Light chain X09.2 <400> 237

Asp He Glu Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly

1015

Glu Lys Vai Thr Met Thr Cys Ser Ala Ser Ser Ser Vai Ser Tyr Met 20 2530

His Trp Tyr Gin Gin Lys Ser Gly Thr Ser Pro Lys Arg Trp lie Tyr 35 4045

- 116 040749

Asp Thr Ser Lys 50

Leu Ala

Ser Gly Vai Pro Gly Arg Phe Ser Gly Ser 55 60

Gly Ser Gly Asn Ser Tyr 65 70

Ser Leu Thr He Ser Ser Vai Glu Ala Glu

80

Asp Asp Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Lys His Pro Leu Thr 85 90 95

Phe Gly Ser Gly Thr Lys Vai Glu He Lys Arg Thr Vai Ala Ala Pro

100 105 110

Ser Vai Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125

Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu

145 150 155 160

Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr Ala 180 185 190

Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys 210 <210> 238 < 2H> 5 < 212> PROTEIN < 213> Artificial sequence <220>

< 223> CDR1 VH X09.2 < 400> 238

Gly Tyr Thr Met Asn

5 <210> 239 <2H> 17 <212> PROTEIN <213> Artificial sequence

- 117 040749 <220>

<223> CDR2 VH Χ09.2 <400> 239

Leu He Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gin Lys Phe Arg

10 15

gly

<210> <2H> <212> <213> 240 10 PROTEIN Artificial sequence <220> <223> CDR3 VH X09.2 <400> 240 Gly Gl^ 'Tyr Asp Gly Arg Gly Phe Asp Tyr 1 5 10

<210> 241 <2H> 10 <212> PROTEIN <213> Artificial sequence <220>

<223> CDR1 VL X09.2 <400> 241

Ser Ala Ser Ser Ser Vai Ser Tyr Met His 15 10 <210> 242 <2H> 7 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR2 VL X09.2 <400> 242

Asp Thr Ser Lys Leu Ala Ser

5 <210> 243 <2H> 9 <212> PROTEIN <213> Artificial sequence <220>

< 223> CDR3 VL X09.2 <400> 243

- 118 040749

Gin Gin Trp Ser Gly Tyr Pro Leu Thr 1 5 <210> 244 <2H> 449 < 212> PROTEIN < 213> Artificial sequence <220>

<223> Heavy chain X09.2 terminal K <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Haa is Asn297 < 400> 244

Gin Vai Gin Leu Gin Gin Ser Gly 1 5

Pro Glu Leu Glu Lys Pro Gly Ala

15

Ser Vai Lys He Ser Cys Lys Ala 20

Ser Gly Tyr Ser Phe Thr Gly Tyr 25 30

Thr Met Asn Trp Vai Lys Gin Ser

40

His Gly Lys Ser Leu Glu Trp He 45

Gly Leu He Thr Pro Tyr Asn Gly

55

Ala Ser Ser Tyr Asn Gin Lys Phe 60

Arg Gly Lys Ala Thr Leu Thr Vai 65 70

Asp Lys Ser Ser Ser Thr Ala Tyr

80

Met Asp Leu Leu Ser Leu Thr Ser

Glu Asp Ser Ala Vai Tyr Phe Cys

95

Ala Arg Gly Gly Tyr Asp Gly Arg 100

Gly Phe Asp Tyr Trp Gly Ser Gly 105 110

Thr Pro Vai Thr Vai Ser Ser Ala

115 120

Ser Thr Lys Gly Pro Ser Vai Phe

125

Pro Leu Ala Pro Ser Ser Lys Ser

130 135

Thr Ser Gly Gly Thr Ala Ala Leu 140

Gly Cys Leu Vai Lys Asp Tyr Phe 145 150

Pro Glu Pro Vai Thr Vai Ser Trp

155 160

Asn Ser Gly Ala Leu Thr Ser Gly 165

Vai His Thr Phe Pro Ala Vai Leu

170 175

- 119 040749

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser 180 185190

Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His Lys Pro 195 200205

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

210 215220

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

225 230 235240

Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser

245 250255

Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp 260 265270

Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

275 280285

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Xaa Ser Thr Tyr Arg Vai 290 295300

Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

305 310 315320

Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys

325 330335

Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr 340 345350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr 355 360365

Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp He Ala Vai Glu Trp Glu

370 375380

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu

385 390 395400

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

405 410415

Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu 420 425430

- 120 040749

Ala Leu His Asn His Tyr Thr Gin

435 440

Lys Ser Leu Ser Leu Ser Pro Gly 445

Lys <210> 245 <2H> 630 <212> PROTEIN <213> Homo sapiens <400> 245

Met Ala Leu Pro Thr Ala Arg Pro

5

Leu Leu Gly Ser Cys Gly Thr Pro 10 15

Ala Leu Gly Ser Leu Leu Phe Leu 20

Leu Phe Ser Leu Gly Trp Vai Gin 25 30

Pro Ser Arg Thr Leu Ala Gly Glu

40

Thr Gly Gin Glu Ala Ala Pro Leu 45

Asp Gly Vai Leu Ala Asn Pro Pro

55

Asn He Ser Ser Leu Ser Pro Arg 60

Gin Leu Leu Gly Phe Pro Cys Ala 65 70

Glu Vai Ser Gly Leu Ser Thr Glu

80

Arg Vai Arg Glu Leu Ala Vai Ala 85

Leu Ala Gin Lys Asn Vai Lys Leu

95

Ser Thr Glu Gin Leu Arg Cys Leu 100

Ala His Arg Leu Ser Glu Pro Pro 105 110

Glu Asp Leu Asp Ala Leu Pro Leu

115 120

Asp Leu Leu Leu Phe Leu Asn Pro 125

Asp Ala Phe Ser Gly Pro Gin Ala

130 135

Cys Thr Arg Phe Phe Ser Arg He 140

Thr Lys Ala Asn Vai Asp Leu Leu 145 150

Pro Arg Gly Ala Pro Glu Arg Gin

155 160

Arg Leu Leu Pro Ala Ala Leu Ala 165

Cys Trp Gly Vai Arg Gly Ser Leu

170 175

Leu Ser Glu Ala Asp Vai Arg Ala 180

Leu Gly Gly Leu Ala Cys Asp Leu 185 190

- 121 040749

Pro Gly Arg Phe Vai Ala Glu Ser Ala Glu Vai Leu Leu Pro Arg Leu

195 200205

Vai Ser Cys Pro Gly Pro Leu Asp Gin Asp Gin Gin Glu Ala Ala Arg

210 215220

Ala Ala Leu Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp

225 230 235240

Ser Vai Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Vai Leu Gly

245 250255

Gin Pro lie lie Arg Ser lie Pro Gin Gly lie Vai Ala Ala Trp Arg 260 265270

Gin Arg Ser Ser Arg Asp Pro Ser Trp Arg Gin Pro Glu Arg Thr lie

275 280285

Leu Arg Pro Arg Phe Arg Arg Glu Vai Glu Lys Thr Ala Cys Pro Ser

290 295300

Gly Lys Lys Ala Arg Glu lie Asp Glu Ser Leu lie Phe Tyr Lys Lys

305 310 315320

Trp Glu Leu Glu Ala Cys Vai Asp Ala Ala Leu Leu Ala Thr Gin Met

325 330335

Asp Arg Vai Asn Ala lie Pro Phe Thr Tyr Glu Gin Leu Asp Vai Leu 340 345350

Lys His Lys Leu Asp Glu Leu Tyr Pro Gin Gly Tyr Pro Glu Ser Vai

355 360365 lie Gin His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp lie 370 375380

Arg Lys Trp Asn Vai Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu

385 390 395400

Vai Asn Lys Gly His Glu Met Ser Pro Gin Ala Pro Arg Arg Pro Leu

405 410415

Pro Gin Vai Ala Thr Leu lie Asp Arg Phe Vai Lys Gly Arg Gly Gin

420 425430

Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr 435 440445

- 122 040749

Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Vai Pro Pro Ser Ser 450 455460

He Trp Ala Vai Arg Pro Gin Asp Leu Asp Thr Cys Asp Pro Arg Gin

465 470 475480

Leu Asp Vai Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gin Asn Met Asn

485 490495

Gly Ser Glu Tyr Phe Vai Lys He Gin Ser Phe Leu Gly Gly Ala Pro

500 505510

Thr Glu Asp Leu Lys Ala Leu Ser Gin Gin Asn Vai Ser Met Asp Leu 515 520525

Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Vai Leu Pro Leu Thr Vai 530 535540

Ala Glu Vai Gin Lys Leu Leu Gly Pro His Vai Glu Gly Leu Lys Ala

545 550 555560

Glu Glu Arg His Arg Pro Vai Arg Asp Trp He Leu Arg Gin Arg Gin

565 570575

Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gin Gly Gly He Pro Asn

580 585590

Gly Tyr Leu Vai Leu Asp Leu Ser Met Gin Glu Ala Leu Ser Gly Thr 595 600605

Pro Cys Leu Leu Gly Pro Gly Pro Vai Leu Thr Vai Leu Ala Leu Leu

610 615620

Leu Ala Ser Thr Leu Ala

625630 <210> 246 <2H>622 <212> PROTEIN <213> Homo sapiens <400> 246

Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro

10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Vai Gin 20 25 30

Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gin Glu Ala Ala Pro Leu

- 123 040749

4045

Asp Gly Vai Leu Ala Asn Pro Asn He Ser Ser Leu Ser Pro Arg 50 5560

Gin Leu Leu Gly Phe Pro Cys Ala Glu Vai Ser Gly Leu Ser Thr Glu 65 70 7580

Arg Vai Arg Glu Leu Ala Vai Ala Leu Ala Gin Lys Asn Vai Lys Leu 85 9095

Ser Thr Glu Gin Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro 100 105110

Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro

115 120125

Asp Ala Phe Ser Gly Pro Gin Ala Cys Thr Arg Phe Phe Ser Arg He 130 135140

Thr Lys Ala Asn Vai Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gin

145 150 155160

Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Vai Arg Gly Ser Leu

165 170175

Leu Ser Glu Ala Asp Vai Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu 180 185190

Pro Gly Arg Phe Vai Ala Glu Ser Ala Glu Vai Leu Leu Pro Arg Leu

195 200205

Vai Ser Cys Pro Gly Pro Leu Asp Gin Asp Gin Gin Glu Ala Ala Arg

210 215220

Ala Ala Leu Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp

225 230 235240

Ser Vai Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Vai Leu Gly

245 250255

Gin Pro lie lie Arg Ser lie Pro Gly lie Vai Ala Ala Trp Arg

260 265270

Gin Arg Ser Ser Arg Asp Pro Ser Trp Arg Gin Pro Glu Arg Thr He

275 280285

Leu Arg Pro Arg Phe Arg Arg Glu Vai Glu Lys Thr Ala Cys Pro Ser

- 124 040749

290 295 300

Gly 305 Lys Lys Ala Arg Glu 310 He asp Glu Ser Leu 315 He Phe Tyr Lys Lys 320 trp Glu Leu Glu Ala 325 Cys Vai asp Ala Ala 330 Leu Leu Ala Thr gin 335 Met asp Arg Vai Asn 340 Ala He Pro Phe Thr 345 Tyr Glu gin Leu asp 350 Vai Leu Lys His Lys 355 Leu asp Glu Leu Tyr 360 Pro gin gly Tyr Pro 365 Glu Ser Vai He gin 370 His Leu gly Tyr Leu 375 Phe Leu Lys Met Ser 380 Pro Glu asp He Arg 385 Lys trp Asn Vai Thr 390 Ser Leu Glu Thr Leu 395 Lys Ala Leu Leu Glu 400 Vai Asn Lys gly His 405 Glu Met Ser Pro gin 410 Vai Ala Thr Leu He 415 asp Arg Phe Vai Lys 420 gly Arg gly gin Leu 425 asp Lys asp Thr Leu 430 asp Thr Leu Thr Ala 435 Phe Tyr Pro gly Tyr 440 Leu Cys Ser Leu Ser 445 Pro Glu Glu Leu Ser 450 Ser Vai Pro Pro Ser 455 Ser He trp Ala Vai 460 Arg Pro gin asp Leu 465 asp Thr Cys asp Pro 470 Arg gin Leu asp Vai 475 Leu Tyr Pro Lys Ala 480 Arg Leu Ala Phe gin 485 Asn Met Asn gly Ser 490 Glu Tyr Phe Vai Lys 495 He gin Ser Phe Leu 500 gly gly Ala Pro Thr 505 Glu asp Leu Lys Ala 510 Leu Ser gin gin Asn 515 Vai Ser Met asp Leu 520 Ala Thr Phe Met Lys 525 Leu Arg Thr asp Ala 530 Vai Leu Pro Leu Thr 535 Vai Ala Glu Vai gin 540 Lys Leu Leu gly Pro His Vai Glu gly Leu Lys Ala Glu Glu Arg His Arg Pro Vai Arg

- 125 -

Claims (25)

CLAIM 1. DLK1 binding conjugate of formula (I): Ab-(DL) _P (I) wherein Ab is an antibody that binds to DLK1; DL represents Where X is selected from the group consisting of: single bond, -CH2- 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, carboxy, C _1-7 alkyl, C _3-7 heterocyclyl , 6is-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 aryl group; (ib) C1.5 saturated aliphatic alkyl; (ic) C _3-6 saturated cycloalkyl; ^R22 R ²³ (id) ^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, said total number of carbon atoms in the R ² group being not more than 5; R ^25b (ie) where one of R ^25a and R ^25b is H and the other is selected from: phenyl, wherein said phenyl is unsubstituted or substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; And (if) ^r24 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; if a single bond is present between C2 and C3, R ² is , where R ^26a and R ^26b is 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 there is a double bond between C2' and C3', R ¹² is selected from the group consisting of: (iia) C ₅ . aryl group, unsubstituted or substituted with one or more substituents selected from the group consisting of: halogen, nitro, cyano, C _1-7 alkoxy, carboxy, C _1-7 alkyl, C _3-7 heterocyclyl , 6is-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 aryl group; (iib) C _1-5 saturated aliphatic alkyl; (iic) C _3-6 saturated cycloalkyl; (iid) where each of R ³¹ , R ³² and R ³³ is independently selected from H, C _1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, said total number of carbon atoms in the R ¹² group being not more than 5; ⁽ⁱⁱ e) where one of R ^35a and R ^35b is H and the other is selected from: phenyl, wherein phenyl is unsubstituted or substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; And (iif) ^r34 where R ³⁴ 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; if a single bond is present between C2' and C3', R ¹² is where R ^36a and R ^36b 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 Cm 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; where C _3-20 heterocyclyl group contains from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms selected from N, O, S; and the C _3-7 heterocyclyl group contains 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms selected from N, O, S. 2. A conjugate according to claim 1, characterized in that X is -CH2-. 3. Conjugate according to any one of claims 1 or 2, characterized in that n is from 1 to 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 from one to three substituent groups, said substituents being selected from methoxy, ethoxy, fluoro, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl; or - 127 040749 (b) cyclopropyl; or (c) a group of formula: while the specified total number of carbon atoms in the group R ² is not more than 3; or (d) a group (e) a group of the formula wherein R ²⁴ is selected from H and methyl. 8. The conjugate according to any one of claims 1-4, characterized in that between C2 and C3 there is one a single bond, 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 1-8, 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 from one to three substituent groups, said substituents being selected from methoxy, ethoxy, fluorine, chlorine, cyano, bisoxymethylene, methylpiperazinyl, morpholino and methylthiophenyl; or (b) cyclopropyl; or (c) a group of formula ^R32 while the specified 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 ^^^R ³⁴ wherein R ³⁴ is selected from H and methyl. 12. The conjugate according to any one of claims 1 to 8, characterized in that between C2' and C3' there is one D^ ^36a single bond, R ¹² is R ^36b 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 alke- - 128 040749 nila, and the alkyl and alkenyl groups are not substituted or substituted. 13. The conjugate according to claim 1, characterized in that DL is: 14. A conjugate according to any one of claims 1 to 13, characterized in that said antibody contains a VH domain containing a VH CDR3 with the amino acid sequence of SEQ ID NO. 7, CDR2 VH with the amino acid sequence of SEQ ID NO. 6 and CDR1 VH with the amino acid sequence of SEQ ID NO. 5. 15. A conjugate according to any one of claims 1 to 14, characterized in that said antibody contains a VH domain containing the sequence of SEQ ID NO. 1. 16. A conjugate according to any one of claims 1 to 15, characterized in that said antibody contains a VL domain containing a VL CDR3 with the amino acid sequence of SEQ ID NO. 10, CDR2 VL with the amino acid sequence of SEQ ID NO. 9 and CDR1 VL with the amino acid sequence of SEQ ID NO. 8. 17. A conjugate according to any one of claims 1 to 16, characterized in that said antibody contains a VL domain containing the sequence of SEQ ID NO. 2. 18. A conjugate according to any one of claims 1 to 17, characterized in that said antibody contains a heavy chain containing the sequence of SEQ ID NO. 3, or a heavy chain containing the sequence of SEQ ID NO. eleven. 19. A conjugate according to any one of claims 1 to 18, characterized in that said antibody contains a light chain containing the sequence of SEQ ID NO. 4. 20. A composition for treating a proliferative disease in a subject, comprising a mixture of antibody-drug conjugates according to any one of claims 1-19, wherein said average drug load per antibody in said mixture of antibody-drug conjugates is from 1 to 4. 21. The use of a conjugate according to any one of claims 1-19 for the treatment of a proliferative disease in a subject. 22. Use according to claim 21, characterized in that said disease is cancer. 23. Use according to claim 22, characterized in that said cancer is a cancer selected from the group consisting of: hepatocellular carcinoma, hepatoblastoma, non-small cell lung cancer, small cell lung cancer, colon cancer, breast cancer, stomach cancer, cancer pancreas, neuroblastoma, adrenal cancer, pheochromocytoma, paraganglioma, medullary thyroid carcinoma, skeletal muscle cancer, liposarcoma, glioma, Wilms tumor, neuroendocrine tumors, acute myeloid leukemia and myelodysplastic syndrome. 24. A pharmaceutical composition for the treatment of a proliferative disease in a subject, comprising a conjugate according to any one of claims 1 to 19 and a pharmaceutically acceptable diluent, carrier or excipient. 25. The pharmaceutical composition of claim 24, further comprising a therapeutically effective amount of a chemotherapeutic agent. - 129 -