EA040749B1 - PYRROLOBENZODIAZEPINE-ANTIBODY CONJUGATES - Google Patents

Description

Cross-reference to related applications

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

Field of technology

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

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)). Subsequently, a number of natural PBBs were reported and more than 10 routes to the synthesis of various analogues were developed (Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. and Thurston, D.E., Chem. Rev. 2011 111 (4), 2815-2864). Members of the family include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chikamycin (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)), mazetramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porotramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), protracarcin (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 tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBBs have the general structure:

They differ in the number, type and position of substituents in both their aromatic rings A and pyrrole rings C, as well as in the degree of saturation of the C ring. The B ring contains either an imine (N=C), a carbinolamine (NH-CH(OH)), or a methyl ether of carbinolamine (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center responsible for DNA alkylation. All of the known natural products have a ^-configuration at the chiral C11a position, resulting in a right-handed twist conformation when viewed from ring C to ring A. This gives them a suitable three-dimensional shape for isohelical conformation with the minor groove of B-form DNA, resulting in a tight 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, and so they are used as antitumor agents.

One pyrrolobenzodiazepine compound is described in 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 compound, also known as SG2000, is shown below:

SG2000

WO 2007/085930 describes the preparation of dimeric PBD compounds containing linker groups for coupling to a cell binding agent such as an antibody. The linker is present in the bridge connecting the monomeric units of the PBD dimer.

Dimeric PBB compounds containing linker groups for coupling 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 portion of the PBB at the C2 position and is typically cleaved by enzyme action 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 portion of the PBB and is typically cleaved by enzyme action 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 for the destruction or inhibition of tumor cells in the treatment of cancer, targets the delivery of the drug moiety to tumors and their intracellular accumulation in them, while systemic administration of these drugs in unconjugated form can lead to unacceptable levels of toxicity to normal cells (Xie et al (2006) Expert. Opin. Biol. Ther. 6(3):281–291; Kovtun et al (2006) Cancer Res. 66(6):3214–3121; Law et al (2006) Cancer Res. 66(4):2328–2337; Wu et al (2005) Nature Biotech. 23(9): 1137–1145; Lambert J. (2005) Current Opin. in Pharmacol. 5:543–549; Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103; Payne, G. (2003) Cancer Cell 3:207–212; Trail et al (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614).

Thus, maximum efficiency with minimum toxicity is required. Efforts to develop and improve ADCs have focused on the selectivity of monoclonal antibodies (mAbs), as well as the mechanism of drug action, drug attachment, drug/antibody ratio (loading), 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 involving tubulin binding, DNA binding, proteasome and/or topoisomerase inhibition. Some cytotoxic drugs are generally inactive or less active when conjugated to large antibodies or protein receptor ligands.

The present inventors have developed specific conjugates of dimeric PBD and an antibody.

The essence of the invention

According to a first aspect of the present invention, there is provided a conjugate of formula (I):

Ab-(DL) _P (I) in which

Ab is an antibody that binds to DLK1;

DL is

Where

X is selected from the group consisting of a single bond, -CH2- and -C2H4-;

n ranges from 1 to 8;

m is 0 or 1;

R ⁷ represents methyl or phenyl;

if there is a double bond between C2 and C3, R ² is selected from a 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, _C1-7 alkyl, _C3-7 heterocyclyl and bis-oxy- _C1-3 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 αalkenyl, C _2-3 αalkenyl and cyclopropyl, wherein the total number of carbon atoms in the R ¹² group is no 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 halogen, methyl, methoxy; pyridyl; and thiophenyl; and

(if) wherein R ²⁴ is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyl; C _2-3 alkynyl; cyclopropyl; phenyl, wherein said phenyl is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl;

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

if a double bond is present 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 α-alkylyl and cyclopropyl, wherein the total number of carbon atoms in the R ¹² group is

does not exceed 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 halogen, methyl, methoxy; pyridyl; and thiophenyl; and (if) R ³⁴ , wherein R ²⁴ is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyl; C _2-3 α-alkylnyl; cyclopropyl; phenyl, wherein said phenyl is 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 , wherein R ^36a and R ^36b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, wherein the alkyl and alkenyl groups are 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 ranges from 1 to 8.

These conjugates were found to exhibit good activity and unexpected tolerability compared to similar conjugates that do not contain the sulfonamide group.

Brief description of the drawings

Fig. 1 shows the cytotoxicity of the conjugate according to the first aspect of the present invention.

- 3 040749 in vitro inventions.

Fig. 2 shows the efficacy of the conjugate according to the first aspect of the present invention in vivo.

Fig. 3 shows the cytotoxicity of the conjugate according to the first aspect of the present invention in A204 and Hep3B cells in two-dimensional and three-dimensional cell cultures in vitro.

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

Fig. 5 shows the efficacy of the conjugate according to the second aspect of the present invention in vivo.

Fig. 6 shows the antitumor activity of the conjugate according to the second aspect of the present invention in the SN12C xenograft model in vivo.

Detailed description of the invention

According to the present invention, there is provided a PBD dimer with a linker attached at position N10 on one of the PBD fragments conjugated to an antibody, as defined below.

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

The linkage between the PBD dimer and the antibody in the present invention is preferably stable outside of cells. Prior to transport or delivery into a cell, the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the antibody remains attached to the drug moiety. Linkers are stable outside of target cells 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. not cleaved, until the conjugate is delivered or transported to its target site; and (iv) maintain the cytotoxic, cytolytic effect or cytostatic effect of the PBD drug moiety. The stability of ADC can be measured using standard analytical techniques such as mass spectroscopy, HPLC and LC/MS separation/analysis techniques.

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

Definition

Deputies.

In this document, the expression optionally substituted refers to the parent group, which may be unsubstituted or which may be substituted.

Unless otherwise specified, as used herein, the term substituted refers to a parent group that bears one or more substituents. The term substituent is used herein in its generally accepted sense and refers to a chemical moiety that is covalently attached or, if desired, fused to the parent group. A wide variety of substituents are well known, and the methods for their formation and introduction into various parent groups are also well known.

Examples of substituents 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 the removal of 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 (e.g., partially unsaturated, fully unsaturated). As used herein, the term C _1-4 alkyl refers to a monovalent moiety obtained by the removal of a hydrogen atom from a carbon atom of a hydrocarbon compound containing from 1 to 4 carbon atoms, which may be aliphatic or alicyclic and which may be saturated or unsaturated (e.g., partially unsaturated, fully 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 ( _C1 ), ethyl (C2), propyl ( _C3 ), butyl (C4), pentyl (C5), hexyl (C6), and heptyl ( _C7 ).

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 ( _C4 ), sec-butyl ( _C4 ), tert-butyl ( _C4 ), isopentyl ( _C5 ), and neopentyl ( _C5 ).

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( _CH3 )= _CH2 ), butenyl ( _C4 ), pentenyl ( _C5 ), and hexenyl ( _C6 ).

_C2-12 alkynyl: As used herein, the term _{C2-12 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).

_C3-12 cycloalkyl: As used herein, the term _{C3_12} cycloalkyl refers to an alkyl group which is also a cyclyl group; i.e., a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom _of a cyclic hydrocarbon (carbocyclic) compound, the _moiety containing from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

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

cyclopropane ( _C3 ), cyclobutane ( _C4 ), cyclopentane ( _C5 ), cyclohexane ( _C6 ), cycloheptane ( _C7 ), methylcyclopropane ( _C4 ), dimethylcyclopropane ( _C5 ), methylcyclobutane ( _C5 ), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7);

unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C3), cyclobutene ( _C4 ), cyclopentene ( _C5 ), cyclohexene ( _C6 ), methylcyclopropene (C4), dimethylcyclopropene ( _C5 ), methylcyclobutene ( _C5 ), dimethylcyclobutene ( _C6 ), methylcyclopentene (C6), dimethylcyclopentene (C7) and dimethylcyclohexene (C7);

saturated polycyclic hydrocarbon compounds:

norcarane (C ₇ ), norpinane (C ₇ ), norbornane (C ₇ ).

_{C3_20} heterocyclyl: As used herein, the term _{C3_20 heterocyclyl} refers to a monovalent moiety obtained by removing _a hydrogen atom from a ring atom of a heterocyclic compound, the moiety containing from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring contains from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

In this case, the prefixes (e.g., _C3-20 , _C3-7 , _C5-6 , etc.) denote the number of ring atoms or a range of ring atoms, whether carbon atoms or heteroatoms. For example, as used herein, the term _{C5-6 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 ( _C3 ), azetidine ( _C4 ), pyrrolidine (tetrahydropyrrole) ( _C5 ), pyrroline (e.g. 3pyrrole, 2,5-dihydropyrrole) ( _C5 ), 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 ( _C4 ), oxolane (tetrahydrofuran) ( _C5 ), oxole (dihydrofuran) ( _C5 ), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepine (C7);

S1: thiirane (C3), thietane (C ₄ ), thiolane (tetrahydrothiophene) (C ₅ ), thiane (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 ( _C5 ), _{dihydrooxazole} ( _C5 ), tetrahydroisoxazole (C5), dihydroisoxazole ( _C5 ), morpholine ( _C6 ), tetrahydrooxazine ( _C6 ), dihydrooxazine ( _C6 ), oxazine ( _C6 );

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

N2O1: oxadiazine (C ₆ );

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

N1O1S1: oxathiazine (C ₆ ).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides in cyclic form, such as furanoses (C5) such as arabinofuranose, lyxofuranose, ribofuranose and xylofuranose, and pyranoses (C6) such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose and talopyranose.

_{C 5_20 aryl: As used herein, the term C 5_20 aryl refers to} a monovalent moiety,

- 5 040749 obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, wherein said moiety 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, wherein said moiety contains from 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 aromatic ring atom of an aromatic compound, wherein said moiety contains from 5 to 10 ring atoms. Preferably, each ring contains from 5 to 7 ring atoms.

In this case, the prefixes (e.g., C _3-20 , C _5-7 , C _5-6 , C _5-10 , etc.) denote the number of ring atoms or a range 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) ( _C6 ), naphthalene ( _C10 ), azulene ( _C10 ), anthracene ( _C14 ), phenanthrene ( _C14 ), 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 indane (e.g., 2,3-dihydro-1H-indene) ( _C9 ), indene ( _C9 ), isoindene ( _C9 ), tetralin (1,2,3,4-tetrahydronaphthalene ( _C10 ), acenaphthene ( _C12 ), fluorene ( _C13 ), phenalene ( _C13 ), acephenanthrene ( _C15 ), and aceanthrene ( _C16 ).

In another embodiment, 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 of 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 ) (e.g. cytosine, thymine, uracil), pyrazine (1,4-diazine) (C ₆ );

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

N4: tetrazole (C5).

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

_C9 (with 2 fused rings), derived from benzofuran ( _O1 ), isobenzofuran ( _O1 ), indole ( _N1 ), isoindole ( _N1 ), indolizine (N1), indoline ( _N1 ), _{isoindoline (N1 )} , purine ( _N4 ) (e.g. adenine, guanine), benzimidazole ( _N2 ), indazole ( _N2 ), benzoxazole ( _N1O1 ), benzisoxazole (N1O1), benzodioxole (O2 ₎ , _benzofurazan ( _N2O1 ), benzotriazole ( _N3 ), benzothiofuran ( _S1 ), benzothiazole ( _N1S1 ), _{benzothiadiazole (N2S )} ;

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 ), quinolizine (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 ), oxanthrene (O ₂ ), phenoxathiin (O ₁ S ₁ ), phenazine (N2), phenoxazine (N ₁ O ₁ ), phenothiazine (N ₁ S ₁ ), thianthrene (S ₂ ), phenanthridine (N ₁ ), phenanthroline (N2), phenazine (N2).

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

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

Hydroxy: -OH.

Ether: -OR, where R is an ether substituent, such as a C _1-7 alkyl group (also called a C _1-7 alkoxy group, which is discussed below), a C _3-20 heterocyclyl group (also called a C 3-20 heterocyclyloxy group), or a C 5-20 aryl group (also called a C _5-20 aryloxy group), preferably a C _1-7 alkyl group.

Alkoxy: -OR, where R is an alkyl group, such as 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, such as _{a C1-7} alkyl group, a _C3-20 heterocyclyl group, or a _C5-20 aryl group, preferably _{a C1-7} alkyl group. Examples of ester groups include, but are not limited to, -C( _{=O)OCH3, -C(=O)OCH2CH3 , -C} (=O)OC( _CH3 ) ₃ , and -C(=O)OPh.

Amino: -NR ¹ R ² , where R ¹ and R ² independently represent amino substituents, such as hydrogen, a C _1-7 alkyl group (also called C _{1-7 alkylamino} or di-C _1.7 αalkylαmino), a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably _H or _{a 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 from 4 to ₈ ring atoms. The 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) ^NR1R2 , where ^R1 and ^R2 are independently amino substituents as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH2, -C(=O) _NHCH3 , -C(=O)N( _CH3 ) ₂ , -C(= ^O ) _NHCH2CH3 , and -C(=O)N(CH2CH3)2, as well as amido groups in which ^R1 and ^R2 , together with the nitrogen atom to which they are attached, form a heterocyclic structure, such 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.

Delta ligand homologous protein 1 (DLK-1) is an EGF-like membrane-bound protein consisting of six tandem EGF-like repeats, a juxtamembrane 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 highly restricted in adults. In contrast, DLK-1 is repetitively 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 represents a promising target for the antibody-drug conjugate (ADC) approach due to its selective expression in a wide range of malignancies and limited expression in normal organs, as well as its expression on HCC cancer stem cells.

HuBa-1-3d.

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

The antibody may also comprise a VL domain. In some embodiments, the antibody comprises a VL domain comprising a VL CDR3 having the amino acid sequence of SEQ ID NO. 10. In some embodiments, the VL domain further comprises a VL CDR2 having the amino acid sequence of SEQ ID NO. 9 and/or a VL CDR1 having the amino acid sequence of SEQ ID NO. 8. In some embodiments, the antibody comprises a VL domain comprising a VL CDR1 having the amino acid sequence of SEQ ID NO. 8, a VL CDR2 having the amino acid sequence of SEQ ID NO. 9, and a VL CDR3 having the amino acid sequence of SEQ ID NO. 10. In preferred embodiments, the antibody comprises a VL domain comprising a VL CDR2 having the amino acid sequence of SEQ ID NO. 10. In preferred embodiments, the antibody comprises a VL domain comprising ...2 having the amino acid sequence of SEQ ID NO. 10. In preferred embodiments, the antibody comprises a VL domain comprising a VL CDR1 having the amino acid sequence of SEQ ID NO. 10. In preferred embodiments, the antibody comprises a VL domain comprising a VL CDR2 having the amino acid sequence of SEQ ID NO. 10. In preferred embodiments, the antibody comprises a VL CDR3 having the amino acid sequence of SEQ ID NO. 10. In some embodiments, the antibody comprises a VL CDR2 having the amino acid sequence of SEQ ID NO. 10. In preferred embodiments, the antibody comprises a VL CDR1 having the amino acid sequence of SEQ ID NO. 10. In some embodiments, the antibody comprises a VL CDR2 having the amino acid sequence of SEQ ID NO. 10. In preferred embodiment

- 7 040749 lysations of the present invention the antibody comprises a VL domain comprising a sequence according to SEQ ID NO. 2.

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

The VH and VL domain(s) may pair to form the antigen-binding site of the antibody, which binds DLK1.

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

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

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

In one aspect, the antibody is an antibody as 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 an antibody as disclosed herein.

Terminology.

As used herein, the term antibody is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), intact antibodies, and antibody fragments, provided that they exhibit the target biological activity, such as the ability to bind DLK1. Antibodies may be murine, human, humanized, chimeric, or of other species. An antibody is a protein produced by the immune system that is capable of recognizing and binding 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 multiple binding sites, also called epitopes, recognized by CDRs on multiple 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 includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest or a portion thereof, such targets including, but not limited to, a cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass or allotype (e.g., human 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) of the immunoglobulin molecule. Immunoglobulins may come from any species, including human, mouse or rabbit.

As used herein, the term "binds DLK1" means that the antibody binds DLK1 with a higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank Accession No. CAA76847, Version No. CAA76847.1 GI:3336842, Accession Update: January 7, 2011, 14:30). In some embodiments, the 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. Antibodies of the present invention can bind DLK1 with high affinity. For example, in some embodiments of the present invention, an antibody can bind DLK1 with 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, a DLK1 polypeptide is assigned to Genbank Accession No. CAA78163, Variant No. CAA78163.1, Accession Update Date: February 2, 2011, 10:34 AM (SEQ ID NO. 12). In one embodiment of the present invention, a nucleic acid encoding

- 8 040749 DLK1 polypeptide, corresponds to Genbank accession number Z12172, variant no. Z12172.1, entry update date: February 2, 2011, 10:34 a.m. According to some embodiments of the present invention, DLK1 comprises the sequence of SEQ ID NO. 13.

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

As used herein, the term monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, 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, an advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The adjective monoclonal indicates the characterization of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be produced by the hybridoma technique first described by Kohler et al (1975) Nature 256:495, or can be produced by recombinant DNA techniques (see US 4,816,567). 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 bearing 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 to or homologous with corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous with corresponding sequences in antibodies derived from another species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the target biological activity (US 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:68516855). Chimeric antibodies include primatized antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey or ape) and human constant region sequences.

As used herein, an intact antibody is an antibody comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., native sequence human constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more effector functions, which refer to those biological activities associated with an Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors such as the B cell receptor and the BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be classified into different classes. There are five major 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 the different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known.

Modification of antibodies

The antibodies disclosed herein may be modified, for example, to make them less immunogenic in a human subject. This may be accomplished 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

Methods 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 by a corresponding sequence from a non-human species, and wherein the modified variable region is linked to at least another portion of another protein, preferably a constant region of a human antibody. The term 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 rodent or non-human species antibodies. The term humanized antibody also includes a variant of an immunoglobulin amino acid sequence or a fragment thereof that comprises a FR comprising substantially the amino acid sequence of a human immunoglobulin and a CDR comprising substantially the amino acid sequence of a non-human immunoglobulin.

Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Alternatively, a humanized antibody is a human antibody that also contains selective non-human (e.g., murine) antibody sequences in place 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 substantially alter its binding and/or biological activity. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.

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

CDR vaccination.

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

In addition, humanized antibodies may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications are made to further improve and maximize the potency of the antibodies. Thus, in general, a humanized antibody will contain all of at least one, and in one aspect, two variable domains, in which all or all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are human immunoglobulin sequences. A 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 involves combining a VH or V _L domain of a particular non-human antibody specific for a particular epitope with a library of human VH or V _L , and the particular human V domains are selected against the antigen of interest. The selected human VH is then combined with the library of VL 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 a final antibody molecule. They are constructed by combining multiple human VH and VL sequence segments in combinations that limit or avoid human T-cell epitopes in the V regions of the final composite antibody. If desired, T-cell epitopes are limited or eliminated by replacing V region segments that contribute to or encode a T-cell epitope with alternative segments that avoid the T-cell epitopes. This method is described in US 2008/0206239 A1.

Deimmunization.

This method involves removing human (or another second species) T-cell epitopes from the V regions of a therapeutic antibody (or other molecule). The V region sequence of a therapeutic

- 10 040 749 ical antibodies are analyzed for the presence of MHC class II binding motifs, for example, by comparison with MHC class II binding motif databases (such as the motif database available at www.wehi.edu.au). Alternatively, MHC class II binding motifs can be identified using computational sweep methods such as those developed by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in these methods, consecutive overlapping peptides from V region sequences are tested to determine their binding energies to MHC class II proteins. These data can then be combined with information on 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 (e.g., human) have been identified, they are eliminated by changing one or more amino acids. The amino acids modified are usually 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 is by substitution, but in some cases, the addition or deletion of an amino acid may be more appropriate.

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

Surface change.

This method includes:

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

(b) obtaining sequence alignments using relative accessibility distributions from X-ray crystallographic structures of a sufficient number of heavy and light chain variable regions of the non-human antibody and the human antibody to yield a set of heavy and light chain framework positions wherein the alignment positions are identical in 98% of a sufficient number of heavy and light chains of the non-human antibody;

(c) determining for the non-human antibody to be humanized a 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 of the human antibody are naturally paired or unpaired;

(e) replacing the group of amino acid residues exposed on the surface of the heavy and light chains identified 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 in step (d);

(f) constructing a three-dimensional model of the variable region of a non-human antibody obtained as a result of the substitution indicated in step (e);

(g) identifying, by comparison of the three-dimensional models constructed 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 complementarity determining regions of the non-human antibody to be humanized;

(h) replacing any human residues identified in step (g) with a parent non-human amino acid residue to define a humanizing group of surface-exposed amino acid residues of the non-human antibody; provided that step (a) need not be performed first, but must be performed before step (g).

Superhumanization.

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

Optimization of the composition of human sequence regions.

This method compares a non-human sequence (e.g., mouse) to the human germline gene repertoire and the differences are assessed as human sequence 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 rather than using a measure of overall identity to generate many diverse humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).

Rearrangement of frame sections.

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

Modification of an antibody using azide.

The antibody can be prepared for conjugation with a drug linker by a three-step method.

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

(2) Trimming of all glycan isoforms (complex, hybrid, high-mannose) by endoglycosidase to release the central GlcNAc.

(3) Enzymatic transfer of an N-acetylgalactose residue bearing an azide group to the central GlcNAc for conjugation to the drug linker.

A review of the above method is given in van Geel, R., et al., Bioconjugate Chemistry, 2015, 26, 2233-2242; DOI: 10.1021/acs.bioconjchem.5b00224. Alternatively, a one-pot method can be used - see examples.

Implementation options

X.

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

According to other embodiments of the present invention, X is _-CH2- .

According to further embodiments of the present invention _{, X is -C2H4- .}

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.

R ⁷ .

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

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

R ² .

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 halogen, nitro, cyano, ether, _C1-7 alkyl, _{C3_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, wherein the total number of carbon atoms in the R ² group is no more than 5;

R ^25b (e) , wherein one of R ^25a and R ^25b is H and the other is selected from: phenyl, wherein phenyl is 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 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 embodiments of the present invention, R ² is preferably phenyl. According to other embodiments of the present invention, R ¹² is preferably 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, for example, a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bonded to the central portion of the PBD through 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 bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred and monosubstituted groups being most preferred. The substituents may be in any position.

If R ² is a C _5-7 aryl group, the single substituent is preferably on a ring atom that is not adjacent to a bond with the rest of the compound, i.e., it is preferably β or γ relative to the rest of the compound. Therefore, if the C _5-7 aryl group is phenyl, the substituent is preferably in the meta or para positions, and is more preferably in the para position.

When R ² is a C _8-10 aryl group, such as quinolinyl or isoquinolinyl, it may bear 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 on ^R2 , when ^R2 is _{a C5.10} aryl group, is an ether, it may, in some embodiments of the present invention, be an alkoxy group, for example, _{a C1.7 alkoxy} group (e.g., methoxy, ethoxy), or it may, in some embodiments of the present invention, be a _C5.7 aryloxy group (e.g., phenoxy, pyridyloxy, furanyloxy). The alkoxy group itself may be further substituted, for example, with an amino group (e.g., dimethylamino).

If the 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 (e.g. methyl, ethyl, propyl, butyl).

If the substituent on R ² , when R ² is a C ₅ . ₁₀ aryl group, is a 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 PBD moiety through a nitrogen atom. These groups can be further substituted, for example, by C _1-4 alkyl groups. If the C6 nitrogen-containing heterocyclyl group is piperazinyl, said further substituent can 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, fluoro, chloro, cyano, bis-oxymethylene, methylpiperazinyl, morpholino and methylthiophenyl. Other particularly preferred substituents for R ² are dimethylaminopropyloxy and carboxy.

Particularly preferred substituted ^R2 groups when ^R2 is a _C5-10 aryl group include, but are not limited to, 4-methoxyphenyl, 3-methoxyphenyl, 4-ethoxyphenyl, 3-ethoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 3,4-bisoxymethylenephenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl and naphthyl. Another possible substituted ^R2 group is 4-nitrophenyl. ^R2 groups of particular interest include 4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylenephenyl.

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

- 13 040749 teniya it can be methyl, ethyl or propyl (n-pentyl or isopropyl). According to some of these embodiments, it can be methyl. According to other embodiments of the present invention, it can be butyl or pentyl, which can 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.

If R ² is , each of R ²¹ , R ²² and R ²³ is independently selected from H, C 3 -saturated alkyl, C _2-3 alkenyl, C _2-3 alkynyl and cyclopropyl, wherein the total number of carbon atoms in the R ² group 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, and 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, wherein the other group is selected from _H , C _1-3 saturated alkyl, C _{2-3 alkenyl} , C _2-3 alkynyl and cyclopropyl.

According to some embodiments of the present invention, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the 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: ·

R ^2Sb

If R ² is , 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. In some embodiments of the present invention, the group that is not H is optionally substituted phenyl. If the optional substituent of phenyl is halogen, it is preferably fluoro. 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 alkenyl;

C _2-3 alkynyl; cyclopropyl; phenyl, wherein phenyl is optionally substituted with a group selected from halogen, methyl, methoxy; pyridyl; and thiophenyl. If the optional substituent of phenyl is halogen, it is preferably fluorine. According to 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 , wherein R ^26a and R ^26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, wherein the alkyl and alkenyl groups are optionally substituted with a group selected from C1-4 alkylamido and C1-4 alkyl ester; or, if one of R ^26a and R ^26b is H, the other is selected from nitrile and C1-4 alkyl ester.

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

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

According to other embodiments of the present invention, 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, wherein the alkyl and alkenyl groups are optionally substituted. In these additional embodiments, even more preferably, the group that is not H is selected from methyl and ethyl.

R ¹² .

The above preferences for R ² apply equally to R ¹² . According to one embodiment of the present invention, DL is

Drug loading

Drug loading is the average amount of drug PBB per antibody, e.g. antibody.

The average amount of drug per antibody in ADC preparations from conjugation reactions can be characterized by conventional means such as UV, reversed-phase HPLC, HIC, mass spectroscopy, ELISA, and electrophoresis. The quantitative distribution of 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 discernible based on antibody-antigen binding and due to the limitation of ELISA detection. Furthermore, ELISA assays for the detection of antibody-drug conjugates do not allow determination of where the drug moieties are attached to the antibody, such as the heavy chain or light chain moieties, or specific amino acid residues. In some cases, separation, purification, and characterization of a homogeneous ADC, where p is some value from an ADC with different drug loading indices, can be accomplished by means such as reversed-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.e., the number of azide groups. For example, an antibody may have only one or two azide groups to which a drug linker can be attached.

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

If more than one nucleophilic or electrophilic group of the antibody reacts with the drug-linker intermediate or linker reagent and then with the drug moiety, the resulting product is a mixture of antibody-drug conjugates with a distribution of drug moieties attached to the antibody, e.g., 1, 2, 3, etc. Liquid chromatographic techniques such as polymer reversed phase (PLRP) and hydrophobic interaction (HIC) can separate the compounds in the mixture based on drug loading. ADC preparations with a single drug loading value (p) can be isolated, however, these single loading value ADCs can still be heterogeneous mixtures because drug moieties can be attached via the linker at different locations on the antibody.

Thus, antibody-drug conjugate compositions of the present invention include mixtures of antibody-drug conjugates in which the antibody comprises one or more PBD drug moieties and in which the drug moieties

- 15 040749 agents can be attached to the antibody at different amino acid residues.

According to one embodiment of the present invention, the average number of dimeric pyrrolobenzodiazepine groups per antibody is in the range of 1 to 8.

According to some embodiments of the present invention, the range is selected from 1-4, 1-4, 24, and 1-3.

According to some embodiments of the present invention, the antibody has one or two dimeric pyrrolobenzodiazepine groups.

Inclusion of other forms

Unless otherwise indicated, the above includes the 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-), a salt or solvate thereof, as well as the conventional protected forms. Similarly, reference to an amino group includes the protonated form (-N+HR1R ² ), a salt or solvate of the amino group, for example, the hydrochloride salt, as well as the conventional protected forms of the amino group. Likewise, reference to a hydroxyl group also includes the anionic form (-O-), a salt or solvate thereof, as well as the conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify and/or handle a corresponding 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 can be anionic (e.g., -COOH can be -COO-), then a salt can 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 Ca2 ⁺ and Mg2 ⁺ , and other cations such as Al3 ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 ⁺ ) and substituted ammonium ions (e.g., NH3R ⁺ , _NH2R2 ⁺ , NHR3 ⁺ , NR4 ⁺ ). Examples of some suitable substituted ammonium ions include those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine ^, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N( _CH3 ) ₄₊ .

If the compound is cationic or contains a functional group that can be cationic (e.g., -NH2 can be _-NH3 ⁺ ), then the salt can 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, glucoheptone, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucinous, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic acid, 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, carboxymethylcellulose.

Solvates

It may be convenient or desirable to prepare, purify and/or process a corresponding solvate of the active compound. As used herein, the term solvate is used in its conventional sense to refer to a complex of a solute (e.g., an active compound, a salt of the active compound) and a solvent. If the solvent is water, the solvate is conveniently referred to as a hydrate, e.g., a monohydrate, a dihydrate, a trihydrate, etc.

The present invention includes compounds in which the solvent is attached via an imine bond of the PBD moiety as illustrated below, wherein the solvent is water or an 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 PBD (as described in the section on R ¹⁰ above). The balance of these equilibria depends on the conditions in which

- 16 040749 of which there are connections, as well as from the structure of the fragment itself.

These specific compounds can be isolated in solid form, for example by lyophilization.

Isomers

Certain compounds of the present invention may exist in one or more specific geometric, optical, enantiomeric, diastereomeric, 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 exo forms; R, S and meso forms; D and L forms; d and l forms; (+) and (-) forms; 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 their combinations, hereinafter collectively referred to as isomers (or isomeric forms).

The term chiral refers to molecules whose mirror images are non-superimposable, while the term achiral refers to molecules whose mirror images are superimposable.

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

A diastereomer refers to a stereoisomer with two or more chiral centers, the molecules of which 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 non-superimposable mirror images of each other.

In the present 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. The compounds of the present invention may contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. It is envisaged 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 denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to denote the sign of rotation of the compound by plane-polarized light, with (-) or l meaning the compound is levorotatory. A compound with the prefix (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer may also be called an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may 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 that is 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 structure-related) isomers (i.e., isomers that differ in the bonds between atoms rather than simply in the positions of the atoms in space). For example, reference to the methoxy group, _-OCH3 , should not be construed as a reference to its structural isomer, the hydroxymethyl group, -CH2OH. Likewise, reference to ortho-chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., _C1.7 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, such as keto, enol and enolate forms, such as in the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hydroxyazo and nitro/acin-nitro.

- 17 040749 ? ,O ch on N ⁺ _ch 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) involve interconversions by proton migration, such as keto-enol and imine-enamine isomerization. Valence tautomers involve interconversions by rearrangement of some 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 ^H1 , ^H2 (D), and ^H3 (T); C may be in any isotopic form including ^C12 , ^C13 , and ^C14 ; O may be in any isotopic form including ^O16 and ^O18 ; and the like.

Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, ^H2 (deuterium, D), ^H3 (tritium), ^C11 , ^C13 , ^C14 , ^N15 , ^F18 , ^P31 , ^P32 , S35, ^Cl36 , and ^I125 . Included are various isotopically labeled compounds of the present invention, such as those into which radioactive isotopes such as ^H3 , ^{C13 ,} and ^C14 are incorporated. Such isotopically labeled compounds may be used in metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or in radioactive treatment of patients. Deuterium labeled or deuterium substituted therapeutic compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetics) properties related to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosing requirements. F ¹⁸ labeled compound may be used for PET or SPECT studies. The isotopically labeled compounds of the present invention and prodrugs thereof 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, particularly deuterium (i.e., ^H2 or D), can provide certain therapeutic advantages due to greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements or an improved therapeutic index. It should be understood that deuterium is considered a substituent in this case. The concentration of such a heavier isotope, particularly deuterium, can be determined by an 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 particular compound includes all such isomeric forms, including (in whole or in part) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are either known in the art or may be readily prepared in a known manner by adaptation of the methods set forth 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 from about 6 hours to about 5 days; and measuring the viability of the cells. In vitro cellular assays are used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC of the present invention.

The potency of antibody-drug conjugates in vitro can be measured using a cell proliferation assay. The CellTiter-Glo® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, WI, USA) homogeneous assay based on recombinant expression of Coleoptera luciferase (U.S. Patents 5,583,024; 5,674,713; and 5,700,670). This cell proliferation assay quantifies the number of viable cells in a 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 6,602,677). The assay

- 18 040749

The 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 the addition of a single reagent (CellTiter-Glo® reagent) directly to cells cultured in serum-supplemented medium. Cell washing, medium removal and multiple pipetting steps are not required. The system can detect as few as 15 cells per well in a 384-well format within 10 min of reagent addition and mixing. Cells can be treated continuously with ADC, or they can be treated and separated from ADC. In general, cells treated briefly, i.e., 3 h, showed similar performance to cells treated continuously.

The homogeneous add-mix-measure format results in cell lysis and the generation of 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 glow-type luminescent signal resulting from a luciferase reaction with a half-life typically greater than five hours, depending on the cell type and medium used. Viable cells are reported as relative luminescence 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 potency of antibody-drug conjugates in vitro can also be measured by cytotoxicity assays. Cultured adherent cells are washed with PBS, separated using trypsin, diluted in complete medium containing 10% FBS, centrifuged, resuspended in fresh medium, and counted using a hemocytometer. Suspension cultures are counted directly. Monodisperse cell suspensions suitable for counting may require mixing the suspension by repeated aspiration to break up cell clumps.

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

A stock solution (1 ml) of ADC (20 μg/ml) is prepared in the appropriate cell culture medium. Serial 10-fold dilutions of the stock ADC are prepared in 15 ml centrifuge tubes by serial transfer of 100 μl into 900 μl of cell culture medium.

Wells with four replicates of each ADC dilution (100 µl) were distributed into 96-well black plates pre-seeded with cell suspension (100 µl) to obtain a final volume of 200 µl. Control wells were supplemented with cell culture medium (100 µl).

If the cell line doubling time exceeds 30 h, ADC incubation lasts 5 days, otherwise a four-day incubation is carried out.

At the end of the incubation period, cell viability is assessed using the Alamar blue assay. AlamarBlue (Invitrogen) is dispensed over the entire plate (20 μl per well) and incubated for 4 h. 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 mean fluorescence intensity in ADC-treated wells compared to the mean fluorescence intensity in control wells.

Application

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

The target site is preferably a population of proliferative cells. The antibody is an antibody to an antigen present in the population of proliferative cells.

According to one embodiment of the present invention, the antigen is absent or present at a reduced level in a population of non-proliferative cells compared to the amount of antigen present in a population of proliferative cells, 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 deliver the RelA compound to the target location.

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

The target location can 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 anti-cancer activity. In particular, the compounds (conjugates) include an antibody conjugated, i.e. covalently attached via a linker, to a moiety of the PBD drug, i.e. a toxin. If the drug is not conjugated to the antibody, the PBD drug has a cytotoxic effect. Thus

- 19 040749 Thus, the biological activity of the PBD drug fragment is modulated by conjugation with an antibody. The antibody-drug conjugates (ADCs) according to the present invention selectively deliver an effective dose of a cytotoxic agent to tumor tissue, as a result of which higher selectivity, i.e. a lower effective dose, can be achieved.

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

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

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

The term proliferative disease refers to the unfavorable or uncontrolled cellular proliferation of excess or abnormal 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 cellular proliferation, including, but not limited to, neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), various cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, bowel 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 diseases, fibroproliferative disorders (e.g., of connective tissue), and atherosclerosis. 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, intestine, colon), breast (mammary), ovary, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.

Disorders of special interest include, but are not limited to, various cancers, 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. Miscellaneous cancers of special 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, skeletal muscle cancer, liposarcoma, glioma, Wilms tumor, neuroendocrine tumors, acute myelogenous leukemia, and myelodysplastic syndrome.

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

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

Disorders of particular interest include, but are not limited to, various cancers, 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 checkpoint disorders, fibrotic disorders (fibrosis), or proliferative diseases such as cancer, particularly metastatic cancer.

It is envisaged that the antibody drug conjugates (ADCs) of the present invention can be used to treat various diseases or disorders, for example, 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, blastocoelic, inflammatory, angiogenic and immunological, including autoimmune, disorders.

Typically, the disease or disorder being treated is a hyperproliferative disorder such as cancer. Examples of cancers being 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, lung adenocarcinoma, and squamous cell lung carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial carcinoma or uterine carcinoma, salivary gland carcinoma, kidney cancer or renal cancer, prostate cancer, vaginal cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

Autoimmune diseases that can be treated with ADC compounds include rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune disorders of the gastrointestinal tract and liver (such as, for example, inflammatory bowel diseases (e.g., 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, opsomeclonal syndrome, myasthenia gravis, neuromyelitis optica, 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, post-transfusion purpura and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing disorders (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant 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 (e.g. Graves' disease and thyroiditis). More preferably, such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis and glomerulonephritis.

Treatment methods

The conjugates of the present invention can be used in a method of 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 a benefit to the patient. Such benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and time course 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 therapies include, but are not limited to, chemotherapy (administration of active agents, including, for example, drugs such as chemotherapeutic agents); surgery; and radiation therapy.

A chemotherapeutic agent is a chemical compound that can be used to treat cancer, regardless of its mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, plant alkaloids, mitotic spindle blockers, 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 (Tarceva®, 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, dichloroplatin(II),

- 21 040749

CAS# 15663-27-1), carboplatin (CAS# 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# 85622-93-1, Temodar®, Temodal®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxu]-N,N-dimethylethanamine, Nolvadex®, Istubal®, Valodex®) and doxorubicin (Adriamycin®), Akti-1/2, HPPD and rapamycin.

Other examples of chemotherapeutic 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™ (without cremophor), albumin-modified nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, Zactima®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (Torizel®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (Telcyta®, Telik), thiotepa and cyclosphosphamide (Cytoxan®, Neosar®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylomelamin; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, calicheamicin gamma-I, calicheamicin-omega-I (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates such as clodronate; esperamicin; and the chromophore portion of neocarzinostatin and related chromophores of the chromoprotein enediyne antibiotics), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, kelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folic acid replacer such as frolinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR, USA); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (including T-2 toxin, verracurin A, roridin A, and anhydrin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (Ara-C); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (Xeloda®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

- 22 040749

Also included within the definition of chemotherapeutic agent are: (i) antihormonal agents whose function is to regulate or inhibit the action of a hormone on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and fareston® (toremifine 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, Megase® (megestrol acetate), Aromasin® (exemestane; Pfizer), formestane, fadrozole, Rivisor® (vorozole), Femara® (letrozole; Novartis) and Arimidex® (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (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, particularly those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as PKC-alpha, Raf, and H-Ras, such as oblimersen (Genasens®, Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g. Angiozyme®) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, such as Allovectin®, Leuvectin®, and Vaxyd®; Proleukin® rIL-2; topoisomerase 1 inhibitors such as Lurtotecan®; Abarelix® rmRH; (ix) antiangiogenic agents such as bevacizumab (Avastin®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of chemotherapeutic agent are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (Avastin®, Genentech); cetuximab (Erbitux®, Imclone); panitumumab (Vectibix®, Amgen), rituximab (Rituxan®, Genentech/Biogen Idec), ofatumumab (Arzerra®, GSK), pertuzumab (Perjeta™, Omnitarg™, 2C4, Genentech), trastuzumab (Herceptin®, Genentech), tositumomab (Bexar, Corixia), and the 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, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pekfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, recivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, takatuzumab tetraxetane, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleuquin, tucuzituzumab, umavizumab, urtoxazumab and visilizumab.

The pharmaceutical compositions according to the present invention and for use according to the present invention may contain, in addition to the active ingredient, i.e. the conjugate compound, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, for example, cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. A tablet may contain a solid carrier or 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. A capsule may contain a solid carrier such as gelatin.

For intravenous, cutaneous or subcutaneous injection or injection into the site of injury, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is free of pyrogens and has a 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 injection, Ringer's injection with lactose. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included if necessary.

Compositions

Although the conjugate compound may be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation.

According to one embodiment of the present invention, the composition is a pharmaceutical composition (e.g., formulation, preparation, drug) comprising a conjugate compound described herein and a pharmaceutically acceptable carrier,

- 23 040749 additive or auxiliary substance.

According to 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 (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweetening agents.

According to one embodiment of the present invention, the composition further comprises other active agents, such as other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks. 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 ed., 1994.

Another aspect of the present invention relates to methods for preparing a pharmaceutical composition comprising mixing at least one [C11]-radiolabeled conjugate or conjugate-like compound as defined herein with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, such as carriers, diluents, excipients, etc. When formulated as discrete units (e.g., 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., that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject (e.g., a human) 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 with respect to compatibility with the other ingredients of the formulation.

The formulations may be prepared by any of the methods well known in the pharmaceutical art. Such methods include the step of combining the active compound with a carrier, which constitutes one or more additional ingredients. Typically, the formulations are prepared by uniformly and intimately combining the active compound with carriers (e.g., liquid carriers, finely divided solid carriers, etc.), and then shaping the product if necessary.

The formulation may be prepared to provide fast or slow release; immediate, delayed, time-controlled or extended release; or a combination thereof.

Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticle). Such liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes which 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, glycerol, 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 injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/mL to about 10 μg/mL, such as from about 10 ng/mL to about 1 μg/mL. The formulations may be presented in sealed single-dose or multi-dose containers, such as ampoules and vials, and may be stored in a sublimated (lyophilized) state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before 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 appropriate dosages of the conjugate compound and compositions containing the conjugate compound may vary among patients. Determining the optimal dosage will typically involve balancing the level of therapeutic benefit with any risk or deleterious side effects. The dosage level selected will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of elimination of the compound, the duration of treatment, other medications,

- 24 040749 the agents, compounds and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health and medical history of the patient. The amount of compound and the route of administration will ultimately be left to the discretion of the physician, veterinarian or clinician, although the dosage will typically be selected to achieve local concentrations at the site of action that achieve the desired effect without causing significant dangerous or harmful side effects.

Administration may be performed in a single dose, continuously, or intermittently (e.g., in divided doses at appropriate intervals) during the course of treatment. Methods for determining the most effective agents and dosages for administration are well known to those skilled in the art and will vary depending on the formulation used for therapy, the purpose of therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations may be performed using a dose level and schedule selected by the attending physician, veterinarian, or clinician.

Typically, a suitable dose of the active compound is within the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram of body weight of the subject per day. If the active compound is a salt, ester, amide, prodrug, or the like, the amount administered is calculated on the basis of 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 dosage regimen: about 100 mg, 3 times per day.

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

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

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

According to one embodiment of the present invention, the conjugate compound is administered to a human patient according to the following dosage regimen: about 100 or about 125 mg, 2 times per day.

The dosages described above can be applied to the conjugate (including the PBD moiety and the linker to the antibody) or to an effective amount of the provided PBD compound, such as the amount of 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 to be treated, as defined above, the severity and course of the disease, whether the molecule is administered prophylactically or therapeutically, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the treating physician. The molecule is administered to the patient in a suitable manner at one time or over several treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of the molecule constitutes the initial intended dosage for administration to the patient, for example, whether by one or more separate administrations 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 approximate dosage of ADC to be administered 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 the target suppression of disease symptoms occurs. An approximate dosing regimen includes a course of administration of an initial loading dose of about 4 mg/kg, followed by 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 readily monitored by routine techniques and assays.

Treatment

As used herein, the term treatment, when applied to the treatment of a condition, generally refers to treatment and therapy, whether in humans or animals (e.g., in veterinary applications), that achieves some intended therapeutic effect, such as inhibition of the progression of a condition, and includes decreasing the rate of progression, delaying the rate of progression, regressing the condition, ameliorating the condition, and curing the condition. Also included is treatment as a prophylactic measure (i.e., prevention, avoidance).

As used herein, the term therapeutically effective amount refers to that amount of an active compound or a material, composition or dosage form containing the active compound that is effective to produce some intended therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with the intended treatment regimen.

- 25 040749

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

Obtaining 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 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, copper-free conjugation, 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 PBD dimer, for example: WO 2011/130598, WO 2013/055987, WO 2014/057074.

Subject/Patient

The subject/patient may be an animal, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g., platypus), a rodent (e.g., guinea pig, hamster, rat, mouse), a murine (e.g., mouse), a lagomorph (e.g., rabbit), avian (e.g., bird), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), porcine (e.g., pig), ovine (e.g., sheep), bovine (e.g., cow), primate, simian (e.g., monkey or ape), ape (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or a human.

In addition, the subject/patient may be 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 essence of the invention

The following numbered embodiments describe certain specifically 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 is

- 26 040749

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 represents 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, wherein the total number of carbon atoms in the R ² group specified is no more than 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) wherein R ²⁴ is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyl; 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 , wherein R ^26a and R ^26b are independently selected from H, F, C1-4 saturated alkyl, C1-3 alkenyl, wherein the alkyl and alkenyl groups are optionally substituted with a group selected from C1-4 alkylamido and C1-4 alkyl ester; or, if one of R ^26a and R ^26b is H, the other is selected from nitrile and C1-4 alkyl ester; if a double bond is present between C2' and C3', R ¹² is selected from the group consisting of:

(iia) a C _5-10 aryl group 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 , wherein 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, wherein the said total number of carbon atoms in the R ¹² group is 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 halogen, methyl, methoxy; pyridyl; and thiophenyl; and (iif) ^R wherein R ²⁴ is selected from: H; C _1-3 saturated alkyl; C _2-3 alkenyl; C _2-3 alkynyl; cyclopropyl; phenyl, wherein 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 , wherein R ^36a and R ^36b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, wherein the alkyl and alkenyl groups are 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. A conjugate according to statement 1 in which X is a single bond.

3. The conjugate of statement 1 in which X is _-CH2- .

4. The conjugate according to statement 1, in which X is -C ₂ H ₄ -.

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

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

10. The compound according to statement 9, wherein R ² is phenyl.

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

12. A compound according to any one of claims 9 to 11, wherein R ² carries from 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, bis-oxymethylene, methylpiperazinyl, morpholino and methylthiophenyl.

14. A compound according to any one of claims 1 to 8, wherein there is a double bond between C2 and C3 and R ² is a C _1-5 saturated aliphatic alkyl group.

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

16. A compound according to any one of claims 1 to 8, wherein there is a double bond between C2 and C3, and R ² is a C _3-6 saturated cycloalkyl group.

17. The compound according to claim 16, wherein R ² is cyclopropyl.

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

19. The compound according to claim 18, wherein the said total number of carbon atoms in the ^R2 group is not more than 4.

20. The compound according to claim 19, wherein the said total number of carbon atoms in the ^R2 group is not more than 3.

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

22. A compound according to any one of claims 18 to 20, wherein two of R ²¹ , R ²² and R ²³ are H, the other group being 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, in which there is a double bond between C2 and C3 and R ²

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

R ²⁴

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

27. The compound according to claim 26, wherein R ²⁴ is selected from H and methyl.

28. A 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. A 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 there is a single bond 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, wherein the alkyl and alkenyl groups are optionally substituted.

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

32. The compound according to claim 31, wherein R ¹² is phenyl.

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

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

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

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

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

38. A compound according to any one of claims 1 to 30, wherein there is a double bond between C2' and C3', and is a C _3-6 saturated cycloalkyl group.

39. The compound according to claim 38, wherein R ¹² is cyclopropyl.

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

_R 12 _R 12

R ¹²

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

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

43. A compound according to any one of claims 40 to 42, wherein one of _R ³¹ , R ³² and R ³³ is H, and the other two groups are selected from H, C _1-3 saturated alkyl, C _{2-3 alkenyl} , C _2-3 alkynyl and cyclopropyl.

44. A compound according to any one of claims 40 to 42, wherein two of R ³¹ , R ³² and R ³³ are H, with

- 29 040749 wherein the other group is selected from H, C1-3 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 there is a double bond between C2' and C3' and R ¹² is a group of the formula:

46. The compound according to statement 45, in which R ¹² is a group:

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

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

49. The compound according to claim 48, wherein R ³⁴ is selected from H and methyl.

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

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

A^ ^p36a is ^R36b , one of R ^36a and R ^36b is H and the other is selected from C _1-4 saturated alkyl, C _2-3 alkenyl, wherein the alkyl and alkenyl groups are optionally substituted.

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

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

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

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

57. The conjugate of any one of claims 1 to 56, wherein said antibody comprises a VH domain containing

- 30 040749 the sequence SEQ ID NO. 1.

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

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

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

61. The conjugate according to any one of claims 1 to 60, wherein said antibody comprises a VL domain comprising the sequence of SEQ ID NO. 2.

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

63. The conjugate of any one of claims 1 to 62, wherein said antibody comprises a heavy chain comprising the sequence of SEQ ID NO. 3 or a heavy chain comprising the sequence of SEQ ID NO. 11.

64. The conjugate according to any one of claims 1 to 63, wherein said antibody comprises a light chain comprising the sequence of SEQ ID NO. 4.

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

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

67. A conjugate according to any one of claims 1 to 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 to 67, wherein said average drug loading 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. The conjugate of any one of claims 1-67 for use in the treatment of a proliferative disease in a subject.

71. The 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 myelogenous leukemia, and myelodysplastic syndrome.

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

74. The pharmaceutical composition according to claim 73, additionally containing a therapeutically effective amount of a chemotherapeutic agent.

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

76. A method for 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 by a two-step process. The first step is a glycan remodeling process in which the native N-linked glycan is trimmed to a central GlcNAc, followed by the addition of an azido-modified GalNAc to form an antibody-GalNAc- _N3 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) was added dropwise. After stirring for 15 min, Et ₃ N (1.42 mL, 1.03 g, 10.2 mmol, 4 equiv) was added dropwise and stirring was continued for another 10 min. A solution of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (1.0 g, 6.1 mmol, 2.4 equiv) in H2O (5 mL) was then added and the reaction mixture was stirred at room temperature for 2 h. After this time, _CHCl3 (50 mL) and H2O (100 mL) were added and the layers were separated. To the aqueous layer in a separatory funnel was added _CH2Cl2 (100 mL) and the pH was adjusted to ₄ with 1 N HCl before separating the layers. _The aqueous layer was extracted twice with _CH2Cl2 ( ₂ x 100 mL), the organic layers were combined, dried ( _Na2SO4 ), filtered and concentrated. The residue was purified by reservoir column chromatography on silica _gel , eluting with _CH2Cl2 to 20% MeOH in _CH2Cl2 . The yield was 0.42 g (1.0 mmol, 39%) 3 as _a colorless sticky wax.

- 32 040749

Synthesis of drug linker.

o o

- 33 040749

ABOUT

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

(a) Palladium tetrakistriphenylphosphine (Pd( _PPh3 ) ₄ , 4.8 mg, 4.15 μmol) was 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 was bubbling with N2, the degassed pyrrolidine solution was added. Weighed Pd( _PPh3 ) ₄ was dissolved in DCM (1 mL) and 0.9 mL of this solution was added. After 50 min of bubbling 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 were dried ( _Na2SO4 ) 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 were passed through SPE columns (HCO3 ^- ) and concentrated. After addition of MeCN (50 ml), the mixture was concentrated again. The obtained residue 2 was used in the next step.

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

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

(b) To a solution of the above residue 2 in CHCl3 (5 mL) was added a solution of 3 (15 mg, 36 μmol, MW 418 g/mol) in _CHCl3 (0.8 mL). The resulting mixture was added to solid EDC.HC1 (4.7 mg, 25 μmol), _CHCl3 (5 mL) was added and the mixture was stirred for 30 min. DCM (30 mL) was added and the resulting mixture was washed with water (30 mL). After separation, the aqueous phase was extracted with DCM (30 mL). _The combined organic layers were dried ( _Na2SO4 ) and concentrated. The residue was purified by RP-HPLC (30-90% MeCN (acid-free) in _H2O (0.01% formic acid)). Before collection, HPLC elution tubes were filled with 5% aqueous (NH4) _HCO3 . The combined HPLC fractions were extracted with DCM ( _3x20 mL). The combined organic layers were dried ( _Na2SO4 ) and concentrated. The product 4 was obtained as a light yellow/white oil (21 mg, 16 µmol, MW 1323 g/mol, 67% over two steps).

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

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

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

Remodeling response.

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 mMtris/C1, 150 MMNaCl, pH=8.0 663.8 No data Antibody to DLK1 in 25 mM Tris/Cl, 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 relative to MAT 4.82 mg/ml His-TnGalNAcT in 25 mM Tris/Cl, 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 equiv. relative to MAT

Purification procedure on protein A.

Protein A binding and elution were 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 an AKTA Prime plus system. The column was prepared and used as follows.

Solution CV Notes 0.1 MNaOH 3 Disinfection 25 mM Tris/Cl, 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 Endotoxin Wash/Removal Stage 25 mM Tris/Cl, 25 mM NaCl, pH=8.0 20 Newt wash/removal stage 0.15 M acetic acid As it happens Collection of peak from 0.1 to necessities 0.1 wholesale units

Following affinity purification on Protein A, a small sample of the product can be recovered using DTT and subsequently analyzed by MS. 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 the modified galactose to the antibody with a substituted central GlcNAc(Fuc) and a minor product (±10% of the total heavy chain) resulting from the transfer of the modified galactose to the antibody with a substituted central GlcNAc (no fucose).

- 35 040749

Buffer replacement after protein A purification.

Next, the buffer in the remodeled and purified GalNAc- _N3 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 from Protein A purification was diluted 1:1 with PBS and 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. The protein concentration was confirmed to be 16.4 mg/mL by UV analysis at A280-320 nm using an extinction coefficient of 1.5, and a total of 2.7 mL was recovered.

Conjugation of 4 with a modified antibody to produce ConjA1

Reaction conditions.

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

The reaction mixture was mixed thoroughly 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 1:1 ratio and re-concentrated to the original volume. This was repeated 12 times before removing the purified ADC mass from the centrifuge device.

Protein concentration was determined by quantitative gel permeation chromatography (SEC) analysis using the antibody calibration curve and the 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 reprobed by quantitative SEC analysis. A sample was removed for testing and the remainder was aliquoted into 1 mL portions and frozen at -80°C.

Analysis of the product showed the following properties:

transparent, colorless, particle-free

5.17 [protein] according to SEC average DAR 1.87 (according to reversed phase chromatography)

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

Glycan remodeling (ADC directed against KAAG1, ConjA2)

Obtaining antibodies.

Approximately 150 mg of anti-KAAG1 antibody (approximately 25 mL at 6.13 mg/mL in PBS, pH 7.4) was buffer exchanged 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 1:1 with 25 mM Tris/Cl, 150 mM NaCl, pH 8.0, then re-concentrated to 12 mL, and this process 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 response.

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 mMtris/C1, 150 mM NaCl, pH=8.0 893 No data MAB to KAAG1 in 25 mM Tris/Cl, 150 mM NaCl, pH=8.0 6000 15 mg/ml

- 36 040749

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

Purification procedure on protein A.

Protein A binding and elution were 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 an AKTA Prime plus system. The column was prepared and used as follows.

Solution CV Notes 0.1 MNaOH 3 Disinfection 25 mM Tris/Cl, 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 Endotoxin washing/removal stage 25 mM Tris/Cl, 25 mM NaCl, pH=8.0 20 Newt wash/removal stage 0.15 M acetic acid As needed Collection of peak from 0.1 to 0.1 opt. units.

Following affinity purification on Protein A, a small sample of the product can be recovered using DTT and subsequently analyzed by MS. 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 the 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 the modified galactose to an antibody with a substituted central GlcNAc (no fucose).

Buffer replacement after protein A purification.

The pH of the Protein A eluate containing remodeled/purified Ab-GalNAc- _N3 was adjusted by adding 1.5 M Tris base at 3.2% v/v, then the buffer was exchanged with PBS and concentrated to »17 mg/mL using Vivaspin 20 centrifugal concentrators (Sigma Z614637). The pH-adjusted pooled product was first diluted 1:1 with PBS and then re-concentrated to the original volume and this process was repeated 6 times. Finally, the buffer-exchanged stock solution was further concentrated to the target value of »17 mg/mL. The protein concentration was confirmed to be 16.5 mg/mL by UV analysis at A280-320 nm using an extinction coefficient of 1.5; a total of 7.9 mL was isolated with a yield of 88%.

Conjugation of 4 with a 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 mixed thoroughly 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.

Cleaning ConjA2.

The filtered conjugation mixture was purified by constant volume diafiltration using a Pellicon 3 30 kDa membrane with a membrane area of »50 g/ ^m2 , a cross-flow of 5.0 ± 0.25 L/min/ ^m2 , a TMP of 1.0 ± 0.2 bar and a total of 12 diavolumes of buffer exchange in PBS at 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 by

- 37 040749

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

Analysis of the product showed the following properties:

transparent, colorless, particle-free

4.9 mg/ml [protein] according to spectroscopy at A280/330 nm average DAR 1.9 (according to reversed 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 antibodies.

Approximately 60 mg of mesothelin antibody was buffer exchanged with 25 mM Tris/Cl, 150 mM NaCl, pH 8.0 using a G25 desalting column; 4 x 2.5 ml at 6 mg/ml loaded on 4 x PD10 desalting columns (GE 17085101). The buffer exchanged antibody was then concentrated to at least 25 mg/ml using a Vivaspin 20 centrifugal concentrator (Sigma Z614637). Protein concentration was confirmed by UV analysis at A280-320 nm using an extinction coefficient of 1.5.

Remodeling response.

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

Solution Volume (µl) Final concentration 25 mMtris/C1, 150 mM NaCl, pH=8.0 663.8 No data Antibody to mesothelin in 25 mM Tris/Cl, 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 relative to 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 equiv. relative to MAT

Purification procedure on protein A.

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

- 38 040749

Solution CV Notes 0.1 MNaOH 3 Disinfection 25 mM Tris/Cl, 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 Endotoxin Wash/Removal Stage 25 mM Tris/Cl, 25 mM NaCl, pH=8.0 20 Newt wash/removal stage 0.15 M acetic acid As needed Collection of peak from 0.1 to 0.1 opt. units.

Following affinity purification on Protein A, a small sample of the product can be recovered using DTT and subsequently analyzed by MS. 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 the 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 the modified galactose to an antibody with a substituted central GlcNAc (no fucose).

Buffer replacement after protein A purification.

Next, the buffer in the remodeled and purified GalNAc-N3-mesothelin antibody is exchanged 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 and 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 of 4 with a modified antibody to produce ConjA3

Reaction conditions.

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

0.3 ml 10 mM 4 in dimethylacetamide.

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

Cleaning ConjA3.

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 1:1 ratio and then re-concentrated to the original volume. This was repeated 12 times before the purified ADC mass was removed from the centrifugal device.

Protein concentration is determined by quantitative SEC analysis using a calibration curve of the 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 removed for testing and the remainder is aliquoted into 1 mL portions and frozen at -80°C.

Cytotoxicity of ConjA1 in vitro.

Flasks containing 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 1:1 ratio and counting clear/blue (live/dead) cells using a Luna II automated cell counter (Logos Biosystems). The cell suspension was diluted to the desired seeding density (20 x 104/mL), dispensed into white 96-well flat-bottomed 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 8x 10-fold dilutions of the ConjA1 stock solution were prepared in a sterile 24-well plate by sequentially transferring 100 µl into 900 µl of cell culture medium. Each ConjA1 dilution was dispensed, 50 µl/well, into quadruple wells of a 96-well plate containing the cell suspension. Control wells received a similar volume of culture medium only.

Following the ConjA1 exposure period, cell viability was measured using a Promega CellTiter-Glo by adding 100 µl/well, mixing for 2 min, and reading the signal on Envision using the luminescence protocol. Data were 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*).

The EC ₅₀ of ConjA1 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 ▼ represents ConjA1 and • represents an otherwise identical ADC control containing the B12 antibody, which is non-specific for DLK1.

In vivo efficacy studies using ConjA1.

Antitumor activity in a patient-derived liver cancer (PDX) xenograft model of LI1097 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 ^mm3 , tumors were harvested and cut into pieces of approximately 2-3 ^mm3 in diameter. Tumors or tumor pieces were washed with ice-cold RPMI1640 media (serum-free) and then placed in ice-cold media for use.

Before 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 right flank with one fragment of the primary human liver tumor LI1097 for tumor development.

After tumor inoculation, the animals were checked for morbidity and mortality daily. The tumor size was measured with a caliper twice a week in two dimensions. The tumor volume was expressed in ^mm3 using the formula: TV=0.5axb2, where a and b represent the long and short diameters of the tumor, respectively.

On study day 12, mice were randomized into 5 groups of 8 mice each; mean tumor volume was ~170 ^mm3 per 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 for up to day 51.

The results are shown in Fig. 2A, in which:

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

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

As shown in Fig. 2A, ConjA1 at 1.0 mg/kg resulted in the greatest tumor growth inhibition, followed by ConjA1 at 0.3 mg/kg. Furthermore, at the highest dose tested, ConjA1 resulted in 3/8 partial responses (PR) and 2/8 complete responses (CR), whereas none of the mice treated with vehicle or isotype control ADC (1 mg/kg, single dose) had PR, CR, or tumor-free survival (TFS).

Antitumor activity in a xenograft model of SK-N-FI neuroblastoma in vivo

Female NOD-SCID mice were six weeks old on the day of implantation. SK-N-FI cells were harvested during the log phase of expansion and resuspended in phosphate-buffered saline with 50% Matrigel. 100 μl (3 x 106 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 weekly to assess body weight and tumor size. Tumor size was measured using digital calipers and calculated according to the following expression:

Tumor volume ( ^mm3 ) = (minor axis)2* (major axis)* π/6

Eighteen days after cancer cell transplantation, 50 mice with tumor volumes ranging from 99.0 ^mm3 to 155.2 ^mm3 (mean 116.2 ^mm3 ) were divided into 5 groups (N=10 in each group). On the day of dosing, subjects were administered the drug by intravenous injection into the tail vein. The endpoint of the study was set as each tumor reaching the endpoint of 1000 ^mm3 or the end of the study (60 days after dosing).

- 40 040749 zy), depending on which came first.

The results are shown in Fig. 2B, in which:

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

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

As shown in Fig. 2B, ConjAl at 1.0 mg/kg resulted in the greatest tumor growth inhibition, followed by ConjAl at 0.5 mg/kg.

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

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

Toxicology study in rats

Rat toxicology study (non-target toxicity assessment, ADC tested does not bind rat Dlk-l).

Method.

ConjAl was evaluated in a single intravenous dose tolerance study in rats. Male Sprague-Dawley rats (n=3/group) were dosed at 5 mg/kg on day 1 and necropsied on day 21 post-dose. Body weight and food intake were monitored frequently with lifelong sampling for clinical pathology (blood on days 8 and 21) and repeat sampling for pharmacokinetic determinations. Macroscopic observations were made at necropsy, with selected organs weighed and preserved for possible histopathologic examination.

Results.

ConjAl was clinically well tolerated at 5 mg/kg with no significant adverse clinical signs. Body weight gain was reduced with animals approximately 15% lighter than controls at the end of the study. White blood cell counts were reduced by day 8 (neutrophil counts were reduced by approximately 95% compared to concurrent controls) with signs of recovery by day 22.

General conclusion.

ConjAl was stable, well tolerated, and showed a favorable pharmacokinetic profile in rats with a half-life of 9 days at 5 mg/kg, indicating that the MTD in rats is at least 5 mg/kg or greater.

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, 1 mM sodium pyruvate and 1% penicillin/streptomycin) in 384-well Elplasia plates pre-coated with pHEMA.

ADC, ConjAl, and B12-1601 compounds were added 24 h after cell seeding, with an initial concentration of 10 mg/mL, in 10-fold serial dilutions for 9 concentrations in quadruplicate. 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 14 days.

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

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

Data were analyzed using Graphpad Prism software.

Results.

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

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/mL];

Fig. 3B shows the 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].

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In vitro cytotoxicity of ConjA2

Flasks containing 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 1:1 ratio and counting clear/blue (live/dead) cells using a Luna II automated cell counter (Logos Biosystems). The cell suspension was diluted to the desired seeding density (20 x 10 ⁴ /ml), dispensed into white 96-well flat-bottomed 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 8x 10-fold dilutions of the stock solution of ConjA2 were prepared in a sterile 24-well plate by serially transferring 100 μl to 900 μl of cell culture medium. Each dilution of ConjA2 was dispensed, 50 μl/well, into quadruple wells of a 96-well plate containing the cell suspension. Control wells received a similar volume of culture medium only.

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

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

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

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

In vivo study of ConjA2 efficacy

Female athymic 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 study day 1.

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 ^5x107 cells/mL. Each mouse was injected subcutaneously (s.c.) into the right flank with ^5x106 cells (0.1 mL of cell suspension), and tumors were monitored when their volumes approached the target range of 100 to 150 ^mm3 . Tumors were measured with calipers in two dimensions, and the volume was calculated using the formula:

Tumor volume (mm ³ ) = w ² x l/2 where w = width and l = length in mm of the tumor. Tumor mass can be estimated by assuming that 1 mg is equivalent to 1 mm ³ of tumor volume.

Sixteen days after tumor implantation, designated study day 1, animals were sorted into groups of 8 mice each with individual tumor volumes ranging from 108 to 144 mm ³ and group mean tumor volumes of 112.5 to 123.8 mm ³ . On study day 1, all treatments were administered intravenously (i.v.) as a single injection (qdx1) by tail vein injection at a dosing volume of 0.2 ml per 20 g body weight (10 ml/kg), adjusted for each individual animal's body weight. Tumors were measured with calipers twice weekly, and each animal was euthanized when its tumor volume reached an endpoint of 1500 mm ³ or at the end of the study, whichever came first. The study was terminated on day 59.

The results are shown in Fig. 5, where:

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

As shown in Fig. 5, ConjA2 at 0.6 mg/kg resulted in significant inhibition of tumor growth.

Antitumor activity of ConjA2 in the SN12C xenograft model in vivo

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

On the day of tumor implantation, each test mouse received 5x10 ⁶ SN12C cells (0.1 ml of cell suspension in 50% Matrigel® (Corning®) matrix in phosphate-buffered saline) implanted subcutaneously in the right flank. Tumor growth was monitored when the average size approached the target range of 100 to 150 mm ³ .

Tumors were measured with calipers in two dimensions and volume was calculated using the formula:

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Tumor volume (mm ³ ) = w ² x l/2 where w = width and l = length in mm of the tumor. Tumor mass can be estimated by assuming that 1 mg is equivalent to 1 mm ³ of tumor volume.

Twenty-three days after tumor implantation, designated study day 1, animals were sorted into nine groups (n=8) with individual tumor volumes ranging from 108 to 172 ^mm3 and group mean tumor volumes of 129 ^mm3 .

On study day 1, all treatments were administered intravenously (i.v.) as a single injection (qdx 1) by tail vein injection at a dosing volume of 0.2 ml per 20 g body weight (10 ml/kg), adjusted for each individual animal's body weight. Tumors were measured with calipers twice weekly, and each animal was euthanized when its tumor volume reached an endpoint of 1000 ^mm3 or at the end of the study, whichever came first. The study was terminated on day 60.

The data are shown in Fig. 6, where it can be seen that ADC (ConjA2) administration reduced tumor growth in a dose-dependent manner.

In vitro cytotoxicity

Flasks containing 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 1:1 ratio and counting clear/blue (live/dead) cells using a Luna II automated cell counter (Logos Biosystems). The cell suspension is diluted to the desired seeding density (20 x 104/mL), dispensed into white 96-well flat-bottomed 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 8x 10-fold dilutions of the stock solution of ConjA3 was prepared in a sterile 24-well plate by serially transferring 100 μl into 900 μl of cell culture medium. Each dilution of ConjA3 was dispensed, 50 μl/well, into quadruple wells of a 96-well plate containing the cell suspension. Control wells were filled with a similar volume of culture medium only.

Following the ConjA3 exposure period, cell viability was measured using a Promega CellTiter-Glo by adding 100 µl/well, mixing for 2 min, and reading the signal on an Envision using the luminescence protocol. Data were analyzed using Graphpad Prism software.

In vivo study of ConjA3 efficacy

Antitumor activity in the OVCAR3 in vivo model.

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 , tumors were harvested and cut into pieces of approximately 2-3 mm ³ diameter.

Tumors or tumor pieces were washed with ice-cold RPMI1640 media (serum-free) and then placed in ice-cold media for use.

Before 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, into the upper right flank with one tumor fragment for tumor development.

After tumor inoculation, the animals are checked daily for morbidity and mortality. The tumor size is measured with a caliper twice a week in two dimensions. The tumor volume is expressed in ^mm3 using the formula: TV=0.5axb2, where a and b are the long and short diameters of the tumor, respectively.

On study day 12, mice are randomized into 5 groups of 8 mice each; mean tumor volume is ~170 ^mm3 per cohort. Mice are dosed with test agents on study day 13. 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.

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Sequences.

SEQ ID NO. 1 [VCO HuBa-l-3d, CDRs underlined]

QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGN TNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQ GTMVTVSS

SEQ ID NO. 2 [VL HuBa-l-3d, CDRs underlined]

DIVMTPSPDSLAVSLGERATINCKSSPSLLNSSNPKNYLAWYPPKPGPPPKLLVYFAST RESGVPDRF SSGSGSGTDFTLTIS SLOAEDVAVYYCOQHYSTPPTFGOGTKLEIK

SEQ ID NO. 3 [HuBa-l-3d heavy chain]

QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGN TNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

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

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSSNQKNYLAWYQQKPGQPPKLLVYFAST RESGVPDRF SSGSGSGTDFTLTIS SLQAEDVAVYYCQQHYSTPPTFGQGTKLEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLS STLTLSKADYEKHKVYACEVTHQGLS SP VTKSFNRGEC

SEQ ID NO. 5 [CDR1 VHHuBa-l-3d]

DYAMH

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

VISTYYGNTNYNPKFKG

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

GGLREYYYAMDY

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

KSSPSLLNSSNPKNYLA

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

FASTRES

SEP ID NP. 10 [CDR3 VLHuBa-l-3d]

PPHYSTPPT

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

QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGN TNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEQ ID NO. 12 [human DLK1 variant 11

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

SEQ ID NO. 13 [human DLK1 variant 21

MTATEALLRVLLLLLAFGHSTYGAECFPACNPQNGFCEDDNVCRCQPGWQGPLCDQCV TSPGCLHGLCGEPGQCICTDGWDGELCDRDVRACSSAPCANNRTCVSLDDGLYECSCA PGYSGKDCQKKDGPCVINGSPCQHGGTCVDDEGRASHASCLCPPGFSGNFCEIVANSCT PNPCENDGVCTDIGGDFRCRCPAGFIDKTCSRPVTNCASSPCQNGGTCLQHTQVSYECL CKPEFTGLTCVKKRALSPQQVTRLPSGYGLAYRLTPGVHELPVQQPEHRILKVSMKELN KKTPLLTEGQAICFTILGVLTSLVVLGTVGIVFLNKCETWVSNLRYNHMLRKKKNLLLQ YNSGEDLAVNIIFPEKIDMTTFSKEAGDEEI

SEQ ID NO. 101 [VH 3 A4, CDRs underlined!

OIOLVOSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGOGLEWIGDINPYNGD TNYNOKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGOGTLVTV ss

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

DIVMTOTPLSLPVTPGEPASISCRSSOSLLHSNGNTYLEWYLQKPGOSPOLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGOGTKLEIK

SEQ ID NO. 103 [heavy chain 3A41

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QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGD TNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQ ID NO. 104 [light chain 3A41

DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.105 [CDR1 VH3A41

GYTFTDDMYS

SEQ ID NO.106 [CDR2 VH3A41

DINPYNGDTN

SEQ ID NO.107 [CDR3 VH3A41

DPGAMDY

SEQ ID NO.108 [CDR1 VL3A41

RSSPSLLHSNGNTYLE

SEQ ID NO.109 [CDR2 VL3A41

TVSNRFS

SEQ ID NO. 110 [CDR3 VL3 A41

FPGSHVPLT

SEQ ID NO. III [heavy chain 3 A4, terminal K1

PIPLVPSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKPAPGPGLEWIGDINPYNGD TNYNPKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGPGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLP SSGLYSLSSVVTVPSSSLGTPTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYN*STYRVVSVLTVLHPDWLNGKEYKCKVSNKALPAPIEKTISKAKGPPREPPVYTLP PSRDELTKNPVSLTCLVKGFYPSDIAVEWESNGPPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWPPGNVFSCSVMHEALHNHYTPKSLSLSPGK

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N* stands for Asn297

SEP ID NO. 112 [KAAG1 person!

MDDDAAPRVEGVPVAVHKHALHDGLRQVAGPGAAAAHLPRWPPPQLAASRREAPPLS QRPHRTQGAGSPPETNEKLTNPQVKEK

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

DVVMTOPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLOKPGOSPKLLIYTVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGOGTKLEIK

SEQ ID NO. 114 [light chain 3 A4-L21

DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 115 [VL 3A4-K4, CDRs underlined!

DIVMTOSPDSLAVSLGERATINCRSSOSLLHSNGNTYLEWYOQKPGOPPKLLIYTVSNRF SGVPDRF SSGSGSGTDFTLTIS SLOAEDVAVYYCFQGSHVPLTFGOGTKVEIK

SEQ ID NO. 116 [light chain 3A4-K41

DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLEWYQQKPGQPPKLLIYTVSNRF SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPLTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 201 [UN XA4, CDR underlined!

OVHLVESGGGVVOPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSH EYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGOG TLVTVSS

SEQ ID NO. 202 [VL XA4, CDRs underlined!

EIVLTOSPATLSLSPGERATLSCRASOSVSSYLAWYOOKPGOAPRLLIYDASNRATGIPA RF SGSGSGTDFTLTIS SLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

SEQ ID NO. 203 [heavy chain XA41

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

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VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQ ID NO. 204 [light chain XA41

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RF SGSGSGTDFTLTIS SLEPEDF AVYYCQQRSNWPLTFGGGTKVEIKRT VAAPS VFIFPP S DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.205 [CDR1 VH XA41

IYGMH

SEQ ID NO.206 [CDR2 VH XA41

VLWYDGSHEYYADSVKG

SEQ ID NO.207 [CDR3 VH XA41

DGDYYDSGSPLDY

SEQ ID NO.208 [CDR1 VL XA41

RASPSVSSYLA

SEQ ID NO.209 [CDR2 VL XA41

DASNRAT

SEQ ID NO.210 [CDR3 VL XA41

PPRSNWPLT

SEQ ID 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

SEQ ID NO. 212 [VH XFT, CDR underlined!

OVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVROAPGKGLEWMGIIDPGDSRTRY SPSFOGOVTISADKSISTAYLOWSSLKASDTAMYYCARGQLYGGTYMDGWGOGTLVT vss

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

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DIALTOPASVSGSPGOSITISCTGTSSDIGGYNSVSWYOQHPGKAPKLMIYGVNNRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIK

SEQ ID NO. 214 [heavy chain XFT1

QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRY SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEP ID NO. 215 [XFT1 Light Chain

DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.216 [XFT CDR1 VH1

GYSFTSYWIG

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

SEQ ID NO.219 [CDR1 VL XFT1

TGTSSDIGGYNSVS

SEQ ID NO. 220 [CDR2 VL XFT1

LMIYGVNNRPS

SEQ ID NO.221 [CDR3 VL XFT1

SSYDIESATP

SEQ ID NO. 222 [heavy chain XFT, 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

SEQ ID NO. 226 [light chain X09]

DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPG RFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.227 CDR1 VH[X09]

GYSFT

SEQ ID NO. 228 [CDR2 UN X09]

LITPYNGAS S YNQKFRG

SEQ ID NO. 229 [CDR3 UN X09]

GGYDGRGFDY

SEQ ID NO.230 [CDR1 VL X09]

SASSSVSYMH

SEQ ID NO.231 [CDR2 VL X09]

- 50 040749

DTSKLAS

SEQ ID NO.232 [CDR3 VL X091

QQWSKHPLT

SEQ ID NO. 233 [heavy chain X09, terminal K1

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEQ ID NO. 234 VCO X09.2, CDR underlined!

OVOLOQSGPELEKPGASVKISCKASGYSFTGYTMNWVKOSHGKSLEWIGLITPYNGASS YNOKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGOGTTVT vss

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

DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYOOKSGTSPKRWIYDTSKLASGVPG RF SGSGSGNS YSLTIS SVEAEDDATYYCOQWSGYPLTFGAGTKLEIK

SEQ ID NO. 236 [heavy chain X09.21

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

N* stands for Asn297

SEQ ID NO. 237 [light chain X09.21

DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPG RFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

- 51 040749

SEO ID NO. 238 [CDR1 UN X09.21

GYTMN

SEP ID NO. 239 [CDR2 UN 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

SEQ ID NO. 244 [heavy chain X09.2, terminal K1

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYN^STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

N* stands for Asn297

SEQ ID NO. 245 [human mesothelium, variant 11

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

SEQ ID NO. 246 [human mesothelium, variant 21

- 52 040749

MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPL DLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPST WSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDEL YPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRF VKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYP KARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTV AEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQ EALSGTPCLLGPGPVLTVLALLLASTLA

- 53 040749

List of sequences.

<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 <150> 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

30

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> HuBa-l-3d Heavy Chain <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 Tyg Tyg 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 Tyr Ser Thr Pro Pro Thr 1 5

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

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

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

Gin 1 Vai Gin Leu Vai 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 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 LLC <210> 32 <400> 32 LLC

- 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

OOO <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

LLC <210>90 <400>90

LLC <210>91 <400>91

OOO <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 ZA4 <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> Xaa 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

30

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 Gly 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, end K <220>

< 221> MOD_RES < 222> (296)..(296) <223> Xaa 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 Vai 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 Phe 60 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 Vai 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

30

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 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

OOO <210>122 <400>122

OOO <210>123 <400>123

OOO <210>124 <400>124

LLC <210>125 <400>125

LLC <210>126 <400>126

LLC <210>127 <400>127

LLC <210>128 <400>128

LLC <210>129 <400>129

LLC <210>130 <400>130

OOO <210>131 <400>131

OOO <210>132 <400>132

OOO <210>133 <400>133

OOO <210>134

- 84 040749 <400>134

LLC <210>135 <400>135

OOO <210>136 <400>136

OOO <210>137 <400>137

LLC <210>138 <400>138

LLC <210>139 <400>139

LLC <210>140 <400>140

OOO <210>141 <400>141

OOO <210>142 <400>142

OOO <210>143 <400>143

OOO <210>144 <400>144

LLC <210>145 <400>145

OOO <210>146 <400>146

OOO <210>147

- 85 040749 <400>147

OOO <210>148 <400>148

LLC <210>149 <400>149

LLC <210>150 <400>150

LLC <210>151 <400>151

OOO <210>152 <400>152

OOO <210>153 <400>153

OOO <210>154 <400>154

LLC <210>155 <400>155

OOO <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> XA4 Light Chain <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 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> Xaa 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> Heavy Chain XET <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Xaa 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

Th

Vai

Trp 160

Asn

Gly

Ala

Leu

165

Th

Gly

Vai

His

170

Th

Phe

Pro

Ala

Vai

175

Leu

Gin

Gly 180

Leu

Tyr

Leu

Vai

Vai

Th

185

Vai

190

Pro

Leu

195

Gly

Th

Gin

Th

Tyr 200

Cys

Asn

Vai

Asn

205

His

Lys

Pro

Asn

210

Th

Lys

Vai

Asp

Lys 215

Lys

Vai

Glu

Pro

Lys 220

Cys

Asp

Lys

Th

225

His

Th

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

Th

Leu

Met

255

Arg

Th

Pro

Glu

260

Vai

Th

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 SerGlyAsnThr70 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 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, End K <220>

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

<400> 222 Gin 1 Vai Glu Leu Vai 5 Gin Ser Gly Ala Vai 10 Lys Lys Pro Gly Glu 15 Ser Leu Lys lie Ser 20 Cys Lys Gly Ser Gly 25 Tyr Ser Phe Thr Ser 30 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

ArgGly65

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

TyrPheCys95

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> Xaa 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 Pro Glu

260

Vai Thru

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, end K <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Xaa 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> Xaa 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, end K <220>

< 221> MOD_RES < 222> (299)..(299) < 223> Xaa 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 Gin 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 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 Gin 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 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 -