EA038267B1 - Pyrrolobenzodiazepine conjugates - Google Patents

Abstract

A compound of formula Ior a pharmaceutically acceptable salt thereof, wherein R6 and R9 are H; R7 is a C1-4alkoxy group; R" is a C3-12alkylene group, which chain may be interrupted by benzene; Y and Y' are O; R6', R7", R9' are selected from the same groups as R6, R7 and R9 respectively; R11b is OH; and RL is a linker for connection to a cell binding agent, such as defined in the claims.

Description

This invention relates to conjugates containing pyrrolobenzodiazepines and related dimers (PBDs) and drug precursor linkers used to prepare such conjugates.

State of the art

Some pyrrolobenzodiazepines (PBDs) have the ability to recognize and bind to specific DNA sequences; the preferred sequence is PuGPu. The first PBD antitumor antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc. 87, 5791-5793 (1965)). Since then, a variety of natural PBDs have been described and more than 10 synthetic methods for the preparation of various analogs have been developed (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeimycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chikamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180, 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), masetramycin (Kuminoto , et al., J. Antibiotics, 33, 665-667 (1980)), neotramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porotramycin (Tsunakawa, et al ., J. Antibiotics, 41, 1366-1373 (1988)), protracarcinol (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91 -97 (1987)), sibanomycin (DC-102) (Hara, et al., J. Antibiotics, 41, 702704 (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)). PBDs have a common structure:

They differ in the number, type and position of substituents in aromatic rings A and pyrrole rings C, as well as in the degree of saturation of ring C. Ring B contains imine (N = C), carbinolamine (NH-CH (OH)) or carbinolamine methyl ester (NH-CH (OMe)) at positions N10-C11, which is an electrophilic center responsible for DNA alkylation. All known natural products have a S-configuration at the chiral position C11a, which ensures their pro-sided twisting when viewed from ring C to ring A. This gives them the appropriate three-dimensional shape for isospirality with the minor groove of the B-form DNA, which leads to an exact match in binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, p. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Soc. 19, 230-237 (1986)). Their ability to form an adduct in the minor groove provides the ability to influence DNA processing, and therefore they are used as antitumor agents.

It has previously been described that the biological activity of these molecules can be enhanced by linking two PBD moieties to each other through their C8 / C'-hydroxyl functional groups via a flexible alkylene linker (Bose, DS, et al., J. Am. Chem. Soc ., 114, 49394941 (1992); Thurston, DE, et al., J. Org. Soc., 61, 8141-8147 (1996)). PBD dimers are suspected to form sequence-selective DNA lesions such as palindromic 5'-Pu-GATC-Py-3 'interstrand cross-linking (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147), which are believed to be primarily responsible for their biological activity.

First dimers (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992)) of the general formula

where n is from 3 to 6.

Compounds where n is 3 and 5 have shown promising in vitro cytotoxicity. However, when studying the antitumor activity using compound n = 3 (DSB-120) (Walton, M., et al., Cancer Chemother Pharmacol. (1996), 38: 431, doi: 10.1007 / s002800050507), it turned out to be less promising. This effect was believed to be due to low tumor selectivity and drug consumption as a result of high protein binding and / or extensive in vivo drug metabolism.

In order to improve these compounds, they were investigated (Gregson, S.J., et al., Chem. Commun., 1999, 797-798, doi: 10.1039 / A809791G) with the inclusion of substituents C2 / C2 ', which should follow the contour of the minor groove of the host. For this connection SG2000 (SJG-136)

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was found to have exceptional cytotoxicity in the picomolar region ... approximately 9000 times more potent than DSB-120.

This compound (also discussed in Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, MC, et al., Cancer, Cancer Research, 64, 6700-6706 ( 2004); and Hartley, JA, et al., Cancer Research, 64, 6693-6699 (2004)) has been investigated in clinical trials as a separate agent, e.g. NCT02034227, studying its use in the treatment of acute myeloid leukemia and chronic lymphocytic leukemia ( see: https://www.clinicaltrials.gov/ct2/show/NCT02034227).

Dimeric PBD compounds containing C2 aryl substituents such as SG2202 (ZC-207) are described in WO 2005/085251:

These compounds have been shown to be very useful cytotoxic agents (Howard, P.W., et al., Bioorg. Med. Chem. (2009), doi: 10.1016 / j.bmcl.2009.09.012).

A PBD review containing ADCs (Mantaj, J., et al., Angew. Chem. Int. Ed. (2016), 55, 2-29; DOI: 10.1002 / anie.201510610) discusses the SAR of PBD dimers. A summary of the SAR is shown in FIG. 3 - In C2-exo and C1-C2 / C2-C3, unsaturation enhances activity. A more detailed discussion can be found in section 2.4, where it says:

DSB-120 has low in vivo activity, which is partly due to its high reactivity with cellular thiol-containing molecules such as glutathione. However, the introduction of C2 / C2'-exo unsaturation, as in SJG-136, resulted in an overall increase in DNA binding affinity and cytotoxicity, as well as a lower reactivity towards cellular nucleophiles with a large amount of agent potentially reaching its target DNA.

2007/085930 describes the preparation of PBD dimeric compounds containing linker groups for binding to a cell binding agent such as an antibody. The specified linker is in the bridge bond of monomeric PBD fragments of this dimer.

Dimeric PBD compounds containing linker groups for binding to a cell binding agent such as an antibody are described in WO 2011/130598. The linker in such compounds is attached to one of the available N10 positions, and is usually cleaved by an enzyme at this linker group. Dimeric PBD compounds are endo- or exo-unsaturated in the C-ring.

WO 2014/057074 and WO 2015/052322 describe specific dimeric PBD conjugates linked through the N10 position in a single monomer, and all of these compounds are endounsaturated at the C-ring.

WO 2014/096365 discloses the connection:

in which the lack of unsaturation in the C-ring is due to the fact that the B-ring is a dilactam and therefore does not have the ability to covalently bind DNA.

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The essence of the invention

This invention provides dimeric drug linkers and PBD conjugates in which the C-ring is not endo- or exo-unsaturated.

The first aspect of this invention includes a compound of formula I r ^l

or a pharmaceutically acceptable salt thereof, wherein R ⁶ and R ⁹ are H;

R ⁷ represents a C _1-4 alkoxy group;

R represents a C _3-12 alkylene group, the chain of which can be interrupted by benzene;

Y and Y 'represent O;

R ⁶ , R ⁷ , R ⁹ are selected from the same groups as R ⁶ , R ⁷ and R ^9, respectively;

R ^11b represents OH;

R ^l is a linker for binding to a cell-binding agent, which is selected from: (IIIa)

where Q is

Q ^X is such that Q is an amino acid residue, a dipeptide residue, or a tripeptide residue;

X represents

where a = 0-5, b = 0-16, c = 0 or 1, d = 0-5;

G ^l is a linker for linking to an antibody or an active fragment thereof; and (IIIb)

where R ^L1 and R ^{L2 are} independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group;

e is 0 or 1; and (a) R ²⁰ is H and R ²¹ is OH or ORA, where RA is C _1-4 alkyl; or (b) R ²⁰ and R ²¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bonded; or (c) R ²¹ is OH or ORA, where RA is C _1-4 alkyl and R ^{20 is} selected from:

(c-i)

(c-ii)

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R ^z (c-iii) *

where R ^{Z is} selected from:

^(zi) (z-ii) OC (= O) CH3;

(z-iii) NO2;

(z-iv) OMe;

(z-v) glucuronide;

(z-vi) -C (= O) -X1-NHC (= O) X2-NH-R ^Z C, where -C (= O) -X1-NH- and -C (= O) -X2-NH - are natural amino acid residues and R ^{ZC is} selected from Me, OMe, OCH2CH ₂ OMe.

It has been found that such drug linkers are rapidly conjugated to ligand units such as antibodies.

A second aspect of this invention provides conjugates of Formula II

L - (D ^L ) _P (II).

where L represents an antibody or an active fragment;

DL is a linker moiety of a drug compound of Formula I '

where R ⁶ , R ⁷ , R ⁹ , R ^11b , Y, R, Y ', R ⁶ ', R ⁷ , R ⁹ ', R ²⁰ and R ²¹ claims 1-4;

RLL is a linker for a connection selected from:

(IIIa ') are as defined in any of a cell binding agent that

where Q and X are as defined in any of paragraphs. 1 and 6;

GLL is a linker associated with an antibody or an active fragment thereof; and (IIIb ')

where RL ¹ and RL ² are as defined in the first aspect; where p is an integer from 1 to 20.

A ligand fragment, described in more detail below, is a targeting agent that binds to a target fragment. For example, a ligand fragment can specifically bind to a cellular component (cell binding agent) or to other target molecules of interest. The ligand fragment can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding antibody fragment, or other binding agent such as an Fc fusion protein.

These conjugates have been found to be highly tolerated, resulting in a high therapeutic index, making them promising candidates for clinical development.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the conjugate of the second aspect of the present invention and a pharmaceutically acceptable diluent, carrier or excipient.

In a fourth aspect of the present invention, there is provided the use of a conjugate for a second ac

- 4,038267 to the invention or pharmaceutical composition according to the third aspect of the present invention for the treatment of a proliferative disease such as cancer in a subject.

Those of skill in the art can readily determine whether a potential conjugate is suitable or unsuitable for treating a proliferative condition for any particular cell type. For example, assays that can conveniently be used to assess the activity provided by a particular compound are described in the Examples below.

Definitions

Deputies.

The expression optionally substituted in this context refers to a parent group that may be unsubstituted or that may be substituted.

Unless otherwise indicated, the term substituted in this context refers to the parent group that has one or more substituents. The term substituent is used in this context in the usual sense and refers to a chemical moiety that is covalently attached or, if possible, fused to the parent group. Numerous substituents are well known, and methods for their preparation and incorporation into various starting groups are also well known.

Examples of substituents are described in more detail below.

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

Examples of saturated alkyl groups include, but are not limited to, methyl (C ₁ ), ethyl (C ₂ ), propyl (C ₃ ), butyl (C ₄ ), pentyl (C5), hexyl (C ₆ ), and heptyl (C ₇ ).

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

Examples of saturated branched alkyl groups include isopropyl (C3), butyl (C _4), sec -butyl (C _4), tert-butyl (C _4), isopentyl (C5), and neo-pentyl (C5).

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

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

C _2-12 alkynyl: The term C _2-12 alkynyl as used herein 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 (—CACH) and 2-propynyl (propargyl, —CH ^ feCH).

C _3-12 cycloalkyl: The term C _3-12 cycloalkyl as used herein refers to an alkyl group which is also a cyclic group, i.e. to a monovalent moiety obtained by abstraction of a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, and said moiety contains 3 to 7 carbon atoms, including 3 to 7 ring etomes.

Examples of cycloalkyl groups include, but are not limited to, groups derived from:

saturated monocyclic hydrocarbon compounds:

cyclopropane (C3), cyclobutane (C ₄ ), cyclopentane (C ₅ ), cyclohexane (C ₆ ), cycloheptane (C ₇ ), methylcyclopropane (C ₄ ), dimethylcyclopropane (C ₅ ), methylcyclobutane (C ₅ ), dimethylcyclobutane (C ₆ ), methylcyclopentane (C ₆ ), dimethylcyclopentane (C ₇ ) and methylcyclohexane (C7);

unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C3), cyclobutene (C ₄ ), cyclopentene (C ₅ ), cyclohexene (C ₆ ), methylcyclopropene (C ₄ ), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C ₆ ), dimethylcyclopentene (C ₇ ) and methylcyclohexene (C ₇ ); and saturated polycyclic hydrocarbon compounds:

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

C _3-20 heterocyclyl: The term C _3-20 heterocyclyl in this context refers to a monovalent moiety obtained by the abstraction of a hydrogen atom from a ring atom of a heterocyclic compound, and said moiety contains 3 to 20 ring atoms, of which 1 to 10 are ring heteroatoms. Preferably, each ring contains 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms.

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In this context, prefixes (eg, C _3-20 , C _3-7 , C _5-6 , etc.) denote the number of ring atoms or a range of the number of ring atoms, whether carbon atoms or heteroatoms. For example, the term C _5-6 heterocyclyl as used herein 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 (C ₄ ), pyrrolidine (tetrahydropyrrole) (C ₅ ), pyrroline (e.g. 3-pyrroline, 2,5-dihydropyrrole) (C ₅ ), 2H-pyrrole or 3H-pyrrole (isopyrrole , isoazole) (C ₅ ), piperidine (C ₆ ), dihydropyridine (C ₆ ), tetrahydropyridine (C ₆ ), azepine (C ₇ );

O ₁ : oxirane (C ₃ ), oxetane (C ₄ ), oxolane (tetrahydrofuran) (C ₅ ), oxol (dihydrofuran) (C ₅ ), ocean (tetrahydropyran) (C ₆ ), dihydropyran (C ₆ ), pyran ( C ₆ ), oxepine (C ₇ );

S1: tyrant (C3), thietane (C ₄ ), thiolane (tetrahydrothiophene) (C ₅ ), thiane (tetrahydrothiopyran) (C ₆ ), thiepan (C7);

O2: dioxolane (C ₅ ), dioxane (C ₆ ) and dioxepane (C ₇ );

O ₃ : trioxane (C ₆ )

N2: imidazolidine (C ₅ ), pyrazolidine (diazolidine) (C ₅ ), imidazoline (C ₅ ), pyrazoline (dihydropyrazole) (C ₅ ), piperazine (C ₆ );

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

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

N2O1: oxadiazine (C ₆ );

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

N1O1S1: oxathiazine (C ₆ ).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides in cyclic form, for example furanose (C ₅ ) such as arabinofuranose, lyxofuranose, ribofuranose and xylofuranose, and pyranose (C ₆ ) such as allopyranose, altropyranose, mannopyranose gulopyranose, idopyranose, galactopyranose, and talopyranose.

C _5-20 aryl: The term C _5-20 aryl as used herein refers to a monovalent moiety obtained by the abstraction of a hydrogen atom from an aromatic ring atom of an aromatic compound, and said moiety contains from 3 to 20 ring atoms. The term C _5-7 aryl in this context refers to a monovalent moiety obtained by the abstraction of a hydrogen atom from an aromatic ring atom of an aromatic compound, and said moiety contains from 5 to 7 ring atoms, and the term C _5-10 aryl in this context refers to a monovalent a fragment obtained by the abstraction of a hydrogen atom from an aromatic ring atom of an aromatic compound, and said fragment contains from 5 to 10 ring atoms. Preferably, each ring contains 5 to 7 ring atoms.

In this context, prefixes (eg, C _3-20 , C _5-7 , C _5-6 , C _5-10 , etc.) denote the number of ring atoms or a range of the number of ring atoms, whether carbon atoms or heteroatoms. For example, the term C _5-6 aryl as used herein refers to an aryl group containing 5 or 6 ring atoms.

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

Examples of carboaryl groups include, but are not limited to, groups derived from benzene (i.e. phenyl) (C ₆ ), naphthalene (C ₁₀ ), azulene (C ₁₀ ), anthracene (C ₁₄ ), phenanthrene (C ₁₄ ) , naphthacene (C ₁₈ ) and pyrene (C ₁₆ ).

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) (C ₉ ), indene (C ₉ ), isoindene (C ₉ ), tetralin (1,2,3,4 tetrahydronaphthalene (C ₁₀ ), acenaphthene (C ₁₂ ), fluorene (C ₁₃ ), phenalene (C ₁₃ ), acetphenanthrene (C ₁₅ ) and aceanthrene (C ₁₀ ).

Alternatively, ring atoms can include one or more heteroatoms, as in heteroaryl groups. Examples of monocyclic heteroaryl groups include, but are not limited to, groups derived from:

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

O1: furan (oxol) (C ₅ );

S1: thiophene (thiol) (C ₅ );

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

N ₂ O1: oxadiazole (furazan) (C ₅ );

N3O1: oxatriazole (C ₅ ).

N1S1: 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 (C ₅ ), triazine (C ₆ );

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N ₄ : tetrazole (C ₅ ).

Examples of fused ring-containing heteroaryl include, but are not limited to:

C ₉ (with 2 fused rings) derived from benzofuran (O ₁ ). isobenzofuran (O ₁ ), indole (N ₁ ), isoindole (N ₁ ), indolizine (N ₁ ), indoline (N ₁ ), isoindoline (N ₁ ), purine (N ₄ ) (eg adenine, guanine), benzimidazole (N ₂ ), indazole (N ₂ ), benzoxazole (N ₁ O ₁ ), benzisoxazole (N ₁ O ₁ ), benzodioxole (O ₂ ), benzofurazan (N2O ₁ ), benzotriazole (N3), benzothiofuran (S ₁ ), benzothiazole (N ₁ S ₁ ), benzothiadiazole (N2S);

C ₁₀ (with 2 condensed rings), derived from chromene (O ₁ ), isochromene (O ₁ ), chroman (O ₁ ), isochroman (O ₁ ), benzodioxane (O ₂ ), quinoline (N ₁ ), isoquinoline (N ₁ ), quinolysine (N ₁ ), benzoxazine (N ₁ O ₁ ), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2) ), pteridine (N ₄ );

Cs (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 (N ₂ ); and

C ₁₄ (with 3 condensed rings), derived from acridine (N ₁ ), xanthene (O ₁ ), thioxanthene (S ₁ ), oxanthrene (O ₂ ), phenoxathiine (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 (N ₂ ).

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

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

Hydroxy: -OH.

An ether: -OR where R is an ether substituent, for example a C _1-7 alkyl group (also referred to as a C _1-7 alkoxy group as described below), a C _3-20 heterocyclyl group (also referred to as a C _3-20 heterocyclyloxy) or C ₅ - ₂₀ aryl group (also referred to as a C _5-20 aryloxy group), preferably a _C1-7 alkyl group.

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

Acetal: -CH (OR ¹ ) (OR ² ), where R ¹ and R ^{2 are} independently substituents of the acetal, for example a C1-7 alkyl group, a C3-20 heterocyclyl group or a C5-20 aryl group, preferably a C1-7 alkyl group, or in the case the cyclic acetal group R ¹ and R ² together with the two oxygen atoms to which they are attached and the carbon atoms to which they are attached form a heterocyclic ring containing from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, C — CH (OMe) 2, —CH (OEt) _2, and —CH (OMe) (OEt).

Hemiacetal: —CH (OH) (OR ¹ ), where R ¹ is a substituent of a hemiacetal, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, CH (OH) (OMe) and —CH (OH) (OEt).

Ketal: -CR (OR ¹ ) (OR ² ), where R ¹ and R ² are as defined for acetals and R is a ketal substituent other than hydrogen, for example a C _1-7 alkyl group, C _3-20 a heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Examples of ketal groups include, but are not limited to, -C (Me) (OMe) ₂ , -C (Me) (OEt) ₂ , -C (Me) (OMe) (OEt), -C (Et) (OMe) 2, —C (Et) (OEt) ₂ and —C (Et) (OMe) (OEt).

Semi-ketal: -CR (OH) (OR ¹ ), where R ¹ is as defined for hemiacetals and R is a substituent of a hemiketal other than hydrogen, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or C _{A 5-20} aryl group, preferably a C _1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -C (Me) (OH) (OMe), -C (Et) (OH) (OMe), -C (Me) (OH) (OEt), and -C (Et ) (OH) (OEt).

Oxo (keto, -one): = O.

Tion (thioketone): = S.

Imino (imine): = NR where R is a substituent of an imino group, for example hydrogen, a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably hydrogen or a C _1-7 alkyl group. Examples of ester groups include, but are not limited to, = NH, = NMe, = NEt, and = NPh.

Formyl (carbaldehyde, carboxaldehyde): -C (= O) H.

Acyl (keto): C (= O) R where R is an acyl substituent, for example a C _1-7 alkyl group (also referred to as C _1-7 alkyl acyl or C _1-7 alkanoyl), C _3-20 heterocyclyl group ( also referred to as C _3-20 heterocyclyl acyl) or C _5-20 aryl group (also referred to as C _5-20 aryl acyl), preferably C _1-7 alkyl group. Examples of acyl groups include, but are not limited to

- 7 038267 this, C (= O) CH ₃ (acetyl), C (= O) CH ₂ CH ₃ (propionyl), C (= O) C (CH ₃ ) ₃ (tert-butyryl) and C (= O ) Ph (benzoyl, phenone).

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

Thiocarboxy (thiocarboxylic acid): -C (= S) SH.

Thiolocarboxy (thiolocarboxylic acid): -C (= O) SH.

Thionocarboxy (thionocarboxylic acid): -C (= S) OH.

Imic acid: -C (= NH) OH.

Hydroxamic acid: -C (= NOH) OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C (= O) OR, where R is an ester substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Examples of ester groups include, but are not limited to, -C (= O) OCH ₃ , -C (= O) OCH ₂ CH ₃ , -C (= O) OC (CH ₃ ) _3, and -C (= O) OPh ...

Acyloxy (reverse ester): -OC (= O) R, where R is an acyloxy substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group ... Examples of acyloxy groups include, but are not limited to, -OC (= O) CH3 (acetoxy), -OC (= O) ODSH3, -OC (= O) C (CH3) s, -OC (= O) Ph, and -OC (= O) CH2Ph.

Oxycarbyloxy: —OC (= O) OR, where R is an ester substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Examples of ester groups include, but are not limited to, —OC (= O) OCH3, —OC (= O) OCH2CH3, —OC (= O) OC (CH3) s, and —OC (= O) OPh.

Amino: -NR1R ² , where R ¹ and R ^{2 are} independently substituents for amino, for example hydrogen, a C1-7 alkyl group (also referred to as a C1-7 alkylamino or di-C1-7 alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group preferably H or a C1-7 alkyl group, or in the case of a cyclic amino group, R ¹ and R ² together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 4 to 8 ring atoms. Amino groups can be primary (-NH2), secondary (-NHR ¹ ) or tertiary (-NHR1R ² ) and in cationic form can be quaternary (- + NR1R2R ³ ). Examples of amino groups include, but are not limited to, —NH2, —NHCH3, —NHC (CH3) 2, —N (CH ₃ ) ₂ , —N (CH2CH ₃ ) 2, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C (= O) NR1R ² , where R ¹ and R ^{2 are} independently substituents for amino, as defined for amino groups. Examples of amido groups include, but are not limited to, -C (= O) NH2, -C (= O) NHCH ₃ , -C (= O) N (CH ₃ ) 2, -C (= O) NHCH2CH _3, and -C (= O) N (CH2CH ₃ ) 2 as well as amido groups in which R ¹ and R ² together with the nitrogen atom to which they are attached form a heterocyclic structure, such as in piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C (= S) NR1R ² , where R ¹ and R ^{2 are} independently substituents for amino, as defined for amino groups. Examples of amido groups include, but are not limited to, -C (= S) NH2, -C (= S) NHCH3, -C (= S) N (CH3) 2, and -C (= S) NHCH2CH3.

Acylamido (acylamino): -NR1C (= O) R ² , where R ¹ is an amide substituent, for example hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group, and R ² represents an acyl substituent, for example a C1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably hydrogen or a C _1-7 alkyl group. Examples atsilamidnyh groups include, but are not limited to, N-NHC (= O) CH _3, -NHC (= O) CH2CH ₃ and NHC (= O) Ph. R ¹ and R ² can together form a cyclic structure, such as in succinimidyl, maleimidyl and phthalamidyl:

suk qin im id il maleimidil phthalimidil

Aminocarbonyloxy: —OC (= O) NR1R ² , where R ¹ and R ^{2 are} independently substituents of the amino group, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC (= O) NH2, —OC (= O) NHMe, —OC (= O) NMe2, and —OC (= O) NEt2.

Ureido: -N (R1) CONR ² R ³ , where R ² and R ^{3 are} independently amino substituents as defined for amino groups and R ¹ is a ureido substituent, for example hydrogen, C _1-7 alkyl group, C _3-20 heterocyclyl a group or a C _5-20 aryl group, preferably hydrogen or a C _1-7 alkyl group. Examples of ureido groups include, but are not limited to, -NHCONH2, -NHCONHMe, -NHCONHEt, -NHCONMe2, -NHCONEt2, -NMeCONH2, -NMeCONHMe, -NMeCONHEt, -NMeNCONMe2, and -NMeCONHEt, -NMeNCONMe2, and -NMeCONHEt.

Guanidino: -NH-C (= NH) NH ₂ .

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Tetrazolyl: A 5-membered aromatic ring containing four nitrogen atoms and one carbon atom

Yes,

Imino: = NR, where R is a substituent of an imino group, for example hydrogen, a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably hydrogen or a C _1-7 alkyl group. Examples of imino groups include, but are not limited to, = NH, = NMe, and = NEt.

Amidine (amidino): -C (= NR) NR ² , where each R is an amidine substituent, for example hydrogen, a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably H or C _1-7 alkyl group. Examples of amidine groups include, but are not limited to, —C (= NH) NH ₂ , —C (= NH) NMe _2, and —C (= NMe) NMe ₂ .

Nitro: -NO ₂ .

Nitroso: -NO.

Azido: -N ₃ .

Cyano (nitrile, carbonitrile): -CN.

Isocyano: -NC.

Cyanato: -OCN.

Isocyanato: -NCO.

Thiocyano (thiocyanato): -SCN.

Isothiocyano (isothiocyanato): -NCS.

Sulfhydryl (thiol, mercapto): -SH.

A thioether (sulfide): -SR where R is a thioether substituent, for example a C _1-7 alkyl group (also referred to as a C _1-7 alkylthio group), a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably A _C1-7 alkyl group. Examples of C _1-7 alkylthio groups include, but are not limited to —SCH3 and —SCH2CH3.

Disulfide: -SS-R where R is a disulfide substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a C _1-7 alkyl group (also referred to as C _{1 -7} alkyl disulfide). Examples of C _1-7 alkyl disulfide groups include, but are not limited to —SSCH3 and —SSCH2CH3.

Sulfin (sulfinyl, sulfoxide): -S (= O) R where R is a sulfinyl substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group group. Examples of sulfinic groups include, but are not limited to,

-S (= O) CH ₃ and -S (= O) CH ₂ CH ₃ .

Sulfone (sulfonyl): -S (= O) ₂ R where R is a sulfonyl substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group including, for example, a fluorinated or perfluorinated C _1-7 alkyl group.

Examples of sulfone groups include, but are not limited to, -S (= O) ₂ CH ₃ (methanesulfonyl, mesyl), -S (= O) ₂ CF ₃ (triflyl), -S (= O) ₂ CH ₂ CH ₃ ( asil), -S (= O) ₂ C ₄ F ₉ (nonaflil), -S (= O) ₂ CH ₂ CF ₃ (tresyl), -S (= O) ₂ CH ₂ CH ₂ NH ₂ (tauryl), -S (= O) ₂ Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brozil), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsil) and 5 -dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): -S (= O) OH, -SO2H.

Sulfonic acid (sulfo): -S (= O) ₂ OH, -SO3H.

Sulfinate (sulfinic acid ester): S (= O) OR, where R is a sulfinate substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group group. Examples of sulfinate groups include, but are not limited to, —S (= O) OCH ₃ (methoxysulfinyl; methyl sulfinate) and —S (= O) OCH ₂ CH ₃ (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): -S (= O) ₂ OR, where R is a sulfonate substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a C _{1- 7} an alkyl group. Examples of sulfonate groups include, but are not limited to, —S (= O) ₂ OCH ₃ (methoxysulfonyl; methyl sulfonate) and —S (= O) ₂ OCH ₂ CH ₃ (ethoxysulfonyl; ethyl sulfonate).

Sulfinyloxy: —OS (= O) R, where R is a substituent of a sulfinyloxy _{group, for example a C 1-7} alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS (= O) CH3 and —OS (= O) CH2CH3.

Sulfonyloxy: -OS (= O) ₂ R, where R is a substituent of a sulfonyloxy group, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably

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C _1-7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS (= O) ₂ CH ₃ (mesylate) and —OS (= O) ₂ CH ₂ CH ₃ (esylate).

Sulfate: -OS (= O) 2OR; where R represents a sulfate substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Examples of sulfate groups include, but are not limited to, —OS (= O) ₂ OCH ₃ and —SO (= O) 2OCH2CH3.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S (= O) NR1R ² , where R ¹ and R ^{2 are} independently substituents of the amino group, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, -S (= O) NH2, -S (= O) NH (CH ₃ ), -S (= O) N (CHs) 2, -S (= O) NH ( CH2CH3), -S (= O) N (CH2CH3) 2 and -S (= O) NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S (= O) 2NR1R ² , where R ¹ and R ^{2 are} independently substituents for amino groups, as defined for amino groups. Examples of sulfonamide groups include, but are not limited to, -S (= O) ₂ NH ₂ , -S (= O) ₂ NH (CH ₃ ), -S (= O) 2N (CH3) 2, -S (= O ) 2NH (CH2CH3), -S (= O) 2N (CH2CH3) 2U -S (= O) 2NHPh.

Sulfamino: —NR1S (= O) 2OH, where R ¹ is an amino substituent as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS (= O) ₂ OH and —N (CH ₃ ) S (= O) ₂ OH.

Sulfonamino: -NR1S (= O) 2R, where R ¹ is an amino group substituent as defined for amino groups and R is a sulfonamino _{group substituent, for example a C 1-7} alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group a group, preferably a C _1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS (= O) ₂ CH ₃ and —N (CH3) S (= O) 2C6H5.

Sulfinamino: -NR1S (= O) R, where R ¹ is a substituent of an amino group as defined for amino groups and R is a substituent of a sulfinamino group, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group a group, preferably a C _1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS (= O) CH ₃ and —N (CH ₃ ) S (= O) C ₆ H ₅ .

Phosphino (phosphine): -PR2 where R is a substituent of a phosphino group, for example -H, C _1-7 alkyl group, C _3-20 heterocyclyl group or C _5-20 aryl group, preferably -H, C _1-7 alkyl group or a C _5-20 aryl group. Examples of phosphino groups include, but are not limited to, —PH ₂ , —P (CH ₃ ) ₂ , —P (CH ₂ CH ₃ ) ₂ , —P (t-Bu) _2, and —P (Ph) ₂ .

Phospho: -P (= O) ₂ .

Phosphinyl (phosphine oxide): -P (= O) R ² , where R is a phosphinyl substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _5-20 aryl group, preferably a C _1-7 alkyl group or a C _5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, -P (= O) (CH ₃ ) ₂ , -P (= O) (CH ₂ Ch ₃ ) ₂ , -P (= O) (t-Bu) _2, and - P (= O) (Ph) ₂ .

Phosphonic acid (phosphono): -P (= O) (OH) ₂ .

Phosphonate (phosphonic ester): -P (= O) (OR) ₂ , where R is a phosphonate substituent, for example -H, C _1-7 alkyl group, C _3-20 heterocyclyl group or C _5-20 aryl group, preferably H, C _1-7 alkyl group or C _5-20 aryl group. Examples of phosphonate groups include, but are not limited to, -P (= O) (OCH ₃ ) ₂ , -P (= O) (OCH ₂ CH ₃ ) ₂ , -P (= O) (Ot-Bu) _2, and - P (= O) (OPh) 2.

Phosphoric acid (phosphonooxy): -OP (= O) (OH) ₂ .

Phosphate (phosphonooxy ester): -OP (= O) (OR) ₂ , where R is a phosphate substituent, for example -H, C _1-7 alkyl group, C _3-20 heterocyclyl group, or C _5-20 aryl group preferably H, C _1-7 alkyl group or C _5-20 aryl group. Examples of phosphate groups include, but are not limited to, -OP (= O) (OCH ₃ ) ₂ , -OP (= O) (oCh ₂ CH ₃ ) ₂ , -OP (= O) (Ot-Bu) _2, and - OP (= O) (OPh) 2.

Phosphorous acid: -OP (OH) ₂ .

Phosphite: —OP (OR) ₂ where R is a phosphite substituent, for example —H, C _1-7 alkyl group, C _3-20 heterocyclyl group or C _5-20 aryl group, preferably H, C _1-7 alkyl group or a C _5-20 aryl group. Examples of phosphite groups include, but are not limited to, —OP (OCH ₃ ) ₂ , —OP (OCH ₂ CH ₃ ) ₂ , —OP (Ot-Bu) _2, and —OP (OPh) ₂ .

Phosphoramidite: —OP (OR1) —NR ² 2 where R ¹ and R ² are phosphoramidite substituents, for example —H, an optionally substituted C1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group preferably —H, C _1-7 alkyl group, or C _5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP (OCH ₂ CH ₃ ) —N (CH ₃ ) ₂ , —OP (OCH ₂ CH ₃ ) —N (i-Pr) _2, and —OP (OCH ₂ CH ₂ CN) -N (i-Pr) ₂ .

Phosphoramidate: —OP (= O) (OR1) —NR ² 2, where R ¹ and R ² are phosphoramidate substituents, for example —H, an optionally substituted C1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _{5- 20} aryl group, preferably —H, C _1-7 alkyl group or C _5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP (= O) (OCH ₂ CH ₃ ) -N (CH ₃ ) ₂ ,

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- OP (= O) (OCH2CH3) -N (i-Pr) 2 and -OP (= O) (OCH2CH2CN) -N (i-Pr) 2.

Alkylene.

C _3-12 alkylene: The term C _3-12 alkylene in this context refers to a bidentate moiety obtained by the abstraction of two hydrogen atoms from the same carbon atom or from two different carbon atoms of a hydrocarbon compound containing from 3 to 12 carbon atoms ( unless otherwise indicated), which can be aliphatic or alicyclic and which can be saturated, partially unsaturated or completely unsaturated. Thus, the term alkylene includes the subclasses of alkenylene, alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C _3-12 alkylene groups include, but are not limited to, - (CH ₂ ) _n -, where n is an integer from 3 to 12, for example -CH2CH2CH2- (propylene), -CH ₂ CH ₂ CH ₂ CH ₂ - (butylene), -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - (pentylene) and -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - (heptylene).

Examples of branched saturated C _3-10 alkylene groups include, but are not limited to, -CH (CH3) CH2-, -CH (CH3) CH2CH2-, -CH (CH3) CH2CH2CH2-, -CH2CH (CH3) CH2-,

- CH2CH (CH3) CH2CH2-, -CH (CH2CH3) -, -CH (CH2CH3) CH2- and -CH2CH (CH2CH3) CH2-.

Examples of linear partially unsaturated C _3-12 alkylene groups (C _3-12 alkenylene and alkynylene groups) include, but are not limited to, -CH = CHCH ₂ -, -CH ₂ -CH = CH ₂ -, -CH = CHCH2-CH2 -, -CH = CHCH2-CH2-CH2-, -CH = CHCH = CH, -CH = CHCH = CHCH2-,

- CH = CHCH = CHCH2-CH2-, -CH = CHCH2-CH = CH, -CH = CHCH2-CH2-CH = CH and -CH2-C ^ C-CH2-.

Examples of branched, partially unsaturated C _3-12 alkylene groups (C _3-12 alkenylene and alkynylene groups) include, but are not limited to, -C (CH ₃ ) = CH, -C (CH ₃ ) = CHCH ₂ -, -CH = CHSN (CHs) - and -C ^ C-CH (CHs) -.

Examples of alicyclic saturated C _3-12 alkylene groups (C _3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (eg, cyclopent-1,3-ylene) and cyclohexylene (eg, cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C _3-12 alkylene groups (C _3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. 4-cyclopentene-1,3-ylene), cyclohexylene (e.g. 2-cyclohexene-1,4 -ylene; 3-cyclohexene-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

In case the C _3-12 alkylene group is interrupted by a heteroatom, the subscript refers to the number of atoms in the chain, including heteroatoms. For example, the chain -C ₂ H ₄ -OC ₂ H ₄ - would be a C ₅ group.

When a C _3-12 alkylene group is interrupted by a heteroatom, the subscript refers to the number of atoms directly in the chain, including the aromatic ring. For example, the chain will be group C ₅ .

Fragment of the ligand.

The ligand fragment can be of any type and includes a protein, polypeptide, peptide, and a non-peptide agent that specifically binds to a target molecule. In some embodiments, the ligand fragment can be a protein, polypeptide, or peptide. In some embodiments, the ligand fragment may be a cyclic polypeptide. These fragments of the ligand may include antibodies or an antibody fragment that contains at least one site that binds the target molecule, lymphokines, hormones, growth factors or any other cell-binding molecule or substance that can specifically bind to the target.

The terms specifically bind and specific binding refer to the binding of an antibody or other protein, polypeptide, or peptide to a particular molecule (eg, antigen). Typically, an antibody or other molecule binds with an affinity of at least about 1x10 ⁷ M ^-1 and binds to a particular molecule with an affinity that is at least twice its binding affinity for a non-specific molecule (e.g. BSA, casein) that is excellent from the specified specific molecule or closely related molecule.

Examples of ligand fragments include those described for use in WO 2007/085930, incorporated herein.

In some embodiments, the ligand fragment is a cell binding agent that binds to an extracellular target on a cell. Such a cell-binding agent can be a protein, polypeptide, peptide, or non-peptide agent. In some embodiments, the cell binding agent can be a protein, polypeptide, or peptide. In some embodiments, the cell binding agent may be a cyclic polypeptide. The cell binding agent can also be an antibody or antigen binding fragment of an antibody. Thus, in one embodiment of the present invention, an antibody-drug conjugate (ADC) is provided.

Cell-binding agent.

The cell binding agent can be of any type and includes peptides and non-peptides. They can include antibodies or an antibody fragment that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, molecules, transport

- 11 038267 nutrient or any other cell-binding molecule or substance. Peptides.

In one embodiment, the cell binding agent is a linear or cyclic peptide containing 4-30, preferably 6-20, consecutive amino acid residues. In this embodiment, it is preferred that one cell binding agent is associated with one monomer or dimer of the pyrrolobenzodiazepine compound.

In one embodiment, the cell binding agent comprises a peptide that binds α _γ β ₆ integrin. This peptide can be selective for α _γ β ₆ over XYS.

In one embodiment, the cell binding agent comprises an A20FMDV-Cys polypeptide. A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, you can use a variant of the sequence A20FMDV-Cys, in which one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are replaced by another amino acid residue. In addition, the specified polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.

Antibodies.

The term antibody is used in this context in its broadest sense and includes, in particular, monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g. bispecific antibodies) and antibody fragments, provided that they exhibit the desired biological activity (Miller et al. (2003) Jour. Of Immunology 170: 4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein made by the immune system that is able to recognize and bind to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001), Immuno Biology, 5th ed., Garland Publishing, New York). A target antigen usually has multiple binding sites, also called epitopes, which are recognized by the complementarity determining regions (CDRs) of various antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen can have more than one corresponding antibody. An antibody comprises a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e. a molecule that contains an antigen binding site that immunospecifically binds to an antigen of the target in question or a portion thereof, and such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins can be obtained from any species of animals, including humans, mice, or rabbits.

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

The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i. E. the individual antibodies constituting the specified population are identical, with the exception of possible natural mutations, which may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, unlike polyclonal antibody preparations, which contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination of other antibodies. The modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of said antibody by any particular method. For example, monoclonal antibodies suitable for use in this invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or can be produced by recombinant DNA techniques (see US Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628; Marks et al. (1991). J. Mol. Biol., 222: 581-597, or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008), Curr. Opinion 20 (4): 450-459).

Monoclonal antibodies in this context include, in particular, chimeric antibodies in which part of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies obtained from certain animal species, or belonging to a particular class or subclass of antibodies, and the rest of the chain ( her) is identical or homologous to the corresponding sequences in antibodies obtained from other animal species, or belonging to other

- 12 038267 class or subclass of antibodies, as well as fragments of such antibodies, provided that they demonstrate the desired biological activity (US 4816567 and Morrison et al. (1984), Proc. Natl. Acad. Sci. USA, 81: 6851-6855 ). Chimeric antibodies include primatized antibodies containing variable domain antigen binding sequences derived from a non-human primate (eg, monkeys or apes) and human constant region sequences.

An intact antibody in this context is an antibody containing the VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. Constant domains can be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more effector functions that relate to biological activity associated with the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis and downregulation of cell surface receptors such as B-cell receptor and BCR.

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

Humanization.

Technologies for reducing the in vivo immunogenicity of a non-human antibody or antibody fragment include technologies called humanization.

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

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

There are many technologies for humanization, including CDR grafting, targeted selection, deimmunization, surface alteration (also known as surface remnant recombination), composite antibodies, human chain composition optimization, and scaffold rearrangement.

CDR vaccination.

In this technology, humanized antibodies are human immunoglobulins (receiving antibody) in which residues from the complementarity determining region (CDR) of the receiving antibody are replaced with residues from the CDRs of non-human species (donor antibody) such as mice, rats, camels, bulls, goats, or rabbits having the required properties (in fact, non-human CDRs are grafted onto a human scaffold). In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues (this can occur, for example, if a particular FR residue has a significant effect on the binding of anti

- 13 038267 genes).

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

Directed selection.

This method consists of combining the VH or VL domain of a given epitope-specific non-human antibody with a human VH or V _L library, and specific human V domains selected for the antigen in question. This selected VH is then combined with the VL library to produce a complete human VHxVL combination. This method is described in the publication Nature Biotechnology (NY), 12, (1994), 899-903.

Composite antibodies.

In this method, two or more segments of amino acid sequences from a human antibody are combined with a finished antibody molecule. They are constructed by combining multiple human VH and VL sequence segments in combinations that restrict or exclude human T cell epitopes in the V regions of the final composite antibody. If necessary, T cell epitopes are restricted or eliminated by replacing V region segments that promote or encode a T cell epitope with alternative segments that eliminate T cell epitopes. This method is described in US 2008/0206239A1.

Deimmunization.

This method involves removing epitopes of a human T cell (or other secondary species) from the V regions of a therapeutic antibody (or other molecule). The sequence of the V region of therapeutic antibodies is analyzed for the presence of MHC class II binding motifs, for example, by comparison with databases of MIS binding motifs (such as the database of these motifs available at www.wehi.edu.au). Alternatively, MHC class II binding motifs can be identified using computational pulling techniques described in Alluvia et al. Mol. Biol. (J. Mol. Biol. 249 244-250 (1995)); in these methods, sequential overlapping peptides from the V region sequences are checked for their binding energy to MHC class II proteins. The findings can then be combined with information on other sequence features that relate to successfully discovered peptides, such as amphipathicity, Rothbard motifs, and cleavage sites for cathepsin B and other processing enzymes.

Once possible epitopes of secondary species (eg, humans) T cells have been identified, they are eliminated by altering one or more amino acids. The modified amino acids are usually found in the T cell epitope itself, but may also be located near the epitope in terms of the primary or secondary structure of the protein (and therefore may not be adjacent to the primary structure). Most typically, such a change is accomplished by substitution, but in some cases it may be more appropriate to add or remove an amino acid.

All changes can be made by recombinant DNA technology so that the final molecule can be expressed from a recombinant host using well known techniques such as site-directed mutagenesis. However, it is also possible to use protein chemistry or any other molecular modification method.

Surface change.

This method includes:

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

(b) creating a sequence alignment using relative availability distributions based on X-ray crystallographic structures of a sufficient amount of heavy and light chains of the variable region of a non-human and a human antibody to obtain a set of framework positions of the heavy and light chains in which the alignment positions are 98% identical with respect to a sufficient amount of heavy and non-human antibody light chains;

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

(d) identifying, by the amino acid residues of a human antibody, the surface of the set of heavy and light chains exposed to the amino acid residues that are most closely identical to the specified surface of the set, exposed to the action of amino acid residues, by determining

- 14,038267 linked in step (c), wherein the heavy and light chains from the human antibody are or are not naturally paired;

(e) substitution, in the amino acid sequence of the non-human antibody to be humanized, the surface of the heavy and light chain set exposed in step (c) to the surface of the heavy and light chain set exposed in step (d );

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

(g) identifying, by comparing the three-dimensional models built in steps (a) and (f), any amino acid residues from the sets identified in steps (c) or (d) that are within 5 angstroms of any atom of any residue regions determining the complementarity of a given non-human antibody to be humanized; and (h) replacing any residues identified in step (g) from human to the original non-human amino acid residue to obtain a humanizing set of non-human antibody surface exposed to amino acid residues; with the proviso that step (a) need not be carried out first, but must be carried out before step (g).

Superhumanization.

This method compares the non-human sequence with the functional repertoire of genes in the human germ line. Those human genes are selected that encode canonical structures identical or closely related to non-human sequences. The selected human genes with the highest homology in the CDRs are selected as FR donors. Finally, non-human CDRs are grafted onto the indicated human FRs. This method is described in WO 2005 / 079479A2.

Optimizing the composition of the human chain.

This method compares the non-human (eg, murine) sequence to the human germline gene repertoire and evaluates the differences as human chain content (HSC), which quantifies the sequence at the level of potential MHC / T cell epitopes. The target sequence is then humanized by maximizing its HSC, instead of using a common identity measurement to create numerous different humanized variants (described in Molecular Immunology, 44, (2007), 1986-1998).

Rearrangement in the frame.

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

Examples of cell binding agents include those described for use in WO 2007/085930, incorporated herein.

Tumor-associated antigens and cognate antibodies for use in embodiments of the present invention are listed below.

Tumor-associated antigens and cognate antibodies (1) BMPR1B (type IB bone morphogenetic protein receptor).

Nucleotide:

Genbank accession number NM_001203.

Genbank version number NM_001203.2 GI: 169790809.

Genbank, entry updated on September 23, 2012 at 02:06 p.m.

Polypeptide:

Genbank accession number NP_001194.

Genbank version number NP_001194.1 GI: 4502431.

Genbank, entry updated on September 23, 2012 at 02:06 p.m.

Cross-references:

101-104 (1994), Oncogene 14 (11): 1377-1382 (1997));

WO 2004/063362 (claim 2); WO 2003/042661 (clause 12);

US 2003/134790-A1 (pp. 38-39); WO 2002/102235 (clause 13; p. 296);

WO 2003/055443 (pp. 91-92); WO 2002/99122 (example 2; pp. 528-530);

WO 2003/029421 (clause 6); WO 2003/024392 (claim 2; Fig. 112);

WO 2002/98358 (claim 1; p. 183); WO 2002/54940 (p. 100-101);

WO 2002/59377 (p. 349-350); WO 2002/30268 (paragraph 27; p. 376);

WO 2001/48204 (example; FIG. 4);

NP_001194, bone morphogenetic protein, type IB / pid = NP_001194.1;

MIM: 603248; AY 065994.

(2) E16 (LAT1, SLC7A5).

Nucleotide:

- 15 038267

Genbank accession number NM_003486.

Genbank version number NM_003486.5 GI: 71979931.

Genbank, entry updated on June 27, 2012 at 12:06 pm

Polypeptide:

Genbank accession number NP_003477.

Genbank version number NP_003477.4 GI: 71979932.

Genbank, entry updated on June 27, 2012 at 12:06 pm

Cross-references:

Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699): 288-291 (1998), Gaugitsch, H. W., et al. (1992) J. Biol. Chem. 267 (16): 11267-11273);

WO 2004/048938 (example 2); WO 2004/032842 (example IV);

WO 2003/042661 (clause 12); WO 2003/016475 (claim 1);

WO 2002/78524 (example 2); WO 2002/99074 (clause 19; pp. 127-129);

WO 2002/86443 (clause 27; p. 222, 393); WO 2003/003906 (clause 10; p. 293);

WO 2002/64798 (clause 33; pp. 93-95); WO 2000/14228 (clause 5; pp. 133-136);

US 2003/224454 (Fig. 3); WO 2003/025138 (clause 12; p. 150);

NP_003477 family of solute carriers 7 (cationic amino acid transporter, y + system), member 5 /pid=NP_003477.3 - Homo sapiens.

MIM: 600182; NM_015923.

(3) STEAP1 (sixth transmembrane epithelial antigen of the prostate).

Nucleotide:

Genbank accession number NM_012449.

Genbank version number NM_012449.2 GI: 22027487.

Genbank, entry update date: September 9, 2012 at 02:57 p.m.

Polypeptide:

Genbank accession number NP_036581.

Genbank version number NP_036581.1 GI: 9558759.

Genbank, entry update date: September 9, 2012 at 02:57 p.m.

Cross-references:

Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R.S., et al. (1999) Proc. Natl. Acad. Sci. Acad. Sci. U.S.A. 96 (25): 14523-14528);

WO 2004/065577 (clause 6); WO 2004/027049 (Fig. 1L); EP 1394274 (example 11); WO 2004/016225 (claim 2); WO 2003/042661 (clause 12);

US 2003/157089 (example 5); US 2003/185830 (example 5); US 2003/064397 (Fig. 2);

WO 2002/89747 (example 5; pp. 618-619); WO 2003/022995 (Example 9; Figs. 13A, 35, Example 53; p. 173, Example 2; Fig. 2A);

sixth transmembrane epithelial antigen of the prostate;

MIM: 604415.

(4) 0772P (CA125, MUC16).

Nucleotide:

Genbank accession number AF361486.

Genbank version number AF361486.3 GI: 34501466.

Genbank, entry updated March 11, 2010 at 07:56 am.

Polypeptide:

Genbank accession number AKK74120.

Genbank version number ALL74120.3 GI: 34501467.

Genbank, entry updated March 11, 2010 at 07:56 am.

Cross-references:

J. Biol. Chem. 276 (29): 27371-27375 (2001)); WO 2004/045553 (claim 14);

WO 2002/92836 (claim 6, FIG. 12); WO 2002/83866 (clause 15; pp. 116-121);

US 2003/124140 (example 16);

GI: 34501467;

(5) MPF (MPF, MSLN, SMR, megakaryocyte stimulation factor, mesothelin).

Nucleotide:

Genbank accession number NM_005823.

Genbank version number NM_005823.5 GI: 293651528.

Genbank, entry update date: September 2, 2012 at 01:47 PM

Polypeptide:

Genbank accession number NP_005814.

Genbank version number NP_005814.2 GI: 53988378.

Genbank, entry update date: September 2, 2012 at 01:47 PM

Cross-references:

- 16 038267

Yamaguchi, N., et al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. USA 96 (20): 1153111536 (1999), Proc. Natl. Acad. Sci. USA 93 (1): 136-140 (1996) J. Biol. Chem. 270 (37): 21984-21990 (1995));

WO 2003/101283 (claim 14); (WO 2002/102235 (clause 13; pp. 287-288);

WO 2002/101075 (claim 4; pp. 308-309); WO 2002/71928 (pp. 320-321);

WO 94/10312 (pp. 52-57); 1M: 601051.

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II, sodium-dependent phosphate transporter 3b).

Nucleotide:

Genbank accession number NM_006424.

Genbank version number NM_006424.2 GI: 110611905.

Genbank, entry update date: July 22, 2012 at 03:39 p.m.

Polypeptide:

Genbank accession number NP_006415.

Genbank version number NP_006415.2 GI: 110611906.

Genbank, entry update date: July 22, 2012 at 03:39 p.m.

Cross-references:

J. Biol. Chem. 277 (22): 19665-19672 (2002), Genomics 62 (2): 281-284 (1999), Feild, J.A. et al. (1999) Biochem. Biophys. Res. Commun. 258 (3): 578-582);

WO 2004/022778 (claim 2); EP 1394274 (example 11); WO 2002/102235 (clause 13; p. 326);

EP 0875569 (claim 1; pp. 17-19); WO 2001/57188 (clause 20; p. 329);

WO 2004/032842 (example IV); WO 2001/75177 (clause 24; pp. 139-140); MIM: 604217.

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Hlog semaphorin 5b, sema 25 domain, seven thrombospondin repeats (type 1 and similar to type 1), transmembrane domain (TM) and short cytoplasmic domain, ( semaphorin) 5B).

Nucleotide:

Genbank accession number AB040878.

Genbank version number AB040878.1 GI: 7959148.

Genbank, entry update date: August 2, 2006 at 05:40 p.m.

Polypeptide:

Genbank accession number BAA95969.

Genbank version number BAA95969.1 GI: 7959149.

Genbank, entry update date: August 2, 2006 at 05:40 p.m.

Cross-references:

Nagase T., et al. (2000) DNA Res. 7 (2): 143-150); WO 2004/000997 (claim 1);

WO 2003/003984 (claim 1); WO 2002/06339 (claim 1; p. 50); WO 2001/88133 (claim 1; pp. 41-43, 48-58);

WO 2003/054152 (clause 20); WO 2003/101400 (clause 11); sample: Q9P283;

Genew; HGNC: 10737.

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA, gene 2700050C12).

Nucleotide:

Genbank accession number AY358628.

Genbank version number AY358628.1 GI: 37182377.

Genbank, entry updated on December 1, 2009 at 04:15 AM.

Polypeptide:

Genbank accession number AAQ88991.

Genbank version number AAQ88991.1 GI: 37182378.

Genbank, entry updated on December 1, 2009 at 04:15 AM.

Cross-references:

Ross et al. (2002) Cancer Res. 62: 2546-2553; US 2003/129192 (clause 2); US 2004/044180 (clause 12);

US 2004/044179 (clause 11); US 2003/096961 (clause 11); US 2003/232056 (example 5);

WO 2003/105758 16 (clause 12); US 2003/206918 (example 5); EP 1347046 (item 1);

WO 2003/025148 (clause 20); GI: 7182378.

(9) ETBR (endothelin type B receptor).

Nucleotide:

Genbank accession number AY275463.

Genbank version number AY275463.1 GI: 30526094.

Genbank, entry updated on March 11, 2010 at 02:26 am.

Polypeptide:

Genbank accession number AAP32295.

Genbank version number AAP32295.1 GI: 30526095.

Genbank, update date of entry: March 11, 201 at 02:26 am.

- 17 038267

Cross-references:

Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177,34-39,1991;

Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178,248-255,1991;

Arai H., et al. Jpn. Circ. J. 56,1303-1307,1992;

Arai H., et al. J. Biol. Chem. 268, 3463-3470, 1993;

Sakamoto A., Yanagisawa M., et al. Biochem. Biophys. Res. Commun. 178,656-663,1991;

Elshourbagy N. A., et al. J. Biol. Chem. 268, 3873-3879, 1993;

Haendler B., et al. J. Cardiovasc. Pharmacol. 20, s1-s4, 1992;

Tsutsumi M., et al. Gene 228, 43-49, 1999;

Strausberg R. L., et al. Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002;

Bourgeois C., et al. J. Clin. Endochnol. Metab. 82,3116-3123,1997;

Okamoto Y., et al. Biol. Chem. 272,21589-21596,1997;

Verheij J.B., et al. Am. J. Med. Genet. 108,223-225,2002;

Hofstra R. M. W., et al. Eur. J. Hum. Genet. 5,180-185,1997;

Puffenberger E. G., et al. Cell 79,1257-1266,1994;

Attie T., et al, Hum. Mol. Genet. 4,2407-2409,1995; Auricchio A., et al. Hum. Mol. Genet. 5: 351-354,1996; Amiel J., et al. Hum. Mol.

Genet. 5, 355-357,1996;

Hofstra R. M. W., et al. Nat. Genet. 12,445-447,1996;

Svensson P. J., et al. Hum. Genet. 103, 145-148,1998;

Fuchs S., et al. Mol. Med. 7,115-124,2001;

Pingault V., et al. (2002) Hum. Genet. 111, 198-206;

WO 2004/045516 (claim 1); WO 2004/048938 (example 2); WO 2004/040000 (clause 151);

WO 2003/087768 (claim 1); WO 2003/016475 (claim 1); WO 2003/016475 (claim 1);

WO 2002/61087 (Fig. 1); WO 2003/016494 (Fig. 6); WO 2003/025138 (clause 12; p. 144);

WO 2001/98351 (claim 1; pp. 124-125); EP 0522868 (claim 8; FIG. 2);

WO 2001/77172 (claim 1; pp. 297-299); US 2003/109676; US 6518404 (Fig. 3); U

S5773223 (n.1α; col. 31-34); WO 2004/001004.

(10) MSG783 (RNF124, hypothetical protein FLJ20315).

Nucleotide:

Genbank accession number NM_017763.

Genbank version number NM_017763.4 GI: 167830482.

Genbank, post update date: July 22, 2012 12:34 pm.

Polypeptide:

Genbank accession number NP_060233.

Genbank version number NP_060233.3 GI: 56711322.

Genbank, post update date: July 22, 2012 12:34 pm.

Cross-references:

WO 2003/104275 (claim 1); WO 2004/046342 (example 2); WO 2003/042661 (clause 12);

WO 2003/083074 (clause 14; p. 61); WO 2003/018621 (claim 1); WO 2003/024392 (claim 2; Fig. 93);

WO 2001/66689 (example 6); LocusID: 54894.

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, sixth prostate transmembrane epithelial antigen 2, sixth prostate transmembrane protein).

Nucleotide:

Genbank accession number AF455138.

Genbank version number AF455138.1 GI: 22655487.

Genbank, entry updated March 11, 2010 at 01:54 am.

Polypeptide:

Genbank accession number AAN04080.

Genbank version number AAN04080.1 GI: 22655488.

Genbank, entry updated March 11, 2010 at 01:54 am.

Cross-references:

Lab. Invest. 82 (11): 1573-1582 (2002)); WO 2003/087306;

US 2003/064397 (claim 1; FIG. 1); WO 2002/72596 (clause 13; pp. 54-55);

WO 2001/72962 (claim 1; FIG. 4B); WO 2003/104270 (clause 11);

WO 2003/104270 (claim 16); US 2004/005598 (clause 22);

WO 2003/042661 (clause 12); US 2003/060612 (clause 12; Fig. 10);

WO 2002/26822 (claim 23; FIG. 2); WO 2002/16429 (clause 12; Fig. 10); GI: 22655488.

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, trasitor receptor potential cation channel 5, subfamily M, member 4).

- 18 038267

Nucleotide:

Genbank accession number NM_017636.

Genbank version number NM_017636.3 GI: 304766649.

Genbank, entry updated on June 29, 2012 at 11:27 am.

Polypeptide:

Genbank accession number NP_060106.

Genbank version number NP_060106.2 GI: 21314671.

Genbank, entry updated on June 29, 2012 at 11:27 am.

Cross-references:

Xu, X.Z., et al. Proc. Natl. Acad. Sci. U.S.A. 98 (19): 10692-10697 (2001), Cell 109 (3): 397-407 (2002), J. Biol. Chem. 278 (33): 30813-30820 (2003));

US 2003/143557 (clause 4); WO 2000/40614 (clause 14; pp. 100-103);

WO 2002/10382 (claim 1; Fig. 9A); WO 2003/042661 (clause 12);

WO 2002/30268 (clause 27; p. 391); US 2003/219806 (clause 4);

WO 2001/62794 (claim 14; FIGS. 1A-D); MIM: 606936.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, growth factor derived from teratocarcinoma).

Nucleotide:

Genbank accession number NM_003212.

Genbank version number NM_003212.3 GI: 292494881.

Genbank, entry updated on September 23, 2012 at 02:27 p.m.

Polypeptide:

Genbank accession number NP_003203.

Genbank version number NP_003203.1 GI: 4507425.

Genbank, entry updated on September 23, 2012 at 02:27 p.m.

Cross-references:

Ciccodicola, A., et al. EMBO J. 8 (7): 1987-1991 (1989) Am. J. Hum. Genet. 49 (3): 555-565 (1991));

US 2003/224411 (claim 1); WO 2003/083041 (example 1); WO 2003/034984 (clause 12);

WO 2002/88170 (clause 2; pp. 52-53); WO 2003/024392 (claim 2; Fig. 58);

WO 2002/16413 (claim 1; pp. 94-95, 105); WO 2002/22808 (claim 2; FIG. 1);

US 5854399 (Example 2; Col 17-18); US 5792616 (Fig. 2); MIM: 187395.

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs. 73792).

Nucleotide:

Genbank accession number M26004.

Genbank version number M26004.1 GI: 181939.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Polypeptide:

Genbank accession number AAA35786.

Genbank version number AAA35786.1 GI: 181940.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Cross-references:

Fujisaku et al. (1989) J. Biol. Chem. 264 (4): 2118-2125); Weis J. J., et al. J. Exp. Med 167, 1047-1066, 1988;

Moore M., et al. Proc. Natl. Acad. Sci. U.S.A. 84,9194-9198, 1987;

Barel M., et al. Mol. Immunol. 35,1025-1031,1998;

Weis J. J., et al. Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986;

Sinha S. K., et al. (1993) J. Immunol. 150, 5311-5320;

WO 2004/045520 (example 4); US 2004/005538 (example 1);

WO 2003/062401 (clause 9); WO 2004/045520 (example 4);

WO 91/02536 (Figures 9.1-9.9); WO 2004/020595 (claim 1);

Accession: Р20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79e, IGb (immunoglobulin-associated, beta), B29).

Nucleotide:

Genbank accession number NM_000626.

Genbank version number NM_000626.2 GI: 90193589.

Genbank, entry update date: June 26, 2012 at 01:53 PM

Polypeptide:

Genbank accession number NP_000617.

Genbank version number NP_000617.1 GI: 11038674.

Genbank, entry update date: June 26, 2012 at 01:53 PM

Cross-references:

Proc. Natl. Acad. Sci. U.S.A. (2003), 100 (7): 4126-4131, Blood (2002), 100 (9): 3068-3076, Muller et al.

- 19 038267 (1992), Eur. J. Immunol. 22 (6): 1621-1625);

WO 2004/016225 (claim 2, FIG. 140); WO 2003/087768, US 2004/101874 (claim 1, p. 102);

WO 2003/062401 (clause 9); WO 2002/78524 (example 2); US 2002/150573 (clause 5, p. 15);

US 5644033; WO 2003/048202 (claim 1, pages 306 and 309); WO 99/58658, US 6534482 (clause 13, Fig. 17A / B);

WO 2000/55351 (clause 11, pp. 1145-1146); MIM: 147245.

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing the phosphatase 1a anchor protein), SPAP1B, SPAP1C).

Nucleotide:

Genbank accession number NM_030764.

Genbank version number NM_030764.3 GI: 227430280.

Genbank, entry update date: June 30, 2012 12:30 pm.

Polypeptide:

Genbank accession number NP_110391.

Genbank version number NP_110391.2 GI: 19923629.

Genbank, entry update date: June 30, 2012 12:30 pm.

Cross-references:

AY358130;

Genome Res. 13 (10): 2265-2270 (2003), Immunogenetics, 54 (2): 87-95 (2002), Blood, 99 (8): 2662-2669 (2002), Proc. Natl. Acad. Sci. U.S.A. 98 (17): 9772-9777 (2001), Xu, M. J., et al. (2001) Biochem. Biophys. Res. Commun. 280 (3): 768-775;

WO 2004/016225 (claim 2); WO 2003/077836; WO 2001/38490 (claim 5; Fig. 18D-1-18D-2); WO 2003/097803 (clause 12); WO 2003/089624 (claim 25); MIM: 606509.

(17) HER2 (ErbB2).

Nucleotide:

Genbank accession number M11730.

Genbank version number M11730.1 GI: 183986.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Polypeptide:

Genbank accession number AAA75493.

Genbank version number AAA75493.1 GI: 306840.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Cross-references:

Coussens L, et al. Science (1985) 230 (4730): 1132-1139);

Yamamoto T., et al. Nature, 319, 230-234, 1986;

Semba K., et al. Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985;

Swiercz J. M., et al. J. Cell Biol. 165,869-880,2004;

Kuhns J. J., et al. J. Biol. Chem. 274,36422-36427,1999;

Cho H.-S., et al. Nature 421,756-760,2003;

Ehsani A., et al. (1993) Genomics 15,426-429;

WO 2004/048938 (example 2); WO 2004/027049 (Fig. 11); WO 2004/009622;

WO 2003/081210; WO 2003/089904 (clause 9); WO 2003/016475 (claim 1); US 2003/118592;

WO 2003/008537 (claim 1); WO 2003/055439 (clause 29; Fig. 1A, B); WO 2003/025228 (clause 37; Fig. 5C);

WO 2002/22636 (example 13; p. 95-107); WO 2002/12341 (clause 68; Fig. 7);

WO 2002/13847 (pp. 71-74); WO 2002/14503 (pp. 114-117); WO 2001/53463 (clause 2; pp. 41-46);

WO 2001/41787 (p. 15); WO 2000/44899 (clause 52; Fig. 7); WO 2000/20579 (claim 3; FIG. 2);

US 5869445 (clause 3; Col 31-38); WO 9630514 (claim 2; pp. 56-61);

EP 1439393 (clause 7); WO 2004/043361 (clause 7); WO 2004/022709; WO 2001/00244 (Example 3; Fig. 4);

accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761. AAA35808.1.

Antibodies.

Abbott - US 20110177095:

for example, an antibody containing CDRs that have generally at least 80% sequence identity to CDRs having the amino acid sequences of SEQ ID NO: 3 (CDR-H1), SEQ ID NO: 4 (CDR-H2), SEQ ID NO: 5 (CDR-H3), SEQ ID NO: 104 and / or SEQ ID NO: 6 (CDR-L1), SEQ ID NO: 7 (CDR-L2) and SEQ ID NO: 8 (CDR-L3), wherein said anti-HER2 antibody or anti-HER2 binding fragment has reduced immunogenicity compared to an antibody having VH of SEQ ID NO: 1 and V _{L of} SEQ ID NO: 2.

Biogen - US 20100119511:

for example, ATCC access numbers: PTA-10355, PTA-10356, PTA-10357, PTA10358;

for example, a purified antibody molecule that binds to HER2 containing all six

CDRs from an antibody selected from the group consisting of BIIB71F10 (SEQ ID NO: 11, 13), BIIB69A09 (SEQ ID NO: 15, 17); BIIB67F10 (SEQ ID NO: 19, 21); BIIB67F11 (SEQ ID NO: 23, 25), BIIB66A12 (SEQ ID NO: 27, 29), BIIB66C01 (SEQ ID NO: 31, 33), BIIB65C10 (SEQ ID NO: 35, 37), BIIB65H09

- 20,038267 (SEQ ID NO: 39, 41) and BIIB65B03 (SEQ ID NO: 43, 45), or CDRs that are identical or that have no more than two changes from said CDRs.

Herceptin (Genentech) - US 6054297; ATCC accession number CRL-10463 (Genentech).

Pertuzumab (Genentech):

US 20110117097:

for example, see SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 23 and 24 and ATCC accession numbers HB-12215, HB-12216, CRL 10463, HB-12697;

US 20090285837 US 20090202546;

for example, ATCC access numbers: HB-12215, HB-12216, CRL 10463, HB-12698;

US 20060088523:

for example, ATCC access numbers: НВ-12215, НВ-12216;

for example, an antibody comprising the variable light and variable heavy chain amino acid sequences in SEQ ID NOs: 3 and 4, respectively;

for example, an antibody comprising a light chain amino acid sequence selected from SEQ ID NOs: 15 and 23 and a heavy chain amino acid sequence selected from SEQ ID NOs: 16 and 24;

US 20060018899:

for example, ATCC access numbers: (7C2) HB-12215, (7F3) HB-12216, (4D5) CRL-10463, (2C4) HB-12697;

for example, an antibody comprising the amino acid sequence of SEQ ID NO: 23, or a deamidated and / or oxidized variant thereof;

US 2011/0159014:

for example, an antibody having a light chain variable domain containing the hypervariable regions of SeQ ID NO: 1, for example, an antibody having a heavy chain variable domain containing the hypervariable regions of SeQ ID NO: 2.

US 20090187007:

Glycotope: TrasGEX antibodies http://www.glycotope.com/pipeline, for example see International Joint Cancer Institute and Changhai Hospital Cancer Cent: HMTI-Fc Ab Gao J., et al. BMB Rep. 2009 Oct 31; 42 (10): 636-41.

Symphogen: US 20110217305.

Union Stem Cell & Gene Engineering, China - Liu HQ., Et al. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 May; 26 (5): 456-8.

(18) NCA (CEACAM6).

Nucleotide:

Genbank accession number M18728.

Genbank version number M18728.1 GI: 189084.

Genbank, entry updated on June 23, 2010 at 08:48 AM.

Polypeptide:

Genbank accession number AAA59907.

Genbank version number AAA59907.1 GI: 189085.

Genbank, entry updated on June 23, 2010 at 08:48 AM.

Cross-references:

Barnett T., et al. Genomics 3, 59-66, 1988;

Tawaragi Y., et al. Biochem. Biophys. Res. Commun. 150, 89-96, 1988;

Strausberg R. L., et al. Proc. Natl. Acad. Sci. 99.16899-16903,2002;

WO 2004/063709; EP 1439393 (clause 7); WO 2004/044178 (example 4);

WO 2004/031238; WO 2003/042661 (clause 12); WO 2002/78524 (example 2);

WO 2002/86443 (clause 27; p. 427); WO 2002/60317 (claim 2);

accession: Р40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728.

(19) MDP (DPEP1).

Nucleotide:

Genbank accession number BC017023.

Genbank version number BC017023.1 GI: 16877538.

Genbank, entry update date: March 6, 2012 at 01:00 p.m.

Polypeptide:

Genbank accession number AAH17023.

Genbank version number AAH17023.1 GI: 16877539.

Genbank, entry update date: March 6, 2012 at 01:00 p.m.

Cross-references:

Proc. Natl. Acad. Sci. U.S.A. 99 (26): 16899-16903 (2002));

WO 2003/016475 (claim 1); WO 2002/64798 (clause 33; pp. 85-87);

- 21 038267

JP 05003790 (Fig. 6-8); WO 99/46284 (Fig. 9); MIM: 179780.

(20) IL20R-alpha (IL20Ra, ZCYTOR7).

Nucleotide:

Genbank accession number AF184971.

Genbank version number AF184971.1 GI: 6013324.

Genbank, entry update date: March 10, 2010 at 10:00 p.m.

Polypeptide:

Genbank accession number AAF01320.

Genbank version number AAF01320.1 GI: 6013325.

Genbank, entry update date: March 10, 2010 at 10:00 p.m.

Cross-references:

Clark H. F., et al. Genome Res. 13,2265-2270,2003;

Mungall A. J., et al. Nature 425, 805-811, 2003;

Blumberg H., et al. Cell, 104, 9-19, 2001;

Dumoutier L, et al. J. Immunol. 167, 3545-3549, 2001;

Parrish-Novak J., et al. J. Biol. Chem. 277,47517-47523,2002;

Pletnev S., et al. (2003) Biochemistry 42: 12617-12624;

Sheikh F., et al. (2004) J. Immunol. 172, 2006-2010;

EP 1394274 (example 11); US 2004/005320 (example 5); WO 2003/029262 (pp. 74-75);

WO 2003/002717 (claim 2; p. 63); WO 2002/22153 (pp. 45-47); US 2002/042366 (pp. 20-21);

WO 2001/46261 (pp. 57-59); WO 2001/46232 (pp. 63-65); WO 98/37193 (claim 1; p. 55-59);

sample: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevikan (BCAN, Vienna).

Nucleotide:

Genbank accession number AF229053.

Genbank version number AF229053.1 GI: 10798902.

Genbank, entry updated March 11, 2010 12:58 pm.

Polypeptide:

Genbank accession number AAG23135.

Genbank version number AAG23135.1 GI: 10798903.

Genbank, entry updated March 11, 2010 12:58 pm.

Cross-references:

Gary S. C., et al. Gene 256, 139-147, 2000;

Clark H. F., et al. Genome Res. 13,2265-2270,2003;

Strausberg R. L., et al. Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002;

US 2003/186372 (clause 11); US 2003/186373 (clause 11); US 2003/119131 (claim 1; Fig. 52);

US 2003/119122 (claim 1; Fig. 52); US 2003/119126 (claim 1); US 2003/119121 (claim 1; Fig. 52);

US 2003/119129 (claim 1); US 2003/119130 (claim 1); US 2003/119128 (claim 1; Fig. 52);

US 2003/119125 (claim 1); WO 2003/016475 (claim 1); WO 2002/02634 (claim 1).

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5).

Nucleotide:

Genbank accession number NM_004442.

Genbank version number NM_004442.6 GI: 111118979.

Genbank, entry updated on September 8, 2012 at 04:43 p.m.

Polypeptide:

Genbank accession number NP_004433.

Genbank version number NP_004433.2 GI: 21396504.

Genbank, entry updated on September 8, 2012 at 04:43 p.m.

Cross-references:

Chan, J. and Watt, V.M., Oncogene 6 (6), 1057-1061 (1991), Oncogene, 10 (5): 897-905 (1995), Annu. Rev. Neurosci. 21: 309-345 (1998) Int. Rev. Cytol. 196: 177-244 (2000));

WO 2003/042661 (clause 12); WO 2000/53216 (claim 1; p. 41); WO 2004/065576 (claim 1);

WO 2004/020583 (clause 9); WO 2003/004529 (pp. 128-132); WO 2000/53216 (claim 1; page 42);

MIM: 600997.

(23) ASLG659 (B7h).

Nucleotide:

Genbank accession number AX092328.

Genbank version number AX092328.1 GI: 13444478.

Genbank, post update date: January 26, 2011, 07:37 am.

Cross-references:

US 2004/0101899 (clause 2); WO 2003104399 (clause 11); WO 2004000221 (Fig. 3);

US 2003/165504 (claim 1); US 2003/124140 (example 2); US 2003/065143 (Fig. 60);

- 22 038267

WO 2002/102235 (clause 13; p. 299); US 2003/091580 (example 2);

WO 2002/10187 (claim 6; Fig. 10); WO 2001/94641 (clause 12; Fig. 7b);

WO 2002/02624 (clause 13; FIGS. 1A, 1B); US 2002/034749 (clause 54; pp. 45-46);

WO 2002/06317 (example 2; p. 320-321, p. 34; p. 321-322);

WO 2002/71928 (pp. 468-469); WO 2002/02587 (Example 1; Fig. 1);

WO 2001/40269 (Example 3; pp. 190-192); WO 2000/36107 (example 2; pp. 205-207);

WO 2004/053079 (clause 12); WO 2003/004989 (claim 1);

WO 2002/71928 (pp. 233-234, 452-453); WO 01/16318.

(24) PSCA (Prostate stem cell antigen precursor).

Nucleotide:

Genbank accession number AJ297436.

Genbank version number AJ297436.1 GI: 9367211.

Genbank, entry updated on February 1, 2011 at 11:25 am.

Polypeptide:

Genbank accession number CAB97347.

Genbank version number AJ297436.1 GI: 9367211.

Genbank, entry updated on February 1, 2011 at 11:25 am.

Cross-references:

Reiter R. E., et al. Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740,1998;

Gu Z., et al. Oncogene 19,1288-1296,2000;

Biochem. Biophys. Res. Commun. (2000) 275 (3): 783-788;

WO 2004/022709; EP 1394274 (example 11); US 2004/018553 (clause 17);

WO 2003/008537 (claim 1); WO 2002/81646 (claim 1; p. 164);

WO 2003/003906 (clause 10; p. 288); WO 2001/40309 (Example 1; Fig. 17);

US 2001/055751 (Example 1; Fig. 1b); WO 2000/32752 (claim 18; FIG. 1);

WO 98/51805 (clause 17; p. 97); WO 98/51824 (clause 10; p. 94); WO 98/40403 (claim 2; FIG. 1B);

accession: 043653; EMBL; AF043498; AAC39607.1.

(25) GEDA.

Nucleotide:

Genbank accession number AY260763.

Genbank version number AY260763.1 GI: 30102448.

Genbank, entry updated March 11, 2010 02:24 am.

Polypeptide:

Genbank accession number AAP14954.

Genbank version number AAP14954.1 GI: 30102449.

Genbank, entry updated March 11, 2010 02:24 am.

Cross-references:

AP14954 lipoma HMGIC fusion-partnerlike protein / pid = AAP14954.1 - Homo sapiens (human);

WO 2003/054152 (clause 20); WO 2003/000842 (claim 1); WO 2003/023013 (example 3, item 20);

US 2003/194704 (clause 45); GI: 30102449.

(26) BAFF-R (B cell activating factor receptor, BLyS 3 receptor, BR3).

Nucleotide:

Genbank accession number AF116456.

Genbank version number AF116456.1 GI: 4585274.

Genbank, entry update date: March 10, 2010 at 09:44 p.m.

Polypeptide:

Genbank accession number AAD25356.

Genbank version number AAD25356.1 GI: 4585275.

Genbank, entry update date: March 10, 2010 at 09:44 p.m.

Cross-references:

BAFF receptor / pid = NP - 443177.1 - Homo sapiens: Thompson, J. S., et al. Science, (5537), 2108-2111 (2001);

WO 2004/058309; WO 2004/011611; WO 2003/045422 (example; pp. 32-33);

WO 2003/014294 (claim 35; Fig. 6B); WO 2003/035846 (clause 70; pp. 615-616);

WO 2002/94852 (Col 136-137); WO 2002/38766 (clause 3; p. 133);

WO 2002/24909 (Example 3; Fig. 3);

MIM: 606269; NP_443177.1; NM_052945_1; AF132600.

(27) CD22 (B cell receptor isoform B CD22, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814).

Nucleotide:

Genbank accession number AK026467.

Genbank version number AK026467.1 GI: 10439337.

Genbank, the date of the update of the entry: September 11, 2006, 11:24 p.m.

- 23 038267

Polypeptide:

Genbank accession number BAB15489.

Genbank version number BAB15489.1 GI: 10439338.

Genbank, the date of the update of the entry: September 11, 2006, 11:24 p.m.

Cross-references:

Wilson et al. (1991). J. Exp. Med. 173: 137-146; WO 2003/072036 (claim 1; FIG. 1); IM: 107266;

NP_001762.1; NM_001771_1.

(27a) CD22 (CD22 molecule).

Nucleotide:

Genbank accession number X52785.

Genbank version number X52785.1 GI: 29778.

Genbank, entry updated on February 2, 2011 at 10:09 am.

Polypeptide:

Genbank accession number CAA36988.

Genbank version number CAA36988.1 GI: 29779.

Genbank, entry updated on February 2, 2011 at 10:09 am.

Cross-references:

Stamenkovic I. et al. Nature 345 (6270), 74-77 (1990).

Other information:

Official symbol: CD22.

Other names: SIGLEC-2, SIGLEC2.

Other abbreviations: B-cell receptor CD22; B-lymphocyte cell adhesion molecule; BL-CAM; antigen CD22; Leu-14 T cell surface antigen; Ig-like sialic acid binding lectin 2; Ig-like lectin 2 that binds sialic acid.

Antibodies.

G5 / 44 (inotuzumab): DiJoseph J. F., et al. Cancer Immunol Immunother. 2005 Jan; 54 (1): 11-24.

Epratuzumab-Goldenberg D. M., et al. Expert. Rev. Anticancer Ther. 6 (10): 1341-53, 2006.

(28) CD79a (CD79A, CD79α alpha), an immunoglobulin-associated alpha, B-cell specific protein that covalently interacts with Ig beta (CD79B) and forms a surface complex with Ig M molecules, 4.84, MW: 25028 TM: 2 [R] Gene chromosome: 19q13.2.

Nucleotide:

Genbank accession number NM_001783.

Genbank version number NM_001783.3 GI: 90193587.

Genbank, entry update date: June 26, 2012, 01:48 p.m.

Polypeptide:

Genbank accession number NP_001774.

Genbank version number NP_001774.1 GI: 4502685.

Genbank, entry update date: June 26, 2012, 01:48 p.m.

Cross-references:

WO 2003/088808, US 2003/0228319; WO 2003/062401 (clause 9);

US 2002/150573 (clause 4, pp. 13-14); WO 99/58658 (clause 13, Fig. 16);

WO 92/07574 (Fig. 1); US5644033;

On et al. (1992) J. Immunol. 148 (5): 1526-1531;

Muller et al. (1992), Eur. J. Immunol. 22: 1621-1625;

Hashimoto et al. (1994) Immunogenetics 40 (4): 287-295;

Preud'homme et al. (1992) Clin. Exp. Immunol. 90 (1): 141-146;

Yu et al. (1992) J. Immunol. 148 (2) 633-637;

Sakaguchi et al. (1988) EMBO J. 7 (11): 3457-3464.

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein coupled receptor that is activated by the chemokine CXCL13, is involved in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly the development of AIDS, lymphoma, myeloma and leukemia);

372 aa, pl: 8.54 MM: 41959 TM: 7 [P] Gene chromosome: 11q23.3.

Nucleotide:

Genbank accession number NM_001716.

Genbank version number NM_001716.4 GI: 342307092.

Genbank, entry update date: September 30, 2012 at 01:49 pm

Polypeptide:

Genbank accession number NP_001707.

Genbank version number NP_001707.1 GI: 4502415.

Genbank, entry update date: September 30, 2012 at 01:49 pm

Cross-references:

WO 2004/040000; WO 2004/015426; US 2003/105292 (example 2);

- 24 038267

US6555339 (example 2); WO 2002/61087 (Fig. 1); WO 2001/57188 (clause 20, p. 269);

WO 2001/72830 (pp. 12-13); WO 2000/22129 (example 1, pp. 152-153, example 2, pp. 254-256);

WO 99/28468 (claim 1, p. 38);

US 54440021 (example 2, column 49-52); WO 94/28931 (pp. 56-58);

WO 92/17497 (claim 7, Fig. 5);

Dobner et al. (1992), Eur. J. Immunol. 22: 2795-2799;

Barella et al. (1995) Biochem. J. 309: 773-779.

(30) HLA-DOB (beta subunit of the MHC class II molecule (antigen 1a), which binds peptides and provides them to CD4 'T lymphocytes); 273 aa, pl: 6.56, MM: 30820. TM: 1 [P] Gene chromosome: 6p21.3.

Nucleotide:

Genbank accession number NM_002120.

Genbank version number NM_002120.3 GI: 118402587.

Genbank, entry updated on September 8, 2012 at 04:46 p.m.

Polypeptide:

Genbank accession number NP_002111.

Genbank version number NP_002111.1 GI: 4504403.

Genbank, entry updated on September 8, 2012 at 04:46 p.m.

Cross-references:

Tonnelle et al. (1985). EMBOJ. 4 (11): 2839-2847;

Jonsson et al. (1989) Immunogenetics 29 (6): 411-413;

Beck et al. (1992) J. Mol. Biol. 228: 433-441;

Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903;

Servenius et al. (1987) J. Biol. Chem. 262: 8759-8766;

Beck et al. (1996) J. Mol. Biol. 255: 1-13;

Naruse et al. (2002) Tissue Antigens 59: 512-519;

WO 99/58658 (clause 13, Fig. 15); US 6153408 (p. 35-38);

US 5976551 (p. 168-170); US 6011146 (p. 145-146);

Kasahara et al. (1989) Immunogenetics 30 (1): 66-68;

Larhammar et al. (1985) J. Biol. Chem. 260 (26): 14111-14119.

P2X5 (ligand-gated ion channel of the purinergic receptor P2X, an ion channel driven by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability);

422aa), pl: 7.63,

MM: 47206 TM: 1 [P] Gene chromosome: 17p13.3.

Nucleotide:

Genbank accession number NM_002561.

Genbank version number NM_002561.3 GI: 325197202.

Genbank, post update date: June 27, 2012 12:41 pm.

Polypeptide:

Genbank accession number NP_002552.

Genbank version number NP_002552.2 GI: 28416933.

Genbank, post update date: June 27, 2012 12:41 pm.

Cross-references:

Le et al. (1997) FEBS Lett. 418 (1-2): 195-199;

WO 2004/047749; WO 2003/072035 (clause 10);

Touchman et al. (2000) Genome Res. 10: 165-173; WO 2002/22660 (claim 20); WO 2003/093444 (claim 1); WO 2003/087768 (claim 1); WO 2003/029277 (p. 82).

CD72 (B cell differentiation antigen CD72, Lyb-2); 359 aa, pl: 8.66, MM: 40225, TM: 1 [P] Gene chromosome: 9p13.3).

Nucleotide:

Genbank accession number NM_001782.

Genbank version number NM_001782.2 GI: 194018444.

Genbank, entry updated on June 26, 2012 at 01:43 pm

Polypeptide:

Genbank accession number NP_001773.

Genbank version number NP_001773.1 GI: 4502683.

Genbank, entry update date: June 26, 2012 at 01:43 pm

Cross-references:

WO 2004042346 (clause 65); WO 2003/026493 (pp. 51-52, 57-58); WO 2000/75655 (pp. 105-106);

Von Hoegen et al. (1990, J. Immunol. 144 (12): 4870-4877;

Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903.

- 25 038267

LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the high leucine (LRR) family, regulates B cell activation and apoptosis; loss of function is associated with increased disease activity in patients with systemic lupus erythematosus);

661 aa, pl: 6.20, MM: 74147 TM: 1 [P] Gene chromosome: 5q12.

Nucleotide:

Genbank accession number NM_005582.

Genbank version number NM_005582.2 GI: 167555126.

Genbank, entry update date: September 2, 2012, 01:50 p.m.

Polypeptide:

Genbank accession number NP_005573.

Genbank version number NP_005573.2 GM67555127.

Genbank, entry update date: September 2, 2012, 01:50 p.m.

Cross-references:

US 2002/193567; WO 97/07198 (clause 11, pp. 39-42);

Miura et al. (1996) Genomics 38 (3): 299-304;

Miura et al. (1998) Blood 92: 2815-2822;

WO 2003/083047; WO 97/44452 (clause 8, pp. 57-61); WO 2000/12130 (pp. 24-26).

(34) FcRH1 (receptor-like protein 1 Fc, a putative receptor for the Fc domain of immunoglobulin, which contains an Ig-like domain of type C2 and an ITAM domain, may play a role in 5.28, MM: 46925 TM: 1 [P] Chromosome of the gene : 1q21-1q22).

Nucleotide:

Genbank accession number NM_052938.

Genbank version number NM_052938.4 GI: 226958543.

Genbank, entry update date: September 2, 2012, 01:43 pm

Polypeptide:

Genbank accession number NP_443170.

Genbank version number NP_443170.1 GI: 16418419.

Genbank, entry update date: September 2, 2012, 01:43 pm

Cross-references:

WO 2003/077836; WO 2001/38490 (claim 6, Fig. 18E-1-18-E-2);

Davis et al. (2001) Proc. Natl. Acad. Sci USA 98 (17): 9772-9777;

WO 2003/089624 (clause 8); EP 1347046 (claim 1); WO 2003/089624 (clause 7).

(35) IRTA2 (translocation-associated immunoglobulin superfamily receptor 2, a putative immunoreceptor with possible involvement in B-cell development and lymphomagenesis; gene deregulation due to translocation occurs in some B-cell malignant diseases); 977 aa, pl:

6.88, MM: 106468, TM: 1 [P] Gene chromosome: 1q21.

Nucleotide:

Genbank accession number AF343662.

Genbank version number AF343662.1 GI: 13591709.

Genbank, entry updated on March 11, 2010 at 01:16 am.

Polypeptide:

Genbank accession number AAK31325.

Genbank version number AAK31325.1 GI: 13591710.

Genbank, entry updated on March 11, 2010 at 01:16 am.

Cross-references:

AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; mice: AK089756, AY158090, AY506558; NP_112571.1; WO 2003/024392 (claim 2, Fig. 97);

Nakayama et al. (2000) Biochem. Biophys. Res. Commun. 277 (1): 124-127;

WO 2003/077836; WO 2001/38490 (claim 3, Fig. 18B-1-18B-2).

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan associated with the EGF / heregulin family of growth factors and follistatin); 374 aa).

Nucleotide:

Genbank accession number AF179274.

Genbank version number AF179274.2 GI: 12280939.

Genbank, entry updated March 11, 2010 at 01:05 am.

Polypeptide:

Genbank accession number AAD55776.

Genbank version number AAD55776.2 GI: 12280940.

Genbank, entry updated March 11, 2010 at 01:05 am.

Cross-references:

NCBI Access: AAD55776, AAF91397, AAG49451, NCBI Reference Sequence:

- 26 038267

NP_057276; NCBI.

Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; AY358907, CAF85723, CQ782436;

WO 2004/074320; JP 2004113151; WO 2003/042661; WO 2003/009814;

EP 1295944 (pp. 69-70); WO 2002/30268 (p. 329); WO 2001/90304; US 2004/249130; US 2004/022727;

WO 2004/063355; US 2004/197325; US 2003/232350; US 2004/005563; US 2003/124579;

Horie et al. (2000) Genomics 67: 146-152;

Uchida et al. (1999) Biochem. Biophys. Res. Commun. 266: 593-602;

Liang et al. (2000) Cancer Res. 60: 4907-12;

Glynne-Jones et al. (2001) Int. J. Cancer. Oct 15; 94 (2): 178-84.

(37) PSMA - FOLH1 (Folate hydrolase (prostate specific membrane antigen) 1).

Nucleotide:

Genbank accession number M99487.

Genbank version number M99487.1 GI: 190663.

Genbank, entry updated on June 23, 2010 at 08:48 AM.

Polypeptide:

Genbank accession number AAA60209.

Genbank version number AAA60209.1 GI: 190664.

Genbank, entry updated on June 23, 2010 at 08:48 AM.

Cross-references:

Israeli R.S., et al. Cancer Res. 53 (2), 227-230 (1993)

Other information:

Official symbol: FOLH1.

Other names: GIG27, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM, PSMA, mGCP.

Other designations:

N-acetylated alpha-linked acid dipeptidase 1;

N-acetylated-alpha-linked acid dipeptidase I;

NAALADase I;

a gene 27 protein that inhibits cell growth;

folipoli-gamma-glutamate carboxypeptidase;

glutamate carboxylase II; glutamate carboxypeptidase 2;

glutamate carboxypeptidase II; membrane glutamate carboxypeptidase;

variant F of the prostate-specific membrane antigen; pteroylpoly-gamma-glutamate carboxypeptidase

Antibodies.

US 7666425:

Antibodies produced by the hybrid having the following ATCC references:

ATCC access number HB-12101, ATCC access number HB-12109, ATCC access number HB-12127 and ATCC access number HB-12126.

A monoclonal antibody selected from the group consisting of 8H12, 3E11, 17G1, 29B4, 30C1 and 20F2 (US 7811564; Moffett S., et al. Hybridoma (Larchmt). 2007 Dec; 26 (6): 363-72).

Cytogen: monoclonal antibodies 7E11-C5 (ATCC accession number HB 10494) and 9H10-A4 (ATCC accession number HB11430) US 5763202.

GlycoMimetics: NUH2 - ATCC access number HB 9762 (US 7135301).

Human Genome Science: HPRAJ70 ATCC Accession Number 97131 (US 6824993); the amino acid sequence encoded by the cDNA clone (HPRAJ70) held by the American Type Culture Collection (ATCC) under accession number 97131.

Medarex: anti-PSMA antibodies without fucosyl residues - US 7875278.

Mouse anti-PSMA antibodies include 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6, 4C8B9, and monoclonal antibodies. Hybridomas secreting 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6, or 4C8B9 are publicly deposited and described in US patent No. 6159508. Corresponding hybridomas have been publicly deposited and described in US patent No. 6107090. In addition, humanized anti-PSMA antibodies, including the humanized version of J591, are described in more detail in PCT publication WO 02/098897.

Other murine anti-human antibodies have been described in the art, such as mAb 107-1A4 (Wang, S. et al. (2001), Int. J. Cancer, 92: 871-876) and mAb 2C9 (Kato, K. et al (2003) Int J. Urol 10: 439-444).

Examples of human anti-PSMA monoclonal antibodies include antibodies 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3, isolated and structurally described as originally described in PCT publications WO 01/09192 and WO 03/064606 and in US Provisional Patent Application Serial No. 60/654125, entitled Human Monoclonal Antibodies to Prostate Specific Membrane Antigen (PSMA), filed February 18, 2005. Amino Acid Follower

27,038267 VH values 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 1-9, respectively. The VL amino acid sequences 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 10-18, respectively.

Other human anti-PSMA antibodies include those described in PCT Publication WO 03/034903 and in US Patent Application No. 2004/0033229.

NW Biotherapeutics: A hybridoma cell line selected from the group consisting of 3F5.4G6 having ATCC accession number HB12060, 3D7-1.I having ATCC accession number HB12309, 4E10-1.14 having ATCC accession number HB12310, 3E11 (ATCC HB12488) , 4D8 (АТСС НВ12487), 3E6 (АТСС НВ12486), 3C9 (АТСС НВ12484), 2C7 (АТСС НВ12490), 1G3 (АТСС НВ12489), 3C4 (АТСС НВ12494), 3C6 (АТСС), НВ12491 (ATCC HB12495), 5C8B9 (ATCC HB12492) and 3G6 (ATCC HB12485) - see US 6150508 mAb 3.9 produced by a hybridoma deposited under the accession number ATCC PTA-3258, or mAb 10.3 produced by a hybridoma deposited in the depository Access ATCC PTA-3347 - US 7850971.

PSMA Development Company - PSMA antibody compositions (US 20080286284, Table 1).

This application is a divisional application from US patent application Serial No. 10/395894, filed March 21, 2003 (US Pat. No. 7,850,971).

University Hospital Freiburg, Germany - mAbs 3 / A12, 3 / E7 and 3 / F11 (Wolf P., et al. Prostate. 2010 Apr 1; 70 (5): 562-9).

(38) SST (somatostatin receptor; note that there are 5 subtypes).

(38 .1) SSTR2 (somatostatin receptor 2).

Nucleotide:

Genbank accession number NM_001050.

Genbank version number NM_001050.2 GI: 44890054.

Genbank, post update date: August 19, 2012, 01:37 p.m.

Polypeptide:

Genbank accession number NP_001041.

Genbank version number NP_001041.1 GI: 4557859.

Genbank, post update date: August 19, 2012, 01:37 p.m.

Cross-references:

Yamada Y., et al. Proc. Natl. Acad. Sci. U.S.A. 89 (1), 251-255 (1992);

Susini C., et al. Ann Oncol. 2006 Dec; 17 (12): 1733-42.

Other information:

Official symbol: SSTR2.

Other designations: SRIF-1; SS2R; type 2 somatostatin receptor.

(38 .2) SSTR5 (somatostatin 5 receptor).

Nucleotide:

Genbank accession number D16827.

Genbank version number D16827.1 GI: 487683.

Genbank, post update date: August 1, 2006, 12:45 pm

Polypeptide:

Genbank accession number BAA04107.

Genbank version number BAA04107.1 GI: 487684.

Genbank, post update date: August 1, 2006, 12:45 pm

Cross-references:

Yamada, Y., et al. Biochem. Biophys. Res. Commun. 195 (2), 844-852 (1993).

Other information:

Official symbol: SSTR5.

Other names: SS-5-R.

Other designations: subtype 5 somatostatin receptor; type 5 somatostatin receptor.

(38 .3) SSTR1.

(38 .4) SSTR3.

(38 .5) SSTR4.

AvB6 - Both subunits (39 + 40).

(39) ITGAV (integrin, alpha V).

Nucleotide:

Genbank accession number M14648 J02826 M18365.

Genbank version number M14648.1 GI: 340306.

Genbank, entry updated on June 23, 2010 at 08:56 am.

Polypeptide:

Genbank accession number AAA36808.

Genbank version number AAA36808.1 GI: 340307.

Genbank, entry updated on June 23, 2010 at 08:56 am.

- 28 038267

Cross-references:

Suzuki S., et al. Proc. Natl. Acad. Sci. U.S.A. 83 (22), 8614-8618 (1986).

Other information:

Official symbol: ITGAV.

Other names: CD51, MSK8, VNRA, VTNR.

Other abbreviations: antigen determined by monoclonal antibody L230; integrin alpha-V; integrin alpha V-beta3; integrin, alpha V (vitronectin receptor, alpha polypeptide, CD51 antigen); the alpha subunit of the vitronectin receptor.

(40) ITGB6 (integrin beta 6).

Nucleotide:

Genbank accession number NM_000888.

Genbank version number NM_000888.3 GI: 9966771.

Genbank, entry update date: June 27, 2012 12:46 pm.

Polypeptide:

Genbank accession number NP_000879.

Genbank version number NP_000879.2 GI: 9625002.

Genbank, entry update date: June 27, 2012 12:46 pm.

Cross-references:

Sheppard D. J., et al. Biol. Chem. 265 (20), 11502-11507 (1990).

Other information:

Official symbol: ITGB6.

Other names: integrin beta-6.

Antibodies.

Biogen: US 7943742 - hybridoma clones 6.3G9 and 6.8G6 were deposited in the ATCC depository with ATCC accession numbers PTA-3649 and -3645, respectively.

Biogen: US 7,465,449 - in some embodiments, the specified antibody contains the same heavy and light chain polypeptide sequences as the antibody produced by the hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10, 6.2A1, 6.2E5, 7.1G10, 7.7G5 or 7.1C5.

Centokor (J&J): US 7550142; US 7163681.

For example, US Pat. No. 7,550,142 is an antibody having human heavy chain and human light chain variable regions containing the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Seattle Genetics: 15H3 (Ryan MC, et al. Cancer Res. April 15, 2012; 72 (8 Supplement): 4630).

(41) CEACAM5 (cell adhesion molecule 5 associated with carcinoembryonic antigen).

Nucleotide:

Genbank accession number M17303.

Genbank version number M17303.1 GI: 178676.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Polypeptide:

Genbank accession number AAB59513.

Genbank version number AAB59513.1 GI: 178677.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Cross-references:

Beauchemin N., et al. Mol. Cell. Biol. 7 (9), 3221-3230 (1987).

Other information:

Official symbol: SEASAM5.

Other names: CD66e, CEA

Other designations: meconium antigen 100.

Antibodies.

AstraZeneca-Medlmmune: US 20100330103; US 20080057063; US 20020142359:

for example, an antibody having complementarity determining regions (CDRs) with the following sequences: heavy chain; CDR1 DNYMH, CDR2 WIDPENGDTE YAPKFRG, CDR3 LIYAGYLAMD Y; and light chain CDR1 - SASSSVTYMH, CDR2 - STSNLAS, CDR3 - QQRSTYPLT.

hybridoma 806.077, deposited with the European Collection of Cell Cultures (ECACC) under the deposit number 96022936.

Research Corporation Technologies, Inc .: US 5047507 Bayer Corporation: US 6013772.

BioAlliance: US 7982017; US 7674605 US 7674605:

an antibody comprising a heavy chain variable region sequence from the amino acid sequence of SEQ ID NO: 1 and a light chain variable region sequence from the amino acid sequence of SEQ ID NO: 2. an antibody comprising a heavy chain variable region sequence from the amino acid sequence of SEQ ID NO: 5, and the light chain variable region sequence from the amino acid sequence of SEQ ID NO: 6.

- 29 038267

Celltech Therapeutics Limited: US 5877293.

The Dow Chemical Company: US 5472693; US 6417337; US 6333405 US 5472693 - for example, ATCC No. CRL-11215 US 6417337 - for example, ATCC CRL-12208 US 6333405 - for example, ATCC CRL-12208.

Immunomedics, Inc: US 7534431; US 7230084; US 7300644; US 6730300; US 20110189085.

an antibody having light chain variable region CDRs that comprise:

CDR1 contains KASQDVGTSVA (SEQ ID NO: 20);

CDR2 contains WTSTRHT (SEQ ID NO: 21) and

CDR3 contains QQYSLYRS (SEQ ID NO: 22);

and CDRs of the variable region of the heavy chain of said anti-CEA, antibodies that contain:

CDR1 contains TYWMS (SEQ ID NO: 23);

CDR2 contains EIHPDSSTINYAPSLKD (SEQ ID NO: 24) and

CDR3 contains LYFGFPWFAY (SEQ ID NO: 25).

US 20100221175; US 20090092598; US; US 20110064653; US 20090185974; US 20080069775.

(42) MET (proto-oncogene met; hepatocyte growth factor receptor).

Nucleotide:

Genbank accession number M35073.

Genbank version number M35073.1 GI: 187553.

Genbank, post update date: March 6, 2012 11:12 am.

Polypeptide:

Genbank accession number AAA59589.

Genbank version number AAA59589.1 GI: 553531.

Genbank, post update date: March 6, 2012 11:12 am.

Cross-references:

Dean M., et al. Nature, 318 (6044), 385-388 (1985).

Other information:

Official symbol: MET.

Other names: AUTS9, HGFR, RCCP2, c-Met.

Other abbreviations: HGF receptor; HGF / SF receptor; SF receptor; hepatocyte growth factor receptor; proto-oncogenic tyrosine kinase met; proto-oncogenic c-Met; scattering factor receptor; tyrosine protein kinase Met.

Antibodies.

Abgenix / Pfizer: US 20100040629, for example, an antibody produced by hybridoma 13.3.2 having American Type Culture Collection (ATCC) accession number PTA-5026;

an antibody produced by hybridoma 9.1.2 having ATCC accession number PTA-5027;

an antibody produced by hybridoma 8.70.2 having ATCC accession number PTA-5028; or an antibody produced by hybridoma 6.90.3 having ATCC accession number PTA-5029.

Amgen / Pfizer: US 20050054019, for example, an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 2 where X2 is glutamate and X4 is serine and a light chain having the amino acid sequence shown in SEQ ID NO: 4 where X8 is alanine, no signal sequences;

an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 6 and a light chain having the amino acid sequence shown in SEQ ID NO: 8 without signal sequences;

an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 10 and a light chain having the amino acid sequence shown in SEQ ID NO: 12 without signal sequences; or an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 14 and a light chain having the amino acid sequence shown in SEQ ID NO: 16 without signal sequences.

Agouron Pharmaceuticals (now Pfizer): US 20060035907 Eli Lilly: US 20100129369.

Genentech: US5686292; US 20100028337; US 20100016241; US 20070129301; US 20070098707; US 20070092520; US 20060270594; US 20060134104; US 20060035278; US 20050233960; US 20050037431.

US 5686292 - for example, ATCC HB-11894 and ATCC HB-11895.

US 20100016241 - for example, ATCC HB-11894 (hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6).

National Defense Medical Center, Taiwan: Lu RM., Et al. Biomaterials. 2011 Apr; 32 (12): 3265-74.

Novartis: US20090175860, for example, an antibody containing the sequences CDR1, CDR2 and CDR3 of the heavy chain 4687, where the sequences CDR1, CDR2 and CDR3 of the heavy chain 4687 are residues 26-35, 50-65 and

- 30 038267

98-102, respectively, from SEQ ID NO: 58; and light chain CDR1, CDR2 and CDR3 sequences 5097, wherein the light chain CDR1, CDR2 and CDR3 sequences of 5097 are residues 24-39, 55-61 and 94-100 of SEQ ID NO: 37.

Pharmacia Corporation: US 20040166544.

Pierre Fabre: US 20110239316, US 20110097262, US 20100115639

Sumsung: US 20110129481 - for example, a monoclonal antibody produced by a hybridoma cell having accession number KCLRF-BP-00219 or accession number KCLRF-BP-00223.

Samsung: US 20110104176 - for example, an antibody produced by a hybridoma cell having accession number: KCLRF-BP-00220.

University of Turin Medical School: DN-30 Pacchiana G., et al. J. Biol. Chem. 2010 Nov 12; 285 (46): 36149-57.

Van Andel Research Institute: Jiao Y., et al. Mol. Biotechnol. 2005 Sep; 31 (1): 41-54.

MUC1 (mucin 1 bound to the cell surface).

Nucleotide:

Genbank accession number J05581.

Genbank version number J05581.1 GI: 188869.

Genbank, entry updated on June 23, 2010 at 08:48 AM.

Polypeptide:

Genbank accession number AAA59876.

Genbank version number AAA59876.1 GI: 188870.

Genbank, entry updated on June 23, 2010 at 08:48 AM.

Cross-references:

Gendler S. J., et al. J. Biol. Chem. 265 (25), 15286-15293 (1990).

Other information:

Official symbol: MUC1.

Other names: RP11-263K19.2, CD227, EMA, H23AG, KL-6, MAM6, MUC-1, MUC-1 / SEC, MUC-1 / X, MUC1 / ZD, PEM, PEMT, PUM.

Other abbreviations: DF3 antigen; antigen H23; DF3 antigen associated with breast carcinoma; mucin associated with carcinoma; episialin; Krebs von den Lungen-6; mucin 1, transmembrane; mucin-1; peanut-reactive bladder mucin; polymorphic epithelial mucin; tumor-associated epithelial mucin; tumor-associated epithelial membrane antigen; tumor-associated mucin.

Antibodies.

AltaRex - Quest Pharma Tech: US 6716966 - for example the Alt-1 antibody produced by the ATCC number PTA-975 hybridoma.

AltaRex - Quest Pharma Tech: US 7147850.

CRT: 5E5 - Sorensen A.L., et al. Glycobiology, vol. 16, no. 2, p. 96-107, 2006; HMFG2 - Burchell J., et al. Cancer Res., 47, 5476-5482 (1987); see WO 2015/159076.

Glycotope GT-MAB: GT-MAB 2.5-GEX (website: http://www.glycotope.com/pipeline/pankomab-gex).

Immunogen: US 7202346, for example, MJ-170 antibody: MJ-170 hybridoma cell line, ATCC accession number. PTA-5286.

Monoclonal antibody MJ-171: MJ-171 hybridoma cell line, accession number ATCC. PTA-5287; monoclonal antibody MJ-172: hybridoma cell line MJ-172, accession number ATCC. PTA-5288; or monoclonal antibody MJ-173: hybridoma cell line MJ-173, accession number ATCC. РТА-5302.

Immunomedics: US 6653104.

Ramot Tel Aviv Uni: US 7897351.

Regents Uni. CA: US 7,183,388; US 20040005647; US 20030077676.

Roche GlycArt: US 8021856.

Russian Cancer Research Center: Imuteran-Ivanov P.K., et al. Biotechnol. J. 2007 Jul; 2 (7): 863-70

Technische Univ Braunschweig: (IIB6, HT186-B7, HT186-D11, HT186-G2, HT200-3A-C1, HT220-M-D1, HT220-M-G8) - Thie H., et al. PLoS One. 2011 Jan 14; 6 (1): e15921.

(44) CA9 (carbonic anhydrase IX).

Nucleotide:

Genbank accession number X66839.

Genbank version number X66839.1 GI: 1000701.

Genbank, entry updated on February 2, 2011 at 10:15 am.

Polypeptide:

Genbank accession number CAA47315.

Genbank version number CAA47315.1 GI: 1000702.

- 31 038267

Genbank, entry updated on February 2, 2011 at 10:15 am.

Cross-references:

Pastorek J., et al. Oncogene 9 (10), 2877-2888 (1994).

Other information:

Official symbol: CA9.

Other names: CAIX, MN.

Other designations: CA-IX; P54 / 58N; RCC-associated antigen G250; RCC associated protein G250; carbonate dehydratase IX; carbonic anhydrase 9;

carbonic anhydrase; membrane antigen MN; pMW1; antigen G250 associated with renal cell carcinoma.

Antibodies.

Abgenix / Amgen: US 20040018198.

Affitel: Anti-CAIX Affitel Molecules (http://www.affibody.com/en/Product-Portfolio/Pipeline/).

Bayer: US 7462696.

Bayer / Morphosys: 3ee9 mAb - Petrul H. M., et al. Mol. Cancer Ther. 2012 Feb; 11 (2): 340-9.

Harvard Medical School: Antibodies G10, G36, G37, G39, G45, G57, G106, G119, G6, G27, G40 and G125.

Xu C., et al. PLoS One. 2010 Mar 10; 5 (3): e9625.

Institute of Virology, Slovak Academy of Sciences (Bayer) - US 5955075, e.g. M75, ATCC access number HB 11128, or MN12, ATCC access number HB 11647.

Institute of Virology, Slovak Academy of Sciences: US 7816493, for example, the monoclonal antibody M75, which is secreted from the VU-M75 hybridoma deposited with the American Type Culture Collection under the ATCC number HB 11128; or monoclonal antibody V / 10 secreted from V / 10-VU hybridoma deposited in the Belgian Coordinated Collection of Microorganisms of the International Depository Organization at Laboratorium voor Moleculaire Bioloqie-Plasmidencollectie (LMBP) at the University of Ghent, Ghent, Belgium, under accession number LMBP 6009CB ...

Institute of Virology, Slovak Academy of Sciences US 20080177046; US 20080176310; US 20080176258; US 20050031623.

Novartis: US 20090252738.

Wilex: US 7691375 - for example, an antibody produced by the hybridoma cell line DSM ASC 2526.

Wilex: US 20110123537; Rencarex: Kennett R. H., et al. Curr Opin Mol. Ther. 2003 Feb; 5 (1): 70-5.

Xencor: US 20090162382.

(45) EGFRvIII (epidermal growth factor receptor (EGFR), transcriptional variant 3.

Nucleotide:

Genbank accession number NM_201283.

Genbank version number NM_201283.1 GI: 41327733.

Genbank, entry update date: September 30, 2012 at 01:47 p.m.

Polypeptide:

Genbank accession number NP_958440.

Genbank version number NP_958440.1 GI: 41327734.

Genbank, entry update date: September 30, 2012 at 01:47 p.m.

Cross-references:

Batra S. K., et al. Cell Growth Differ, 1995; 6: 1251-1259.

Antibodies.

US 7628986 and US 7736644 (Amgen):

for example, a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 142 and variants, and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 144 and variants.

US 20100111979 (Amgen):

for example, an antibody comprising a heavy chain amino acid sequence comprising:

CDR1 consisting of a sequence selected from the group consisting of amino acid sequences for the CDR1 region of antibodies 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16) and 333 (SEQ ID NO: 17);

CDR2 consisting of a sequence selected from the group consisting of amino acid sequences for the CDR2 region of antibodies 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13) , 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16) and 333 (SEQ ID NO: 17); and

CDR3 consisting of a sequence selected from the group consisting of amino acid sequences for the CDR3 region of antibodies 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211

32 038267 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16) and 333 (SEQ ID NO: 17).

US 20090240038 (Amgen):

for example, an antibody having at least one of the heavy or light chain polypeptides that contains an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 142, SEQ ID NO: 144, and any combination thereof.

US 20090175887 (Amgen):

for example, an antibody having a heavy chain amino acid sequence selected from the group consisting of an antibody heavy chain amino acid sequence 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO : 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16) and 333 (SEQ ID NO: 17).

US 20090156790 (Amgen), for example, an antibody having a heavy chain polypeptide and a light chain polypeptide, wherein at least one of the heavy or light chain polypeptides comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of from: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 142, SEQ ID NO: 144, and any combination thereof.

US 20090155282, US 20050059087 and US 20050053608 (Amgen), for example, an antibody having a heavy chain amino acid sequence selected from the group consisting of the antibody heavy chain amino acid sequence 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 ( SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16) and 333 (SEQ ID NO: 17).

MR1-1 (US 7129332; Duke):

for example, a variant antibody having SEQ ID NO: 18 with substitutions S98PT99Y in VH CDR3 and F92W in VL CDR3.

L8A4, H10, Y10 (Wikstrand CJ., Et al. Cancer Res. 1995 Jul 15; 55 (14): 3140-8; Duke).

US 20090311803 (Harvard University):

for example, SEQ ID NO: 9 for the variable region of the heavy chain of an antibody and SEQ ID NO: 3 for the amino acid sequence of the variable region of the light chain

US 20070274991 (EMD72000, also known as matuzumab; Harvard University):

for example SEQ ID NO: 3 and 9 for light chain and heavy chains, respectively.

US 6129915 (Schering):

for example SEQ ID NO: 1, 2, 3, 4, 5 and 6.

mAb CH12 - Wang H., et al. FASEB J. 2012 Jan; 26 (1): 73-80 (Shanghai Cancer Institute).

RAbDMvIII - Gupta P., et al. BMC Biotechnol. 2010 Oct 7; 10:72 (Stanford University Medical Center).

mAb Ua30 - Ohman L., et al. Tumour Biol. 2002 Mar-Apr; (2): 61-9 (Uppsala University).

Han D. G., et al. Nan Fang Yi Ke Da Xue Xue Bao. 2010 Jan; 30 (1): 25-9 (Xi'an Jiaotong University).

(46) CD33 (CD33 molecule:

Nucleotide:

Genbank accession number M_23197.

Genbank version number NM_23197.1 GI: 180097.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Polypeptide:

Genbank accession number AAA51948.

Genbank version number AAA51948.1 GI: 188098.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Cross-references:

Simmons D., et al. J. Immunol. 141 (8), 2797-2800 (1988).

Other information:

Official symbol: CD33.

Other names: SIGLEC-3, SIGLEC3, p67.

Other names: CD33 antigen (gp67); gp67; myeloid cell surface antigen CD33; Ig-like sialic acid binding lectin 3; Ig-like sialic acid binding lectin 3.

Antibodies.

H195 (lintuzumab) - Raza A., et al. Leuk Lymphoma. 2009 Aug; 50 (8): 1336-44; US 6759045 (Seattle Genetics / lmmunomedics).

mAb OKT9: Sutherland, D.R. et al. Proc Natl Acad Sci USA, 78 (7): 4515-4519, 1981, Schneider, C., et al. J. Biol. Chem. 257, 8516-8522 (1982) mAb E6: Hoogenboom, H. R., et a .. J. Immunol. 144, 3211-3217 (1990).

- 33 038267

US 6590088 (Human Genome Sciences:

for example SEQ ID NOs: 1 and 2 and ATCC accession number 97521.

US 7557189 (Immunogen):

for example, an antibody or a fragment thereof containing a heavy chain variable region that contains three CDRs having the amino acid sequences of SEQ ID NO: 1-3, and a light chain variable region containing three CDRs having the amino acid sequences of SEQ ID NO: 4-6.

(47) CD19 (CD19 molecule).

Nucleotide:

Genbank accession number NM_001178098.

Genbank version number NM_001178098.1 GI: 296010920.

Genbank, post update date: September 10, 2012 12:43 pm.

Polypeptide:

Genbank accession number NP_001171569.

Genbank version number NP_001171569.1 GI: 296010921.

Genbank, post update date: September 10, 2012 12:43 pm.

Cross-references:

Tedder T. F., et al. J. Immunol. 143 (2): 712-7 (1989).

Other information:

Official symbol: CD19.

Other names: B4, CVID3.

Other designations: B-lymphocyte antigen CD19; B4 antigen of the surface of B-lymphocytes; T cell surface antigen Leu-12; differentiation antigen CD19.

Antibodies.

Immunogen: HuB4 - Al-Katib A. M., et al. Clin Cancer Res. 2009 Jun 15; 15 (12): 4038-45.

4G7: Kugler M., et al. Protein Eng Des Sel. 2009 Mar; 22 (3): 135-47, for example, the sequences in FIG. 3 publications by Knappik, A. et al. Mol. Biol. 2000 Feb; 296 (1): 57-86.

AstraZeneca / Medlmmune: MEDI-551 - Herbst R., et al. J. Pharmacol. Exp. Ther. 2010 Oct; 335 (1): 21322.

Glenmark Pharmaceuticals: GBR-401 - Hou S., et al. Mol. Cancer. November 2011 (Meeting Abstract Supplement) C164.

US 7109304 (Immunomedics):

for example, an antibody comprising the sequence hA19Vk (SEQ ID NO: 7) and the sequence hA19VH (SEQ ID NO: 10).

US 7902338 (Immunomedics):

for example, an antibody or antigen-binding fragment thereof that contains the sequences of the complementarity determining regions of the CDR light chain CDR1 SEQ ID NO: 16 (KASQSVDYDGDSYLN); CDR2 of SEQ ID NO: 17 (DASNLVS); and CDR3 SEQ ID NO: 18 (QQSTEDPWT) and CDRs of the heavy cerium sequence CDR1 SEQ ID NO: 19 (SYWMN); CDR2 SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3 SEQ ID NO: 21 (RETTTVGRYYYAMDY), and also contains the framework region (FR) of the human antibody and constant region sequences with one or more amino acid residues of the framework region substituted from the corresponding sequences of the original framework region murine antibody, and wherein said substituted FR residues comprise a serine-to-phenylalanine substitution at residue 91 of the Kabat heavy chain variable region.

Medarex: MDX-1342 - Cardarelli P.M., et al. Cancer Immunol. Immunother. 2010 Feb; 59 (2): 257-65.

MorphoSys / Xencor: MOR-208 / XmAb-5574 - Zalevsky J., et al. Blood. 2009 Apr 16; 113 (16): 3735-43.

US 7968687 (Seattle Genetics):

an antibody or antigen-binding fragment containing the variable domain of the heavy chain containing the amino acid sequence of SEQ ID NO: 9, and the variable domain of the light chain containing the amino acid sequence of SEQ ID NO: 24.

4G7 chim - Lang P., et al. Blood. 2004 May 15; 103 (10): 3982-5 (University of Tubingen).

For example, FIG. 6 and SEQ ID NO: 80 in US 20120082664.

Zhejiang University School of Medicine: 2E8 - Zhang J., et al. J. Drug Target. 2010 Nov; 18 (9): 675-8.

(48) IL2RA (interleukin 2 receptor, alpha); NCBI reference sequence:

NM_000417.2).

Nucleotide:

Genbank accession number NM_000417.

Genbank version number NM_000417.2 GI: 269973860.

Genbank, entry update date: 09.09.2012 at 04:59 p.m.

Polypeptide:

- 34 038267

Genbank accession number NP_000408.

Genbank version number NP_000408.1 GI: 4557667.

Genbank, entry update date: 09.09.2012 at 04:59 p.m.

Cross-references:

Kuziel W. A., et al. J. Invest. Dermatol. 94 (6 SUPPL), 27S-32S (1990).

Other information:

Official symbol: IL2RA.

Other names: RP11-536K7.1, CD25, IDDM10, IL2R, TCGFR.

Other abbreviations: the alpha subunit of the FIL-2 receptor; IL-2-RA; the alpha subunit of IL-2R; IL2RA; TAC antigen; the alpha subunit of the interleukin-2 receptor; p55.

Antibodies.

US 6383487 (Novartis / UCL: Baxilisimab [Simulect]).

US 6521230 (Novartis / UCL: Baxilisimab [Simulect]):

n an antibody having an antigen binding site that contains at least one domain containing a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8, and a CDR3 having the amino acid sequence of SEQ ID NO: nine; or said CDR1, CDR2 and CDR3 in the sequence as a whole contain an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, 8 and 9 in the sequence as a whole.

Daclizumab - Rech AJ., Et al. Ann N Y Acad Sci. 2009 Sep; 1174: 99-106 (Roche).

(49) AXL (receptor tyrosine kinase AXL).

Nucleotide:

Genbank accession number M76125.

Genbank version number M76125.1 GI: 292869.

Genbank, entry updated on June 23, 2010 at 08:53 am.

Polypeptide:

Genbank accession number AAA61243.

Genbank version number AAA61243.1 GI: 29870.

Genbank, entry updated on June 23, 2010 at 08:53 am.

Cross-references:

O'Bryan J. P., et al. Mol. Cell. Biol. 11 (10), 5016-5031 (1991); Bergsagel P. L., et al. J. Immunol. 148 (2), 590-596 (1992).

Other information:

Official symbol: AXL.

Other names: JTK11, UFO.

Other designations: AXL oncogene; AXL transforming sequence / gene; AXLoncogene; UFO receptor tyrosine protein kinase.

Antibodies.

YW327.6S2 - Ye X., et al. Oncogene. 2010 Sep 23; 29 (38): 5254-64.

(Genentech).

BergenBio: BGB324 (http://www.bergenbio.com/BGB324).

(50) CD30 - TNFRSF8 (8 member of the tumor necrosis factor receptor superfamily).

Nucleotide:

Genbank accession number M83554.

Genbank version number M83554.1 GI: 180095.

Genbank, entry updated on June 23, 2010 at 08:53 am.

Polypeptide:

Genbank accession number AAA51947.

Genbank version number AAA51947.1 GI: 180096.

Genbank, entry updated on June 23, 2010 at 08:53 am.

Cross-references:

Durkop H., et al. Cell 68 (3), 421-427 (1992)

Other information:

Official symbol: TNFRSF8.

Other names: CD30, D1S166E, Ki-1.

Other abbreviations: CD30L receptor; Ki-1 antigen; cytokine receptor CD30; lymphocyte activation antigen CD30; 8 is a member of the tumor necrosis factor receptor superfamily.

BCMA (B-cell maturation antigen) - TNFRSF17 (17th member of the tumor necrosis factor receptor superfamily).

Nucleotide:

Genbank accession number Z29574.

Genbank version number Z29574.1 GI: 471244.

- 35 038267

Genbank, entry update date: February 02, 2011 10:40 am.

Polypeptide:

Genbank accession number CAA82690.

Genbank version number CAA82690.1 GI: 471245.

Genbank, entry update date: February 02, 2011 10:40 am.

Cross-references:

Laabi Y., et al. Nucleic Acids Res. 22 (7), 1147-1154 (1994).

Other information:

Official symbol: TNFRSF17.

Other names: ВСМ, ВСМА, CD269.

Other abbreviations: Other abbreviations: B cell maturation antigen; B cell maturation factor; B cell maturation protein; 17 member of the tumor necrosis factor receptor superfamily.

(52) CT Ags -CTA (testicular cancer antigens).

Cross-references:

Fratta E., et al. Mol Oncol. 2011 Apr; 5 (2): 164-82; Lim S.H., at al. Am. J. Blood Res. 2012; 2 (1): 29-35.

(53) CD174 (Lewis Y) - FUT3 (fucosyltransferase 3 (galactoside-3 (4) -c-fucosyltransferase, Lewis blood group).

Nucleotide:

Genbank accession number NM000149.

Genbank version number NM000149.3 GI: 148277008.

Genbank, entry updated on June 26, 2012 at 04:49 p.m.

Polypeptide:

Genbank accession number NP_000140.

Genbank version number NP_000140.1 GI: 4503809.

Genbank, entry updated on June 26, 2012 at 04:49 p.m.

Cross-references:

Kukowska-Latallo, J. F., et al. Genes Dev. 4 (8), 1288-1303 (1990).

Other information:

Official symbol: FUT3.

Other names: CD174, FT3B, FucT-III, LE, Les.

Other designations: FT Lewis; alpha (1,3 / 1,4) fucosyltransferase; Lewis blood group alpha-4-fucosyltransferase; fucosyltransferase III; galactoside-3 (4) -c-fucosyltransferase.

CLEC14A (C-type lectin domain family member A of 14; Genbank accession number NM175060).

Nucleotide:

Genbank accession number NM175060.

Genbank version number NM175060.2 GI: 371123930.

Genbank, entry update date: April 01, 2012, 03:34 p.m.

Polypeptide:

Genbank accession number NP_778230.

Genbank version number NP_778230.1 GI: 28269707.

Genbank, entry update date: April 01, 2012, 03:34 p.m.

Other information:

Official symbol: CLEC14A.

Other names: UNQ236 / PRO269, C14orf27, CEG1, EGFR-5.

Other abbreviations: member A of family 14 of the C-type pectin domain; CIECT and a protein containing an EGF-like domain; epidermal growth factor receptor 5.

(55) GRP78 - HSPA5 (heat shock protein 5 70 kDa (glucose-regulated protein, 78 kDa).

Nucleotide:

Genbank accession number NM005347.

Genbank version number NM005347.4 GI: 305855105.

Genbank, entry update date: September 30, 2012 at 01:42 p.m.

Polypeptide:

Genbank accession number NP_005338.

Genbank version number NP_005338.1 GI: 16507237.

Genbank, entry update date: September 30, 2012 at 01:42 p.m.

Cross-references:

Ting J., et al. DNA, 7 (4), 275-286 (1988).

Other information:

Official symbol: HSPA5.

Other names: BIP, GRP78, MIF2.

Other designations: glucose-regulated protein 78 kDa; protein grp78, which binds cavity Ca (2+) endoplasmic reticulum; a protein that binds the heavy chain of immunoglobulin.

- 36 038267 (56) CD70 (CD70 molecule) L08096.

Nucleotide:

Genbank accession number L08096.

Genbank version number L08096.1 GI: 307127.

Genbank, entry updated on June 23, 2012 at 08:54 am.

Polypeptide:

Genbank accession number AAA36175.

Genbank version number AAA36175.1 GI: 307128.

Genbank, entry updated on June 23, 2012 at 08:54 am.

Cross-references:

Goodwin R. G., et al. Cell, 73 (3), 447-456 (1993).

Other information:

Official symbol: CD70.

Other names: CD27L, CD27LG, TNFSF7.

Other designations: ligand CD27; CD27-L; antigen CD70; Ki-24 antigen; surface antigen CD70; 7 member of the superfamily of tumor necrosis factor (ligand); 7 member of the tumor necrosis factor ligand superfamily.

Antibodies.

MDX-1411 vs. CD70 (Medarex).

h1F6 (Oflazoglu, E., et al., Clin Cancer Res. 2008 Oct 1; 14 (19): 6171-80; Seattle Genetics). For example, see US 20060083736, SEQ ID NO: 1, 2, 11 and 12 and FIG. 1.

Stem cell specific antigens.

For example: 5T4 (see line (63) below), CD25 (see line (48) below), CD32.

Polypeptide:

Genbank accession number ABK42161.

Genbank version number ABK42161.1 GI: 117616286.

Genbank, post update date: July 25, 2007, 03:00 p.m.

LGR5 / GPR49.

Nucleotide:

Genbank accession number NM_003667.

Genbank version number NM_003667.2 GI: 24475886.

Genbank, entry update date: July 22, 2012, 03:38 p.m.

Polypeptide:

Genbank accession number NP_003658.

Genbank version number NP_003658.1 GI: 4504379.

Genbank, entry update date: July 22, 2012, 03:38 p.m.

Prominin / SO 133.

Nucleotide:

Genbank accession number NM_006017.

Genbank version number NM_006017.2 GI: 224994187.

Genbank, entry update date: September 30, 2012 at 01:47 p.m.

Polypeptide:

Genbank accession number NP_006008.

Genbank version number NP_006008.1 GI: 5174387.

Genbank, entry update date: September 30, 2012 at 01:47 p.m.

(58) ASG-5.

Cross-references:

(Smith L.M., et al. AACR 2010 Annual Meeting (abstract # 2590); Gudas J.M., et al. AACR 2010, Annual Meeting (abstract # 4393).

Antibodies.

Ahtu-AGS-5 antibody: M6.131 (Smith, L.M., et.al AACR 2010, Annual Meeting (abstract # 2590).

(59) ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase 3).

Nucleotide:

Genbank accession number AF005632.

Genbank version number AF005632.2 GI: 4432589.

Genbank, entry update date: March 10, 2010 at 09:41 p.m.

Polypeptide:

Genbank accession number AAC51813.

Genbank version number AAC51813.1 GI: 2465540.

Genbank, entry update date: March 10, 2010 at 09:41 p.m.

Cross-references:

Jin-Hua P., et al. Genomics, 45 (2), 412-415 (1997).

- 37 038267

Other information:

Official symbol: ENPP3.

Other names: RP5-988G15.3, B10, CD203c, NPP3, PD-IBETA, PDNP3.

Other designations: E-NPP 3; dJ1005H11.3 (phosphodiesterase I / nucleotide pyrophosphatase 3); dJ914N13.3 (phosphodiesterase I / nucleotide pyrophosphatase 3); 3 member of the ectonucleotide pyrophosphatase / phosphodiesterase family; gp130RB13-6; beta phosphodiesterase I; phosphodiesterase I / nucleotide pyrophosphatase 3; beta-phosphodiesterase-I.

(60) PRR4 (proline-rich 4 (lacrimal)).

Nucleotide:

Genbank accession number NM_007244.

Genbank version number NM_007244.2 GI: 154448885.

Genbank, entry update date: June 28, 2012 at 12:39 p.m.

Polypeptide:

Genbank accession number NP_009175.

Genbank version number NP_009175.2 GM54448886.

Genbank, entry update date: June 28, 2012 at 12:39 p.m.

Cross-references:

Dickinson D. P., et al. Invest. Ophthalmol. Vis. Sci. 36 (10), 2020-2031 (1995).

Other information:

Official symbol: PRR4.

Other names: LPRP, PROL4.

Other abbreviations: lacrimal proline-rich protein; proline-rich protein 4 associated with nasopharyngeal carcinoma; proline-rich polypeptide 4; proline-rich protein 4.

(61) GCC - GUCY2C (guanylate cyclase 2C (thermostable enterotoxin receptor).

Nucleotide:

Genbank accession number NM_004963.

Genbank version number NM_004963.3 GI: 222080082.

Genbank, entry update date: September 30, 2012 at 01:50 p.m.

Polypeptide:

Genbank accession number NP_004954.

Genbank version number NP_004954.2 GI: 222080083.

Genbank, entry update date: September 30, 2012 at 01:50 p.m.

Cross-references:

De Sauvage F. J., et al. J. Biol. Chem. 266 (27), 17912-17918 (1991); Singh S., et al. Biochem. Biophys. Res. Commun. 179 (3), 1455-1463 (1991).

Other information:

Official symbol: GUCY2C.

Other names: DIAR6, GUC2C, MUCIL, STAR.

Other designations: GC-C; STA receptor; guanylyl cyclase C; hSTAR; thermostable enterotoxin receptor; intestinal guanylate cyclase.

(62) Liv-1-SLC39A6 (6 member of solute carrier family 39 (zinc transporter)).

Nucleotide:

Genbank accession number U41060.

Genbank version number U41060.2 GI: 12711792.

Genbank, entry updated on November 30, 2009 at 04:35 p.m.

Polypeptide:

Genbank accession number AAA96258.

Genbank version number AAA96258.2 GI: 12711793.

Genbank, entry updated on November 30, 2009 at 04:35 p.m.

Cross-references:

Taylor K. M., et al. Biochim Biophys Acta. 2003 Apr 1; 1611 (1-2): 16-30.

Other information:

Official symbol: SLC39A6.

Other names: LIV-1.

Other abbreviations: estrogen-regulated LIV-1 protein; ZIP-6; estrogen-regulated protein LIV-1; 6 member of the family of solute carriers 39 (metal ion transporter); 6 member of the family of solute carriers 36; zinc transporter ZIP6; zrt- and lrt-like protein 6.

(63) 5T4, trophoblastic glycoprotein, TPBG - TPBG (trophoblastic glycoprotein).

Nucleotide:

Genbank accession number AJ012159.

Genbank version number AJ012159.1 GI: 3805946.

Genbank, entry update date: February 01, 2011 at 10:27 am.

- 38 038267

Polypeptide:

Genbank accession number CAA09930.

Genbank version number CAA09930.1 GI: 3805947.

Genbank, entry update date: February 01, 2011 at 10:27 am.

Cross-references:

King K. W., et al. Biochim. Biophys. Acta, 1445 (3), 257-270 (1999).

Other information:

Official symbol: TPBG.

Other names: 5T4, 5T4AG, M6P1.

Other designations: 5T4 oncofetal antigen; oncofetal trophoblastic glycoprotein 5T4; oncotrophoblastic glycoprotein 5T4.

See WO 2015/155345.

(64) CD56 - NCMA1 (nerve cell adhesion molecule 1).

Nucleotide:

Genbank accession number NM_000615.

Genbank version number NM_000615.6 GI: 336285433.

Genbank, entry updated on September 23, 2012 at 02:32 p.m.

Polypeptide:

Genbank accession number NP_000606.

Genbank version number NP_000606.3 GI: 94420689.

Genbank, entry updated on September 23, 2012 at 02:32 p.m.

Cross-references:

Dickson, G., et al., Cell, 50 (7), 1119-1130 (1987).

Other information:

Official symbol: NCAM1.

Other names: CD56, MSK39, NCAM antigen recognized by monoclonal antibody 5.1H11; nerve cell adhesion molecule, NCAM.

Antibodies.

Immunogen: HuN901 (Smith S. V., et al. Curr Opin. Mol. Ther. 2005 Aug; 7 (4): 394-401).

For example, see humanized mouse N901 antibody. See fig. 1b and 1e in Roguska, M.A., et al. Proc. Natl. Acad. Sci. USA, Feb 1994; 91: 969-973.

(65) CanAg (CA242 tumor associated antigen).

Cross-references:

Haglund C., et al. Br. J. Cancer 60: 845-851, 1989; Baeckstrom D., et al. J. Biol. Chem. 266: 21537-21547,1991.

Antibodies.

huC242 (Tolcher A. W. et al., J. Clin. Oncol. 2003 Jan 15; 21 (2): 211-22; Immunogen).

For example, see US 20080138898A1, SEQ ID NO: 1 and 2.

(66) FOLR1 (folate receptor 1).

Nucleotide:

Genbank accession number J05013.

Genbank, version number J05013.1 GM82417.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Polypeptide:

Genbank accession number AAA35823.

Genbank version number AAA35823.1 GI: 182418.

Genbank, entry updated on June 23, 2010 at 08:47 am.

Cross-references:

Elwood P. C., et al. J. Biol. Chem. 264 (25), 14893-14901 (1989).

Other information:

Official symbol: FOLR1.

Other names: FBP, FOLR.

Other designations: FR-alpha; FBP of KB cells; folate binding protein in adults; folate-binding protein; folate receptor alpha; folate receptor in adults; antigen MOv18 associated with ovarian tumor.

Antibodies.

M9346A - Whiteman K. R., et al. Cancer Res. April 15, 2012; 72 (8 Supplement): 4628 (Immunogen).

(67) GPNMB (glycoprotein (transmembrane) nmb).

Nucleotide:

Genbank accession number X76534.

Genbank version number X76534.1 GI: 666042.

Genbank, entry update date: February 02, 2011 at 10:10 am.

- 39 038267

Polypeptide:

Genbank accession number CAA54044.

Genbank version number CAA54044.1 GI: 666043.

Genbank, entry update date: February 02, 2011 at 10:10 am.

Cross-references:

Weterman M. A., et al. Int. J. Cancer 60 (1), 73-81 (1995).

Other information:

Official symbol: GPNMB.

Other names: UNQ1725 / PRO9925, HGFIN, NMB.

Other abbreviations: NMB glycoprotein; glycoprotein nmb-like protein; osteoaktivin; transmembrane glycoprotein HGFIN; transmembrane glycoprotein NMB.

Antibodies.

Celldex Therapeutics: CR011 (Tse K. F., et al. Clin. Cancer Res. 2006 Feb 15; 12 (4): 1373-82).

For example, see EP1827492B1, SEQ ID NO: 22, 24, 26, 31, 33 and 35.

(68) TIM-1-HAVCR1 (hepatitis A virus cell receptor 1).

Nucleotide:

Genbank accession number AF043724.

Genbank version number AF043724.1 GI: 2827453.

Genbank, entry update date: March 10, 2010 at 06:24 p.m.

Polypeptide:

Genbank accession number AAC39862.

Genbank version number AAC39862.1 GI: 2827454.

Genbank, entry update date: March 10, 2010 at 06:24 p.m.

Cross-references:

Feigelstock D., et al. J. Virol. 72 (8), 6621-6628 (1998).

Other information:

Official symbol: HAVCR1.

Other names: HAVCR, HAVCR-1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1.

Other abbreviations: protein 1 domain of T-cell immunoglobulin and mucin domain; T-cell membrane protein 1; renal damage molecule 1.

(69) RG-1 / prostate tumor target Mindin - Mindin / RG-1.

Cross-references:

Parry R., et al. Cancer Res. 2005 Sep 15; 65 (18): 8397-405.

(70) B7-H4 - VTCN1 (inhibitor 1 of T-cell activation containing a V-shaped domain)

Nucleotide:

Genbank accession number BX648021.

Genbank version number BX648021.1 GI: 34367180.

Genbank, entry update date: 02 February 2011 08:40 am.

Cross-references:

Sica G. L., et al. Immunity. 2003 Jun; 18 (6): 849-61.

Other information:

Official symbol: VTCN1.

Other names: RP11-229A19.4, B7-H4, B7H4, B7S1, B7X, B7h.5, PRO1291, VCTN1.

Other designations: member of the H4 family B7; member of 1 superfamily B7; co-stimulatory molecule of B7x T cells; co-stimulatory molecule of B7x T cells; inhibitor 1 of activation of T cells containing the V-shaped domain; immune co-stimulating protein B7-H4.

PTK7 (protein tyrosine kinase 7 PTK7).

Nucleotide:

Genbank accession number AF447176.

Genbank version number AF447176.1 GI: 17432420.

Genbank, entry updated on November 28, 2008 at 01:51 PM

Polypeptide:

Genbank accession number AAL39062.

Genbank, version number AAL39062.1 GM7432421.

Genbank, entry updated on November 28, 2008 at 01:51 PM

Cross-references:

Park S. K., et al. J. Biochem. 119 (2), 235-239 (1996).

Other information:

Official symbol: PTK7.

Other names: CCK-4, CCK4.

Other abbreviations: colon carcinoma kinase 4; inactive protein tyrosine kinase 7; pseudoreceptor 7 tyrosine kinase; protein 7, similar to protein tyrosine kinase.

- 40 038267

CD37 (molecule CD37).

Nucleotide:

Genbank accession number NM_001040031.

Genbank version number NM_001040031.1 GI: 91807109.

Genbank, entry update date: July 29, 2012 at 02:08 p.m.

Polypeptide:

Genbank accession number NP_001035120.

Genbank version number NP_001035120.1 GI: 91807110.

Genbank, entry update date: July 29, 2012 at 02:08 p.m.

Cross-references:

Schwartz-Albiez R., et al. J. Immunol. 140 (3), 905-914 (1988).

Other information:

Official symbol: CD37.

Other names: GP52-40, TSPAN26.

Other names: CD37 antigen; cell differentiation antigen 37; leukocyte antigen CD37; leukocyte surface antigen CD37; tetraspanin-26; tspan-26.

Antibodies.

Boehringer Ingelheim: mAb 37.1 (Heider K. H., et al. Blood. 2011 Oct 13; 118 (15): 4159-68).

Trubion: CD37-SMIP (G28-1 scFv-Ig) ((Zhao X., et al. Blood. 2007; 110: 2569-2577).

For example, see US 20110171208A1 SEQ ID NO: 253.

Immunogen: K7153A (Deckert J., et al. Cancer Res April 15, 2012; 72 (8 Supplement): 4625) (73) CD138-SDC1 (syndecan 1).

Nucleotide:

Genbank accession number AJ551176.

Genbank version number AJ551176.1 GI: 29243141.

Genbank, entry update date: February 01, 2011 at 12:09 p.m.

Polypeptide:

Genbank accession number CAD80245.

Genbank version number CAD80245.1 GI: 29243142.

Genbank, entry update date: February 01, 2011 at 12:09 p.m.

Cross-references:

O'Connell F. P., et al. Am. J. Clin. Pathol. 2004 Feb; 121 (2): 254-63.

Other information:

Official symbol: SDC1.

Other names: CD138, SDC, SYND1, syndecan.

Other names: CD138 antigen; heparan sulfate proteoglycan, fibroblast growth factor receptor; syndecan proteoglycan 1; syndecan-1.

Antibodies.

Biotest: chimerized MAb (nBT062) - (Jagannath S., et al. Poster ASH # 3060, 2010; WIPO patent application WO 2010/128087).

For example, see US20090232810, SEQ ID NO: 1 and 2.

Immunogen: B-B4 (Tassone P., et al. Blood 104_3688-3696).

For example, see US20090175863A1, SEQ ID NO: 1 and 2.

CD74 (CD74 molecule, major histocompatibility complex, class II invariant chain).

Nucleotide:

Genbank accession number NM_004355.

Genbank version number NM_004355.1 GI: 343403784.

Genbank, entry update date: September 23, 2012 at 02:30 p.m.

Polypeptide:

Genbank accession number NP_004346.

Genbank version number NP_004346.1 GI: 10835071.

Genbank, entry update date: September 23, 2012 at 02:30 p.m.

Cross-references:

Kudo, J., et al. Nucleic Acids Res. 13 (24), 8827-8841 (1985).

Other information:

Official symbol: CD74.

Other names: DHLAG, HLADG, II, Ia-GAMMA.

Other designations: CD74 antigen (an invariant major histocompatibility complex polypeptide associated with a class II antigen); HLA class II histocompatibility antigen gamma chain; an invariant chain associated with HLA-DR antigens; HLA-DR-gamma; Ia-associated invariant chain; gamma chain HLA-DR MHC; the gamma chain of class II antigens; p33.

Antibodies.

- 41 038267

Immunomedics: hLL1 (Milatuzumab,) - Berkova Z., et al. Expert Opin Investig Drugs. 2010 Jan; 19 (1): 141-9).

For example, see US20040115193, SEQ ID NO: 19, 20, 21, 22, 23 and 24.

Genmab: HuMax-CD74 (see website).

(75) Claudins - CL (claudins).

Cross-references:

Offner S., et al. Cancer Immunol Immunother. 2005 May; 54 (5): 431-45;

Suzuki H., et al. Ann NY Acad Sci. 2012 Jul; 1258: 65-70).

In humans, 24 members of this family are described - see literature references.

(76) EGFR (epidermal growth factor receptor).

Nucleotide:

Genbank accession number NM_005228.

Genbank version number NM_005228.3 GI: 41927737.

Genbank, entry update date: September 30, 2012 at 01:47 p.m.

Polypeptide:

Genbank accession number NP_005219.

Genbank version number NP_005219.2 GI: 29725609.

Genbank, entry update date: September 30, 2012 at 01:47 p.m.

Cross-references:

Dhomen N.S., et al. Crit. Rev. Oncog .. 2012; 17 (1): 31-50.

Other information:

Official symbol: EGFR.

Other names: ERBB, ERBB1, HER1, PIG61, mENA.

Other designations: homologue of the viral oncogene of avian erythroblast leukemia (v-erb-b); a protein 40 that inhibits cell growth; protein 61, causing cell proliferation; proto-oncogene c-Erb-1; receptor tyrosine protein kinase erbB-1.

Antibodies.

BMS: Cetuximab (Erbitux) - Broadbridge V.T., et al. Expert Rev Anticancer Ther. 2012 May; 12 (5): 555-65.

For example, see US 6217866 - ATTS Depository Number 9764.

Amgen: Panitumumab (vectibix) - Argiles G., et al. Future Oncol. 2012 Apr; 8 (4): 373-89.

For example, see US 6,235,883, SEQ ID NO: 23-38.

Genmab: Zalutumumab - Rivera F., et al. Expert Opin Biol Ther. 2009 May; 9 (5): 667-74.

YM Biosciences: Nimotuzumab - Ramakrishnan M.S., et al. MAbs. 2009 Jan-Feb; 1 (1): 41-8.

For example, see US5891996, SEQ ID NO: 27-34.

(77) Her3 (ErbB3) - ERBB3 (homologue 3 of the viral oncogene of erythroblast leukemia v-erb-b2 (birds)).

Nucleotide:

Genbank accession number M34309.

Genbank version number M34309.1 GI: 183990.

Genbank, entry updated on June 23, 2010 at 08:47 p.m.

Polypeptide:

Genbank accession number AAA35979.

Genbank version number AAA35979.1 GI: 306841.

Genbank, entry updated on June 23, 2010 at 08:47 p.m.

Cross-references:

Plowman, G. D., et al., Proc. Natl. Acad. Sci. U.S.A. 87 (13), 4905-4909 (1990).

Other information:

Official symbol: ERBB3.

Other names: ErbB-3, HER3, LCCS2, MDA-BF-1, c-erbB-3, c-erbB3, erbB3-S, p180-ErbB3, p45sErbB3, p85-sErbB3.

Other designations: protooncogene-like protein c-ErbB-3; receptor tyrosine protein kinase erbB-3; cell surface receptor tyrosine kinase type HER3

Antibodies.

Merimack Pharma: MM-121 (Schoeberl B., et al. Cancer Res. 2010 Mar 15; 70 (6): 2485-2494).

For example, see US 2011028129, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and 8.

(78) RON - MST1R (macrophage stimulating receptor 1 (c-met-related tyrosine kinase)).

Nucleotide:

Genbank accession number X70040.

Genbank version number X70040.1 GI: 36109.

Genbank, entry update date: February 02, 2011, 10:17 p.m.

Polypeptide:

- 42 038267

Genbank accession number CCA49634.

Genbank version number CCA49634.1 GI: 36110.

Genbank, entry update date: February 02, 2011, 10:17 p.m.

Cross-references:

Ronsin C., et al. Oncogene 8 (5), 1195-1202 (1993).

Other information:

Official symbol: MST1R.

Other names: CD136, CDw136, PTK8, RON.

Other abbreviations: MSP receptor; MST1R, option RON30; MST1R, option RON62; protein tyrosine kinase 8 PTK8; RON, option E2E3; c-met-related tyrosine kinase; macrophage-stimulating protein receptor; p185-Ron; soluble version 1 RON; soluble version 2 RON; soluble version 3 RON; soluble version 4 RON.

(79) EPHA2 (EPH receptor A2).

Nucleotide:

Genbank accession number BC037166.

Genbank version number BC037166.2 GI: 33879863.

Genbank, entry update date: March 6, 2012 at 01:59 p.m.

Polypeptide:

Genbank accession number AAN37166.

Genbank version number AAN37166.1 GI: 22713539.

Genbank, entry update date: March 6, 2012 at 01:59 p.m.

Cross-references:

Strausberg R. L., et al. Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002)

Other information:

Official symbol: ERNA2.

Other names: ARCC2, STRA, STRP1, ECK.

Other abbreviations: ephrin type A receptor 2; receptor protein tyrosine kinase of epithelial cells; soluble version 1 EPHA2; ECK receptor tyrosine protein kinase.

Antibodies.

Medimmune: 1C1 (Lee J. W., et al. Clin Cancer Res. 2010 May 1; 16 (9): 2562-2570).

For example, see US 20090304721A1, fig. 7 and 8.

(80) CD20 - MS4A1 (transmembrane 4 domains, subfamily A, member 1).

Nucleotide:

Genbank accession number M27394.

Genbank version number M27394.1 GI: 179307.

Genbank, entry updated November 30, 2009 11:16 am.

Polypeptide:

Genbank accession number AAA35581.

Genbank version number AAA35581.1 GI: 179308.

Genbank, entry updated November 30, 2009 11:16 am.

Cross-references:

Tedder T. F., et al. Proc. Natl. Acad. Sci. U.S.A. 85 (1), 208-212 (1988).

Other information:

Official symbol: MS4A1.

Other names: B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7.

Other abbreviations: B-lymphocyte CD20 antigen; B1 antigen of the cell surface of B-lymphocytes; antigen CD20; CD20 receptor; antigen Leu-16 of the surface of leukocytes.

Antibodies.

Genentech / Roche: Rituximab - Abdulla N.E., et al. BioDrugs. 2012 Apr 1; (2): 71-82.

For example, see US 5736137, ATCC deposit number HB-69119.

GSK / Genmab: Ofatumumab- Nightingale G., et al. Ann Pharmacother. 2011 Oct; 45 (10): 1248-55.

For example, see US 20090169550A1, SEQ ID NO: 2, 4 and 5.

Immunomedics: Veltuzumab - Goldenberg D.M., et al. Leuk Lymphoma. 2010 May; 51 (5): 747-55.

For example, see US7919273B2, SEQ ID NO: 1, 2, 3, 4, 5 and 6.

(81) Tenascin C - TNC (tenascin C).

Nucleotide:

Genbank accession number NM_002160.

Genbank version number NM_002160.3 GI: 340745336.

Genbank, entry updated on September 23, 2012 at 02:33 p.m.

Polypeptide:

Genbank accession number NP_002151.

Genbank version number NP_002151.2 GI: 153946395.

- 43 038267

Genbank, entry updated on September 23, 2012 at 02:33 p.m.

Cross-references:

Nies D. E., et al. J. Biol. Chem. 266 (5), 2818-2823 (1991); Siri A., et al. Nucleic Acids Res. 19 (3), 525-531 (1991).

Other information:

Official symbol: TNC.

Other names: 150-225, GMEM, GP, HXB, JI, TN, TN-C.

Other designations: GP 150-225; cytotactin; glioma-associated extracellular matrix antigen; hexabrachion (tenascin); muscle tendon antigen; neuronectin; tenascin; tenascinC, isoform 14 / AD1 / 16.

Antibodies.

Philogen: G11 (von Lukowicz T., et al. J. Nucl. Med. 2007 Apr; 48 (4): 582-7) and F16 (Pedretti M., et al. Lung Cancer. 2009 Apr; 64 (1) : 28-33).

For example, see US7968685, SEQ ID NO: 29, 35, 45 and 47.

(82) FAP (fibroblast activation protein alpha).

Nucleotide:

Genbank accession number U09278.

Genbank version number U09278.1 GI: 1888315.

Genbank, entry updated on June 23, 2010 09:22 am.

Polypeptide:

Genbank accession number AAB49652.

Genbank version number AAB49652.1 GI: 1888316.

Genbank, entry updated on June 23, 2010 09:22 am.

Cross-references:

Scanlan, M. J., et al. Proc. Natl. Acad. Sci. U.S.A. 91 (12), 5657-5661 (1994).

Other information:

Official symbol: FAP.

Other names: DPPIV, FAP A.

Other abbreviations: 170 kDa membrane bound melanoma gelatinase; integral membrane serine protease; separaza.

(83) DKK-1 (Dickkopf 1 homologue (Xenopus laevis).

Nucleotide:

Genbank accession number NM_012242.

Genbank version number NM_012242.2 GI: 61676924.

Genbank, entry update date: September 30, 2012, 01:48 p.m.

Polypeptide:

Genbank accession number NP_036374.

Genbank version number NP_036374.1 GI: 7110719.

Genbank, entry update date: September 30, 2012, 01:48 p.m.

Cross-references:

Fedi P. et al. J. Biol. Chem. 274 (27), 19465-19472 (1999).

Other information:

Official symbol: DKK1.

Other names: UNQ492 / PRO1008, DKK-1, SK.

Other abbreviations: dickkopf-related protein-1; dickkopf-1-like; dickkopf-like protein 1; dickkopf-related protein 1; hDkk-1.

Antibodies.

Novartis: BHQ880 (Fulciniti M, et al. Blood. 2009 Jul 9; 114 (2): 371-379).

For example, see US 20120052070A1, SEQ ID NO: 100 and 108.

(84) CD52 (CD52 molecule).

Nucleotide:

Genbank accession number NM_001803.

Genbank version number NM_001803.2 GI: 68342029.

Genbank, entry update date: September 30, 2012, 01:48 p.m.

Polypeptide:

Genbank accession number NP_001794.

Genbank version number NP_001794.2 GI: 68342030.

Genbank, entry update date: September 30, 2012, 01:48 p.m.

Cross-references:

Xia M. Q., et al. Eur. J. Immunol. 21 (7), 1677-1684 (1991).

Other information:

Official symbol: CD52.

- 44 038267

Other names: CDW52.

Other designations: SAMRATH-1 antigen; CD52 antigen (CAMRATH-1 antigen); CDW52 antigen (CAMRATH-1 antigen); antigen 1 Cambridge pathology; epididymal secretory protein E5; he5; human epididymis-specific protein 5.

Antibodies.

Alemtuzumab (Campat) - Skoetz N., et al. Cochrane Database Syst Rev. 2012 Feb 15; 2: CD008078.

For example, see Drugbank Acc. No. DB00087 (BIOD00109, BTD00109).

(85) CS1 - SLAMF7 (7th member of the SLAM family).

Nucleotide:

Genbank accession number NM_021181.

Genbank version number NM_021181.3 GI: 1993571.

Genbank, entry updated on June 29, 2012 at 11:24 am.

Polypeptide:

Genbank accession number NP_067004.

Genbank version number NP_067004.3 GI: 19923572.

Genbank, entry updated on June 29, 2012 at 11:24 am.

Cross-references:

Boles K.S., et al. Immunogenetics 52 (3-4), 302-307 (2001).

Other information:

Official symbol: SLAMF7.

Other names: UNQ576 / PRO1138, 19A, CD319, CRACC, CS1.

Other abbreviations: 19A24 protein; CD2, subpopulation 1; CD2-like receptor that activates cytotoxic cells; CD2-like receptor for the activation of cytotoxic cells; membrane protein FOAP-12; new LY9 (antigen of 9 lymphocytes) -like protein; protein 19A.

Antibodies.

BMS: elotuzumab / Hul_uc63 (Benson D.M., et al. J. Clin. Oncol. 2012 Jun 1; 30 (16): 2013-2015).

For example, see US 20110206701, SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 and 16.

Endoglin - ENG (endoglin).

Nucleotide:

Genbank accession number AF035753.

Genbank version number AF035753.1 GI: 3452260.

Genbank, entry update date: March 10, 2010 at 06:36 p.m.

Polypeptide:

Genbank accession number AAC32802.

Genbank version number AAC32802.1 GI: 3452261.

Genbank, entry update date: March 10, 2010 at 06:36 p.m.

Cross-references:

Rius C., et al. Blood 92 (12) 4677-4690 (1998).

Official symbol: ENG.

Other information:

Other names: RP11-228B15.2, CD105, END, HHT1, ORW, ORW1.

Other names: CD105 antigen.

Annexes A1 - ANXA1 (Annexes A1).

Nucleotide:

Genbank accession number X05908.

Genbank version number X05908.1 GI: 34387.

Genbank, entry update date: February 02, 2011 at 10:02 am.

Polypeptide:

Genbank accession number CCA29338.

Genbank version number CCA29338.1 GI: 34388.

Genbank, entry update date: February 02, 2011 at 10:02 am.

Cross-references:

Wallner B. P., et al. Nature 320 (6057), 77-81 (1986).

Other information:

Official symbol: ANXA1.

Other names: RP11-71A24.1, ANX1, LPC1.

Other designations: annexation I (lipocortin I); annexin-1; calpactin II; calpactin-2; chromobindin-9; lipocortin I; p35; a protein that inhibits phospholipase A2.

(88) V-CAM (CD106) - VCAM1 (vascular endothelial adhesion molecule 1).

Nucleotide:

Genbank accession number M60335.

Genbank version number M60335.1 GI: 340193.

- 45 038267

Genbank, entry updated on June 23, 2010 at 08:56 am.

Polypeptide:

Genbank accession number AAA61269.

Genbank version number AAA61269.1 GI: 340194.

Genbank, entry updated on June 23, 2010 at 08:56 am.

Cross-references:

Hession C., et al. J. Biol. Chem. 266 (11), 6682-6685 (1991).

Other information: Official symbol: VCAM1.

Other names: CD106, INCAM-100

Other names: CD106 antigen; vascular cell adhesion protein 1. Antibody sequences

Anti-integrin α _γ β ₆

RHAB6.2

QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYA PKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTAVPNLRGDLQVLAQKVAGPGFTL

RHCB6.2

QVQLVQSGAEVKKPGASVKVSCKASGYTFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYA PKFQGRVTITTDTSASTAYMELSSLRSEDTAVYYCARGTPTAVPNLRGDLQVLAQKVAGPGYPL YWV

RHF

QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYA PKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS

RHFB6

QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYA PKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTAVPNLRGDLQVLAQKVAGPYGF □T

RHAYIOObP

QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYA PKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTGPYPFDYWGQGTLVTVSS

RKF

ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSG SGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK

RKFL36L50

ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWLQQKPGQAPRLLIYLTSNLASGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK

RKC

EIVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK

Anti-POPs

CD33 Hum195VH

- 46 038267

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYN QKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS

CD33 Hum195VK

DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVP SRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK

Anmu-CD19

CD19 B4 with resurfaced VH

QVQLVQPGAEWKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTNYN QNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTVSS

CD19 B4 with altered CC surface

EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFS GSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK

Anti-Neg2

VH chain of herceptin

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS

VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

VL chain of herceptin

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

Anmu-CD25

VK simulect (also known as basiliximab)

QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKPGTSPKRWIYDTSKLASGVPARFS GSGSGTSYSLTISSMEAEDAATYYCHQRSSYTFGGGTKLEIK

UN simulekt

QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQRPGQGLEWIGAIYPGNSDTSYNQK FEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVSS

Anti-PSMA

Deimmunized UN '1

EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTYNQK FEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSS

- 47 038267

Deimmunized UK ‘1

DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPSRFS GSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK

Deimmunized UN 1 '5

EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATH YAESVKGRVTISRDDSKSIVYLQMNNLRAEDTGVYYCTRRWNNFWGQGTTVTVSS

Deimmunized VH2 '5

EVKLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATH YAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS

Deimmunized VH3 '5

EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATH YAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS

Deimmunized VH4 '5

EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATH YAESVKGRFTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS

Deimmunized VK1 '5

NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFT GSGSATDFTLTISSLQTEDLADYYCGQSYTFPYTFGQGTKLEMK

Deimmunized VK2 '5

NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFS GSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK

Deimmunized VK3 '5

NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRF SGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK

Deimmunized VK4 '5

NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRF SGSGSGTDFTLTISSLQAEDEADYYCGQSYTFPYTFGQGTKLEIK

Deimmunized UK DI '5

- 48 038267

NIVMTQFPKSMSASAGERMTLTCKASENVGTYVSWYQQKPTQSPKMLIYGASNRFTGVPDRF SGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFGGGTKLEMK

Deimmunized VH DI '5

EVKLEESGGGLVQPGGSMKISCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNFATH

YAESVKGRVIISRDDSKSSVYLQMNSLRAEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RHA '5

EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFATH YAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RHB '5

EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATH YAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RHC '5

EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATH YAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RHP '5

EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFATH YAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RHE '5

EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATH YAESVKGRFTISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RHF '5

EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATH YAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RHG '5

EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATH YAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS

Humanized RKA '5

DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSRFS GSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

- 49 038267

Humanized RKB '5

DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSRFS

GSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

Humanized RKC '5

DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFS

GSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

Humanized RKD '5

DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFS

GSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

Humanized RKE '5

NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPDRFT

GSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

Humanized RKF '5

NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFS

GSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

Humanized RKG '5

NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPDRFT

GSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

The parent antibody can also be a fusion protein containing an albumin binding peptide sequence (Dennis et al. (2002), Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins, J. Biol. Chem. 277: 35035-35043; WO 01/45746 ). Antibodies of this invention include fusion proteins with ABP sequences described in publications:

(i) Dennis et al. (2002) J. Biol. Chem. 277: 35035-35043 in tab. III and IV, p. 35038;

(ii) US 2004/0001827 in [0076] and (iii) WO 01/45746 at p. 12, 13, and all of these publications are incorporated herein by reference.

In one embodiment, the antibody is formulated to target a tumor-associated ave6 antigen.

The cell binding agent can be labeled, for example, to facilitate detection or purification of the agent, either prior to incorporation into the conjugate or as part of the conjugate. The label can be a biotin label. In another embodiment, the cell binding agent may be radiolabeled.

Binding of the linker fragment to the ligand fragment.

The ligand moiety is linked to the linker moiety through a disulfide bond.

In one embodiment, the bond between the ligand moiety and the drug linker is between the thiol group of the cysteine residue in the ligand moiety and the maleimide group in the drug linker moiety.

Cysteine residues of the linker moiety may be available for contacting with a functional group of the linker moiety to form a bond. In other embodiments, for example, if the ligand fragment is an antibody, the thiol groups of the antibody may be part of interchain disulfide bonds. These interchain bonds can be converted to free thiol groups, for example, by treating the antibody with a DTT agent prior to reacting with the functional group of the linker moiety.

In some embodiments, a cysteine residue is introduced into the heavy or light chain of an antibody. Positions for insertion of cysteine by substitution in heavy or light chains of an antibody include those described in US Published Application No. 2007-0092940 and International Patent Publication WO 2008070593, which are incorporated herein.

Treatment methods.

The compounds of this 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 of Formula II. The term therapeutically effective amount means an amount sufficient to provide a beneficial effect to a patient. Such a beneficial effect may be the relief of at least one symptom. The actual amount administered, as well as the frequency and schedule of administration, depend on the nature and severity of the pathological condition to be treated. Prescribing treatment, such as determining the dose, is the responsibility of general practitioners and other medical personnel.

- 50 038267

The conjugate can be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the pathological condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (administration of active agents, including, for example, drugs); surgical operations; and radiation therapy.

Pharmaceutical compositions according to this invention and for use in accordance with this invention may contain, in addition to the active ingredient, i. E. a conjugate of Formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials 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 exact nature of the carrier or other material depends on the route of administration, which can be oral, or by injection, such as cutaneous, subcutaneous, or intravenous.

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

For intravenous, cutaneous or subcutaneous injection or injection into the lesion, the active ingredient must be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity and stability. Suitable solutions can be prepared by those skilled in the art using, for example, isotonic media such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be added as needed.

These conjugates can be used to treat proliferative disease and autoimmune disease. The term proliferative disease refers to unwanted or uncontrolled cell proliferation of excess or abnormal cells that is undesirable, such as neoplastic or hyperplastic growth in vitro or in vivo.

Examples of proliferative pathological conditions include, but are not limited to, benign, precancerous and malignant cell proliferation, including, but not limited to, neoplasms and tumors (e.g., histocytoma, glioma, astrocytoma, osteoma), cancer (e.g., lung cancer, small cell carcinoma 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), leukemia, psoriasis, bone disease, fibroproliferative disorders (eg, connective tissue) and atherosclerosis. Other cancers of interest include, but are not limited to, hematological diseases; malignant diseases such as leukemias and lymphomas such as non-Hodgkin's lymphoma and subtypes such as DLBCL, marginal cell lymphoma, mantle cell lymphoma and follicular lymphoma, Hodgkin's lymphoma, AML and other cancers from B or T cells.

Examples of autoimmune diseases include the following: rheumatoid arthritis, autoimmune demyelinating diseases (eg, multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grais disease e.g. Crohn's disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine insufficiency, Schmidt's syndrome, autoimmune uveitis, Adison's disease, Thyroiditis Hashimoto's, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenia purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dyskinesia, sclerodactylia and female anemia) , ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, abortion, antiphospholipid syndrome, allergic alveolitis syndrome, multiform alveolitis , Cushing's syndrome, autoimmune chronic active hepatitis, pulmonary allergy of poultry farmers, toxic epidermal necrolysis, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion uyu reaction, Takayasu arteritis, rheumatic polymial

- 51 038267 giyu, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sumpter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Kaplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, edomyocardial ectomyocardial fibrosis , fetal erythroblastosis, eosinophilic fasciitis, Schulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuchs cyclitis, IgA nephromathy, Henoch-Schonlein purpura, graft versus host disease, graft rejection, cardiomyopathy, lymphoma syndrome polychondritis, cryoglobulinemia, Waldenstrom's macroglobulinemia, Evans syndrome, and autoimmune gonadal insufficiency.

In some embodiments, the autoimmune disease is a B-lymphocyte disorder (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1 lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease) or Th2 lymphocytes (eg, atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic host versus graft disease ). In general, disorders affecting dendritic cells include Th1 lymphocyte or Th2 lymphocyte disorders. In some embodiments, the autoimmune disorder is a T cell mediated immunological disorder.

In some embodiments, the amount of conjugate administered is from about 0.01 to about 10 mg / kg per dose. In some embodiments, the amount of conjugate administered is from about 0.01 to about 5 mg / kg per dose. In some embodiments, the amount of conjugate administered is from about 0.05 to about 5 mg / kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 5 mg / kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 4 mg / kg per dose. In some embodiments, the amount of conjugate administered is from about 0.05 to about 3 mg / kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 3 mg / kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 2 mg / kg per dose.

Drug load.

Drug loading (p) is the average amount of PBD drugs per cell-binding agent, eg, antibody. When the compounds of the invention are linked to cysteines, the drug content can be from 1 to 8 drug units (D) per cell binding agent, i.e. 1, 2, 3, 4, 5, 6, 7 and 8 drug moieties are covalently linked to a cell-binding agent. Conjugate compositions include a set of agents that bind to a cell, for example, antibodies conjugated to a drug in an amount of 1 to 8. If the compounds of the invention are bound to lysines, then the drug loading can be from 1 to 80 drug units (D) per one a cell-binding agent, although an upper limit of 40, 20, 10, or 8 may be preferred. Conjugate compositions include a combination of agents that bind to a cell, for example, antibodies conjugated to a drug in an amount of 1 to 80, 1 to 40, 1 to 20, 1 to 10, or 1 to 8.

The average amount of drugs per antibody in ADC preparations obtained from conjugation reactions can be determined by standard methods such as UV, reverse phase HPLC, GIH, mass spectroscopy, enzyme-linked immunosorbent assay and electrophoresis. It is also possible to quantify the distribution of ADC in terms of p. Using an enzyme-linked immunosorbent assay, the average p-value of a particular ADC preparation can be determined (Hamblett et al. (2004), Clin. Cancer Res. 10: 7063-7070; Sanderson et al. (2005), Clin. Cancer Res. 11: 843852 ). However, the distribution of p (drug) values cannot be determined by antibody-antigen binding and because of the ELISA detection limit. In addition, the enzyme-linked immunosorbent assay for detecting antibody-drug conjugates does not provide a determination of the positions at which drug fragments are attached to the antibody, such as heavy chain or light chain fragments, specific amino acid residues. In some cases, the separation, purification and characterization of a homogeneous ADC, in which p is a determined value based on an ADC with a different drug content, can be accomplished using, for example, reverse phase HPLC or electrophoresis. Such technologies are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number of attachment sites in the antibody. For example, an antibody can have only one or more cysteine thiol groups, or can have only one or more sufficiently reactive thiol groups to which a linker can be attached. Higher drug levels, for example p> 5, can lead to aggregation, instability, toxicity, or decreased cell permeability of some antibody-drug conjugates.

- 52 038267

Typically, fewer drug moieties are conjugated in an antibody conjugation reaction than the maximum amount theoretically possible. An antibody may contain, for example, a plurality of lysine residues that do not interact with a drug linker (A or B). Only the most reactive lysine groups can react with the amino-reactive linker reagent. Also, only the most reactive cysteine thiol groups can react with the thiol-reactive linker reagent. In general, antibodies do not contain many, if any, free and reactive cysteine thiol groups that can be linked to a drug moiety. Most of the cysteine thiol residues in the antibodies of these compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partial or complete reduction conditions. The drug loading (drug / antibody ratio) in the ADC can be controlled in several different ways, including (i) limiting the molar excess of the drug linker (A or B) relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, and (iii) conditions partial or limited reduction to modify the cysteine thiol.

Some antibodies contain reducible interchain disulfides, i.e. cysteine bridges. Antibodies can be rendered reactive for conjugation with linker reagents by treating them with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by reacting lysines with 2-iminothiolane (Trout's reagent), which converts the amine to a thiol. Reactive thiol groups can be introduced into an antibody (or a fragment thereof) by constructing one, two, three, four, or more cysteine residues (eg, producing mutant antibodies containing one or more unnatural cysteine amino acid residues). US 7,521,541 describes the construction of antibodies by introducing reactive cysteine amino acids.

Cysteine amino acids can be engineered at reactive antibody sites that do not form intrachain or intermolecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26 (8): 925-932; Dornan et al. (2009), Blood 114 (13 ): 2721-2729; US 7521541; US 7723485; WO 2009/052249). Engineered cysteine thiols can interact with linker reagents or drug-linker reagents of this invention that contain thiol-reactive electrophilic groups such as maleimide or alpha-haloamides to form ADCs with cysteine engineered antibodies and PBD drug fragments. Thus, the position of the drug fragment can be designed, controlled and known. Drug loading can be controlled as thiol groups of engineered cysteine typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Construction of an IgG antibody to introduce a cysteine amino acid through one site substitution of the heavy or light chain provides two new cysteines in the symmetrical antibody. A drug loading of about 2 can be achieved with almost complete homogeneity of the ADC conjugate product.

If more than one nucleophilic or electrophilic group of an antibody is contacted with an intermediate drug-linker, or with a linker reagent, and then with a drug fragment, the resulting product is a mixture of ADC compounds with a distribution of drug fragments attached to the antibody, for example, 1, 2, 3, etc. Liquid chromatography techniques such as polymer reverse phase (POF) chromatography and hydrophobic interaction chromatography (HIC) can separate compounds in a mixture based on drug loading. It is possible to isolate ADC preparations with a single drug loading value (p), however, such a single ADC content value can still mean a heterogeneous mixture, since drug fragments can be attached via a linker at different antibody sites.

Thus, the antibody-drug conjugate compositions of this invention include mixtures of antibody-drug conjugates in which the antibody has one or more PBD drug fragments and in which the drug fragments can be linked to the antibody via different amino acid residues.

In one embodiment, the average number of dimeric pyrrolobenzodiazepine groups per cell binding agent is 1 to 20. In some embodiments, the range is selected from the ranges 1 to 8, 2 to 8, 2 to 6, 2 to 4 and from 4 to 8.

In some embodiments, there is one dimeric pyrrolobenzodiazepine group per cell binding agent.

General synthesis methods.

The synthesis of PBD compounds is described in detail in the following references, the contents of which are incorporated herein by reference:

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a) WO 00/12508 (pp. 14-30);

b) WO 2005/023814 (p. 3-10);

c) WO 2004/043963 (pp. 28-29) and

d) WO 2005/085251 (pp. 30-39).

Synthesis method

Compounds of this invention of Formula I, wherein R ²⁰ and R ²¹ form a carbon nitrogen double bond between the nitrogen and carbon atoms to which they are bonded, can be prepared from a compound of Formula 2 to ¹

oh ϋ

Formula 2 where R ⁶ , R ⁷ , R ⁹ , R ⁶ , R ⁷ , R ⁹ , R ^11b , Y, Y and R are as defined for compounds of formula I and R ^ll is a precursor R ^L is the specified method especially suitable for compounds of formula I, wherein R ^L is of formula IIIa.

For such compounds, R ^{LL is} typically a moiety of R ^L , such as a group of formula IIIa '

In such a case, the reaction involves the addition of a GL group. The second necessary step is to delete the Prot ° group.

Compounds of formula 2 can be prepared by deprotection of the R ^LL group of compounds of formula 3

R "-Pro1

oh oh

Formula 3 wherein R ⁶ , R ⁷ , R ⁹ , R ⁶ , R ⁷ , R ⁹ , R ^11b , Y, Y and R are as defined for compounds of formula I, R ^LL-Prot is a protected version of R ^LL, and Prot ^N is a nitrogen protecting group (eg, Fmoc, Boc) that is orthogonal to the R ^LL protecting group.

Compounds of formula 3 can be prepared by ring closure in compounds of formula 4

where the specified ring closure is carried out by oxidation, for example, according to Swern. Compounds of Formula 4 can be synthesized from compounds of Formula 5

R ⁶

R

Formula 5 by the stepwise addition of two protecting groups, which can be accomplished by simple protection of the amino group, which will lead to an imino bond in the final compound (for example, Fmoc, Boc), followed by the setting of the desired protecting group on the other amino group.

Compounds of formula I wherein R ^L is of formula IIIb can be synthesized in the same manner, although the entire R ^L group can be set starting from the compound of formula 5 instead of using a protected precursor.

Compounds of formula 5 can be synthesized by known methods such as described in WO 2011/130598.

Alternatively, compounds of Formula 4 can be synthesized via the monomeric route as described in Example 3.

Compounds of this invention of formula I wherein R ²⁰ and R ²¹ do not form a nitrogen double bond

- 54 038267 carbon between the nitrogen and carbon atoms to which they are attached can be obtained by modifications of the above synthetic sequences.

Synthesis of drug conjugates.

Conjugates can be prepared as previously described. Antibodies can be conjugated to drug linkers as described in Doronina et al., Nature Biotechnology, 2003, 21, 778784). Briefly, antibodies (4-5 mg / ml) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris- (carboxyethyl) phosphine hydrochloride (TCEP) at 37 ° C. The progress of the reaction, in which the interchain disulfides are reduced, is monitored by reaction with 5,5'-dithio-bis- (2-nitrobenzoic acid) and the reaction is continued until the desired thiol / mAb ratio is reached. The reduced antibody is then cooled to 0 ° C and alkylated using 1.5 eq. maleimide linker of the drug per thiol group of the antibody. After 1 hour, the reaction is stopped by adding 5 eq. N-acetylcysteine. The stopped drug linker is removed by gel filtration on a PD-10 column. The ADC is then sterile filtered through a 0.22 µm syringe filter. Protein concentration can be determined by spectral analysis at 280 and 329 nm, respectively, correcting for the contribution of drug absorption at 280 nm. Size exclusion chromatography can be used to determine the degree of antibody aggregation, and RP-HPLC can be used to determine the residual NAC-quenched drug linker content.

Additional preferences.

The following preferences can be applied to all aspects of the present invention described above, or they can be related to one aspect. Preferences can be combined with each other in any combination.

In some embodiments, R ⁶ , R ⁷ , R ^9, and Y are selected from the same groups as R ⁶ , R ⁷ , R ^9, and Y, respectively. In some of these embodiments, R ⁶ , R ⁷ , R ^9, and Y are the same as R ⁶ , R ⁷ , R ^9, and Y, respectively.

N10'-C11 '.

In some embodiments, R ²⁰ is H and R ²¹ is OH, ORA, where RA is C1-4 alkyl. In some of these embodiments, R ²¹ is OH. In other of these embodiments, R ²¹ is ORA, where RA is C1-4 alkyl. In some of these embodiments, RA is methyl.

In some embodiments, R ²⁰ is H and R ²¹ is H.

In some embodiments, where R ²⁰ is (d-iii), there may be an additional nitro group on the benzene ring, for example, ortho to R ^z .

In some embodiments, R ²¹ is OH or ORA, where RA is C _1-4 alkyl and R ^{20 is} selected from:

- 55 038267

-C (= O) -X1-NHC (= O) X ₂ -NH- is a dipeptide. The amino acids in the dipeptide can be any combination of naturally occurring amino acids and unnatural amino acids. The dipeptide is the site of action for cathepsin-mediated cleavage.

In one embodiment, the dipeptide, —C (= O) —X ₁ —NHC (= O) X ₂ —NH— is selected from:

-Phe-Lys-,

-Val-Ala-,

-Val-Lys-,

-Ala-Lys-,

-Val-Cit-,

-Phe-Cit-,

-Leu-Cit-,

-Ile-Cit-,

- 56 038267

-Phe-Arg-,

-Trp-Cit-, where Cit is citrulline.

Preferably the dipeptide, -C (= O) -X1-NHC (= O) X ₂ -NH-, is selected from:

-Phe-Lys-,

-Val-Ala-,

-Val-Lys-,

-Ala-Lys-,

-Val-Cit-.

Most preferably the dipeptide, -C (= O) -X1-NHC (= O) X2-NH-, is -Phe-Lys or -Val-Ala-.

Other dipeptide combinations can be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, incorporated herein by reference.

In one embodiment, the amino acid side chain is chemically protected as needed, for example the amino group or carboxy group of the amino acid side chain can be derivatized.

In one embodiment, the amino group NH2 of a side chain amino acid such as lysine is a derivatized form selected from the group consisting of NHR and NRR '.

In one embodiment, the carboxy group COOH of a side chain amino acid such as aspartic acid is a derivatized form selected from the group consisting of COOR, CONH2, CONHR, and CONRR '.

In one embodiment, the amino acid side chain is chemically protected, if necessary. The side chain protecting group can be as described below. The present inventors have found that protected amino acid sequences can be cleaved by enzymes. For example, a dipeptide sequence containing a Boc-protected side chain Lys residue is cleaved by cathepsin.

Amino acid side chain protecting groups are well known in the art and are described in the Novabiochem catalog. Additional protecting group strategies are outlined in Protecting Groups in Organic Synthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those amino acids that have reactive side chain functionality:

Arg: Z, Mtr, Tos;

Asn: Trt, Xan;

Asp: Bzl, tert-Bu;

Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;

Glu: Bzl, tert-Bu;

Gin: Trt, Xan;

His: Boc, Dnp, Tos, Trt;

Lys: Boc, Z-Cl, Fmoc, Z, Alloc;

Ser: Bzl, TBDMS, TBDPS;

Thr: Bz;

Trp: Boc;

Tyr: Bzl, Z, Z-Br.

In one embodiment, the side chain guard is selected to be orthogonal to the group provided as or as part of the cover group where it is present. Thus, removal of the side chain protecting group does not remove the covering group or any functionality of the protecting group that is part of the covering group.

In other embodiments of the invention, the selected amino acids are amino acids lacking reactive side chain functionality. For example, amino acids can be selected from Ala, Gly, Ile, Leu, Met, Phe, Pro, and Val.

In the present invention, it is particularly preferred that if L ¹ contains a dipeptide, then C (= O) -X1-NHC (= O) X2-NH- is the same dipeptide.

Other preferred R ²⁰ groups include:

- 57 038267

Dimeric bond.

In some embodiments, both Y and Y 'are O.

In some embodiments, R is an unsubstituted ^^ alkylene group. In some such embodiments R is a C3, C5 or C ₇ alkylene. In particular, R can be C3 or C5 alkylene.

In other embodiments, R is a group of formula

where r is 1 or 2.

The phenylene group can be replaced with a pyridylene group.

R ⁶ to R ⁹ .

In some embodiments, R ⁹ is H.

In some embodiments, R ^{6 is} selected from H, OH, OR, SH, NH ₂ , nitro, and halogen, and can be selected from H or halogen. In some of these embodiments, R ⁶ is H.

In some embodiments, R ^{7 is} selected from H, OH, OR, SH, SR, NH2, NHR, NRR ', and halogen. In some such embodiments, R ^{7 is} selected from H, OH, and OR, wherein R is selected from optionally substituted C _1-7 alkyl, C _3-10 heterocyclyl, and C _5-10 aryl groups. More preferably, R may be a C _1-4 alkyl group which may or may not be substituted. A suitable substituent is a C _5-6 aryl group (eg phenyl). Particularly preferred 7-position substituents are OMe and OCH ₂ Ph. Other particularly suitable substituents are dimethylamino (ie —NMe ₂ ); - (OC ₂ H ₄ ) qOMe, where q is from 0 to 2; nitrogen-containing C ₆ heterocyclyls, including morpholino, piperidinyl, and N-methylpiperazinyl.

These options for implementation and preferences refer to R ⁹ , R ⁶ and R ^7, respectively.

R ^11b .

In some embodiments, R ^11b is OH.

In some embodiments, R ^11b is ORA, where R ^A is C _1-4 alkyl. In some of these embodiments, R ^A is methyl.

In some embodiments of the first aspect of this invention, compounds of formula Ia, Ib, or Ic are provided:

where R ^{1a is} selected from methyl and benzyl;

RL and R ^11b are as defined above.

These options for implementation and preferences also relate to the second aspect of the present invention.

Linker (R ^l ).

In some embodiments, R ^L is of Formula IIIa.

In some embodiments, R ^LL has formula IIIa '.

G4

G ^l can be selected from:

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(G ^L1 - ¹ ) O Λ (G ^L4 ) О Hal N — I H ⁵ Where Hal = 1, Br, Cl (G ^L1 - ² ) G (G ^L5 ) 0 Hal — 0 H (G ^L2 ) bn about (G ^L6 ) 0 ⁰ '° / Λ — N (G ^L3 - ¹ ) s— ε ^λ 0 (NO ₂ ) where the NO2 group is optional (G ^L7 ) (G ^L3 - ² ) s— 0 (ΝΟ ₂ ) where the group ΝΟ2 is optional (G ^L8 ) Have (G ^L3 - ³ ) s— ο ₂ νΛΛ / where the group ΝΟ2 is optional (G ^L9 ) Ν ₃ ^ (G ^L “) гГ ο ₂ νλ = / where the group ΝΟ2 is optional

where Ar represents a C _5-6 arylene group, for example phenylene.

In some embodiments, G ^{L is} selected from G ^L1-1 and G ^l1-2 . In some of these embodiments, G ^l is G ^L1-1 G ^ll .

G ^ll can be selected from:

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where Ar represents a C ₅ _ ₆ arylene group, for example phenylene.

In some embodiments, G ^{LL is} selected from G ^LL1-1 and G ^LL1-2 . In some of these embodiments, G ^LL is G ^LL1-1 X.

X represents:

where a = 0-5, b = 0-16, c = 0 or 1, d = 0-5;

a can be 0, 1, 2, 3, 4, or 5.

In some embodiments, a is from 0 to 3. In some of these embodiments, a is 0 or 1. In other embodiments, a is 0.

b can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b is 0 to 12. B In some embodiments, b is 0 to 8 and can be 0, 2, 4, or 8.

c can be 0 or 1.

d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is from 0 to 3. In some such embodiments, d is 1 or 2. In other embodiments, d is 2.

In some embodiments of group X, a is 0, c is 1 and d is 2, b can be from 0 to 8. In some such embodiments, b is 0, 4, or 8.

Q.

In one embodiment, Q is an amino acid residue. The amino acid can be a naturally occurring amino acid or a unnatural amino acid.

In one embodiment, Q is selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, wherein Cit is citrulline.

In one embodiment, Q contains a dipeptide residue. The amino acids in the dipeptide can be any combination of naturally occurring amino acids and unnatural amino acids. In some embodiments, the dipeptide comprises naturally occurring amino acids. If the linker is a cathepsin-labile linker, then the dipeptide is a cathepsin-mediated cleavage site. In such a case, the dipeptide is a recognition site for cathepsin.

In one embodiment, Q is selected from:

CO-Phe-Lys-NH,

CO-Val-Ala-NH,

- 60 038267 ^CO -Val-Lys- ^NH , ^CO -Ala-Lys- ^NH ,

CO-Val-Cit-NH, CO -Phe-Cit- NH CO -Leu-Cit- NH, CO -lle-Cit- NH, CO-Phe-Arg-NH and CO-Trp-Cit-NH, wherein Cit is is citrulline. Preferably Q is selected from: CO-Phe-Lys-NH,

CO-Val-Ala-NH, CO -Val-Lys- NH.

^CO -Ala-Lys- ^NH , ^CO -Val-Cit- ^NH .

Most preferably, Q is selected from: CO-Phe-Lys-NH, CO-Val-Cit-NH and

CO-Val-Ala-NH.

Other promising dipeptide combinations include:

CO-Gly-Gly-NH,

CO-Pro-Pro-NH and ^CO- Val-Glu- ^NH .

Other dipeptide combinations can be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, incorporated herein by reference.

In some embodiments, Q ^X is a tripeptide residue. The amino acids in the tripeptide can be any combination of naturally occurring amino acids and unnatural amino acids. In some embodiments, the tripeptide comprises naturally occurring amino acids. If the linker is a cathepsin-labile linker, then the tripeptide is a cathepsin-mediated cleavage site. In such a case, the tripeptide is a recognition site for cathepsin.

In one embodiment, the amino acid side chain is chemically protected, if desired. The side chain protecting group can be as described below. Protected amino acid sequences can be cleaved by enzymes. For example, a dipeptide sequence containing a Boc-protected side chain Lys residue is cleaved by cathepsin.

Amino acid side chain protecting groups are well known in the art and are described in the Novabiochem catalog as well as described elsewhere herein.

In some embodiments, RL is of Formula IIIb. In some embodiments, R ^LL has formula IIIb '.

R ^l1 and RL ^{2 are} independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group.

In some embodiments, both RL1 and RL ² are H.

In some embodiments, R ^l1 is H and RL ² is methyl.

In some embodiments, both RL1 and RL ² are methyl.

In some embodiments, R ^l1 and RL ^2, together with the carbon atom to which they are attached, form a cyclopropylene group.

In some embodiments, R ^l1 and RL ^2, together with the carbon atom to which they are attached, form a cyclobutylene group.

In some embodiments in Group IIIb, e is 0. In other embodiments, e is 1 and the nitro group can be at any available position on the ring. In some of these embodiments, it is in the ortho position. In other such embodiments, it is in the para position.

In one specific embodiment, the first aspect of this invention comprises a compound of Formula Id

- 61 038267

where, Q is selected from: (a) -CH2-;

(b) -C3H6- and

^(c)

In one specific embodiment of the second aspect of the present invention, the drug compound linker (DL) has the formula (Id ')

where Q is selected from: (a) -CH2-;

(b) -C3H6- and

^(c)

In some embodiments of this invention, the C11 substituent may be in the following stereochemical position relative to adjacent groups:

In other embodiments, the C11 substituent may be in the following stereochemical position relative to adjacent groups:

Examples of

General information.

Flash chromatography was performed on a Biotage Isolera 1 ™ instrument using gradient elution starting with 88% hexane / EtOAc or 99.9% DCM / MeOH until all UV active components were completely eluted from the column (detection at 214 and 254 nm ). When significant elution of the UV active material was observed, the gradient was manually stopped. The purity of the fractions was checked by thin layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel with a fluorescent indicator on aluminum plates. TLC visualization was performed using UV light or iodine vapor, unless otherwise indicated. Extraction and chromatographic solvents were purchased and used without further purification from VWR, UK. All chemicals were purchased from Sigma-Aldrich or TCI Europe unless otherwise noted. The pegylated reagents were purchased from Quanta Biodesign, USA, through Stratech, UK.

1H and ¹³ C NMR spectra were recorded on a Bruker Avance® 400 spectrometer. Splitting constants are reported in Hertz (Hz). Chemical shifts were recorded in parts per million (ppm) downfield relative to the tetramethylsilane signal. The spin multiplicities were recorded as s (singlet), br.s (broadened singlet), d (doublet), t (triplet), and m (multiplet).

The following analytical LC / MS conditions were used (to check the progress of the reaction and determine the purity): Electrospray mass spectrometry in positive ionization mode was performed on a Shimadzu Nexera® / Prominence® LCMS-2020 instrument. The mobile phase used solvent A (H ₂ O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic sour

- 62 038267 you). Gradient for a typical 3 minute chromatogram: The original composition 5% B was held for 25 seconds, then the concentration was increased from 5 to 100% B over 1 minute 35 seconds. This composition was held for 50 s at 100% B, then returned to 5% B within 5 s and held for 5 s. The total duration of the gradient cycle was 3.0 minutes. Gradient for a typical 15 min chromatogram: Initial 5% B was held for 1 min, then increased from 5 to 100% B in 9 min. This composition was held for 2 min at 100% B, then returned to 5% B for 10 s and held at 5% B for 2 min 50 s. The total duration of the gradient cycle was 15.0 minutes. The flow rate was 0.8 ml / min (for a 3-minute chromatogram) and 0.6 ml / min (for a 15-minute chromatogram). Detection was performed at 254 nm. Columns: Waters Acquity UPLC® VEN Shield RP18, 1.7 μm, 2.1x50 mm @ 50 ° C, equipped with a Waters Acquity UPLC® VEN Shield RP18 VanGuard, 130A, 1.7 μm, 2.1x5 mm (standard 3- minute cycle); and ACE Excel 2 C18-AR, 2 μm, 3.0x100 mm, equipped with Waters Acquity UPLC® VEN Shield RP18 VanGuard, 130A, 1.7 μm, 2.1x5 mm (15 min cycle).

Used the following preparative HPLC conditions. Reverse phase ultrafast high performance liquid chromatography (UPLC) was performed on a Shimazdzu Prominence® instrument using a Phenomenex® Gemini NX column, 5 μm, C18 (at 50 ° C), 150x21.2 mm. Solvent A (H ₂ O with 0.1% formic acid) and solvent B (CH ₃ CN with 0.1% formic acid) were used as eluents. All UPLC experiments were performed using the following gradient conditions.

Method A: Initial 13% B was increased to 60% B in 15 minutes, then increased to 100% B in 2 minutes. Hold for 1 min at 100% B, then return to 13% B in 0.1 min and hold for 1.9 min. The total duration of the gradient cycle was 20.0 minutes. The flow rate was 20.0 ml / min, and detection was performed at 254 and 280 nm.

Method B: The original 13% B content was increased to 70% B in 17 minutes, held at this B content for 2 minutes, then returned to 13% B in 0.1 minutes and held for 1.9 minutes. The total duration of the gradient cycle was 20.0 minutes. The flow rate was 20.0 ml / min and detection was performed at 223 nm.

Method C: Initial 13% B was increased to 75% B in 15 minutes, then increased to 100% B in 2 minutes. Kept for 1 min at 100% B, then returned to 13% B in 0.1 min and held for 1.9 min. The total duration of the gradient cycle was 20.0 minutes. The flow rate was 20.0 ml / min, and detection was performed at 254 and 280 nm.

Example 1.

- 63 038267

(a) ((Propane-1,3-diyl-bis- (oxy)) - bis- (5-methoxy-2-niΊro-4,1-phenylene)) - bis-S) -2- (hydroxymethyl) pyrrolidine -1-yl) methanone) (I2).

DMF (12 drops) was added to a stirred suspension of bis-nitrobenzoic acid I1 (10 g, 21.5 mmol) and oxalyl chloride (5.6 ml, 8.2 g, 64.5 mmol) in anhydrous DCM (150 ml). After initial foaming, the reaction slurry turned into a solution and the mixture was allowed to stir at room temperature for 16 hours. Most of the solvent was removed by evaporation in vacuo and the resulting concentrated solution was redissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was filtered off under vacuum, washed with cold diethyl ether, and dried for 1 h in a vacuum oven at 40 ° C. The solid acid chloride was added in portions to a stirred suspension of (S) - (+) - 2-pyrrolidinemethanol (5.0 g, 4.9 ml, 49.5 mmol) and TEA (15.0 ml, 10.9 g, 108 mmol ) in DCM (100 ml) at -40 ° C (dry ice / CH ₃ CN). After stirring for 1 h, the reaction was complete as determined by LC / MS with the desired product exclusively at 1.33 min retention time, ESI + m / z 655 [M + Na] ⁺ , 633 [M + H] ⁺ . The mixture was diluted with DCM (100 ml) and washed with 1N. HCl (2x50 ml), saturated NaHCO ₃ (3x40 ml), saturated aqueous sodium chloride solution (50 ml), dried (MgSO ₄ ), filtered and the solvent evaporated in vacuo to give the purified product 12 as a yellow solid (13.6 d, 100% yield).

(b) ((2S, 2'S) - (4,4 '- (Propane-1,3-diyl-bis (oxy)) - bis- (5-methoxy-2-niΊrobenzoyl)) - bis- (pyrrolidine1, 2-diyl)) - bis- (methylene) diacetate (I3).

A solution of Ac ₂ O (4.47 ml, 4.83 g, 47.3 mmol) in dry DCM (25 ml) was added dropwise to stirring

- 64 038267 to an injected solution of bis-alcohol 12 (13.6 g, 21.5 mmol), DMAP (263 mg, 2.15 mmol) and pyridine (4.17 ml, 4.08 g, 51.6 mmol) in dry DCM (125 ml) at 0 ° C (ice / acetone) under argon. The reaction mixture was left to warm and after 1 h at room temperature, LC / MS analysis indicated the completion of the reaction and complete conversion to the desired product with a retention time of 1.55 min, ESI + m / z 740 [M + Na] +, 717 [M + H] +. The mixture was diluted with DCM (20 ml) and washed with 1N. HCl (2x100 ml), H2O (25 ml), saturated aqueous sodium chloride solution (50 ml), dried (MgSO ₄ ), filtered and the solvent was evaporated in vacuo to give the crude bis-acetate 13 as a yellow solid (14.4 d, 94% yield), which was of satisfactory purity for transfer to the next step without further purification.

(c) ((2S, 2'S) - (4,4 '- (Propane-1,3-diyl-bis (oxy)) - bis- (2-amino-5-methoxybenzoyl)) - bis- (pyrrolidine1, 2-diyl)) - bis- (methylene) diacetate (I4).

A 10% Pd-C sample (132 mg) was carefully treated with EtOAc (10 ml) to give a suspension that was added to a solution of nitro compound I3 (1.32 g, 1.84 mmol) in EtOAc (20 ml) and EtOH (30 ml) in the hydrogenation vessel. Using a Parr® apparatus, the mixture was treated with hydrogen gas to 10 psi and shaken at room temperature, then degassed under vacuum, this process was repeated two more times. The vessel was filled with hydrogen gas to 45 psi, shaken and pressurized while consuming hydrogen. LC / MS analysis showed incomplete reaction after 3 hours and was allowed to shake at 45 psi for 3 days (weekend) after which satisfactory conversion to product was achieved, retention time = 1.32 min. ESI + m / z 657 [M + H] +. The reaction mixture was degassed under vacuum and then filtered through a celite® pad. The filtrate was evaporated in vacuo, the resulting residue was redissolved in DCM (30 ml), dried (MgSO ₄ ), filtered and the solvent was evaporated in vacuo to give crude bis-aniline I4 as a yellowish foam (1.1 g, 91% yield), which contained 8% impurities, but was used in the next stage without further purification.

(d) ((S) -1 - (4- (3 - (4 - ((S) -2- (Acetoxymethyl) pyrrolidine-1 -carbonyl) -5 - ((tert-butoxycarbonyl) - amino) -2- methoxyphenoxy) propoxy) -2-amino-5-methoxybenzoyl) pyrrolidin-2-yl) methyl acetate (I5).

Boc ₂ O (330 mg, 1.51 mmol) was added to a stirred solution of bis-aniline I4 (1.1 g, 1.68 mmol) in dry THF (10 ml). The reaction mixture was heated and stirred at 75 ° C for 16 h. LC / MS analysis revealed the desired mono-Boc product I5 at a retention time of 1.58 min, I% = 50, ESI + m / z 779 [M + Na] ⁺ , 757 [M + H] +. Together with unreacted starting material at a retention time of 1.32 min, I% = 30, and bis-Boc material at a retention time of 1.81 min, I% = 21, ESI + m / z 879 [M + Na] +, 857 [M + H] +. The reaction mixture was left to cool to room temperature and THF was evaporated in vacuo. Purification with Isolera ™ (DCM / MeOH, SNAP Ultra 50 g, 100 ml / min) gave the mono-Boc product I5 as an orange foam (519 mg, 46% yield based on Boc ₂ O, eluted at 97% DCM / MeOH) unreacted bis-aniline I4 (285 mg, eluted at 95% DCM / MeOH) and bis-Boc (248 mg, eluted at 98% DCM / MeOH).

(e) ((S) -1- (4- (3- (4 - ((S) -2- (Acetoxymethyl) pyrrolidine-1-carbonyl) -5 - ((((4 - ((S) -2 - ((S) -2- (((allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl) oxy) carbonyl) amino) -2methoxyphenoxy) propoxy) -2 - ((tert-butoxycarbonyl) amino) -5 -methoxybenzoyl) pyrrolidine-2yl) methyl acetate (17).

Triphosgene (380 mg , 1.28 mmol). After stirring for 10 min under argon, LC / MS analysis showed complete conversion to isocyanate (sampling in MeOH to yield methyl carbamate, retention time 1.66 min, ESI + m / z 837 [M + Na] +, 815 [M + H] +). The mixture was treated with additional TEA (740 μl, 539 mg, 5.34 mmol) followed by the addition of linker 16 (1.34 g, 3.56 mmol). After 2 h stirring under argon, LC / MS showed satisfactory conversion to carbamate I7 (retention time 1.74 min, (ESI +) m / z 1182 [M + Na] +, 1160 [M + H] +). The mixture was diluted with DCM (80 ml) and washed with saturated NH ₄ Cl solution (2x30 ml), H2O (30 ml), saturated aqueous sodium chloride solution (50 ml), dried (MgSO ₄ ), filtered and evaporated in vacuo to give the crude product. Purification with Isolera ™ (Hexane / EtOAc, SNAP Ultra 100 g, 100 ml / min) gave the purified carbamate I7 / elution at 65% hexane / EtOAc) as a yellow foam (2.95 g, 71% yield).

(f) tert-Butyl (5- (3- (5 - ((((4 - ((S) -2 - ((S) -2 - (((allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido ) benzyl) oxy) carbonyl) αmino) -4 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl) -2methoxyphenoxy) propoxy) -2 - ((S) -2- (hydroxymethyl) purrolidine-1- carbonyl) -4-methoxyphenyl) carbamate (I8).

Solid K ₂ CO ₃ (1.75 g, 12.7 mmol) was added to a stirred solution of the acetate protected compound I7 (2.93 g, 2.53 mmol) in MeOH (60 ml) and H2O (12 ml). After stirring for 1 h at room temperature, the reaction was considered complete as determined by LC / MS with the desired product at a retention time of 1.57 min, ESI + m / z 1098 [M + Na] +, 1076 [M + H] +. MeOH

- 65 038267 was removed by evaporation in vacuo and the resulting residue was partitioned between water (75 ml) and DCM (75 ml). The layers were separated and the aqueous phase was extracted with DCM (3x25 ml). The combined organic layers were washed with water (3x50 ml), saturated aqueous sodium chloride solution (60 ml), dried (MgSO ₄ ), filtered and evaporated in vacuo to give the crude product. Purification with Isolera ™ (DCM / MeOH, SNAP Ultra 100 g, 100 ml / min) with bis-alcohol I8 (elution at 97% DCM / MeOH) as a white foam (2.44 g, 90% yield).

(g) 4 - ((S) -2 - ((S) -2 - (((Allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl (11S, 11aS) -8- (3 - ((( 11S, 11aS) -10- (tert-butoxycarbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-1H-pyrrolo [2,1-c | [1, 4] benzodiazepin8-yl) oxy) propoxy) -11-hydroxy-7-methoxy-5-oxo2,3,11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepine-10 ( 5H) -carboxylate (I9).

A solution of anhydrous DMSO (710 μl, 780 mg, 9.99 mmol) in dry DCM (20 ml) was added dropwise to a stirred solution of oxalyl chloride (2.72 ml of a 2.0 M solution in DCM, 5.44 mmol) in dry DCM (20 ml) at -45 ° C (dry ice / CH3CN) under argon. After stirring for 15 minutes at -45 ° C, the reaction mixture was treated dropwise with a solution of bis-alcohol I8 (2.44 g, 2.27 mmol) in dry DCM (30 ml). After stirring at -45 ° C for an additional 1 h, the reaction mixture was treated dropwise with a solution of TEA (3.16 ml, 2.29 g, 22.7 mmol) in dry DCM (20 ml). The reaction mixture was allowed to warm to room temperature over 1.5 h and diluted with DCM (100 ml), then washed with saturated NH ₄ Cl (2x50 ml), saturated NaHCO ₃ (50 ml), water (30 ml), saturated aqueous chloride solution sodium (50 ml), dried (MgSO ₄ ), filtered and evaporated in vacuo to give the crude product. Purification with Isolera ™ (DCM / MeOH, SNAP Ultra 100 g, 100 ml / min) gave cyclized compound I9 (elution at 95.7% DCM / MeOH) as a yellowish foam (1.61 g, 66% yield): LC / MS I9 retention time 1.46 min, ESI + m / z 1072 [M + H] +, 1094 [M + Na] +.

(h) 4 - ((S) -2 - ((S) -2-Amino-3-methylbutanamido) propanamido) benzyl (11S, 11aS) -8- (3 - (((11S, 11aS) -10 (tert -butoxycarbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo [2,1c] [1,4] benzodiazepin-8-yl) oxy ) nropoxy) -11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1Hpyrrolo [2,1-c] [1,4] benzodiazepine-10 (5H) -carboxylate (I10) ...

Pd (PPh ₃ ) ₄ (6.47 mg, 5 , 6 μmol). After stirring for 4 h under argon at room temperature, LC / MS analysis showed complete reaction with the desired product observed at 1.10 min retention time, ESI +, m / z 1010 [M + Na] +, 988 [ M + H] +. The reaction mixture was diluted with DCM (30 ml), then washed with saturated NH ₄ Cl (2x20 ml), saturated aqueous sodium chloride solution (30 ml), dried (MgSO ₄ ), filtered and evaporated in vacuo to give the crude product. Trituration with diethyl ether followed by evaporation in vacuo gave the crude amine I10 (261 mg, 95% yield), which was used in the next step without further purification or analysis.

(i) tert-Butyl (11S, 11aS) -8- (3 - (((11S, 11aS) -10 - (((4 - ((2S, 5S) -37- (2,5-dioxo-2, 5-dihydro-1H-pyrrole-1yl) -5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34triazageheptatriacontanamido ) benzyl) oxy) carbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-1H-pyrrolo [2,1 -c] [1,4] benzodiazepine-8 -yl) oxy) propoxy) -11 -hydroxy-7-methoxy-5-oxo 2,3,11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepine-10 (5H) - carboxylate (I11).

To a stirred solution of MAL-dPEG® ₈ -acid (172 mg, 0.29 mmol, Stratech Scientific Limited) and amine I10 (261 mg, 0.26 mmol) in dry DCM (10 ml) at room temperature was added EDCI (56 mg , 0.29 mmol). The reaction mixture was stirred under argon for 2.5 h, after which LC / MS analysis showed complete conversion to the desired product with a retention time of 1.38 min, ESI + m / z 1585 [M + Na] +, 1563 [M + H] +. The reaction mixture was diluted with DCM (30 ml) and washed with H2O (20 ml), saturated aqueous sodium chloride solution (2x20 ml), dried (MgSO ₄ ), filtered and evaporated in vacuo to give the crude product. Purification of Isolera ™ (DCM / MeOH, SNAP Ultra 25 g, 75 ml / min) afforded amide I11 (elution at 91% DCM / MeOH) as a white foam (277 mg, 67% yield).

(j) 4 - ((2S, 5S) -37- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -5-isopropyl-2-methyl-4,7,35- trioxo10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontmanamido) benzyl (11S, 11aS) -11-hydroxy-7methoxy-8- (3 - (((S) - 7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo [2,1-c | [1,4] benzodiazepine-8yl) oxy) propoxy) -5-oxo-2,3, 11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepine-10 (5H) carboxylate (1).

Solution 95: 5 v / v TFA / H ₂ O (2 ml) was added to a crude sample of Boc-protected compound I11 (262 mg, 0.17 mmol) at 0 ° C (ice / acetone). After stirring at 0 ° C for 3 h, the reaction was considered complete by LC / MS, peak of the desired product at 1.30 min retention time, ESI + m / z 1445 [M + H] +. The reaction mixture was kept cold and added dropwise to a cooled saturated aqueous NaHCO ₃ solution (100 ml). The mixture was extracted with DCM (3x30 ml) and the combined organic layers were washed with saturated aqueous sodium chloride solution (30 ml), dried (MgSO ₄ ), filtered and evaporated in vacuo to give crude

- 66 038267 puppy product. Purification of Isolera ™ (CHCl ₃ / MeOH, SNAP Ultra 25 g, 25 ml / min) gave 1 (elution at 89.6% CHCl ₃ / MeOH) as a yellow foam (170 mg, 70% yield). Further purification by preparative HPLC (Method A) gave 1 as a light yellow foam (105 mg, 43% yield).

LC / MS (15 min run) retention time 5.25 min, ESI + m / z 1445 [M + H] ⁺ ;

¹ H NMR (400 MHz, d6-DMSO) δ 9.92 (s, 1H), 8.16 (d, 1H, J = 6.8 Hz), 7.99 (t, 1H, J = 5.7 Hz), 7.86 (d, 1H, J = 8.6 Hz), 7.80 (d, 1H, J = 4.5 Hz), 7.64-7.50 (m, 2H), 7, 34 (s, 1H), 7.24-7.13 (m, 2H), 7.06 (s, 1H), 7.00 (s, 2H), 6.88 (s, 1H), 6.75 (s, 1H), 6.53-6.41 (m, 1H), 5.52-5.41 (m, 1H), 5.13 (d, 1H, J = 12.2Hz), 4, 93-4.77 (m, 1H), 4.42-, 34 (m, 1H), 4.30-3.90 (m, 6H), 3.80-3.60 (m, 4H), 3 , 80 (s, 3H), 3.79 (s, 3H), 3.60 (t, 4H, J = 7.3 Hz), 3.533.46 (m, 28H), 3.41-3.33 ( m, 1H), 3.32-3.29 (m, 2H, overlapped with H2O), 3.19-3.12 (m, 2H), 2.48-1.60, m, 15H), 1.35 -1.20 (m, 3H), 0.87 (d, 3H, J = 6.6 Hz), 0.83 (d, 3H, J = 6.8 Hz).

Example 2.

- 67 038267

(a) ((Pentane-1,5-diyl-bis (oxy)) - bis- (5-methoxy-2-nitro-4,1-phenylene)) - bis - (((S) -2- ( hydroxymethyl) pyrrolidin-1-yl) methanone) (I13).

DMF (5 drops) was added to a stirred suspension of bis-nitrobenzoic acid I12 (4.05 g, 8.192 mmol, 1.0 eq.) And oxalyl chloride (12.3 ml of a 2 M solution, 24.57 mmol, 3.0 eq. ) in anhydrous CH ₂ Cl ₂ (65 ml). After initial foaming, the reaction suspension turned into a solution and the mixture was allowed to stir at room temperature for 16 h. The reaction mixture was evaporated in vacuo and the resulting solid was triturated with Et ₂ O and dried in a vacuum oven at 40 ° C for 3 h. Solid the acid chloride was added in portions to a stirred suspension of (S) - (+) - 2 pyrrolidinemethanol (1.78 ml, 18.02 mmol, 2.2 eq.) and iso-Pr ₂ NEt (7.13 ml, 40.96 mmol, 5.0 eq.) In CH ₂ Cl ₂ (65 ml) at -40 ° C (dry ice / CH ₃ CN). After stirring for 1 h, the reaction temperature reached 0 ° C and was completed as determined by LC / MS with the extremely desired product at a retention time of 1.44 min, ESI + m / z 661 [M + H] +, 683 [M + Na] +. The mixture was diluted

- 68 038267

CH2C12 (100 ml) and washed sequentially with H2O, 1N. NaOH and HCl (50 ml), dried (MgSO ₄ ), filtered and the solvent was evaporated in vacuo to give pure product I13 as a yellow foam (4.44 g, 82% yield), which was used without further purification.

(b) ((Pentane-1,5-diyl-bis- (oxy)) - bis- (5-methoxy-2-nitro-4,1-phenylene)) - bis - (((S) -2- ( ((tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-1-yl) methanone) (I14).

Imidazole (2.74 g, 40.32 mmol, 6.0 eq.) Was added in portions, then TBSCI (3.04 g, 20.16 mmol, 3.0 eq.) To a stirred solution of bis-alcohol I13 (4, 44 g, 6.720 mmol, 1.0 eq.) In an argon atmosphere. After 90 min, the reaction mixture was filtered and the filtrate was washed with H2O, dried over MgSO ₄ and evaporated in vacuo. Purification by flash column chromatography (50-80% EtOAc in hexanes) gave I14 as a yellow foam (4.84 g, 5.443 mmol, 81% yield)

Retention time LC / MS = 2.18 min, ESI + m / z 889 [M + H] +, 911 [M + Na] ⁺ .

(c) ((Pentane-1,5-diyl-bis- (oxy)) - bis- (2-amino-5-methoxy-4,1-phenipene)) - bis - (((S) -2- ( ((tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-1-yl) methanone) (I15).

Zn powder was added to a stirred solution of bis-nitro compound I14 (1.32 g, 1.84 mmol) in MeOH (40 ml) at 0 ° C. 5% HCO2H in MeOH was added dropwise at 0 ° C and the mixture was stirred for 2 h. The reaction mixture was diluted with EtOAc and washed with saturated NaHCO ₃ solution, the organic phase was dried over MgSO ₄ and evaporated in vacuo. Purification by flash column chromatography (0-2% MeOH in CHCl ₃ ) gave product I15 as a pale yellow foam (3.098 mmol, 3.736 mmol, 69% yield).

Retention time LC / MS = 2.09 min, ESI + m / z 415 [M + 2H] ²⁺ , 829 [M + H] +, 851 [M + Na] +.

(d) tert-Butyl (5 - ((5- (5-amino-4 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1carbonyl) -2-methoxyphenoxy) pentyloxy) - 2 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine 1 -carbonyl) -4-methoxyphenyl) carbamate (I16).

Boc ₂ O (734 mg, 3.362 mmol) was added to a stirred solution of bis-aniline I15 (3.098 g, 3.736 mmol) in dry THF (20 ml). The reaction mixture was stirred for 16 h and evaporated in vacuo. Purification by flash column chromatography (30-50% EtOAc in hexane) gave the monoBoc product I16 as a yellow foam (1.474 g, 47% yield based on Boc ₂ O), unreacted bis-aniline I15 (1.043 g, 30% yield) and bis-Boc (419 mg, 15% yield, retention time LC / MS = 2.37 min).

Retention time LC / MS I16 = 2.25 min, ESI + m / z 929 [M + H] +, 951 [M + Na] ⁺ .

(e) mret-Butyl (5 - ((5- (5 - (((4 - ((S) -2 - ((S) -2 - (((allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl) oxy) carbonyl) amino) -4 - ((8) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1carbonyl) -2-hydroxyphenoxy) pentyloxy) -2 - ((S) - 2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine 1 -carbonyl) -4-methoxyphenyl) carbamate (I17).

Triphosgene (169 mg, 0.5710 mmol, 0.36 eq.) Was added to a stirred solution of the monoproduct Boc I16 (1.474 g, 1.586 mmol, 1.0 eq.) And Et ₃ N (486 μL, 3.489 mmol, 2.2 eq.) in dry CH ₂ Cl ₂ (9 ml) at 10 ° C. After stirring for 10 min under argon, LC / MS analysis showed complete conversion to isocyanate (sampling in MeOH to yield methyl carbamate, retention time 2.30 min, ESI + m / z 1009 [M + Na] +, 987 [ M + H] +). A solution of I6 (898 mg, 2.379 mmol, 1.5 eq.) And Et ₃ N (332 μl, 2.379 mmol, 1.5 eq.) In dry CH ₂ Cl ₂ (14 ml) was added. The reaction mixture was gradually warmed to room temperature and stirred for 16 hours. 15 minute LC / MS analysis indicated consumption of starting material. The reaction mixture was filtered through a SiO ₂ pad (5% MeOH eluted with CH ₂ Cl ₂ ) to remove excess I6. Flash column chromatography (20-80% EtOAc in hexane) gives I17 as a yellow foam (1.439 g, 68% yield). Retention time LC / MS = 2.26 min (3 min run) and 10.43 min (15 min run) ESI + m / z 1355 [M + Na] ⁺ , 1333 [M + H] +. A small amount of urea dimer was observed (retention time LC / MS = 12.11 min, ESI + m / z 1906 [M + Na] ⁺ ), which was removed in subsequent purification steps.

(f) tert-Butyl (5 - ((5- (5 - (((4 - ((S) -2 - ((S) -2 - (((allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl) oxy) carbonyl) amino) -2-hydroxy-4 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl) phenoxy) pentyloxy) -2 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl) -4-methoxyphenyl) carbamate (I18).

Acetic acid (124 μl, 2.160 mmol, 2.0 eq.) Was added to a 1 M solution of TBAF (3.2 ml, 3,200 mmol, 3.0 eq.) And then added to a stirred solution of I17 in THF (67 ml) at 0 ° C. The reaction mixture was warmed to room temperature and stirred for 16 hours. LC / MS indicated the reaction was complete. TBAF (1.00 ml of 1 M solution, 1 mmol, 1.0 eq.) Was added and the reaction mixture was stirred for another 24 h. The reaction mixture was evaporated in vacuo and purified with Isolera ™ (0-5% MeOH in CH ₂ Cl ₂ ) to give I18 as a pale yellow foam (916 mg, 77% yield).

Retention time LC / MS = 1.62 min, ESI + m / z 1126 [M + Na] ⁺ , 1104 [M + H] +.

(g) 4 - ((S) -2 - ((S) -2 - (((Allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl (11S, 11aS) -8 - ((5 - (( (11 S, 11aS) - 10- (tert-butoxycarbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-III-benzo [e] pyrrolo [1,2 -a] [1,4] diazepin-8-yl) oxy) pentyloxy) -11-hydroxy-7-methoxy-5oxo-2,3,11,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2 -a] [1,4] diazepine-10 (5H) -carboxylate (I19).

IBX (1.14 g, 1.825 mmol, 2.2 eq.) Was added to a stirred solution of diol I18 (916 mg,

- 69 038267

0.8296 mmol, 1.0 eq.) In DMSO. The reaction mixture was warmed to 35 ° C and stirred for 60 h. H ₂ O was added and the aqueous layer was extracted several times with CHCl ₃ . The organic extracts were combined, washed with saturated aqueous NaHCO ₃ and dried over MgSO ₄ . Purification of Isolera ™ (1-8% MeOH in CH ₂ Cl ₂ ) gave I19 as an orange foam (908 mg, 99% yield).

Retention time LC / MS = 1.50 min, ESI + m / z 1122 [M + Na] +, 1100 [M + H] +.

(h) 4 - ((S) -2 - ((S) -2-Amino-3-methylbutanamido) propanamido) benzyl (11S, 11aS) -8 - ((5 - ((((11S, 11aS) -10 ( tert-butoxycarbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1Hbenzo [e] pyrrolo [1,2-a] [1,4] diazepine- 8-yl) oxy) pentyloxy) -11-hydroxy-7-methoxy-5-oxo-2,3,11,11atetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine- 10 (5H) -carboxylate (I20).

Pd (PPh ₃ ) ₄ (15.8 mg, 13.64 μmol, 0.050 eq.) Was added to a stirred solution of pyrrolidine (56 μl, 0.6818 mmol, 2.5 eq.) And I19 (300 mg, 0.2727 mmol) in CH ₂ Cl ₂ (10 ml) under argon. After 30 min, saturated aqueous NH ₄ Cl solution was added, the mixture was vigorously stirred and transferred to an Isolute® phase separator. The collected organic phase was evaporated in vacuo to give an orange foam I20 which was used without further purification.

(i) tert-Butyl (11S, 11aS) -8 - ((5 - (((11S, 11aS) -10 - (((4 - ((2S, 5S) -37- (2,5-dioxo-2 , 5-dihydro-1H-pyrrol1-yl) -5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6, 34triazageptatriacontanamido) benzyl) oxy) carbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-1H-benzo [e] pyrrolo [1,2-a] [1, 4] diazepin-8-yl) oxy) pentyloxy) -11-hydroxy-7-methoxy-5oxo-2,3,11,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1, 4] diazepine-10 (5H) -carboxylate (I21).

EDCl was added to a stirred solution of MAL-dPEG® _8- acid (360 mg, 0.6079 mmol, Stratech Scientific Limited) and amine I20 (608 mg, 0.5526 mmol) in CH ₂ Cl ₂ (15 ml) at room temperature. HCl (117 mg, 0.29 mmol). The reaction mixture was stirred under argon for 24 h, after which LC / MS analysis indicated complete consumption of I20. The reaction mixture was diluted with CH ₂ Cl ₂ and sequentially washed with saturated aqueous NH ₄ Cl with saturated aqueous NaHCO ₃ , dried over MgSO ₄ and evaporated in vacuo to give the crude product. Purification of Isolera ™ (4-16% MeOH in CH ₂ Cl ₂ ) gave amide I21 as a white solid (77 mg, 79% purity (UV integration at 223 nm), 8.8% crude yield; 107 mg, purity 88%, 12% crude yield; 224 mg, 86% purity, 25% crude yield).

(j) 4 - ((2S, 5S) -37- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -5-isopropyl-2-methyl-4,7,35- trioxo10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido) benzyl (11S, 11aS) -11-hydroxy-7methoxy-8- (3 - (((S) - 7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepine-8yl) oxy) propoxy) -5-oxo-2,3, 11,11a-tetrahydro-1H-pyrrolo [2,1-c] [1,4] benzodiazepine-10 (5H) carboxylate (2).

Ice-cooled solution 95: 5 v / v TFA / H ₂ O (3 ml) was added to a crude sample of Boc-protected compound I21 (107 mg, 67.51 μmol) at 0 ° C (ice / saturated aqueous sodium chloride solution). After stirring at 0 ° C for 30 h, the reaction was considered complete by LC / MS, peak of the desired product at 1.38 min retention time, ESI + m / z 737 [M + 2H] ²⁺ , 748 [M + H + Na] ²⁺ ; 1472 [M + H] +. The reaction mixture was kept cold and added dropwise to a cooled saturated aqueous NaHCO ₃ solution. The mixture was extracted with CH ₂ Cl ₂ , then 10% MeOH in CH ₂ Cl ₂ , the combined organic layers were dried over MgSO ₄ and evaporated in vacuo to give the crude product. This process was repeated for other batches of I21, the crude products were combined and purified by preparative HPLC (Method B) to give Compound 2 as a white solid after lyophilization (126 mg, 33% yield, 96% purity by UV at 223 nm).

LC / MS (30 min run) retention time = 10.96 min, ESI + m / z 1472 [M + H] +.

- 70 038267

Example 3.

- 72 038267 (a) I23.

This step can be performed as indicated in the literature (see, for example, WO 2005085259A2; or Wells, et al., Bioorganic & Medicinal Chemistry Letters, 18 (2008), 2147-2151). The method includes hydrogenation in a Parr apparatus at room temperature with 10% Pd / C in EtOH. The output is quantitative. Ethanol was removed by re-evaporation (EtOH followed by DCM).

(b) tert-Butyl (11S, 11aS) -8 - ((3- (bromomethyl) benzyl) oxy) -7-methoxy-5-oxo-11 - ((tetrahydro-2Hpyran-2-yl) oxy) -2 , 3,11,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine-10 (5H) -carboxylate (I25).

A mixture of phenol I23 (4 g, 8.91 mmol, 1 eq.), 1,3-bis- (bromomethyl) benzene I24 (9.42 g, 35.7 mmol, 4 eq.), Potassium carbonate (1.23 g, 8.91 mmol, 1 eq.) and acetone (40 ml) were heated at 60 ° C for 5 h. After completion of the reaction as confirmed by LC / MS, the solids were removed by filtration and the filtrate was evaporated to dryness in vacuo. The residue was purified by chromatography (Biotage Isolera, 100 g Ultra, EtOAc / hexane gradient 30/70 to 80/20 at 12CV). Yield 4.25 g (75%). LC / MS, 3 min method, 1.82 min (ESI +) m / z (relative intensity) 631.15 ([M + H] +, 100), bifurcated peak: THP diastereoisomers.

1H NMR (400 MHz, DMSO-de) δ 7.67-7.27 (m, 4H), 7.20-6.57 (m, 2H), 5.72-5.57 (m, 1H), 5.24-4.84 (m, 3H), 4.72 (s, 2H), 3.91-3.73 (m, 4H), 3.61-3.33 (m, 4H), 2, 20-1.75 (m, 4H), 1.74-1.57 (m, 2H), 1.55-1.01 (m, 13H).

I28 is known in the literature (see WO 2013053872A1, compound 2, p. 60).

(c) (S) - (2- (Hydroxymethyl) pyrrolidin-1-yl) (5-methoxy-2-nitro-4 - ((triisopropylsilyl) oxy) - phenyl) methanone (I29).

EDCI (12.4 g, 65 mmol, 1.2 eq.) Was added to a solution of acid I28 (20 g, 54.1 mmol, 1 eq.) And hydroxybenzotriazole hydrate (8.05 g, 59.5 mmol, 1, 1 eq.) In dichloromethane (200 ml) at 0 ° C. The cold bath was removed and the reaction was left for 30 min at room temperature, after which a solution of (S) -pyrrolidin-2-ylmethanol (5.87 ml, 59.5 mmol, 1.1 eq.) And triethylamine (11, 32 ml, 81.1 mmol, 1.5 eq.) In dichloromethane (100 ml) at -10 ° C under argon. The reaction mixture was left stirring at room temperature for 40 min to 1 h and monitored by LC / MS and TLC (EtOAc). The solids were removed by filtration through celite and the organic phase was washed with cold aqueous 0.1 M HCl until the pH reached a value of 4 or 5. The organic phase was then washed with water, then with saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution. The organic layer was dried over magnesium sulfate, filtered, and excess solvent was removed on a rotary evaporator under reduced pressure. The residue was subjected to flash column chromatography (Isolera Biotage, 340 g Ultra; gradient 25/75 ethyl acetate / hexane to 100/0 ethyl acetate / hexane at 6 CV). Excess solvent was removed on a rotary evaporator under reduced pressure to give pure product I29 as a pale yellow foam (15.7 g, 64%).

LC / MS 1.92 min (ESI +) m / z (relative intensity) 453.15 ([M + H] +, 30%; 328.15, 100%);

1H NMR (400 MHz, chloroform-d) δ 7.70 (s, 1H), 6.77 (s, 1H), 4.57-4.24 (m, 2H), 4.01-3.69 ( m, 5H), 3.25-3.06 (m, 2H), 2.18 (dt, J = 7.5, 5.6 Hz, 1H), 1.96-1.62 (m, 3H) , 1.42-1.18 (m, 3H), 1.10 (d, J = 7.4Hz, 18H).

(d) (S) - (2 - (((tert-Butyldimethylsilyl) oxy) methyl) pyrrolidin-1-yl) (5-methoxy-2-nitro-4- ((triisopropylsilyl) oxy) phenyl) methanone (I30) ...

tert-Butyldimethylsilyl chloride (10.39 g, 68.9 mmol, 2 eq.) was added to a solution of alcohol I29 (15.6 g, 34.5 mmol, 1 eq.) and imidazole (5.87 g, 86.2 mmol , 2.5 eq.) In DCM (100 ml). The reaction mixture was stirred overnight at room temperature. The reaction mixture was washed sequentially with water (300 ml), 0.5 M citric acid (200 ml), saturated aqueous sodium chloride solution (100 ml) and dried (MgSO ₄ ). Filtration and removal of excess solvent gave the crude product which was subjected to flash column chromatography (Biotage Isolera, KP-Sil 340 g; 10/90 v / v ethyl acetate / hexane to 30/70 v / v ethyl acetate / hexane) with isolating the silyl ether I30 as a thick yellow oil. Yield: 18.9 g, 97%.

LC / MS 2.32 min (ESI +) m / z (relative intensity) 567.55 ([M + H] +, 100%) (e) (S) - (2-amino-5-methoxy-4- ((triisopropylsilyl) oxy) phenyl) (2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-1-yl) methanone (I31).

A solution of nitro compound I30 (18.9 g, 33.3 mmol, 1 eq.) In ethyl acetate (200 ml) in 10% Pd / C (10% w / w 1.89 g) was hydrogenated under pressure (45 lb / Inch) on a Parr apparatus for 6 h. The reaction mixture was filtered through celite to remove Pd / C and the filter pad was washed with ethyl acetate. Excess solvent was removed on a rotary evaporator under reduced pressure, followed by drying under high vacuum to give amine I31 as a thick oil.

LC / MS, 3 min method, 2.28 min (ESI +) m / z (relative intensity) 537.30 ([M + H] +, 100);

1H NMR (400 MHz, chloroform-d) δ 6.73 (s, 1H), 6.24 (s, 1H), 4.54-4.13 (m, 3H), 4.07-3.80 ( m, 1H), 3.79-3.61 (m, 4H), 3.50 (dd, J = 9.2, 4.2Hz, 2H), 2.10-1.97 (m, 2H) , 1.92 (dt, J = 11.7, 6.2 Hz, 1H), 1.80-1.65 (m, 1H), 1.24 (ddt, J = 13.7, 9.9, 6.4 Hz, 3H), 1.09 (d, J = 7.3 Hz, 18H), 0.90 (s, 9H), 0.04 (d, J = 2.8 Hz, 6H).

- 73 038267 (f) Allyl ((S) -1 - (((S) -1 - ((4 - ((((2 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-1 - carbonyl) -4-methoxy-5 - ((triisopropylsilyl) oxy) phenyl) carbamoyl) oxy) methyl) phenyl) amino) -1oxopropan-2-yl) amino) -3-methyl-1-oxobutane-2 -yl) carbamate (I32).

Triethylamine (10.1 ml, 72.4 mmol, 2.2 eq.) Was added to a stirred solution of amine I31 (17.68 g, 32.9 mmol, 1 eq.) And triphosgene (3.51 g, 11.8 mmol, 0.36 equiv.) in dry tetrahydrofuran (180 ml) at 5 ° C (ice bath). The isocyanate production reaction was monitored by periodically removing aliquots from the reaction mixture and adding methanol, and performing LC / MS analysis. After completion of isocyanate formation, a suspension of alloc-Val-Ala-PABOH I6 (18.6 g, 49.4 mmol, 1.5 eq.) And triethylamine (6.88 mL, 49.4 mmol, 1 , 5 eq.) In dry tetrahydrofuran (70 ml). The reaction mixture was left stirring at 40 ° C for 4 hours. The solids were filtered off. Excess solvent was removed on a rotary evaporator under reduced pressure. The resulting residue was dried over silica gel and subjected to manual flash column chromatography; 40/60 rpm ethyl acetate / hexane up to 70/30 v / v ethyl acetate / hexane. Pure fractions were collected and combined, and excess eluent was removed on a rotary evaporator under reduced pressure to give product I32 8.17 g (26.4%).

LC / MS, 3 min method, 2.29 min (ESI +) m / z (relative intensity) 962.45 ([M + Na] ⁺ , 100; 940.40 ([M + H] ⁺ , 30);

? H NMR (400 MHz, chloroform-d)? 8.95 (s, 1H), 8.53 (s, 1H), 7.78 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 6.80 (s, 1H), 6.71 (d, J = 7.5 Hz, 1H), 5.89 ( tt, J = 10.8, 5.3 Hz, 1H), 5.44-5.15 (m, 3H), 5.10 (s, 2H), 4.66 (p, J = 7.2 Hz , 1H), 4.62-4.53 (m, 2H), 4.32 (s, 1H), 4.08-3.86 (m, 2H), 3.74 (s, 4H), 3, 52 (dd, J = 27.4, 7.6 Hz, 2H), 2.15 (d, J = 6.8 Hz, 1H), 2.09-1.85 (m, 3H), 1.71 (s, 1H), 1.46 (d, J = 7.0 Hz, 3H), 1.29 (dq, J = 15.0, 7.4 Hz, 3H), 1.11 (d , J = 7.4 Hz, 18H), 0.95 (dd, J = 14.1, 6.8 Hz, 6H), 0.89 (s, 9H), 0.02 (d, J = 13, 1 Hz, 6H).

(9) Allyl ((S) -1 - (((S) -1 - ((4 - ((((2 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1carbonyl ) -5-hydroxy-4-methoxyphenyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) 3-methyl-1-oxobutan-2-yl) carbamate (I33).

Lithium acetate (50 mg, 0.49 mmol) was added to a solution of compound I32 (7 g, 7.44 mmol, 1 eq.) In wet dimethylformamide (61.2 ml, 50: 1 DMF / water). After 4 hours, the reaction was complete. Excess DMF was removed in vacuo, the residue was diluted with ethyl acetate (300 ml) and washed with 0.5 M aqueous citric acid (100 ml), water (300 ml) and saturated aqueous sodium chloride solution (100 ml). The organic layer was dried over magnesium sulfate, filtered, and excess ethyl acetate was removed on a rotary evaporator under reduced pressure. The resulting residue was subjected to flash column chromatography (Biotage Isolera 100 g Ultra; gradient, 40/60 to 80/20 v / v ethyl acetate / hexane at 8 CV). Pure fractions were collected and combined, and excess eluent was removed on a rotary evaporator under reduced pressure to give product I33 (5.13 g, 88%).

LC / MS, 3 min method, 1.82 min (ESI +) m / z (relative intensity) 784.40 ([M + H] +, 100);

1H NMR (400 MHz, chloroform-d) δ 9.06 (s, 1H), 8.62 (s, 1H), 7.78 (s, 1H), 7.45 (d, J = 8.2 Hz , 2H), 7.357.18 (m, 2H), 6.92 (d, J = 7.5 Hz, 1H), 6.80 (s, 1H), 6.50 (s, 1H), 5.89 (ddd, J = 16.2, 10.7, 5.4 Hz, 1H), 5.44 (d, J = 8.1 Hz, 1H), 5.37-5.15 (m, 2H), 5.14-5.01 (m, 2H), 4.67 (p, J = 7.1 Hz, 1H), 4.63-4.50 (m, 2H), 4.33 (s, 1H) , 4.13-3.89 (m, 2H), 3.81 (s, 3H), 3.74-3.33 (m, 3H), 2.24-1.84 (m, 4H), 1 , 69 (d, J = 21.2 Hz, 1H), 1.43 (d, J = 7.0 Hz, 3H), 1.07-0.71 (m, 15H), 0.23-0, 20 (m, 6H).

(h) tert-Butyl (11S, 11aS) -8 - ((3 - ((5 - ((((4 - ((S) -2 - ((S) -2 - (((allyloxy) carbonyl) amino ) -3-methylbutanamido) npropanamido) benzyl) oxy) carbonyl) αmino) -4 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1-carbonyl) -2-methoxyphenoxy) methyl) benzyl) oxy) -7-methoxy-5-oxo-11 - ((tetrahydro-2Hpyran-2-yl) oxy) -2,3,11,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2- a] [1,4] diazepine-10 (5H) -carboxylate (I34).

Potassium carbonate (582 mg, 4.21 mmol, 1.1 eq.) Was added to a solution of I25 (2.66 g, 4.21 mmol, 1.1 eq.) And phenol I33 (3 g, 3.82 mmol, 1 eq.) In acetone (18 ml). The reaction mixture was stirred for 4 h at 63 ° C. The solids were removed by filtration through cotton wool. Acetone was removed on a rotary evaporator under reduced pressure.

The resulting residue was subjected to flash column chromatography (Biotage isolera, 100 g Ultra, silica gel; gradient, 50/50 to 100/0 v / v ethyl acetate / hexane at 8 CV, elution 83%). The purified fractions were collected and combined, and excess eluent was removed on a rotary evaporator under reduced pressure to give product I34 (4.71 g, 92%).

LC / MS, 3 min method, 2.08 min (ESI +) m / z (relative intensity) 1335.15 ([M + H] +, 50);

1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.20 (s, 1H), 8.13 (d, J = 7.0 Hz, 1H), 7.68-7 , 50 (m, 3H), 7.50-7.37 (m, 3H), 7.32 (d, J = 8.2 Hz, 2H), 7.28-7.01 (m, 2H), 6.86 (s, 2H), 5.90 (ddd, J = 16.0, 10.7, 5.2 Hz, 1H), 5.64 (t, J = 9.8 Hz, 1H), 5 , 30 (d, J = 17.2 Hz, 1H), 5.23-4.84 (m, 8H), 4.57-4.36 (m, 3H), 4.11 (s, 1H), 3.95-3.59 (m, 9H), 3.56-3.34 (m, 4H), 1.94 (d, J = 34.0 Hz, 10H), 1.74-1.06 ( m, 21H), 1.01-0.59 (m, 15H), 0.03 (s, 6H).

(i) tert-Butyl (11S, 11aS) -8 - ((3 - ((5 - ((((4 - ((S) -2 - ((S) -2 - (((allyloxy) carbonyl) amino ) -3-methylbutanamido) npropanamido) benzyl) oxy) carbonyl) αmino) -4 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl) -2methoxyphenoxy) methyl) benzyl) oxy) -7-methoxy -5-oxo-11 - ((tetrahydro-2H-pyran-2-yl) oxy) -2,3,11,11a

- 74 038267 tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine-10 (5H) -carboxylate (I35).

Tetra-n-butylammonium fluoride (1 M, 6.94 ml, 6.94 mmol, 2 eq.) Was added to a solution of I34 (4.63 g, 3.47 mmol, 1 eq.) In tetrahydrofuran (28 ml). The starting material was completely consumed after 1 hour. The reaction mixture was diluted with ethyl acetate (30 ml) and washed sequentially with water and saturated aqueous sodium chloride solution. The organic phase was dried over magnesium sulfate, filtered, and excess ethyl acetate was removed on a rotary evaporator under reduced pressure. The resulting residue was subjected to flash column chromatography (Biotage isolera, 50 g Ultra; gradient, 98/2 to 90/10 v / v ethyl acetate / methanol at 4 CV, elution with 10% methanol). The purified fractions were collected and combined, and the excess eluent was removed on a rotary evaporator under reduced pressure to give product I35 (4.23 g, quantitative).

LC / MS, 3 min method, 1.75 min (ESI +) m / z (relative intensity) 1220.30 ([M + H] +, 100);

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.98 (s, 1H), 9.17 (s, 1H), 8.13 (d, J = 7.0 Hz, 1H), 7.70 -7.49 (m, 3H), 7.51-7.27 (m, 6H), 7.21 (d, J = 8.8 Hz, 1H), 7.15-6.58 (m, 3H ), 5.90 (dt, J = 10.9, 5.5 Hz, 1H), 5.66 (d, J = 9.3 Hz, 1H), 5.38-4.82 (m, 9H) , 4.73 (t, J = 5.8 Hz, 1H), 4.59-4.34 (m, 3H), 4.05 (dd, J = 15.4, 8.3 Hz, 1H), 3.96-3.68 (m, 8H), 3.66-3.32 (m, 6H), 2.16-1.72 (m, 8H), 1.63 (d, J = 9.8 Hz, 3H), 1.54-1.02 (m, 18H), 0.86 (dd, J = 18.2, 6.7 Hz, 6H).

(j) 4 - ((S) -2 - ((S) -2 - (((Allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl (11S, 11aS) -8 - ((3 - (( ((11S, 11aS) -10- (tert-butoxycarbonyl) -7-methoxy-5-oxo-11 - ((tetrahydro-2H-pyran-2yl) oxy) -2,3,5,10,11,11a- hexahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8-yl) oxy) methyl) benzyl) oxy) -11-hydroxy-7-methoxy-5-oxo-2, 3,11,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine10 (5H) -carboxylate (I36).

Stabilized IBX 45% (2.72 g, 4.36 mmol, 1.2 eq.) Was added to a solution of I35 (4.44 g, 3.64 mmol, 1 eq.) In DMSO (2.6 mL). The reaction mixture was left stirring overnight. Added another 0.2 eq. IBX (450 mg, 0.73 mmol, 0.2 eq.) And the solution was left stirring for an additional 18 h until LC / MS observed the reaction was complete. The solution was precipitated in water (250 ml) and filtered. The product was dissolved in DCM and the residual white solid was removed by filtration. The organic phase was washed with aqueous NaHCO ₃ , water, saturated aqueous sodium chloride and dried over magnesium sulfate. The dichloromethane was removed on a rotary evaporator under reduced pressure. The resulting residue was subjected to flash column chromatography (Biotage Isolera 100 g g Ultra; gradient, 99/1 to 92/8 v / v ethyl acetate / hexane at 10 CV). The purified fractions were collected and combined, and excess eluent was removed on a rotary evaporator under reduced pressure to give product I36 (3.04 g, 69%).

LC / MS 15 min, Ace method Excel 2, 7.89 and 7.97 min (THP diastereoisomers) (ESI +) m / z (relative intensity) 1218.30 ([M] ⁺ , 100);

1H NMR (400 MHz, DMSO-d ₆ ) δ 9.93 (s, 1H), 8.11 (d, J = 6.9 Hz, 1H), 7.68-7.27 (m, 6H), 7.27-7.01 (m, 4H), 7.01-6.32 (m, 3H), 6.02-5.81 (m, 1H), 5.71-5.57 (m, 1H ), 5.57-5.40 (m, 1H), 5.29 (d, J = 17.2 Hz, 1H), 5.214.78 (m, 8H), 4.58-4.32 (m, 3H), 3.99-3.68 (m, 8H), 3.58-3.31 (m, 8H), 2.23-1.72 (m, 9H), 1.72-1.04 ( m, 18H), 0.85 (dd, J = 18.0, 6.7 Hz, 6H).

(k) 4 - ((S) -2 - ((S) -2-Amino-3-methylbutanamido) propanamido) benzyl (11S, 11aS) -8 - ((3 - (((((11S, 11aS) 10- (tert-butoxycarbonyl) -7-methoxy-5-oxo-11 - ((tetrahydro-2H-pyran-2-yl) oxy) -2,3,5,10,11,11ahexahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8-yl) oxy) methyl) benzyl) oxy) -11-hydroxy-7 methoxy-5-oxo-2,3,11,11a-tetrahydro-1H- benzo [e] pyrrolo [1,2-a] [1,4] diazepine-10 (5H) -carboxylate (I37).

tetrakis- (Triphenylphosphine) palladium (0) (11 mg, 0.01 mmol, 0.02 eq.) was added to a solution of I36 (600 mg, 0.49 mmol, 1 eq.) and pyrrolidine (51 μl, 0.62 mmol, 1.25 equiv.) in dry dichloromethane (10 ml). The reaction mixture was purged with argon three times and stirred for 20 min at room temperature. The reaction mixture was then diluted with dichloromethane (50 ml) and washed sequentially with saturated aqueous ammonium chloride (50 ml) and saturated aqueous sodium chloride (30 ml). The organic phase was dried over magnesium sulfate, filtered and the excess dichloromethane was removed using a rotary evaporator under reduced pressure. The resulting residue I37 was used as a crude mixture for the next reaction.

LC / MS, 3 min method, 1.29 min (ESI +) m / z (relative intensity) 1134.35 ([M + H] +, 80).

(l) tert-Butyl (11S, 11aS) -8 - ((3 - ((((11S, 11aS) -10 - (((4 - ((2S, 5S) -37- (2,5-dioxo 2,5-dihydro-1H-pyrrol1-yl) -5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6 , 34-triazaheptatriacontanamido) benzyl) oxy) carbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1Hbenzo [e] pyrrolo [1,2-a] [1,4] diazepin-8-yl) oxy) methyl) benzyl) oxy) -7-methoxy-5-oxo-11 - ((tetrahydro2H-pyran-2-yl) oxy) -2,3,11,11a -tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine-10 (5H) -carboxylate (I38).

1-Ethyl-3- (3'-dimethylaminopropyl) carbodiimide (94 mg, 0.79 mmol, 1 eq.) Was added to a solution of crude I37 (558 mg, 0.49 mmol, 1 eq.) And MaI- (PEG) _8- acids (292 mg, 0.49 mmol, 1 eq.) In chloroform (12 ml). The reaction mixture was degassed three times with argon and stirred for 2 h, after which the presence of starting material was no longer observed by LC / MS. Reaction mixture

- 75 038267 was diluted with dichloromethane and washed successively with water and saturated aqueous sodium chloride solution. The organic phase was dried over magnesium sulfate, filtered, and the excess dichloromethane was removed using a rotary evaporator under reduced pressure. The resulting residue was subjected to flash column chromatography (Biotage Isolera 50g Ultra; 98/2 to 90/10 v / v DCM / methanol 10 CV). Pure fractions were collected and combined and excess eluent removed using a rotary evaporator under reduced pressure to give product I38 (485 g, 58%).

LC / MS Method 3 min, 1.58 min (ESI +) m / z (relative intensity) 1709.30 ([M + H] +, 100);

Ή MR (400 MHz, DMSOC) δ 9.88 (s, 1H), 8.13 (d, J = 7.0 Hz, 1H), 8.06-7.92 (m, 1H), 7.85 (d, J = 8.6 Hz, 1H) 7.68-7.04 (m, 9H), 6.99 (s, 2H), 6.89 (d, J = 15.0 Hz, 2H), 6.52 (s, 1H), 5.66 (d, J = 9.4 Hz, 1H), 5.47 (d, J = 8.0 Hz, 1H), 5.26-4.75 (m , 6H), 4.49-4.31 (m, 1H), 4.20 (t, J = 7.6 Hz, 1H), 3.80 (d, J = 11.9 Hz, 6H), 3 , 59 (t, J = 7.2 Hz, 4H), 3.55-3.41 (m, 32H), 3.41-3.30 (m, 11H), 3.21-3.09 (m , 3H), 2.48-2.28 (m, 4H), 2.18-1.08 (m, 24H), 0.84 (dd, J = 15.0, 6.7Hz, 5H).

(m) 4 - ((2S, 5S) -37- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -5-isopropyl-2-methyl-4,7,35- trioxo10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido) benzyl (11S, 11 aS) -11-hydroxy-7 methoxy-8 - ((3 - ((( (S) -7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8yl) oxy) methyl) benzyl ) oxy) -5-oxo-2,3,11,11 a-tetrahydro-1 H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine10 (5H) -carboxylate (3).

Cold TFA / water mixture (6 ml) was added to I38 (460 mg, 0.27 mmol, 1 eq.) And the resulting solution was stirred at 0 ° C for 2 h. The reaction mixture was neutralized with saturated aqueous NaHCO ₃ (200 ml) and dichloromethane (50 ml). The DCM layer was washed sequentially with water and saturated aqueous sodium chloride solution. The organic phase was dried over magnesium sulfate, filtered, and the excess dichloromethane was removed using a rotary evaporator under reduced pressure. The resulting residue was subjected to flash column chromatography (Biotage Isolera 50g Ultra; 98/2 to 88/12 v / v DCM / methanol 10 CV). Pure fractions were collected, combined (154 mg, 38%) and further purified by preparative reverse phase HPLC (Method C) (gradient up to 75/25 acetonitrile / water, 0.02% formic acid) to give pure compound 3 (78 mg, 19% ).

LC / MS Method 15 min, Ace-Excel2 6.18 min (ESI +), m / z (relative intensity) 1506.70 ([M + H] +, 100);

Ή NMR (400 MHz, DMSO-d6) δ 10.07-9.79 (m, 1H), 8.14 (d, J = 7.2 Hz, 1H), 7.98 (t, J = 5, 6 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.78 (d, J = 4.4 Hz, 1H), 7.67-7.30 (m, 7H) , 7.28-7.05 (m, 3H), 6.99 (s, 2H), 6.98-6.85 (m, 2H), 6.58-6.47 (m, 1H), 5 , 59-5.34 (m, 1H), 5.32-4.77 (m, 6H), 4.48-4.30 (m, 1H), 4.28-4.08 (m, 1H) , 3.88-3.75 (m, 5H), 3.75-3.55 (m, 6H), 3.55-3.42 (m, 28H), 3.42-3.32 (m, 6H), 3.14 (q, J = 5.8Hz, 2H), 2.48-2.16 (m, 6H), 2.081.77 (m, 6H), 1.36-1.17 (m , 4H), 0.84 (dd, J = 15.4, 6.7 Hz, 6H).

Example 4.

- 76 038267

(a) ((Propane-1,3-diyl-bis- (oxy) -bis- (5-methoxy-2-nitro-4,1-phenylene)) - bis - (((S) -2- (hydroxymethyl ) pyrrolidin-1-yl) methanone) (I2).

DMF (12 drops) was added to a stirred suspension of I1 (10 g, 21.5 mmol) and oxalyl chloride (5.6 ml, 8.2 g, 64.5 mmol) in anhydrous DCM (150 ml). After initial bubbling, the reaction suspension turned into a solution and the reaction mixture was allowed to stir at room temperature for 16 hours. Most of the solvents were removed using a rotary evaporator under reduced pressure. The resulting concentrated solution was redissolved in a minimal amount of anhydrous DCM and triturated with diethyl ether. The yellow precipitate was collected by vacuum filtration, washed with cold diethyl ether, and dried for 1 h in a vacuum oven at 40 ° C. The acid chloride was added portionwise to a stirred suspension of (S) - (+) - 2 pyrrolidinemethanol (5.0 g, 4.9 ml, 49.5 mmol) and TEA (15.0 ml, 10.9 g, 108 mmol) in anhydrous DCM (100 ml) at -40 ° C (dry ice / CH ₃ CN). The resulting solution was stirred for an additional 60 min, diluted with DCM (100 ml) and washed with 1N HCl. HCl (2x50 ml), saturated NaHCO ₃ (3x40 ml), aqueous saturated sodium chloride solution (50 ml), dried (MgSO ₄ ) and the solvent was removed in vacuo to give pure product I2 as a yellow solid (13.6 g, output 100%).

LC / MS (Method A) retention time 1.33 min (ESI +), m / z 655 [M + Na] +, 633 [M + H] + (see Appendix).

Ή NMR (400 MHz, DMSO-d6) 1.68-1.80 (m, 2H), 1.80-2.00 (m, 6H), 2.27 (d, 2H), 3.05-3 , 25 (m, 4H), 3.37-3.48 (m, 2H), 3.56-3.76 (m, 2H), 3.92 (s, 6H), 4.09 (dd, 2H ), 4.25-4.31 (m, 4H), 4.82 (t, 2H), 7.08 (s, 2H), 7.73 (s, 2H).

(b) ((Propane-1,3-diyl-bis- (oxy)) - bis- (5-methoxy-2-nitro-4,1-phenylene)) - bis - (((S) -2- ( ((tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-1-yl) methanone) (I3 9).

- 77 038267

TBS-CI (8.12 g, 53.90 mmol) was added to a solution of I2 (15.5 g, 24.50 mmol) and imidazole (4.17 g, 61.25 mmol) in DCM (300 ml) at room temperature. temperature in a nitrogen atmosphere. The mixture was stirred at room temperature for 12 hours. Water (200 ml) was added, the organic layer was removed and the aqueous phase was extracted with DCM (2x300 ml). The combined organic phases were dried (Na ₂ SO ₄ ) and evaporated in vacuo to give a dark residue, which was purified by column chromatography (0 to 2% methanol / DCM). The pure fractions were evaporated in vacuo to give I39 as a brown solid (17.0 g, 81% yield).

LC / MS (Method A) retention time 1.83 min (ESI +), m / z 861 [M + H] +.

Ή NMR (400 MHz, DMSO-d ₆ ) 0.09 (s, 12H), 0.91 (s, 18H), 1.70-1.81 (m, 2H), 1.87-1.99 ( m, 6H), 2.222.30 (m, 2H) 3.10 (t, 4H), 3.40-3.51 (m, 2H), 3.59-3.67 (m, 2H), 3, 88-3.95 (m, 2H), 3.91 (s, 6H), 4.28 (t, 6H), 6.96 (s, 2H), 7.72 (s, 2H).

(c) ((Propane-1,3-diyl-bis (oxy)) - bis- (2-amino-5-methoxy-4,1-phenylene)) bis (((S) -2 - ((( tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-1-yl) methanone) (I40).

Zinc (25.8 g, 394.8 mmol) and saturated NH ₄ Cl solution (150 ml) were added to a solution of I39 (17 g, 19.74 mmol) in EtOH (300 ml) at room temperature. The resulting mixture was stirred at 50 ° C for 3 h, cooled and filtered through a pad of celite, which was then washed with EtOAc (300 ml) and water (300 ml). The organic layer was separated and the aqueous phase was extracted with EtOAc (3x400 ml). The combined organic phases were dried (MgSO ₄ ) and evaporated in vacuo to give a yellow residue, which was purified by column chromatography (0 to 5% methanol / DCM). Pure fractions were evaporated to dryness to give I40 as a yellow solid (13.00 g, 82% yield).

LC / MS (Method A) retention time 2.30 min (ESI +) m / z 802.2 [M + H] + Ή NMR (400 MHz,

DMSO-d ₆ ) 0.06 (s, 12H), 0.85 (s, 18H), 1.52-1.78 (m, 2H), 1.81-2.00 (m, 6H), 2 , 14-2.22 (m, 2H), 3.41 (d, 4H), 3.61-3.75 (m, 4H), 3.63 (s, 6H), 4.01-4.16 (m, 6H), 4.98-5.22 (m, 4H), 6.40 (s, 2H), 6.66 (s, 2H).

(d) Allyl-5- (3- (5-amino-4 - ((S) -2 - (((tert-butyldimethylsilyl) oxy)) methyl) pyrrolidine-1-carbonyl) 2-methoxyphenoxy) nropoxy) -2 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1-carbonyl) 4-methoxyphenyl) carbamate (I41).

Allyl chloroformate (784 μL, 0.9 g, 7.36 mmol) was added dropwise to a solution of I40 (5.9 g, 7.36 mmol) and pyridine (715 μL, 0.7 g, 8.84 mmol) in DCM (100 ml) at 0 ° C. The reaction mixture was allowed to warm to room temperature and stirred for an additional 2 minutes. The reaction mixture was washed with 0.5M HCl (50 ml), saturated aqueous sodium hydrogen carbonate solution (50 ml) and saturated aqueous sodium chloride solution (50 ml). The solvent was removed under reduced pressure and the resulting oil was purified by column chromatography; (Initial elution with 50% ethyl acetate / heptane removed the bis-alloc protected amine, followed by elution with ethyl acetate to remove the desired mono-alloc protected product (I41). Finally, any unreacted starting material was removed with 5% methanol / DCM). Pure fractions were evaporated under reduced pressure to give I41 as a yellow solid (3.5 g, 54% yield).

LC / MS (Method B) retention time 2.41 min (ESI +), m / z 886.5 [M + H] + (e) Allyl (5- (3- (5 - ((((4- ((S) -2 - ((S) -2) - (((allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl) oxy) carbonyl) amino) -4 - ((S) -2- ( ((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1carbonyl) -2-methoxyphenoxy) propoxy) -2 - ((S) -2 - ((((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1carbonyl) -4- methoxyphenyl) carbamate (I42).

Triphosgene (0.41 g, 1.4 mmol) was added to a stirred solution of I41 (3.5 g, 3.95 mmol) in anhydrous THF (70 mL) at room temperature under argon. Triethylamine (1.2 ml, 0.87 g, 8.6 mmol) was added and the resulting mixture was stirred for 10 minutes. LC / MS analysis showed complete conversion to isocyanate (sampling in MeOH to yield methyl carbamate, retention time 2.48 min, (ESI +) m / z 944.4 [M + H] +). A mixture of I6 (1.64 g, 4.35 mmol) and triethylamine (0.83 ml, 0.6 g, 5.9 mmol) in anhydrous THF (30 ml) was added. The reaction mixture was stirred under argon at 40 ° C for 2 h. The solvent was removed in vacuo and the residue was purified by column chromatography (0.5-2.5% methanol / DCM) to give I42 as a white solid (3, 58 g, yield 70%).

LC / MS (Method B) retention time 2.45 min, (ESI +) m / z 1290.0 [M + H] +.

(f) Allyl (5- (3- (5 - ((((4 - ((S) -2 - ((S) -2) - (((allyloxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl) oxy) carbonyl) amino) -4 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl) -2methoxyphenoxy) propoxy) -2 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl ) -4-methoxyphenyl) carbamate (I43).

Tetra-n-butylammonium fluoride (1 M, 6.1 ml, 6.1 mmol) was added to a solution of I42 (3.58 g, 2.78 mmol) in tetrahydrofuran (35 ml) at room temperature. The resulting mixture was stirred for 60 minutes, then evaporated to dryness under reduced pressure. The residue was purified by column chromatography (2 to 5% methanol / DCM) to give I43 as a white foam (2.95 g, 98% yield).

LC / MS (Method B) retention time 1.70 min, (ESI +) m / z 1061.3 [M + H] +.

- 78 038267 (g) Allyl (11S, 11aS) -8- (3 - (((11S, 11aS) -10 - ((4 - ((S) -2 - ((S) -2 - ((αlyloxy ) carbonyl)) amino) -3methylbutanamido) propanamido) benzyl) oxy) carbonyl) -11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-1H-benzo [e] pyrrolo [ 1,2-a] [1,4] diazepin-8-yl) oxy) propoxy) -11-hydroxy-7-methoxy-5-oxo 2,3,11,11a-tetrahydro-1H-benzo [e] pyrrolo [ 1,2-a] [1,4] diazepine-10 (5H) -carboxylate (I44).

Stahl solution for aerobic oxidation of TEMPO 0.2 M in MeCN (5.47 ml, 1.1 mmol), and then tetrakis-acetonitrile copper (I) triflate (0.41 g, 1.1 mmol) was added to solution I43 ( 2.9 g, 2.74 mmol) in DCM (30 ml) and acetonitrile (6 ml) and stirred at 35 ° C for 36 h under air. The reaction mixture was washed with water (25 ml), dried (biotage phase separator) and evaporated to dryness under reduced pressure. The residue was purified by column chromatography (3 to 6% methanol / DCM) to afford the oxidized product I44 as a white solid (2.46 g, 85% yield).

LC / MS (Method B) retention time 1.60 min, (ESI +) m / z 1057.1 [M + H] ⁺ .

(h) 4 - ((S) -2 - ((S) -2-Amino-3-methylbutanamido) propanamido) benzyl (11S, 11aS) -11-hydroxy-7methoxy-8- (3 - (((S) -7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8yl) oxy) propoxy) -5-oxo -2,3,11,11 a-tetrahydro-1 H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine-10 (5H) carboxylate (I45).

Pd (Ph ₃ P) ₄ (10 mg, 5 mol%) was added to a solution of I44 (200 mg, 0.19 mmol) and pyrrolidine (40 μL, 0.34 g, 0.48 mmol) in DCM (10 ml ) at room temperature. The resulting solution was stirred for 30 minutes. The reaction mixture was washed with saturated ammonium chloride (10 ml), dried (biotage phase separator) and evaporated to dryness under reduced pressure. Then the residue was incubated in high vacuum for 4 h to remove traces of pyrrolidine. The resulting almost white solid was used in the next step without further purification (160 mg, 97% yield).

LC / MS (Method B) retention time 1.17 min, (ESI +) m / z 871.1 [M + H] ⁺ .

(i) 4 - ((2S, 5S) -37- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -5-isopropyl-2-methyl-4,7,35- trioxo10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido) benzyl (11S, 11 aS) -11-hydroxy-7methoxy-8- (3 - (((S) -7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8yl) oxy) propoxy) -5-oxo -2,3,11,11 a-tetrahydro-1 H-benzo [e] pyrrolo [1,2-a] [1,4] diazepine-10 (5H) carboxylate (1).

EDCl.HCl (46 mg, 0.24 mmol) was added to a solution of I45 and MaI-PEG _8- acid (130 mg, 0.22 mmol) in CHCl ₃ (10 ml) and stirred at room temperature for 2 h. LC / MS showed that 78% of the starting material was still present. Another 2 equiv. EDCl.HCl was added in portions to drive the reaction to completion. The reaction mixture was washed with water (10 ml), dried (Biotage PS) and evaporated to dryness under reduced pressure to give a yellow solid which was purified by preparative HPLC to give product 1 as an off-white solid (90 mg, 34% yield).

LC / MS (Method B) retention time 1.47 min, (ESI +) m / z 1445.9 [M + H] ⁺ .

Example 5.

(i) 4 - ((29S, 32S) -1-Azido-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28, 31 - diazatritriacontane-33-amido) benzyl (11S, 11aS) -11-hydroxy-7-methoxy-8- (3 - (((S) -7-methoxy-5-oxo2,3,5,11 a-) tetrahydro-1 H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8-yl) oxy) propoxy) -5-oxo-2,3,11,11atetrahydro-1 H-benzo [ e] pyrrolo [1,2-a] [1,4] diazepin-10 (5H) -carboxylate (4)

EDCl.HCl (27 mg, 0.14 mmol) was added to a solution of I45 and azido-PEG _8- acid (49 mg, 0.10 mmol) in CHCl ₃ (6 ml) and stirred at room temperature for 1 h. Solvent evaporated under reduced pressure to give a yellow foam. Purification by preparative HPLC

- 79 038267 gave product 4 as an off-white solid (20 mg, 17% yield).

LC / MS (Method B) retention time 6.01 min, (ESI +) m / z 1320 [M + H] ⁺ .

(ii) (R) -2 - ((3-Nitropyridin-2-yl) disulfanyl) propyl (11S, 11aS) -11-hydroxy-7-methoxy-8- (3 - (((S) 7-methoxy) -5-oxo-2,3,5,11 a-tetrahydro-1 H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8-yl) oxy) propoxy) -5oxo-2 , 3,11,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-10 (5H) -carboxylate (5)

VOS

140146

147 ns

149 (a) tert-Butyl (5- (3- (5-amino-4 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1 carbonyl) -2-methoxyphenoxy) propoxy) -2 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1carbonyl) -4-methoxyphenyl) carbamate (I46).

Boc-anhydride (0.5 ml, 2.3 mmol, 1.0 eq.) Was added to a solution of I40 (1.9 g, 2.3 mmol, 1.0 eq.) In THF (50 ml) and stirred at 55 ° C for 5 h. The solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography (50-100% ethyl acetate / hexane) to give the product as a yellow solid, 1.7 g (80%).

LC / MS (Method 1): h.p. 2.48 min, m / z (902.5) M + H.

(b) tert-Butyl (2 - ((S) -2 - (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1-carbonyl) -5- (3- (4 ((S) -2 - (( (tert-butyldimethylsilyl) oxy) methyl) pyrrolidine-1-carbonyl) -2-methoxy-5 - ((((R) -2 - ((3nitropyridin-2-yl) disulfaneyl) propoxy) carbonyl) amino) phenoxy) propoxy ) -4-methoxyphenyl) carbamate (I47).

Triphosgene (0.135 g, 0.455 mmol, 0.35 eq.) Was added to a solution of (2R) -2 - [(3-hutpo-2pyridyl) disulfanyl] propan-1-ol (0.316 g, 1.28 mmol, 1.05 eq.) and pyridine (111 mg, 1.4 mmol, 1.15 eq.) in anhydrous dichloromethane (5 ml) and stirred at room temperature for 30 min. Then the resulting solution was added to a solution of I46 (1.10 g, 1.22 mmol, 1.0 eq.) And pyridine (106 mg, 1.34 mmol, 1.1 eq.) In anhydrous dichloromethane (10 ml) and stirred at room temperature for 60 minutes. The solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography (40-50% ethyl acetate / hexane) to give the product as a yellow foam, 1.21 g (85%).

LC / MS (Method 1): h.p. 2.53 min, m / z (1174.5) M + H.

(c) tert-Butyl (2 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl) -5- (3- (4 - ((S) -2- (hydroxymethyl) pyrrolidine-1-carbonyl) -2-methoxy-5 - ((((R) -2 - ((3-nitropyridin-2-yl) disulfanyl) propoxy) carbonyl) amino) phenoxy) propoxy) -4-methoxyphenyl) carbamate (I48).

I47 (1.21 g, 1.03 mmol) was dissolved in a mixture of acetic acid (5 ml), THF (1 ml), methanol (1 ml) and water (2 ml). The resulting mixture was stirred at room temperature for 90 minutes, then evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (50 ml), washed with water (50 ml), then with saturated aqueous NaHCO ₃ (50 ml), dried (MgSO ₄ ) and evaporated under reduced pressure. The residue was purified by column chromatography (4% methanol / DCM) to give the product as a yellow solid, 0.97 g (100%).

LC / MS (Method 1): h.p. 1.87 min, m / z (946.0) M + H.

(d) tert-Butyl (11S, 11aS) -11-hydroxy-8- (3 - (((11S, 11aS) -11-hydroxy-7-methoxy-10 - (((R) -2 - ((3 ) nitropyridin-2-yl) disulfaneyl) propoxy) carbonyl) -5-oxo-2,3,5,10,11,11 a-hexahydro-1Hbenzo [e] pyrrolo [1,2-a] [1,4] diazepin-8-yl) oxy) propoxy) -7-methoxy-5-oxo-2,3,11,11 a-tetrahydro-1Hbenzo [e] pyrrolo [1,2-a] [1,4] diazepine -10 (5H) -carboxylate (I49).

Stahl solution for aerobic oxidation TEMPO (2.05 ml, 0.4 mmol, 0.2 mol / L) and then tetrakisacetonitrile copper (I) triflate (0.15 g, 0.40 mmol) were added to solution I48 (0 , 97 g, 1.0 mmol) in DXM

- 80 038267 (20 ml, 312.0 mmol). The resulting mixture was stirred at 35 ° C for 15 hours. The organic phase was washed with water (25 ml), dried (biotage) and evaporated to dryness under reduced pressure, and purified by column chromatography (3-6% methanol / DCM) to give the product as a white solid, 0.77 g (79%).

LC / MS (Method 1): h.p. 1.70 min, m / z (941.9) M + H.

(f) (R) -2 - ((3-Nitropyridin-2-yl) disulfanyl) propyl (11S, 11aS) -11-hydroxy-7-methoxy-8- (3 - (((S) 7-methoxy) -5-oxo-2,3,5,11a-tetrahydro-1H-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-8-yl) oxy) propoxy) -5oxo-2,3 , 11,11a-tetrahydro-3-benzo [e] pyrrolo [1,2-a] [1,4] diazepin-10 (5H) -carboxylate (5).

Trifluoroacetic acid (4.5 ml) was added to water (0.5 ml) and cooled to 0 ° C. This solution was then added to I49 (0.75 g, 0.80 mmol) and the resulting mixture was stirred at 0 ° C for 2 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (10 ml) and the reaction mixture was neutralized by the addition of saturated an aqueous solution of NaHCO ₃ . After drying (biotage) and evaporation under reduced pressure, the residue was purified by column chromatography (4-6% methanol / DCM) to give the product as a bright yellow solid, 0.6 g (91%).

LC / MS (Method 2): h.p. 6.04 min, m / z (824.0) M + H.

Analytical LC / MS conditions for example 5 (ii).

Positive mode electrospray mass spectrometry was performed using a Waters Aquity H-grade SQD2. Solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) were used as the mobile phase.

Method 1.

Gradient for a typical 3 minute chromatogram: The original composition 5% B was held for 25 seconds, then the concentration was increased from 5 to 100% B over 1 minute 35 seconds. This composition was held for 50 s at 100% B, then returned to 5% B within 5 s and held for 5 s. The total duration of the gradient cycle was 3.0 minutes. The flow rate was 0.8 ml / min. Detection was performed at 254 nm. Column: Waters Acquity UPLC® VEN Shield RP18, 1.7 μm, 2.1x50 mm @ 50 ° C, equipped with a Waters Acquity UPLC® VEN Shield RP18 VanGuard Guard Column, 130A, 1.7 μm, 2.1x5 mm.

Method 2.

Gradient for a typical 15 minute chromatogram: Initial 5% B was held for 1 minute, then increased from 5 to 100% B in 9 minutes. This composition was held for 2 min at 100% B, then returned to 5% B within 10 s and held at 5% B for 2 min 50 s. The total duration of the gradient cycle was 15.0 minutes. The flow rate was 0.8 ml / min (for a 3-minute chromatogram) and 0.6 ml / min (for a 15-minute chromatogram). Detection was performed at 254 nm. Column: ACE Excel 2 C18-AR, 2 μm, 3.0x100 mm, equipped with Waters Acquity UPLC® VEN Shield RP18 VanGuard Guard Column, 130A, 1.7 μm, 2.1x5 mm.

Example 6. Conjugation.

Conjugate-HER-1.

mM dithiothreitol (DTT) solution in phosphate buffered saline pH 7.4 (PBS) was added (80 mol eq / antibody, 16 μmol, 0.32 ml 50 mM solution) to 12 ml Herceptin antibody solution (30 mg , 0.2 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 2.5 mg / ml. The reconstitution mixture was heated at 25 ° C for 4 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 3 μmol, 0.08 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 1.5 mg / ml (or until complete re-oxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 1 was added as a solution in DMSO (10 molar equiv. / Antibody, 1.0 μmol in 1.5 ml DMSO) to 13.5 ml of the resulting reoxidized antibody solution (15 mg, 0.1 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 3 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (15 μmol, 0.150 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

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UPLC analysis on the Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile on a reduced sample of HER-1 conjugate at 214 and 330 nm (specific for compound 1 ) showed a mixture of light and heavy chains attached to several molecules of compound 1, which is consistent with a drug-to-antibody ratio (DAR) of 1.74 molecules of compound 1 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of HER-1 conjugate at 280 nm showed a monomer purity of over 99%. SEC analysis by HPLC showed a final ADC concentration of 1.39 mg / ml in 7.8 ml, resulting in a weight of 10.8 mg ADC (72% yield).

Conjugate-HER-2.

mM dithiothreitol (DTT) solution in phosphate buffered saline with pH 7.4 (PBS) was added (80 mol eq / antibody, 55.5 μmol, 1.11 ml of 50 mM solution) to 11.8 ml of Herceptin antibody solution (104 mg , 0.69 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 4.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 20 mol eq. / Antibody, 12.4 μmol, 0.25 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 rpm) shaking with an antibody concentration of ~ 2.4 mg / ml (or until complete reoxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 2 was added as a solution in DMSO (10 mol eq. / Antibody, 1.03 μmol in 1.40 ml of DMSO) to 14 ml of the resulting reoxidized antibody solution (15.5 mg, 0.103 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 1.5 h at 25 ° C, and then the conjugation was stopped with N-acetylcysteine (5.15 μmol, 0.051 ml at 100 mm).

Excess free drug was removed with a tangential flow filtration (TFF) unit using mPES, a 30 kDa MidiKros® fiber filter with a surface area of 115 cm ² , in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile on a reduced sample of HER-2 conjugate at 214 and 330 nm (specific for compound 2 ) showed a mixture of light and heavy chains attached to several molecules of compound 2, which is consistent with a drug-to-antibody (DAR) ratio of 1.85 molecules of compound 2 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of HER-2 conjugate at 280 nm showed a monomer purity greater than 98%. SEC analysis by HPLC showed a final ADC concentration of 0.88 mg / ml in 8.5 ml, resulting in an ADC weight of 7.5 mg (48% yield).

Conjugate-HER-3.

A mM solution of tris- (2-carboxyethyl) phosphine hydrochloride (TCEP) in phosphate buffered saline pH 7.4 (PBS) was added (40 mol eq / antibody, 40 μmol, 0.08 ml at 50 mM) to 1.39 ml of Herceptin antibody solution (15 mg, 0.1 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 4.0 mg / ml. The reconstitution mixture was heated at 37 ° C for 2 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was changed by dialysis using a 50 kDa MWCO cassette to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent for 16 hours at room temperature. A 50 mM dehydroascorbic acid solution (DHAA, 25 mol eq. / Antibody, 2.5 μmol, 0.04 ml of a 50 mM solution) was added in

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DMSO and the mixture was left to react for reoxidation for 2 h at room temperature with moderate (60 rpm) shaking with an antibody concentration of ~ 1.5 mg / ml. Due to incomplete oxidation, another 0.04 ml of 50 mM DHAA was added and further shaken at room temperature for 2 hours. Thereafter, complete reoxidation of cysteine thiols was observed by HPLC to re-form interchain cysteine disulfides. The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 3 was added as a solution in DMSO (10 mol eq. / Antibody, 0.8 μmol in 1.1 ml DMSO) to 11 ml of the resulting solution of reoxidized antibody (12 mg, 0.08 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 1 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (3.2 μmol, 0.032 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C. UPLC analysis on the Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile on a reduced sample of HER-3 conjugate at 214 and 330 nm (specific for compound 3 ) showed a mixture of light and heavy chains attached to multiple molecules of compound 3, which is consistent with a drug-to-antibody (DAR) ratio of 1.78 molecules of compound 3 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate, pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of HER-3 conjugate at 280 nm showed a monomer purity of over 94%. SEC analysis by HPLC showed a final ADC concentration of 1.14 mg / ml in 8.2 ml, resulting in a weight of 9.3 mg ADC (62% yield).

Conjugate-R347-1.

mM dithiothreitol (DTT) solution in phosphate buffered saline with pH 7.4 (PBS) was added (80 mol eq / antibody, 697 μmol, 13.87 ml of 50 mM solution) to 44.36 ml of R347 antibody solution ( 1300 mg, 8.67 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 5.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the buffer for the reduced antibody was exchanged using a tangential flow filtration (TFF) unit using mPES, a 30 kDa MidiKros® fiber filter with a surface area of 235 cm ² to a reoxidation buffer containing PBS at pH 7.4 and 1 mM EDTA to remove excess reducing agent. The recovered antibody was centrifuged for 3 minutes at 4000 rpm and then filtered using a 0.22 μm membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 130 μmol, 2.6 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of 5.0 mg / ml (or until complete re-oxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 1 was added as a solution in DMSO (10 molar equiv. / Antibody, 86.7 μmol in 23.4 ml DMSO) to 330 ml of the resulting reoxidized antibody solution (1300 mg, 8.67 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 3 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (433 μmol, 4.33 ml at 100 mM).

Excess free drug was removed with a tangential flow filtration (TFF) unit using mPES, a 30 kDa MidiKros® fiber filter with a surface area of 235 cm ² , in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was formulated with 25 mM histidine, 200 mM sucrose, pH 6.0. The ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 78 ° C.

UPLC analysis on the Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile on a reduced sample of conjugate-R347-1 at 214 and 330 nm (specific for compound 1 ), showed a mixture of light and heavy chains attached to several molecules of compound 1, which agrees

- 83 038267 with a drug-to-antibody ratio (DAR) of 1.82 molecules of Compound 1 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), the ADC sample at 280 nm showed a monomer purity of over 99%. SEC analysis by HPLC showed a final R347-1 conjugate concentration of 10.11 mg / ml in 113 ml, resulting in an ADC weight of 1141 mg (88% yield).

Conjugate-Y347-2.

mM dithiothreitol (DTT) solution in phosphate buffered saline with pH 7.4 (PBS) was added (80 mol eq / antibody, 213 μmol, 4.3 ml of 50 mM solution) to 13.7 ml of R347 antibody solution ( 400 mg, 2.67 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 5.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the buffer for the reduced antibody was exchanged using a tangential flow filtration (TFF) unit using mPES, a MidiKros® 30 kDa fiber filter with a surface area of 115 cm ² to a reoxidation buffer containing PBS at pH 7.4 and 1 mM EDTA to remove excess reducing agent. The recovered antibody was centrifuged for 3 minutes at 4000 rpm and then filtered using a 0.22 μm membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 40 μmol, 0.8 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 3.0 mg / ml (or until complete re-oxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 2 was added as a solution in DMSO (10 molar equiv. / Antibody, 26.7 μmol in 15.5 ml DMSO) to 330 ml of the resulting solution of reoxidized antibody (400 mg, 2.67 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 1.5 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (125 μmol, 1.25 ml at 100 mM).

Excess free drug was removed with a tangential flow filtration (TFF) unit using mPES, a 30 kDa MidiKros® fiber filter with a surface area of 115 cm ² , in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was formulated with 25 mM histidine, 200 mM sucrose, pH 6.0. The ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 78 ° C.

UPLC analysis on the Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile on a reduced sample of conjugate-R347-2 at 214 and 330 nm (specific for compound 2 ) showed a mixture of light and heavy chains attached to several molecules of compound 2, which is consistent with a drug-to-antibody (DAR) ratio of 1.8 molecules of compound 2 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate with a pH of 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of conjugate-R347-2 at 280 nm showed a monomer purity of more than 99%. SEC analysis by HPLC showed a final ADC concentration of 3.06 mg / ml in 93 ml, resulting in a weight of 284 mg ADC (71% yield).

Conjugate-R347-3.

mM dithiothreitol (DTT) solution in phosphate buffered saline with pH 7.4 (PBS) was added (80 mol eq / antibody, 240 μmol, 4.8 ml of 50 mM solution) to 15.36 ml of R347 antibody solution ( 450 mg, 3.0 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 5.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the buffer for the reduced antibody was exchanged using a tangential flow filtration (TFF) unit using mPES, a 30 kDa MidiKros® fiber filter with a surface area of 235 cm ² to a reoxidation buffer containing PBS at pH 7.4 and 1 mM EDTA to remove excess reducing agent. The recovered antibody was centrifuged for 3 minutes at 4000 rpm and then filtered using a 0.22 μm membrane filter. Added 50 mM dehydroascor solution

- 84 038267 binic acid (DHAA, 15 mol.eq. / Antibody, 45 μmol, 0.9 ml of a 50 mM solution) in DMSO and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 rpm ) shaking with an antibody concentration of ~ 3.5 mg / ml (or until complete reoxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 3 was added as a solution in DMSO (10 mol eq. / Antibody, 30.0 μmol in 13.0 ml DMSO) to 330 ml of the resulting reoxidized antibody solution (450 mg, 3.0 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 3 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (150 μmol, 1.5 ml at 100 mM).

Excess free drug was removed with a tangential flow filtration (TFF) unit using mPES, a 30 kDa MidiKros® fiber filter with a surface area of 235 cm ² , in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was formulated with 25 mM histidine, 200 mM sucrose, pH 6.0. The ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 78 ° C.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile of the reconstituted conjugate sample at 214 and 330 nm (specific for compound 3) showed a mixture of lungs and heavy chains attached to multiple molecules of compound 3, which is consistent with the drug to antibody ratio (DAR) of 1.82 molecules of compound 3 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate with a pH of 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of the I347-3 conjugate at 280 nm showed a monomer purity of more than 99%. SEC analysis by HPLC showed the final ADC concentration of 2.35 mg / ml in 174 ml, the resulting weight of the I347-3 conjugate was 409 mg (91% yield).

Conjugate-HLL2-1.

mM dithiothreitol (DTT) solution in phosphate buffered saline pH 7.4 (PBS) was added (80 mol eq / antibody, 53.3 μmol, 1.07 ml 50 mM solution) to 11.8 ml antibody solution HLL2 (100 mg, 0.6 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 5.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 9 μmol, 0.18 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 3.0 mg / ml (or until complete re-oxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 1 was added as a solution in DMSO (10 mol eq. / Antibody, 1.2 μmol in 0.58 ml DMSO) to 7 ml of the resulting solution of reoxidized antibody (18 mg, 0.12 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 3 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (6 μmol, 0.06 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on the Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile on a reduced sample of conjugate-HLL2-1 at 214 and 330 nm (specific for compound 1 ) showed a mixture of light and heavy chains attached to several molecules of compound 1, which is consistent with a drug-to-antibody ratio (DAR) of 1.74 molecules of compound 1 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a column

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Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm (with 4 μm guard column 3.0x20 mm) eluting at 0.3 ml / min with sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of conjugate-HLL2-1 at 280 nm, showed a monomer purity of more than 98%. SEC analysis by HPLC showed a final ADC concentration of 1.6 mg / ml in 7.5 ml, resulting in a weight of 12 mg ADC (67% yield).

Conjugate-HLL2 -2.

mM dithiothreitol (DTT) solution in phosphate buffered saline pH 7.4 (PBS) was added (80 mol eq / antibody, 53.3 μmol, 1.07 ml 50 mM solution) to 11.8 ml antibody solution HLL2 (100 mg, 0.6 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 5.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 9 μmol, 0.18 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 3.0 mg / ml (or until complete re-oxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 2 was added as a solution in DMSO (10 molar equiv. / Antibody, 1.2 μmol in 0.58 ml DMSO) to 7 ml of the resulting solution of reoxidized antibody (18 mg, 0.12 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 3 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (6 μmol, 0.06 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile, a reconstituted conjugate sample at 214 and 330 nm (specific for compound 2) showed a mixture light and heavy chains attached to multiple molecules of compound 2, which is consistent with a drug-to-antibody ratio (DAR) of 1.78 molecules of compound 2 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of the conjugate-HLL2-2 at 280 nm showed a monomer purity of more than 98%. SEC analysis by HPLC showed the final ADC concentration of 1.56 mg / ml in 8.0 ml, the resulting weight of the conjugate-HLL2-2 was 12.5 mg (69% yield).

Conjugate-HLL2-3.

mM dithiothreitol (DTT) solution in phosphate buffered saline pH 7.4 (PBS) was added (80 mol eq / antibody, 53.3 μmol, 1.07 ml 50 mM solution) to 11.8 ml antibody solution HLL2 (100 mg, 0.6 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 5.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 9 μmol, 0.18 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 3.0 mg / ml (or until complete re-oxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 3 was added as a solution in DMSO (10 molar equiv. / Antibody, 1.2 μmol in 0.58 ml DMSO) to 7 ml of the resulting reoxidized antibody solution (18 mg, 0.12 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 3 h at 25 ° C and then the conjugation was stopped using

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N-acetylcysteine (6 μmol, 0.06 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile, a reconstituted conjugate sample at 214 and 330 nm (specific for compound 3), showed a mixture light and heavy chains attached to multiple molecules of compound 3, which is consistent with a drug-to-antibody (DAR) ratio of 1.79 molecules of compound 3 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of the conjugate-HLL2-3 at 280 nm showed a monomer purity of more than 98%. SEC analysis by HPLC showed a final ADC concentration of 1.73 mg / ml in 8.2 ml, resulting in a weight of the HLL2-3 conjugate of 14.2 mg (79% yield).

Conjugate-CD7 9b-1.

mM dithiothreitol (DTT) solution in phosphate buffered saline pH 7.4 (PBS) was added (80 mol eq / antibody, 53.6 μmol, 1.07 ml 50 mM solution) to 13.9 ml antibody solution CD79b (100 mg, 0.67 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 4.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 4 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 9 μmol, 0.18 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 2.0 mg / ml (or until complete reoxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 1 was added as a solution in DMSO (10 molar equiv. / Antibody, 1.2 μmol in 1.0 ml DMSO) to 9.0 ml of the resulting reoxidized antibody solution (18 mg, 0.12 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 2 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (4.8 μmol, 0.048 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile, a reconstituted conjugate sample at 214 and 330 nm (specific for compound 1), showed a mixture light and heavy chains attached to multiple molecules of compound 1, which is consistent with a drug-to-antibody ratio (DAR) of 1.90 molecules of compound 1 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of the conjugate-CD79b-1 at 280 nm showed a monomer purity greater than 98%. SEC analysis by HPLC showed a final ADC concentration of 2.00 mg / ml in 7.85 ml, resulting in a weight of the CD79b-1 conjugate of 15.7 mg (79% yield).

Conjugate-CD7 9b-2.

mM dithiothreitol (DTT) solution in phosphate buffered saline pH 7.4 (PBS) was added (80 mol eq / antibody, 53.6 μmol, 1.07 ml 50 mM solution) to 13.9 ml antibody solution CD79b (100 mg, 0.67 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 4.0 mg / ml. The reducing mixture was heated at 25 ° C for 4 h (or until complete recovery, according to HPLC) in an orbital shaker

- 87 038267 with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 9 μmol, 0.18 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 2.0 mg / ml (or until complete reoxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 2 was added as a solution in DMSO (10 molar equiv. / Antibody, 1.2 μmol in 1.0 ml DMSO) to 9.0 ml of the resulting reoxidized antibody solution (18 mg, 0.12 μmol) to a final concentration in DMSO 10% (v / v). The solution was shaken for 2 h at 25 ° C and then the conjugation was stopped with N-acetylcysteine (4.8 μmol, 0.048 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile, a reconstituted conjugate sample at 214 and 330 nm (specific for compound 2) showed a mixture light and heavy chains attached to multiple molecules of compound 2, which is consistent with a drug-to-antibody ratio (DAR) of 1.87 molecules of compound 2 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), a sample of the conjugate-CD79b-2 at 280 nm showed a monomer purity greater than 98%. SEC analysis by HPLC showed a final ADC concentration of 2.25 mg / ml in 5.9 ml, resulting in a weight of the CD79b-2 conjugate of 13.3 mg (66% yield).

Conjugate-10-E mM solution of dithiothreitol (DTT) in phosphate buffered saline with pH 7.4 (PBS) was added (80 mol eq / antibody, 75 μmol, 1.5 ml of 50 mM solution) to 26.4 ml of 1C1 antibody solution (140 mg, 0.93 μmol) in reducing buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) with a final antibody concentration of 5.0 mg / ml. The reconstitution mixture was heated at 25 ° C for 3.5 h (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 mol eq. / Antibody, 14 μmol, 0.28 ml of a 50 mM solution) in DMSO was added and the mixture was left to react for reoxidation for 16 h at room temperature at moderate (60 v / min) shaking with an antibody concentration of ~ 3.0 mg / ml (or until complete re-oxidation of cysteine thiols with the formation of interchain cysteine disulfides, according to UHPLC). The reoxidation mixture was centrifuged for 3 minutes at 4000 rpm and then filtered through a sterilizing filter with a 0.22 μm membrane. Compound 1 was added as a solution in DMSO (10 molar equiv. / Antibody, 1.3 μmol in 0.6 ml DMSO) to 6 ml of the resulting solution of reoxidized antibody (20 mg, 0.133 μmol) to a final concentration in DMSO of 10% (v / v). The solution was shaken for 4 hours at 25 ° C and then the conjugation was stopped with N-acetylcysteine (6.65 μmol, 0.067 ml at 100 mm).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was formulated with 25 mM histidine, 200 mM sucrose, pH 6.0. The ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at -78 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile, a reconstituted conjugate sample at 214 and 330 nm (specific for compound 1), showed a mixture light and heavy chains attached to several molecules of compound 1, which is consistent with

- 88 038267 by carrying a drug-for-antibody (DAR) amounting to 1.86 molecules of Compound 1 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), the ADC sample at 280 nm showed a monomer purity of over 98%. SEC analysis by HPLC showed a final ADC concentration of 1.49 mg / ml in 8.0 ml, resulting in an ADC weight of 11.9 mg (60% yield).

Conjugate-HER-1 ++ (high DAR).

mM dithiothreitol (DTT) solution in phosphate buffered saline with pH 7.4 (PBS) was added (100 mol.eq. / antibody, 6.7 μmol, 0.13 ml of 50 mM solution) to 5 ml of antibody solution (10 mg, 67 μmol) in reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 2.0 mg / ml. The resulting mixture was incubated at 25 ° C overnight (or until complete recovery by HPLC) in an orbital shaker with moderate (60 rpm) shaking. After cooling to room temperature, the reduced antibody buffer was exchanged by spin filtration using a Vivaspin spin filter with MWCO 50 kDa to conjugation buffer containing PBS pH 7.4 and 1 mM EDTA to remove excess reducing agent. Compound 1 was added as a solution in DMSO (20 molar equiv. / Antibody, 1.34 μmol in 0.4 ml DMSO) to 4.0 ml of the resulting solution of reduced antibody (10 mg, 67 nmol) to a final concentration in DMSO 10 % (v / v). The solution was shaken for 1 h at 25 ° C and then the conjugation was stopped with an excess of N-acetylcysteine (6.7 μmol, 67 μl at 100 mM).

The resulting ADC was purified using a preparative size exclusion column (GE Sephadex 26/60) mounted on an AKTA Start instrument using PBS buffer, pH 7.4. Fractions were collected and analyzed for monomeric content using a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min filtered through sterilizing filter with SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v) at 280 nm. Fractions with a monomer content> 92% were pooled and then concentrated using 50 kDa MWCO vivaspin in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate and after complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile of the reconstituted conjugate sample at 214 and 330 nm (specific for compound 1) showed a mixture of lungs and heavy chains attached to multiple molecules of compound 1, which is consistent with the drug to antibody ratio (DAR) of 7.41 molecules of compound 1 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), the ADC sample at 280 nm showed a monomer purity of 95%. SEC analysis by HPLC showed a final ADC concentration of 1.44 mg / ml in 4.5 ml, resulting in a weight of 6.5 mg ADC (65% yield).

Conjugate-HER-1 + (average DAR).

A mM solution of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in phosphate buffered saline pH 7.4 (PBS) was added (2 mol eq / antibody, 0.134 μmol, 13.3 μl at 10 mM) to 4 ml of an antibody solution (10 mg, 67 nmol) in a reducing buffer containing PBS and 1 mM ethylenediamine tetraacetic acid (EDTA) with a final antibody concentration of 2.5 mg / ml. The reconstitution mixture was heated at 37 ° C for 2 h in an orbital shaker with moderate (60 rpm) shaking. Compound 1 was added as a solution in DMSO (15 molar equiv. / Antibody, 1.0 μmol, 0.1 ml in 10 mM in 0.3 ml DMSO) and the resulting mixture was shaken for 1.5 h at 25 ° C. and then the conjugation was stopped with N-acetylcysteine (5 μmol, 50 ml at 100 mM).

Excess free drug was removed by spin filtration using a Vivaspin spin filter with MWCO 50 kDa in a buffer containing PBS at pH 7.4. Free drug removal was monitored by RP-HPLC using undiluted conjugate. After complete removal of free drug, the ADC was filtered through a 0.22 μm Mustang filter in a sterile atmosphere and then stored at 4 ° C.

UPLC analysis on a Shimadzu Prominence system, which was carried out using a Phenomenex Aeris 3.6 μm XB-C18 150x2.1 mm column, eluting with a gradient of water and acetonitrile, recovered

- 89,038,267 conjugate sample at 214 and 330 nm (specific for compound 1) showed a mixture of light and heavy chains attached to several molecules of compound 1, which is consistent with a drug-to-antibody ratio (DAR) of 4. 2 molecules of compound 1 per antibody.

UPLC analysis on a Shimadzu Prominence system, which was performed using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150 mm column (with a 4 μm 3.0x20 mm guard column), eluting at a rate of 0.3 ml / min with sterile filtered SEC buffer, containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v), the ADC sample at 280 nm showed a monomer purity of over 99%. SEC analysis by HPLC showed a final ADC concentration of 2.05 mg / ml in 3.6 ml, resulting in a weight of the ADC of 7.36 mg (74% yield).

Example 7. In vitro analysis.

Method for determining the cytotoxicity of MTS.

Cell concentration and viability from subconfluent (80-90% confluence) cell culture in a T75 flask was measured by trypan blue staining and counted with a LUNA-II ™ automatic cell counter. Cells were diluted to 2x10 ⁵ / ml, dispensed (50 μl / well) into 96-well flat bottom plates.

A stock solution (1 ml) of the antibody-drug conjugate (ADC) for testing (20 μg / ml) was prepared by diluting the filter-sterilized ADC in cell culture medium. A series of 8x 10-fold dilutions of the original ADC was performed in a 24-well plate by serial transferring 100 μl in 900 μl of cell culture medium. Diluted ADC was dispensed (50 μl / well) in 4 replicates into the wells of a 96-well plate containing 50 μl of the cell suspension plated previously. Control wells were filled with 50 μl of cell culture medium. A 96-well plate containing cells and ADC was incubated at 37 ° C in a CO2 incubator for the exposure time.

At the end of the incubation period, cell viability was measured using the MTS assay. MTS (Promega) was placed (20 μl per well) in each well and incubated for 4 h at 37 ° C in a CO2 incubator. Absorbance in the wells was measured at 490 nm. Percent cell survival was calculated from the mean absorbance in 4 wells treated with ADC compared to the mean absorbance in 4 control, untreated wells (100%). IC _{50 was} determined from dose response data using a GraphPad Prism using a non-linear curve-smoothing algorithm: a variable slope sigmoidal dose response curve.

ADC incubation times were 4 days with SK-BR-3, MDA-MB-468, WSU-DLCL2 and SU-DHL-4, 5 days for Granta519, 6 days for BJAB, and 7 days for NCI-N87. MDA-MB-468, NCI-N87, WSU-DLCL2 and SU-DHL-4 were grown in RPMI 1640 medium with Glutamax + 10% (v / v) fetal bovine serum HyClone ™, Granta519 in DMEM + Glutamax with 10% (v / v) fetal bovine serum HyClone ™, SK-BR-3 in McCoys 5A with 10% (v / v) fetal bovine serum HyClone ™ and BJAB in RPMI 1640 + Glutamax with 20% (v / v) vol.) fetal bovine serum HyClone ™.

Method for determining the cytotoxicity of CellTiter-Glo.

Cell concentration and viability from subconfluent (80-90% confluence) cell culture in a T75 flask was measured by trypan blue staining and counted with a LUNA-II ™ automatic cell counter. Cells were diluted and distributed at 1500 cells per well (50 μl suspension / well) into white 96-well flat bottom plates.

A stock solution (1 ml) of the antibody-drug conjugate (ADC) for testing (20 μg / ml) was prepared by diluting the filter-sterilized ADC in cell culture medium. A series of 8x 10-fold dilutions of the original ADC was performed in a 24-well plate by serial transferring 100 μl in 900 μl of cell culture medium. Diluted ADC was dispensed (50 μl / well) in 4 replicates into the wells of a 96-well plate containing 50 μl of the cell suspension plated previously. Control wells were filled with 50 μl of cell culture medium. A 96-well plate containing cells and ADC was incubated at 37 ° C in a CO2 incubator for the exposure time.

At the end of the incubation period, cell viability was measured using the CellTiterGlo assay. CellTiter-Glo (Promega) was placed at 100 μl per well and shaken for 2 minutes before stabilizing at room temperature for 10 minutes. Then read the luminescence in each well. Percent cell survival was calculated from the mean absorbance in 4 wells treated with ADC compared to the mean absorbance in 4 control, untreated wells (100%). IC _{50 was} determined from dose response data using a GraphPad Prism using a non-linear curve-smoothing algorithm: a variable slope sigmoidal dose response curve.

The incubation time of the ADC for PC-3 was 6 days. PC-3 was grown in RPMI 1640 medium with Glutamax + 10% (v / v) HyClone ™ fetal bovine serum.

- 90 038267

Results.

ECso (μg / ml) SU-DHL-4 GRANTA-519 BJAB WSU-DLCL2 Conjugate-CO79b-1 1,216 49.69 0.2211 > 100 Conjugate-CO79b-2 0.4781 0.1124 0.005712 0.7559 Conjugate-B347-2 > 10

All SU-DHL-4, GRANTA519, BJAB and WSU-DL-CL2 cell lines express CD79b.

ECso (μg / ml) SK-BR-3 NCI-N87 MDA-MB-468 Conjugate-Neg-1 0.06953 0.9677 3.492 Conjugate-Neg-3 0.0175 0.02502 2,322 Conjugate-Neg-2 0.07427 0.08078 0.9533 Conjugate-HEP-1 ++ 0.1108 12.18 Conjugate-HEP-1 + 0.3226 > 10

Cell lines SK-BR-3 and NCI-N87 express Her2. The cell line MDA-MB-468 does not express HER2.

ECso (μg / ml) RS-3 Conjugate-1C1-1 0.5408 Conjugate-R347-1 > 100

Example 8 In vitro assay (i) Daudi.

Analyzed conjugates: conjugate-HLL2-1; conjugate-HLL2-2; conjugate-HLL2-3.

Ten weeks old female CB.17 SCID mice were injected with 0.1 ml of 1 x 10 ⁷ Daudi cells subcutaneously in the right flank. When the tumors reached an average size of 100-150 mm ³ , the test compounds were administered to the animals. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was a tumor size of 1500 mm ³ or 60 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone.

The ADC concentration was adjusted to give the following doses:

Conjugate Doses (mg ADC / kg body) Conjugate HLL2-1 0.6 or 1.8 Conjugate HLL2-2 0.3 or 1 Conjugate HLL2-3 0.1 or 0.3

All regimens were reasonably tolerated with minimal weight loss.

Conjugate-HLL2-1: The median time to endpoint (TTE) for vehicle-dosed control mice was 27.8 days, which corresponds to the maximum possible tumor growth delay (TGD) of 32.2 days (116%) for 60- day study. One non-treatment death was recorded on day 28 and this animal was excluded from the analysis.

The 0.6 mg / kg regimen resulted in a TGD of 48.1 days (73%), which is statistically significant compared to controls (p <0.001). In addition, this design had five out of nine 59-regression responses, consisting of two partial and three complete regressions. Six animals reached the endpoint of 1500 mm ³ , with 3 surviving after 60 days. The three survivors had an average tumor volume of 221 mm ³ . One death not related to treatment was reported and this animal was excluded from the analysis.

The 1.8 mg / kg regimen resulted in the highest possible, significant TGD (versus controls, p <0.001), and had eight out of ten survivors at day 60 and resulted in a survival advantage that was statistically significantly different from control mice. injected with vehicle (p <0.001). Eight animals showed complete regression responses. Five of these animals were tumor free after 60 days.

Both treatment regimens provided statistically significant survival benefits over controls (p <0.001 for both 0.6 and 1.8 mg / ml).

Conjugate-HLL2-2: Median time to endpoint (TTE) for vehicle-dosed control mice was 27.8 days, which corresponds to the maximum possible tumor growth delay (TGD) of 32.2 days (116%) for 60- day study.

Both 0.3 and 1 mg / kg regimens resulted in the maximum achievable TGD (32.2 days, 116%). Both of these results were statistically significant compared to the control group (p <0.001 for each regimen).

- 91 038267

80% of animals injected with 0.3 mg / kg showed regression responses consisting of two partial responses and six complete responses, four of which remained tumor free at the end of the study. Four animals dosed with 0.3 mg / kg reached the tumor volume endpoint, resulting in six surviving animals being pooled from the tumor at the end of the study.

100% of animals injected with 1 mg / kg showed regression responses and all animals were tumor free on day 60.

Both treatment regimens resulted in significant differences in overall survival compared to controls (control vs. either 0.3 or 1 mg / kg, p <0.0001).

Conjugate-HLL2-3: The median time to endpoint (TTE) for vehicle-dosed control mice was 27.8 days, which corresponds to the maximum possible tumor growth delay (TGD) of 32.2 days (116%) for 60- day study.

The 0.1 and 0.3 mg / kg regimens resulted in TGDs of 12.9 (46%) and 24.6 days (88%), respectively. Both of these results were statistically significant compared to the control group (p <0.001 for each regimen).

30% regression responses were observed in animals receiving the 0.1 mg / ml regimen with three partial responses. All animals in this group reached the tumor volume endpoint by day 60.

70% of animals injected with 0.3 mg / kg showed regression responses consisting of three partial responses and four complete responses, one of which remained tumor free at the end of the study. Four animals dosed with 0.3 mg / kg reached the tumor volume endpoint, resulting in six surviving animals being pooled from the tumor at the end of the study. Seven animals in this group reached the tumor volume endpoint, leaving three survivors for 60 days with an MTV of 750 mm ³ at the end of the study.

Both treatment regimens resulted in significant differences in overall survival compared to controls (control vs. either 0.1 or 0.13 mg / kg, p <0.0001).

(ii) JIMT-1.

Analyzed conjugates: Conjugate-Her-3.

10 weeks old female CB.17 SCID mice were injected with 0.1 ml of 1x10 ⁷ JIMT-1 cells in 50% Matrigel subcutaneously in the right flank. When the tumors reached an average size of 100-150 mm ³ , the test compounds were administered to the animals. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was the tumor size of 1000 mm ³ or 59 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone. The ADC concentration was adjusted to give 1 or 3 mg ADC / kg body weight in a single dose.

All regimens were reasonably tolerated with minimal weight loss. The median time to endpoint (TTE) for vehicle-dosed control mice was 48.4 days, which corresponds to the maximum possible tumor growth delay (TGD) of 10.6 days (22%) for the 59 day study.

A dose-dependent effect was observed where in animals receiving a dose of 1 mg / kg, the average tumor volume remained unchanged until 34 days, then progressed, while in animals receiving a dose of 3 mg / kg, there was a slight decrease in tumor size to MTV 81 mm ³ . Both regimens resulted in a maximum TGD of 10.6 days (22%) compared to the control group (P <0.001 for both treatment groups).

The 1 mg / kg regimen resulted in nine survivors in the study with an MTV of 650 mm ³ and no objective regression responses.

The 3 mg / kg regimen resulted in nine survivors with 20% objective regression responses consisting of two partial responses. The MTV of survivors in the study was 108 mm ³ . Treatment regimens did not differ significantly from each other (p> 0.05).

Both treatment regimens resulted in significant differences in overall survival compared to controls (control versus either 1 or 3 mg / kg, p <0.0001).

(iii) NCI-N87.

Analyzed conjugates: Conjugate-Her-3.

Ten weeks old female CB.17 SCID mice were injected with 0.1 ml of 1x10 ⁷ NCI-N87 cells in 50% Matrigel subcutaneously in the right flank. When the tumors reached an average size of 100-150 mm ³ , the test compounds were administered to the animals. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was a tumor volume of 800 mm ³ or 81 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone. The ADC concentration was adjusted to inject 0.3 or

- 92,038267 mg ADC / kg body weight per dose.

All regimens were reasonably tolerated with minimal weight loss. Six out of ten control tumors reached 800 mm ³ with time to endpoints (TTE) ranging from 36.8 to 81.0 days. The median time to endpoint (TTE) for vehicle-dosed control mice was 77 days, which corresponds to the maximum possible tumor growth delay (TGD) of 4 days (5%) for the 81-day study. Four control animals survived with a mean tumor volume (MTV) of 696 mm ³ .

The 0.3 and 1 mg / kg regimens resulted in TGDs of 0.5 (1%) and 4.0 days (5%), respectively. Both of these results were not statistically significant compared to controls or to each other (p> 0.05). No objective regressions were recorded in both groups. Five animals injected with 0.3 mg / kg and seven animals injected with 1 mg / kg survived with MTV 550 mm ³ in both groups.

None of the treatment regimens resulted in statistically significant survival benefits over controls (p> 0.05 for both treatment groups), and there was no significant difference between treatment groups (p> 0.05).

(iv) NCI-N87.

Analyzed conjugates: conjugate-Her-1, conjugate-Her-3.

Eight week old female CB.17 SCID mice were injected with 0.1 ml of 1 x 10 ⁷ NCI-N87 cells in 50% Matrigel subcutaneously in the right flank. When the tumors reached an average size of 100-150 mm ³ , the test compounds were administered to the animals. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was a tumor volume of 800 mm ³ or 78 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone.

The ADC concentration was adjusted to give the following doses:

Conjugate Doses (mg ADC / kg body) Conjugate Neg-1 1, 3 or 6 Conjugate Neg-3 0.3, 1 and 3

All regimens were reasonably tolerated with minimal weight loss.

Conjugate-Her-1: The median time to endpoint (TTE) for vehicle-dosed control mice was 42 days, which corresponds to the maximum possible tumor growth delay (TGD) of 36 days (86%) for the 60-day study.

The 0.6 and 1 mg / kg regimens resulted in TGDs of 9.9 (24%) and 11.6 days (28%), respectively. None of these results were statistically significant compared to controls (p> 0.05). All animals in both groups reached an endpoint of 800 mm ³ .

The mg / kg regimen resulted in the highest possible, significant TGD (versus controls, p <0.001), and had eight out of ten survivors at day 60 and resulted in a survival advantage that was statistically significantly different from the treated control mice carrier (p <0.0001). One animal reached the endpoint of 800 mm ³ on day 78, leaving nine survivors with mean tumor volumes of 550 mm ³ .

No regressive reactions were observed with the 1, 3, or 6 mg / kg regimens.

Conjugate-Her-3: The median time to endpoint (TTE) for vehicle-dosed control mice was 42.0 days, which corresponds to a maximum possible tumor growth delay (TGD) of 36.0 days (86%) for 78- day study.

The TGDs for 0.3, 1 and 3 mg / kg were 15.1 (36%), 30.6 (73%), and 36.0 (86%) days, respectively. There were significant differences for 1 and 3 mg / kg compared to controls (p <0.001 for both treatment groups, but not for 0.3 mg / kg (p> 0.05). No regressive reactions were observed in animals treated with 0 , 3 and 1 mg / kg ADC Ninety percent of regression responses were observed in animals treated with 3 mg / kg, which consisted of eight partial responses and one complete response that remained tumor free at the end of the study. mg / kg reached the endpoint of 800 mm ³ Five animals injected with 1 mg / kg reached the end point, leaving five survivors after 78 days.They had an MTV of 486 mm ^3. All ten animals injected with 3 mg / kg survived the study with MTV 63 mm ³ .

The 1 and 3 mg / kg regimens resulted in significant differences in survival compared to controls (p <0.001 for both treatment groups). The 0.3 mg / kg treatment group was not statistically significant compared to control (p> 0.05). Both groups 1 and 3 mg / kg were significantly different from each other from the 0.3 mg / kg group (p <0.001 and p <0.0001, respectively).

- 93 038267 (v) NCI-N87.

Analyzed conjugates: Conjugate-Her-2.

Eight week old female CB.17 SCID mice were injected with 0.1 ml of 1 x 10 ⁷ NCI-N87 cells in 50% Matrigel subcutaneously in the right flank. When the tumors reached an average size of 100-150 mm ³ , the test compounds were administered to the animals. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was a tumor volume of 800 mm ³ or 79 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone. The ADC concentration was adjusted to give 1 or 2 mg ADC / kg body weight in a single dose.

All regimens were reasonably tolerated with minimal weight loss. The median time to endpoint (TTE) for vehicle-dosed control mice was 49.6 days, which corresponds to the maximum possible tumor growth delay (TGD) of 29.4 days (59%) for the 79 day study.

The 1 and 2 mg / kg regimens resulted in TGDs of 7.6 (15%) and 23.6 days (48%), respectively. Both of these results were statistically significant compared to the control group (p <0.05 and p <0.001, respectively).

Eight animals injected with 1 mg / kg reached an endpoint of 800 mm ³ , leaving two survivors for 79 days with an MTV of 694 mm ³ . Seven animals in the group, which received 2 mg / ml, achieved the endpoint tumor volume to day 79, leaving three survivors at the end of the study with MTV 600 mm ^3.

Both treatment regimens resulted in significant differences in overall survival compared to controls (control versus 1 mg / kg, p <0.05; control versus 2 mg / kg, p <0.001).

Regression responses were not recorded with either design.

(vi) NCI-N87.

Analyzed conjugates: conjugate-Her-1, conjugate-1 ++.

Eight week old female CB.17 SCID mice were injected with 0.1 ml of 1x10 ⁷ NCI-N87 cells in 50% Matrigel subcutaneously in the right flank. When the tumors reached an average size of 100-150 mm ³ , the test compounds were administered to the animals. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was a tumor volume of 800 mm ³ or 81 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone.

The ADC concentration was adjusted to give the following doses:

Conjugate Doses (mg ADC / kg body) Conjugate Neg-1 6, 18, 6 (once a week x 3), 8 (once a week x 3) Conjugate HER-1 ++ 6

All regimens were reasonably tolerated with minimal weight loss.

The median time to endpoint (TTE) for vehicle-dosed control mice was 40.2 days, which corresponds to the maximum possible tumor growth delay (TGD) of 40 days (86%) for the 80-day study.

Conjugate-Her-1: single dose regimens of 6 and 18 mg / kg resulted in the maximum possible delay in tumor growth. In the 6 mg / kg single dose regimen, the mean tumor volume for 5 mice was 600 mm ³ , with no visible regression reactions. In a single dose regimen of 18 mg / kg, there were ten survivors with an average tumor volume of 36 mm ³ . There were 4 partial regressions and 6 complete regressions.

Three weekly dosing regimens of 6 and 8 mg / kg resulted in the maximum possible delay in tumor growth. In a three-week dosing regimen of 6 mg / kg, the mean tumor volume for 9 mice at the end of the study was 245 mm ³ with two partial regressions. In a three week dosing regimen of 8 mg / kg, there were ten survivors with an average tumor volume of 92 mm ³ . There were 7 partial regressions, 3 complete regressions, and 2 tumor-free survivors.

Conjugate-HER-1 ++: The 6 mg / kg single dose regimen resulted in the maximum possible delay in tumor growth. There were ten survivors with an average tumor volume of 161 mm ³ . There were 5 partial regressions, 5 complete regressions, and 2 tumor-free survivors.

(vii) NCI-N87.

Conjugates analyzed: conjugate-Her-1, conjugate-Her-1 +, conjugate-Her-1 ++. Female CB.17 SCID mice at the age of eight weeks were injected with 0.1 ml of 1 x 10 ⁷ NCI-N87 cells in 50% Matrigel subcutaneously in the right side. When the tumors reached an average size of 100-150 mm ³ , the animals were started

- 94 038267 to introduce test compounds. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was a tumor volume of 800 mm ³ or 83 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone.

The ADC concentration was adjusted to give the following doses:

Conjugate Doses (mg ADC / kg body) Conjugate Neg-1 6, 18 Conjugate HER-1 + 3, 6 Conjugate HER-1 ++ 1.5, 3

All regimens were reasonably tolerated with minimal weight loss.

The median time to endpoint (TTE) for vehicle-dosed control mice was 36.8 days, which corresponds to a maximum possible tumor growth delay (TGD) of 46.2 days (126%) for the 80-day study.

Conjugate-Her-1: Regimens 6 and 18 mg / kg resulted in delayed tumor growth by 45.9 days (125%) and 46.2 days (126%). In the 6 mg / kg regimen, the mean tumor volume for 4 mice was 564 mm ³ , with no visible regression reactions. In the 18 mg / kg regimen, there were nine survivors with an average tumor volume of 108 mm ³ . There were 9 partial regressions and 1 complete regression.

Conjugate-HER-1 ++: the 1.5 and 3 mg / kg regimen resulted in delayed tumor growth by 45.9 days (125%) and 46.2 days (126%). In the 1.5 mg / kg regimen, there were two survivors with an average tumor volume of 634 mm ³ . In the 3 mg / kg regimen, there were ten survivors with an average tumor volume of 451 mm ³ .

Conjugate-HER-1 +: regimens 3 and 6 mg / kg resulted in tumor growth retardation by 46.2 days (126%). In the 3 mg / kg regimen, there were seven survivors with an average tumor volume of 600 mm ³ . In the 6 mg / kg regimen, there were ten survivors with an average tumor volume of 256 mm ³ . There were two partial regressions.

(vii) JIMT1.

Analyzed conjugates: conjugate-Her-1, conjugate-1 ++.

Eight week old female CB.17 SCID mice were injected with 0.1 ml of 1 x ^{10 7} JIMT1 cells in 50% Matrigel subcutaneously in the right flank. When the tumors reached an average size of 100-150 mm ³ , the test compounds were administered to the animals. The mice were weighed twice a week. Tumor size was measured twice a week. The animals were monitored individually. The end point of the experiment was the tumor size of 1000 mm ³ or 60 days, whichever came first.

Groups of 10 xenograft mice were injected intravenously with 0.2 ml per 20 g body weight of antibody-drug conjugate (ADC) in phosphate buffered saline (vehicle) or 0.2 ml per 20 g body weight with vehicle alone.

The ADC concentration was adjusted to give the following doses:

Conjugate Doses (mg ADC / kg body) Conjugate hl er-1 18, 24 Conjugate HER-1 ++ 4, 6, 8

All regimens were reasonably tolerated with minimal weight loss.

The median time to endpoint (TTE) for vehicle-dosed control mice was 37.5 days, which corresponds to the maximum possible tumor growth delay (TGD) of 22.5 days (60%) for the 60-day study.

Conjugate-Her-1: regimens 18 and 24 mg / kg resulted in delayed tumor growth by 5.3 days (14%) and 3.2 days (9%). In the 18 mg / kg regimen, all animals reached an endpoint of 1000 mm ³ . In the 24 mg / kg regimen, there was one surviving animal with a tumor volume of 968 mm ³ . Both regimens resulted in a significant overall difference in survival compared to controls (P <0.01).

Conjugate-HER-1 ++: Schemes 4, 6, and 8 mg / kg resulted in a 4.4-day delay in tumor growth (12%). 6.9 days (18%) and 17.7 days (47%). In the 4 mg / kg regimen, all animals reached an endpoint of 1000 mm ³ . In the 6 mg / kg regimen, there was one surviving animal with a tumor volume of 650 mm ³ . In the 8 mg / kg regimen, there was one surviving animal with a tumor volume of 847 mm ³ . All regimens resulted in a significant overall difference in survival compared to controls (P <0.01).

- 95 038267

Example 9. Toxicological analysis.

A single dose toxicity study was used to determine the maximum tolerated dose (MTD) and safety profile of the ADC, which was conjugate-R347-1; conjugate-R347-2; conjugate-R347-3.

Male Sprague-Dawley rats (Envigo, Inc) received a single slow intravenous bolus injection of ADC via the tail vein. The vehicle used for dilution was a vehicle containing 25 mM histidine-HCl, 7% sucrose, 0.02% polysorbate 80 and having a pH of 6.0. Various parameters were assessed during the study, including mortality, exercise, observation of cage behavior, body weight, change in body weight, clinical pathology (clinical chemistry, hematology and coagulation) and indicators of macropathology. All animals were euthanized on study day 29 (SD).

Conjugate-R347-1.

Group (mg / kg) N 2 5 5 4 7 5 6 ten 5 eight 16 5 ten twenty 5 12 25 5

Tolerability was determined based on endpoint toxicity criteria, including minor reductions in hematologic parameters, microscopic assessments, and bone marrow suppression. Based on microscopic changes in the higher dose animals, the maximum tolerated dose (MTD) in rats after a single dose was considered to be 25 mg / kg.

Conjugate-R347-2.

Group Dose (mg / kg) N 2 2 5 3 5 5 4 eight 5

Tolerability was determined based on endpoint toxicity criteria. ADC was carried up to 8 mg / kg in rats after a single dosing. The findings included dose-dependent weight loss and bone marrow suppression.

Conjugate-R347-3.

Group (mg / kg) N 5 1 5 nine 2.5 5 eleven 4 5

Tolerability was determined based on endpoint toxicity criteria. ADC was carried up to 4 mg / kg in rats after a single dose. Results included dose-dependent weight loss and bone marrow suppression.

Therapeutic index.

The therapeutic index of each ADC / drug linker was calculated using the following equation:

TI = MTD in rats (mg / kg) / MED in a mouse model of potency (mg / kg).

Drug linker MTD (mg / kg) 1 25 2 eight 3 4

NCI-N87 MED (mg / kg) TI 6 4.2 2 4 1 4

Claims (17)

CLAIM (1) BMPR1B; 1. Compound of formula I or a pharmaceutically acceptable salt thereof, wherein R ⁶ and R ⁹ are H; R ⁷ represents a C _1-4 alkoxy group; R represents a C _3-12 alkylene group, the chain of which can be interrupted by benzene; Y and Y 'represent O; R ⁶ , R ⁷ , R ⁹ are selected from the same groups as R ⁶ , R ⁷ and R ^9, respectively; R ^11b represents OH; RL is a linker for binding to a cell binding agent, which is selected from: (IIIa): where Q is Q ^X is such that Q is an amino acid residue, a dipeptide residue, or a tripeptide residue; X represents where a = 0-5, b = 0-16, c = 0 or 1, d = 0-5; GL is a linker for binding to an antibody or an active fragment thereof; and (IIIb): where R ^L1 and R ^{L2 are} independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1; and (a) R ²⁰ is H and R ²¹ is OH or ORA, where R ^A is C _1-4 α-alkyl; or (b) R ²⁰ and R ²¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bonded; or (c) R ²¹ is OH or OR ^A , where R ^A is C _1-4 alkyl and R ^{20 is} selected from: ^(c-i) ό. .о (c-ii) * ^: o. ^ o (s ^- w) * - 97 038267 where R ^{Z is} selected from: (z-i) (z-ii) OC (= O) CH3; (z-iii) NO2; (z-iv) OMe; (z-v) glucuronide; (z-vi) -C (= O) -X1-NHC (= O) X ₂ -NH-R ^Z C, where -C (= O) -X1-NH- and -C (= O) -X2- NH- are naturally occurring amino acid residues and R ^{ZC is} selected from Me, OMe, OCH2CH ₂ OMe. (2) E16; 2. A compound according to claim 1, wherein R represents: (i) a C _3-7 alkylene group, the chain of which can be interrupted by benzene; or (ii) a group of the formula where r is 1 or 2. (3) STEAP1; 3. A compound according to any one of claims 1, 2, in which R ²⁰ and R ²¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bonded. (4) 0772P; 4. A compound according to claim 1 having the formula Ia, Ib or Ic: where R ^1a represents methyl; RL and R ^11b are as defined in claim 1. (5) MPF; 5. A compound according to any one of claims 1 to 4, in which RL has formula IIIa and Q is a dipeptide residue selected from: CO-Phe-Lys-NH, CO-Val-Ala-NH, CO-Val-Lys-NH, CO-Ala-Lys-NH, CO-Val-Cit-NH, CO-Phe-Cit-NH, CO-Leu-Cit-NH, CO-Ile-Cit-NH, CO-Phe-Arg-NH and CO-Trp-Cit-NH. (6) Napi3b; 6. A compound according to any one of claims 1-5, in which: a) R ^l has formula IIIa and a is 0; and / or b) R ^l has the formula IIIa and b is from 0 to 8; and / or c) R ^l has formula IIIa and d is 2. - 98 038267 (7) Sema 5b; 7. A compound according to any one of claims 1-6, in which RL has formula IIIa and GL is selected from: (G ^L1 - ¹ ) O (G ^L4 ) O Hal N — i H * where Hal = 1, Br, Cl (CH-2) η (G ^L5 ) O Ha 1— ° 4 (G ^L2 ) ο EN ο ( G ^L6 ) о 0 N Λ ky / o (G ^L3 ' ¹ ) S— (ΝΟ ₂ ) where group ΝΟ ₂ is optional (G ^L7 ) B G --- (G ^L3 - ² ) s— 0 (NO ₂ ) wherein the group NO ₂ is optional ^{(G L8) V (G L3} - ³⁾ o _2/0 where the group NO ₂ is optional (G ^{L9) N} 3 ^ (G ^{L3 '4)} ss ^ 05 ο ₂ ν - \ = Ύ where the group NO ₂ is optional where Ar represents a C _5-6 arylene group. (8) PSCA hlg; 8. The compound of claim 7, wherein GL is GL ^1-1 . (9) ETBR; 9. A compound according to claim 1, wherein the compound has the formula Id - 99 038267 where Q is selected from: (a) -CH2-; (b) -C3H6- and (c) ... (10) MSG783; - 101 038267 (11) STEAP2; 10. Conjugate of formula (II) L - (D ^L ) _P (II), where L is an antibody or an active fragment; DL is a linker moiety of a drug compound of Formula I ' where R ⁶ , R ⁷ , R ⁹ , R ^11b , Y, R, Y ', R ⁶ , R ⁷ , R ⁹ , R ²⁰ and R ²¹ are as defined in any one of claims 1 to 4; RLL is a cell binding agent linker selected from: (IIIa '): where Q and X are as defined in any one of claims 1 and 6; and GLL is a linker associated with an antibody or an active fragment thereof; and (IIIb '): where RL ¹ and RL ² are as defined in claim 1; p is an integer from 1 to 20. 11. The conjugate of claim 10, wherein the GLL is selected from: - 100 038267 (GLL1-1) 0 cba $ 0 (G ^LL6 ) о СВА? ___ // (С ^Ш ' ² ) 0 Ar _y CBA | y O (GL4 CBAg (G ^LL2 ) O O (GLL8-1) CBA Λ ^ Ν ₄ ^N C ^ ₍ G ^ll 3- ¹ ) (G ^ll 8-2) _Ν CBA (G ^ll 3-2) ^CBA P ^ (G ^LL9 - ¹ )% CBA (G ^LL4 ) CBA? ____ ί \ n Ζ ^ Ν 0 Y (G ^LL9 - ² ) .Ν <CBA (GLL5) 0 CBA 5 ___ // ° h where Ar is C _{5- 6} arylene group. (12) TrpM4; 12. The conjugate of claim 11, wherein G ^LL is G ^LL1-1 (13) CRIPTO; 13. The conjugate of claim 10, wherein D ^L is of formula (Id '). where Q is selected from: (a) -CH2-; (b) -C3H6- and (c) ... (14) CD21; 14. A conjugate according to any one of claims 10 to 13, wherein the ligand fragment is an antibody or an active fragment thereof that binds to one or more tumor-associated antigens or cell surface receptors selected from (1) to (88): 15. The conjugate according to any one of paragraphs. 10-14, where p is an integer from 1 to 8. (15) CD79b; 16. A pharmaceutical composition comprising a conjugate according to any one of claims 10-15 and a pharmaceutically acceptable diluent, carrier or excipient. (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20R-alpha; (21) Brevikan; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29) CXCR5; (30) HLA-DOB; (31) P2X5; (32) CD72; (33) LY64; (34) FcRH1; (35) IRTA2; (36) TENB2; (37) PSMA-FOLH1; (38) SST; (38 .1) SSTR2; (38 .2) SSTR5; (38 .3) SSTR1; (38 .4) SSTR3; (38 .5) SSTR4; (39) ITGAV; (40) ITGB6; (41) CEACAM5; (42) MET; (43) MUC1; (44) CA9; (45) EGFRvIII; (46) CD33; (47) CD19; (48) IL2RA; (49) AXL; (50) CD30-TNFRSF8; (51) BCMA-TNFRSF17; (52) CT Ags - CTA; (53) CD174 (Lewis Y) - FUT3; (54) CLEC14A; (55) GRP78-HSPA5; (56) CD70; (57) stem cell specific antigens; (58) ASG-5; (59) ENPP3; (60) PRR4; (61) GCC-GUCY2C; (62) Liv-1-SLC39A6; (63) 5T4; (64) CD56-NCMA1; (65) CanAg; (66) FOLR1; (67) GPNMB; (68) TIM-1-HAVCR1; - 102 038267 (69) RG-1 / prostate tumor target Mindin - Mindin / RG-1; (70) B7-H4-VTCN1; (71) PTK7; (72) CD37; (73) CD138-SDC1; (74) CD74; (75) Claudins - CL; (76) EGFR; (77) Her3; (78) RON-MST1R; (79) EPHA2; (80) CD20-MS4A1; (81) Tenascin C-TNC; (82) FAP; (83) DKK-1; (84) CD52; (85) CS1-SLAMF7; (86) Endoglin - ENG; (87) Annexin A1-ANXA1; (88) V-CAM (CD106) -VCAM1. 17. The use of a conjugate according to any one of claims 10-15 or a pharmaceutical composition according to claim 16 for the treatment of cancer in a subject.