GB2531715A

GB2531715A - Novel drug conjugates

Info

Publication number: GB2531715A
Application number: GB1418984.9A
Authority: GB
Inventors: Frigerio Mark; Godwin Antony
Original assignee: Polytherics Ltd
Current assignee: Abzena UK Ltd
Priority date: 2014-10-24
Filing date: 2014-10-24
Publication date: 2016-05-04
Also published as: GB201418984D0

Abstract

A conjugate comprising a protein, peptide and/or polymer attached to a maytansine containing payload via a linker, characterised in that the maytansine-containing payload consists of at least two maytansine moieties linked to each other through a non-degradable bridging group, with the proviso that when the conjugate comprises a protein or peptide, the linker attaching the payload to the protein or peptide is degradable. Also disclosed is a conjugating reagent that comprises said maytansine containing payload, a maytansine-containing compound as defined therein and a process for the preparation of said conjugate.

Description

Novel Drug Conjugates

Field of invention

This invention relates to novel drug conjugates comprising cytotoxic payloads linked to a protein or peptide capable of binding to a partner or target. The invention also relates to novel conjugating reagents incorporating cytotoxic payloads and functional groups capable of reaction with at least one nucleophile present in a peptide or protein and to compounds for use as payloads in drug conjugates.

Background of the invention

Much research has been devoted in recent years to the conjugation of a wide variety of payloads, for example diagnostic, therapeutic and labelling agents, to peptides and proteins for a wide range of applications. The protein or peptide itself may have therapeutic properties, and/or it may be a binding protein.

Peptides and proteins have potential use as therapeutic agents, and conjugating is one way of improving their properties. For example, water soluble, synthetic polymers, particularly polyalkylene glycols, are widely used to conjugate therapeutically active peptides or proteins. These therapeutic conjugates have been shown to alter pharmacokinetics favourably by prolonging circulation time and decreasing clearance rates, decreasing systemic toxicity, and in several cases, displaying increased clinical efficacy. The process of covalently conjugating polyethylene glycol, PEG, to proteins is commonly known as "PEGylation".

Binding proteins, particular antibodies or antibody fragments, are frequently conjugated. The specificity of binding proteins for specific markers on the surface of target cells and molecules has led to their extensive use either as diagnostic or therapeutic agents in their own right or as carriers for payloads which may include diagnostic and therapeutic agents. Such proteins conjugated to labels and reporter groups such as fluorophores, radioisotopes and enzymes find use in labelling and imaging applications, while conjugating to cytotoxic agents and chemotherapy drugs to produce antibody-drug conjuagtes (ADCs) allows targeted delivery of such agents to specific tissues or structures, for example particular cell types or growth factors, minimising the impact on normal, healthy tissue and significantly reducing the side effects associated with chemotherapy treatments. Such conjugates have extensive potential therapeutic applications in several disease areas, particularly in cancer.

The specificity of binding proteins for specific markers on the surface of target cells and molecules has led to their extensive use as carriers for a variety of diagnostic and therapeutic agents. For example, such proteins conjugated to labels and reporter groups such as fluorophores, radioisotopes and enzymes find use in labelling and imaging applications, while conjugating to cytotoxic agents and chemotherapy drugs allows targeted delivery of such agents to specific tissues or structures, for example particular cell types or growth factors, minimising the impact on normal, healthy tissue and significantly reducing the side effects associated with chemotherapy treatments. Such conjugates have extensive potential therapeutic applications in several disease areas, particularly in cancer.

It is important for optimised efficacy and to ensure dose to dose consistency that the number of conjugated moieties per binding protein is the same, and that each moiety is specifically conjugated to the same amino acid residue in each binding protein. Accordingly, a number of methods have been developed to improve the homogeneity of such conjugates. Liberatore et al, Bioconj. Chem 1990, 1, 36-50, and del Rosario et al, Bioconj. Chem. 1990, 1, 51-59 describe the use of reagents which may be used to cross-link across the disulfide bonds in proteins, including antibodies. WO 2005/007197 describes a process for the conjugating of polymers to proteins, using novel conjugating reagents having the ability to conjugate with both sulfur atoms derived from a disulfide bond in a protein to give novel thioether conjugates.

Two antibody drug conjugates have received regulatory approval: one is brentuximab vedotin, in which the drug is an auristatin, and one is trastuzumab emtansine, in which the drug is a maytansine. In both these commercially-available conjugates, the linkage of the drug to the antibody uses a linker based on maleimide. Maleimides are widely used in conjugating reagents. However, as with many other conjugating reagents, the use of maleimides presents a number of difficulties: control of the conjugating reaction is difficult, leading to products having low homogeneity, and stability of the resulting conjugates can be a problem.

Maytansines are a class of cytotoxic compounds that includes maytansine itself, a natural product isolated from the east African shrub Maytenus serrata, and related compounds known as maytansinoids (e.g. DM1, ansamitocin P-3). Maytansine and its analogues are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis.

However, due to their cytotoxicity, research has been directed to the development of conjugates containing maytansines, with a view to reducing side-effects/toxicity, improving drug delivery, or improving potency. For example, W02009/134976 discloses antibody-drug conjugates containing hydrophilic linkers incorporating a polyethylene glycol spacer, wherein the drug may, amongst other possibilities, be a maytansinoid. W02011/039721 discloses maytansinoids and their use to prepare conjugates with an antibody. Antibody-drug conjugates containing maytansines, such as trastuzumab emtansine (T-DM1), are currently in development for the treatment of various diseases including the treatment of cancer.

WO 2014/064424 discloses the use of maytansines as cytotoxic drug payloads in drug conjugates. Each maytansine is linked to a protein or peptide capable of binding to a partner or target via a linker. The linker may be a branched linker having a branching element derived from an aspartate, glutamate or similar residue coupled to two or more maytansine moieties via linker portions that degrade to release the maytansine drug payloads under physiological conditions. J. Med. Chem., 2006, 49(14), 43924408, Chari et aL, discloses maytansinoid dimers linked via short degradable bridging groups comprising 4 or 6 chain carbon atoms and a disulphide group that cleaves under physiological conditions.

There remains a need for improved means of delivering maytansines using conjugates with good stability and homogeneity, which can be prepared effectively, and which demonstrate 25 the required efficacy. We have now found a novel way of using maytansines which gives improved potency compared with previous proposals.

Summary of the invention

The present invention provides a conjugate comprising a protein, peptide and/or polymer attached to a maytansine-containing payload via a linker, a conjugating reagent useful in forming such conjugates and a maytansine-containing compound for use as payload. The maytansine-containing payloads and compounds of the invention consists of at least two maytansine moieties linked to each other through a non-degradable bridging group.

The present invention provides in a first aspect a maytansine-containing compound, in which at least two maytansine moieties (D) are linked to each other through a bridging group (Bd). The bridging group (Bd) is non-degradable under physiological conditions. Advantageously, the bridging group (Bd) has at least 3 chain carbon atoms and optionally contains poly(ethylene glycol) spacers in addition to the 3 chain carbon atoms. Advantageously, no two heteroatoms are adjacent to one another in the bridging group. Advantageously, the bridging group does not include the moiety: -C(0)-CH(NR1X)-(CH2)b-C(0)-, where b is 1, 2 or 3, R1 is selected from hydrogen and C1 to C6 alkyl, and X is any group. The maytansinecontaining compound of the first aspect, in which two maytansine moieties are present, may be represented by the following formula (I): D-Bd-D (I) In a second aspect, the invention provides a conjugating reagent, which contains a functional group capable of reaction with a peptide or protein and/or a functional group capable of reacting with a polymer, the payload being attached to the functional group(s) via one or more linkers, characterised in that the conjugation reagent comprises a maytansine-containing payload consisting of at least two maytansine moieties linked to each other through a nondegradable bridging group, with the proviso that when the conjugating reagent comprises a functional group capable of reaction with at a peptide or protein, the linker attaching the payload to the functional group capable of reaction with at a peptide or protein is degradable.

When the conjugating reagent contains a functional group capable of reaction with at least one nucleophile present in a peptide or protein, the functional group including at least one leaving group which is lost on reaction with said nucleophile, the maytansine-containing payload consisting of at least two maytansine moieties linked to each other through a non-degradable bridging group is advantageously attached to the functional group capable of reaction with at least one nucleophile present in a peptide or protein via a degradable linker. The conjugating reagent optionally contains a functional group capable of reaction with at least one nucleophile present in a peptide or protein, the functional group advantageously including at least one leaving group which is lost on reaction with said nucleophile, characterised in that the conjugation reagent comprises a maytansine-containing payload consisting of at least two maytansine moieties, especially two maytansine moieties, linked to each other through a non-degradable bridging group, and in that the payload is attached to the functional group capable of reaction with at least one nucleophile present in a peptide or protein via a linker, especially a degradable linker. The linker is suitable for linking the bridging group to a protein or peptide capable of binding to a partner or target. Preferably, the bridging group (Bd) of the maytansine-containing payload (D2Bd) is connected to a degradable linker (Lkd) that includes a degradable group which breaks under physiological conditions. The degradable group may, for example, be sensitive to hydrolytic conditions, especially acidic conditions; be susceptible to degradation under reducing conditions; or be susceptible to enzymatic degradation.

The maytansine-containing conjugating reagent of the second aspect, in which two maytansine moieties are present, may be represented by the following formula (II): D2Bd-Lk-F (II) in which D2Bd represents a maytansine-containing payload consisting of two maytansine moieties linked to each other through a non-degradable bridging group, Lk is a linker, especially a degradable linker (Lkd), and F represents a functional group capable of reaction with a peptide or protein and/or a functional group capable of reacting with a polymer.

The present invention further provides a process for the conjugation of a peptide, protein and/or a polymer, which comprises reacting said peptide, protein and/or a polymer with a conjugating reagent of the second aspect of the invention. When the conjugating reagent is reacted with a peptide or polymer, said conjugating reagent is advantageously capable of reaction with at least one nucleophile present in said peptide or protein, said reagent advantageously containing at least one leaving group which is lost on reaction with said nucleophile.

The invention also provides in a third aspect a conjugate comprising a protein, peptide and/or polymer attached to a maytansine-containing payload via a linker, characterised in that the maytansine-containing payload consists of at least two maytansine moieties linked to each other through a non-degradable bridging group. When the conjugate comprises a protein or peptide, the linker attaching the payload to the protein or peptide is advantageously degradable. The conjugate of the third aspect of the invention may, for example may, for 30 example, comprise a maytansine-containing drug moiety (D2Bd) linked via a linker (Lk), especially a degradable linker (Lkd), to a protein or peptide (Ab) capable of binding to a partner or target, wherein the maytansine-containing drug moiety (D2Bd) comprises at least two maytansine moieties (D) linked to each other through a non-degradable bridging group (Bd). A maytansine-containing conjugate of the third aspect of the invention, in which two maytansine moieties (D) are present, may be represented by the following formula (III): D2Bd-Lk-Ab (III) The linker (Lk) is advantageously a degradable linker (Lk') that includes a degradable group which cleaves under physiological conditions separating the maytansine-containing drug moiety (D2Bd) comprising at least two maytansine moieties (D) linked to each other through a bridging group (Bd) from the protein or peptide (Ab) capable of binding to a partner or target. The degradable group may, for example, be sensitive to hydrolytic conditions, especially acidic conditions; be susceptible to degradation under reducing conditions; or be susceptible to enzymatic degradation. The non-degradable bridging group (Bd) contains no groups that are susceptible to cleavage under the same conditions as those under which the degradable group in the degradable linker cleaves.

The maytansine-containing compound of the first aspect of the invention, may, for example, comprise or consist of at least two maytansine moieties (D), especially two maytansine moieties, linked to each other through a bridging group (Bd) having at least 3 chain carbon atoms, especially at least 7 chain carbon atoms, and optional poly(ethylene glycol) units in addition to the chain carbon atoms, with the proviso that no two heteroatoms are adjacent to one another in the bridging group and with the proviso that the bridging group does not include the moiety: -C(0)-CH(NRIX)-(CH2)b-C(0)-, where b is 1, 2 or 3, RI is selected from hydrogen and CI to C6 alkyl, and X is any group. Optionally, the bridging group incorporates from 0 to 8 carbonyl groups, especially from 2 to 8 carbonyl groups. The bridging group optionally incorporates from 0 to 4 unsaturated carbon-carbon double bonds; and/or from 0 to 4 C3 to C10 aryl or heteroaryl groups in the chain. Optionally, the chain is interspersed with from 0 to 11, especially from 2 to 11, chain heteroatoms selected from N, 0 and S, with the proviso that no two heteroatoms are adjacent to one another. Advantageously, a chain carbon atoms in the bridging group is substituted with a pendant connecting group selected from amine, carboxy, alkyne, azide, hydroxyl or thiol. Advantageously the bridging group includes at least one amide linkage in the chain.

The present inventors have found that compounds comprising two or more maytansine moieties linked together via a bridging group of the invention is a particularly effective way of delivering maytansines. In particular it has been found that a payload comprising two maytansine moieties linked together by the bridging groups of the invention is more potent than when two maytansine moieties are delivered separately by an antibody drug conjugate. It is believed that the bridging group of the invention is less susceptible to degradation under physiological conditions than the groups previously used to link maytansine moieties and therefore the maytansine moieties remain bound together. This is believed to lead to the surprising increase in potency. Throughout this Specification and claims, except where the context required otherwise, "degradable" should be understood to mean "degradable under physiological conditions" and "non-degradable" should be understood to mean "nondegradable under physiological conditions".

Detailed description of the invention Bridging Groups The bridging group is advantageously non-degradable and does not cleave under physiological conditions. Where the bridging group is attached to two maytansine moieties, the bridging group is advantageously bonded at either end of a chain to a maytansine moiety.

The bridging group contains at least 3 chain carbon atoms, especially at least 5 carbon atoms, such as at least 7 carbon atoms, in a chain. The bridging group preferably contains from 7 to 51 chain carbon atoms, for example from 9 to 41 chain carbon atoms, or from 11 to 37 chain carbon atoms, especially from 13 to 35, such as from 15 to 33 chain carbon atoms. The bridging group advantageously comprises an odd number of carbon atoms. For the avoidance of doubt, any optional ethylene glycol groups present in the bridging group are in addition to the chain carbon atoms and carbon atoms in ethylene glycol groups do not count towards the numbers of chain carbon atoms specified herein. The bridging group preferably includes an alkyl chain, for example an alkyl chain including at least two, especially at least 3, adjacent sp3 carbon atoms. The bridging group may also include chain carbon atoms in unsaturated, aryl or heteroaryl groups, for example in addition to an alkyl chain. For the avoidance of doubt, any chain carbon atoms in any optional unsaturated, aryl or heteroaryl groups count towards the numbers of chain carbon atoms specified herein.

The bridging group in the maytansine-containing compound of the first aspect of the invention advantageously comprises a pendant connecting group. The connecting group is preferably attached to a chain carbon atom of the bridging group (Bd) either directly or via a spacer group, such as a pendant alkyl chain, e.g. a pendant C1 to C6 alkyl chain. Preferably, the connecting group is a group that allows attachment of the bridging group (Bd) to a degradable linker (Lkd) enabling incorporation of the maytansine-containing drug moiety into a conjugating reagent of the second aspect of the invention or a drug conjugate of the third aspect of the invention. Exemplary pendant connecting groups include amine, such as primary amines, e.g. -NHR in which R is selected from hydrogen C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl or heteroaryl; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl esters, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; thiol; hydroxyl; azide and alkyne, e.g. C2 to C6 alkyne. Thus, the bridging group of the invention is preferably substituted with at least one pendant amine, carboxy, alkyne, azide, hydroxyl or thiol group. Amine and carboxy groups have been found to be particularly suitable, especially amine such as -NH2.

In addition to the at least 3 chain carbon atoms, especially at least 7 chain carbon atoms, the bridging group optionally includes poly(ethylene glycol)-containing spacers. Preferably, the bridging group includes from 0 to 4 poly(ethylene glycol)-containing spacers, especially 0 or 2 poly(ethylene glycol)-containing spacers. The poly(ethylene glycol)-containing spacers comprise 2 or more repeating ethylene glycol -(CH2-CH2-0)-units. Preferably each poly(ethylene glycol)-containing spacer consists of from 2 to 20 ethylene glycol units. The ethylene glycol units are advantageously interspersed in the chain of at least 3 carbon atoms, for example interspersed in an alkyl chain.

Advantageously, the chain of at least 3 carbon atoms, especially at least 7 chain carbon atoms, of the bridging group incorporates from 0 to 8 carbonyl groups, especially from 0 to 6 carbonyl groups, such as from 2 to 8 carbonyl groups, especially from 2 to 6 carbonyl groups.

Advantageously, the chain of at least 3 carbon atoms, especially at least 7 chain carbon atoms, of the bridging group incorporates 0 to 4 unsaturated carbon-carbon double bonds, especially 0 to 2 unsaturated carbon-carbon double bonds. Advantageously, the chain of at least 3 carbon atoms, especially at least 7 chain carbon atoms, of the bridging group incorporates and from 0 to 4 C3 to C10 aryl or heteroaryl groups, especially 0 to 2 C3 to C10 aryl or heteroaryl groups in the chain.

Advantageously, the chain of the bridging group is interspersed by from 0 to 11 chain heteroatoms selected from N, 0 and S, with the proviso that no two heteroatoms are adjacent to one another. The 0 to 11 chain heteroatoms selected from N, 0 and S are in addition to any oxygen atoms present in optional ethylene glycol units interspersed in the chain. The bridging group may, for example, include from 2 to 11 or 2 to 9 chain heteroatoms, especially from 4 to 6 chain heteroatoms, in addition to any oxygen atoms present in any optional poly(ethylene glycol) spacers. The bridging group may, for example, include at least 2 chain nitrogen atoms, especially from 2 to 6 chain nitrogen atoms, in addition to any oxygen atoms present in any optional poly(ethylene glycol) spacers. For the avoidance of doubt, any oxygen atoms present in the bridging group in ethylene glycol groups of optional poly(ethylene glycol) spacers are in addition to the interspersed heteroatoms in the chain and such oxygen atoms in ethylene glycol groups do not count towards the numbers of chain heteroatoms atoms specified herein. Advantageously, the bridging group includes from 2 to 4 amide linkages in the chain, i.e. from 2 to 4 of the from 0 to 11 chain heteroatoms are advantageously nitrogen atoms that are included as part of an amide linkage.

Advantageously, the bridging group is symmetrical, especially where the bridging group is linked to two maytansine moieties. Preferably the bridging group is symmetrical with a pendant connecting group being in the centre of the bridging group, e.g. on a central carbon atom equally spaced from the ends of the bridging group.

The bridging group advantageously comprises one or more amide linkages in the chain, especially amide linkages of the structure -C(0)-NR-in which R is selected from hydrogen C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl, especially hydrogen or methyl. The bridging group may, for example, comprise at least two amide linkages, for example from 2 to 6 amide linkages, especially 2 or 4 amide linkages. The bridging unit may, for example, includes at least 2, such as 2 or 4 amide linkages of the formula -NR-C(0)-in which R is selected from hydrogen and C1 to C6 alkyl, especially hydrogen. From 2 to 4 of the 0 to 6 carbonyl groups are advantageously included as part of an amide linkage.

The bridging group (Bd) may, for example, comprise two or more alkyl chains linked via amide linkages. Advantageously, the bridging group comprises a central portion and two peripheral portions attached to either end of the central portion via amide linkages.

Advantageously, the bridging group is symmetrical and comprises a central portion and two identical peripheral portions attached to either end of the central portion via amide linkages. The central portion of the bridging group may for example be of the formula -NR-C(0)-AleC(0)-NR-in which R is selected from hydrogen C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl, especially hydrogen or methyl, preferably hydrogen, and Ake is an alkyl chain that includes a pendant connecting group. Suitable pendant connecting groups are as described above. Other than the pendant connecting group, the alkyl chain (Ak') of the central portion of the bridging group may be unsubstituted or substituted with 0 to 3 substituent groups, especially 0 to 2 substituent groups. Suitable substituent groups for the central alkyl chain, the carbon atoms of which are chain carbon atoms in the bridging group (Bd), are discussed below. The central alkyl chain (Ake) typically includes from 3 to 11 carbon atoms, especially from 3 to 7 chain carbon atoms, such as 5 chain carbon atoms. The central alkyl chain (Ale) typically includes an odd number of chain carbon atoms. The pendant connecting group is typically attached to the central chain carbon atom. The central alkyl chain (Ake) is typically symmetrical.

Where the bridging group is linked to two maytansine moieties, the bridging group (Bd) is advantageously linked to the maytansine moiety at each end via a carbonyl group. Where the bridging group (Bd) is linked to more than two maytansine moieties, the bringing group is advantageously branched, with the ends of the branches advantageously being linked to a maytansine moiety via a carbonyl group. The carbonyl group may be part of the maytansine moiety (e.g. if the maytansine moiety is of the structure (A), (B), (C), or (D) described below) or part of the bridging group (e.g. where the maytansine moiety is of the structure (E) described below it is preferred that the bridging group terminates at each end in a carbonyl group). Preferably, the carbonyl group that links each maytansine moiety to the bridging group is connected to a terminal alkyl chain (Ale), terminal alkyl chains (Akt) being included at each end of the bridging group (Bd). The terminal alkyl chains (Akt) of the bridging group may be unsubstituted or substituted with 0 to 4 substituent groups, especially 0 to 2 substituent groups. Suitable substituent groups for the terminal alkyl chain, the carbon atoms of which are chain carbon atoms in the bridging group (Bd), are discussed below. The terminal alkyl chains (Akt) typically include from 1 to 9 carbon atoms, especially from 3 to 7 chain carbon atoms such as 5 chain carbon atoms, not including the carbonyl carbon atom by which the terminal alkyl chain is linked to the maytansine moiety. The terminal alkyl chains (Akt) are advantageously linked to the adjoining section of the bridging group (Bd) via amide linkages. The terminal alkyl chains (Akt) may, for example be connected to the central alkyl chain (Ale) via an amide linkage. Alternatively, there may be an intermediate moiety (Lk') between the terminal alkyl chains (Akt) and the central portion of the bridging group (Bd).

The intermediate moiety (LW) may, for example, include unsaturated, aryl or heteroaryl groups or include a poly(ethylene glycol) unit, e.g. in addition to one or more alkyl chain portions. The intermediate moiety (LW) advantageously includes a poly(ethylene glycol) unit. The intermediate moiety (LW) is advantageously linked to the terminal alkyl chains (AW) and to the central alkyl chain (Al() via amide linkages.

Optionally, the at least 3, for example from 7 to 41, chain carbon atoms in the bridging group (Bd), including carbon atoms in any unsaturated, aryl or heteroaryl groups incorporated in an alkyl chain, are substituted with 0 to 8 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C2 to C6 alkyne; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; -NR1R2, wherein each of Wand R2 is independently selected from hydrogen and C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl; azide; thiol; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl esters, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; and C3 to C10 aryl or heteroaryl groups. The C3 to C10 aryl or heteroaryl substituent groups may themselves be optionally substituted with 1 to 4 substituent groups each independently selected from hydroxyl; halo; Cl to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl, C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR1R2, wherein each of RI and R2 is independently selected from hydrogen C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl. The substituent groups on the chain carbon atoms advantageously comprise at least one pendant connecting group substituent as described above, for example, at least one amine, carboxy, alkyne, azide, hydroxyl or thiol group. Preferably, at least one sp3 carbon atom in the bridging group is substituted with amine, such as primary amines, e.g. -NHR in which R is selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6haloalkyl esters, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; thiol; hydroxyl; azide and alkyne, e.g. C2 to C6 alkyne. More preferably at least one sp3 carbon atom in the bridging group is substituted with -NHR in which R is selected from hydrogen C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl or heteroaryl groups, especially -N112.

Chain N atoms in the bridging group, such as N atoms interspersed in an alkyl chain, may be unsubstituted (i.e. secondary amine groups attached to hydrogen) or substituted with C1 to C6 alkyl or C2 to C6 alkenyl both optionally substituted with halo or hydroxyl or C3 to C10 aryl. Preferably, the chain of at least 7 carbon atoms includes at least 2, such as 2 or four amide linkages in the chain.

In one embodiment, the bridging group is a chain of at least 3 carbon atoms especially at least 7 carbon atoms, is interspersed with from 2 to 11 heteroatoms selected from N, 0 and S in the chain and including at least 2 chain nitrogen atoms, preferably at least 4 chain nitrogen atoms, with the proviso that no two heteroatoms are adjacent to one another, the chain of at least 3 carbon atoms incorporating from 2 to 8 carbonyl groups, especially from 2 to 6 10 carbonyl groups; from 0 to 4 unsaturated carbon-carbon double bonds, especially 0 to 2 unsaturated carbon-carbon double bonds; and from 0 to 4 C3 to C10 aryl or heteroaryl groups, especially 0 to 2 C3 to C10 aryl or heteroaryl groups, wherein the chain of at least 3 carbon atoms advantageously includes at least 2, such as 2 or 4 amide linkages in the chain. In another embodiment, the bridging group is a chain of at least 3 carbon atoms, especially at least 7 carbon atoms, is interspersed with 0 to 4 poly(ethylene glycol) spacers each comprising from 2 to 20 ethylene glycols units and interspersed with from 2 to 11 chain heteroatoms selected from N, 0 and S in the chain and including at least 2 chain nitrogen atoms, preferably at least 4 chain nitrogen atoms, with the proviso that no two heteroatoms arc adjacent to one another, the chain of at least 3 carbon atoms incorporating from 2 to 8 carbonyl groups, especially from 2 to 6 carbonyl groups, from 0 to 4 unsaturated carbon-carbon double bonds, especially 0 to 2 unsaturated carbon-carbon double bonds, and from 0 to 4 C3 to Cm aryl or heteroaryl groups, especially 0 to 2 C3 to Cm aryl or heteroaryl groups. In a further embodiment, the bridging group is a chain of at least 7 carbon atoms interspersed with 0, 2 or 4 poly(ethylene glycol) spacers each comprising from 2 to 20 ethylene glycols units and interspersed with from 2 to 11 chain heteroatoms selected from N, 0 and S and including at least 2 chain nitrogen atoms, preferably at least 4 chain nitrogen atoms, with the proviso that no two heteroatoms are adjacent to one another, the chain of at least 7 carbon atoms incorporating from 2 to 8 carbonyl groups, especially from 2 to 6 carbonyl groups, from 0 to 4 unsaturated carbon-carbon double bonds, especially 0 to 2 unsaturated carbon-carbon double bonds, and from 0 to 4 C3 to Co aryl or heteroaryl groups, especially 0 to 2 C3 to Cm aryl or heteroaryl groups, wherein the chain of at least 7 carbon atoms includes at least 2, such as 2 or 4 amide linkages in the chain.

The bridging group advantageously does not include a group that degrades under physiological conditions to cleave the bridging group. Preferably, the bridging group does not include a dipeptide portion having 2 or more adjacent amino acid residues. Preferably, the bridging group does not include: o x in which X is any group. Preferably the bridging group does not include a group susceptible to hydrolysis under acidic conditions, such as a group selected from hydrazones, semicarbazones, thiosemicarboazones, cis-acotinic amides, orthoesters and ketals in the chain. Preferably the bridging group does not include: <s, H in the chain. Preferably, the bridging group does not include: in the chain. Preferably, the bridging group does not include:

H

in which A is an amino acid residue, in the chain.

Advantageously, the bridging group does not include in the chain: * the moiety: -NH-CH(X)-C(0)-NH-CH(X)-C(0)-in which X is any group, especially a dipeptide portion having 2 or more adjacent amino acid residues, * a group selected from a hydrazone, semicarbazone, thiosemicarboazone, cis-acotinic amide, orthoester or ketal; or <N residue.

* in which A is an amino acid Preferably, the maytansine-containing compound of the first aspect of the invention comprises two maytansine moieties (D) linked via a bridging group having a chain of at least 3 carbon atoms, especially at least 7 carbon atoms; wherein the chain carbon atoms are interspersed with from 0 to 11 heteroatoms, especially at least 2 heteroatoms, selected from N, 0 and S, such as at least 2 chain nitrogen atoms, with the proviso that no two heteroatoms are adjacent to one another; wherein the chain carries at least one pendant amine, carboxy, alkyne, azide, hydroxyl or thiol connector group, wherein the chain optionally includes at least 2, especially from 2 to 4, amide linkages; wherein optional poly(ethylene glycol) spacers are interspersed in the chain in addition to the chain carbon atoms; and wherein, the chain does not include the moiety: -C(0)-CH(NRIX)-(CH2)b-C(0)-, where b is 1, 2 or 3, R1 is selected from hydrogen and C1 to C6 alkyl, and X is any group.

Optionally the maytansine-containing compound is of the formula (I): D-Ak-D (I) in which: each D represents a maytansine moiety; and Ak is an alkyl chain, wherein the alkyl chain is substituted with 0 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C2 to C6 alkyne; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; -NRIR2, wherein each of RI and R2 is independently selected from hydrogen and Cl to C6 alkyl, C1 to C6 haloalkyl, and C3 to C10 aryl or heteroaryl; azide; thiol; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl ester, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; and C3 to Ci0 aryl or heteroaryl groups, the C3 to C10 aryl or heteroaryl substituent groups may themselves be optionally substituted with 1 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR1R2, wherein each of R1 and R2 is independently selected from hydrogen, Cl to C6 alkyl, C1 to C6 haloalkyl, and C3 to C10 aryl or heteroaryl; wherein the alkyl chain is interspersed with from 0 to 6, especially from 2 to 6, carbonyl groups from 0 to 4 unsaturated carbon-carbon double bonds and from 0 to 4 C3 to Co aryl or heteroaryl groups, wherein the two maytansine moieties (D) are separated by a chain of at least 3 carbon atoms, especially at least 7 carbon atoms, including sp3 carbon atoms in the alkyl chain and carbon atoms in any carbonyl groups, unsaturated carbon-carbon double bonds and aryl or heteroaryl groups interspersed in the alkyl chain; wherein alkyl chain is interspersed with from 0 to 11, especially from 2 to 11 heteroatoms, selected from N, 0 and S, with the proviso that no two heteroatoms are adjacent to one another; and wherein the alkyl chain carries at least one pendant amine, carboxy, alkyne, azide, hydroxyl or thiol connector group as described above; and wherein the alkyl chain does not include the moiety: -C(0)-CH(NR1X)-(CH2)1,-C(0)-, where b is 1, 2 or 3, Rl is selected from hydrogen and C1 to C6 alkyl, and X is any group.

Optionally, the maytansine-containing compound is of the formula (Ia): D-Ak1-[PEG]"-A1(2-[PEG]"-Ak3-D (Ia) in which: each D represents a maytansine moiety; [PEG]" is a poly(ethylene glycol) comprising from 2 to 20 ethylene glycol units; and Akl, Ak2 and Ak3 are each alkyl groups containing at least one carbon atom wherein Aki, Ak2 and Ak3 together have at least 7, for example from 7 to 51, chain carbon atoms substituted with 0 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to Co alkenyl; C2 to C6 alkyne; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; -NR1R2, wherein each of R1 and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to Cc, haloalkyl, and C3 to C10 aryl or heteroaryl; azide; thiol; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl ester, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; and C3 to C10 aryl or heteroaryl groups, the C3 to C,0 aryl or heteroaryl substituent groups may themselves be optionally substituted with 1 to 4 substituent groups each independently selected from hydroxyl; halo; C, to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR1R2, wherein each of le and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl, and C3 to C,0 aryl or heteroaryl; wherein Ak1, Ak2 and Ale are together interspersed with from 0 to 6 carbonyl groups, from 0 to 4 unsaturated carbon-carbon double bonds and from 0 to 4 C3 to C10 aryl or heteroaryl groups, wherein Ak1, Ak2 and Ale are together interspersed with from 0 to 11 heteroatoms selected 5 from N, 0 and S, with the proviso that no two heteroatoms are adjacent to one another; wherein Ak1, Ak2 and Aka together have at least 7, for example from 7 to 51, chain carbon atoms including sp3 carbon atoms and carbon atoms in any carbonyl groups, unsaturated carbon-carbon double bonds and aryl or heteroaryl groups interspersed in the alkyl chain; wherein the alkyl chain carries at least one pendant connector group as described above; 10 and wherein the alkyl chain does not include the moiety: -C(0)-CH(NRIX)-(CH2)0-C(0)-, where b is 1, 2 or 3, R1 is selected from hydrogen and C1 to C6 alkyl, and X is any group.

Optionally, the maytansine-containing compound is of the formula (Ib): D1C(0)1t-Akt-N(R)-C(0)-LkI-N(R)-C(0)-Ake-C(0)-N(R)-Lkl-C(0)-N(R)-AWIC(0) 1t-D (lb) in which: each D represents a maytansine moiety; t is 0 to 1, t being 0 if the maytansine moiety includes a carbonyl group at the point where it is attached to the bridging group; Akt represents an alkyl chain having from 1 to 9 chain carbon atoms, especially from 3 to 7 chain carbon atoms such as 5 chain carbon atoms, substituted with 0 to 4, especially 0 to 2, substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C2 to C6 alkyne; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; -NR'R2, wherein each of R1 and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl, and C3 to C10 aryl or heteroaryl; azide; thiol; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl ester, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; and C3 to C10 aryl or heteroaryl groups, the C3 to C,0 aryl or heteroaryl substituent groups may themselves be optionally substituted with 1 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR1142, wherein each of R' and R2 is independently selected from hydrogen, C1to C6 alkyl, C1 to C6 haloalkyl, and C3 to C10 aryl or heteroaryl; Lk' is a linking group which advantageously includes at least one unsaturated, aryl or heteroaryl groups or includes a poly(ethylene glycol) unit, especially a poly(ethylene glycol) unit; and Ake represents an alkyl chain having an odd number of chain carbon atoms from 1 to 9, especially from 3 to 7, such as 5 the central carbon atom being substituted with a pendant connecting group selected from an amine, such as a primary amine; a carboxylic acid, including activated carboxylic acids such as acid chlorides and acid anhydrides; an ester; and and a thiol, the alkyl chain being additionally substituted with 0 to 3, especially 0 to 2, substituent groups each independently selected from hydroxyl; halo; C1 to Co alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C2 to C6 alkyne; Cl to Co alkoxy optionally substituted with halo or hydroxyl; -NR1R2, wherein each of R1 and R2 is independently selected from hydrogen,C1 to Co alkyl, C1 to C6 haloalkyl, and C3 to Cio aryl or heteroaryl; azide; thiol; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl ester, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; and C3 to C10 aryl or heteroaryl groups, the C3 to C10 aryl or heteroaryl substituent groups may themselves be optionally substituted with 1 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR1R2, wherein each of R1 and R2 is independently selected from hydrogen and C1 to C6 alkyl and C1 to C6 haloalkyl.

Optionally, the maytansine-containing compound is of the formula (Ic): D-[C(0)]1-Akt-N(R)-C(0)-Akc-C(0)-N(R)-Ale-[C(0)]t-D (Ic) in which: each D represents a maytansine moiety; t is 0 to 1, t being 0 if the maytansine moiety includes a carbonyl group at the point where it is attached to the bridging group; Ale represents an alkyl chain having from 1 to 9 chain carbon atoms, especially from 3 to 7 chain carbon atoms such as 5 chain carbon atoms, substituted with 0 to 4, especially 0 to 2, substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C2 to C6 alkyne; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; -NR1R2, wherein each of R1 and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to Co haloalkyl, and C3 to Cio aryl or heteroaryl; azide; thiol; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to -C6 haloalkyl ester, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; and C3 to C10 aryl or heteroaryl groups, the C3 to C10 aryl or heteroaryl substituent groups may themselves be optionally substituted with 1 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR1R2, wherein each of RI and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl, and C3 to C10 aryl or heteroaryl; and Ake represents an alkyl chain having an odd number of chain carbon atoms from 1 to 9, especially from 3 to 7, such as 5, wherein the central carbon atom of the alkyl chain of Ake is substituted with a pendant connecting group selected from amine, such as primary amines, e.g. -NHR in which R is selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl and C4 to C10 aryl or heteroaryl; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl esters, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; thiol; hydroxyl; azide and alkyne, e.g. C2 to C6 alkyne, either directly or via a pendant C1 to C6 alkyl group, and wherein the alkyl chain of Ale is additionally substituted with 0 to 3, especially 0 to 2, substituent groups each independently selected from hydroxyl; halo; Cl to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C2 10 C6 alkyne; C1 to C5 alkoxy optionally substituted with halo or hydroxyl; -NRIR2, wherein each of RI and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl, and C3 to C10 aryl or heteroaryl; azide; thiol; carboxy, including carboxylic acid, ester e.g. C1 to C6 alkyl or C1 to C6 haloalkyl ester, and activated carboxylic acids such as acid chlorides and acid anhydrides, especially carboxylic acid; and C3 to C10 aryl or heteroaryl groups, the C3 to C10 aryl or heteroaryl substituent groups may themselves be optionally substituted with 1 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl; C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR1R2, wherein each of R' and R2 is independently selected from hydrogen and C1 to C6 alkyl and C1 to C6 haloalkyl.

Maytansine moieties The maytansine-containing compound of the first aspect of the invention, the maytansinecontaining conjugating reagent of the second aspect of the invention, and the maytansinecontaining conjugate of the third aspect of the invention comprises at least two, for example 2, 3 or 4, preferably two, maytansine moieties (D) linked to each other through a bridging group (Bd). D represents a maytansine moiety (i.e. the bridging group (Bd) is bonded to the residue of a maytansine). The tenn "maytansine" includes compounds such as maytansine itself, maytansinoids such as 15-methoxyansamitocin P-3, and derivatives thereof.

Preferably the maytansine is a compound containing substructure (A) Rd (A), in which X represents 0 or S; Ra represents hydrogen or Ci_alkyl; Rb represents hydrogen, hydroxy, Ci_alkoxy or ChalkylC(0)0-; Rc represents hydrogen, hydroxy, ehalkoxy or Ch alkylC(0)0-; Rd represents hydrogen or Chalkyl; Re represents halogen or hydrogen, and Rf represents hydrogen or Chalkyl.

Preferably X represents 0. Preferably Ra represents Chalkyl, especially methyl. Preferably Rb represents hydrogen. Preferably Rc represents hydrogen or methoxy, more preferably hydrogen. Preferably Rd represents Chalkyl, especially methyl. Preferably Re represents chlorine or hydrogen, especially chlorine. Preferably Rf represents Chalkyl, especially methyl.

More preferably, the maytansine comprises substructure (B) Re Rf 0 Rd 0

XH H Ram

H

N Rb (B),

in which X and Ra-Rf have the meanings set out above.

Still more preferably, the maytansine comprises substructure (C) or (D) most preferably (C). MeO (C),

In some preferred embodiments, the maytansine includes the following group (E) bonded to the ester carbonyl carbon atom of substructure (A), (B), (C) or (D): (E), For example the maytansine may comprise substructure (F): MeO In some preferred embodiments, the maytansine includes one of the following groups bonded to the ester carbonyl carbon atom of substructure (A), (B), (C) or (D): In some preferred embodiments, the maytansine contains group (L) bonded to the ester carbonyl carbon atom of substructure (A), (B), (C) or (D): 0 (H), 0 (I),

SH

(J), (14 Examples of specific preferred maytansines include: MeO (M) (Maytansine), MeO (N), MeO (0) (Ansamitocin P-3), Me0 (P) (Mertansine, DM1), MeO (Q) (S-methyl DM1), Me0 SH (R) (DM4), and (S) (15-methoxyansamitocin P-3).

The bridging group (Bd) may be bonded to the maytansine moiety (D) at any suitable point. Where the maytansine moiety corresponds to a maytansine comprising group (E), Bd may for example be bonded to the nitrogen atom of group (E), e.g.: Me0 (T).

In the conjugating reagent and conjugates of the second and third aspect of the invention the payload (D2Bd-) comprises two maytansine moieties (D) linked via a bridging group (Bd).

The conjugating reagent and conjugates may comprise a single payload (D2Bd-) comprising two maytansine moieties (D) or multiple payloads, for example from 2 to 9 payloads (D2Bd-), 10 each payload (D2Bd-) comprising two maytansine moieties (D) linked via a bridging group (Bd).

Conjugating reagents The conjugating reagent of the second aspect of the invention comprises a maytansine-containing payload consisting of at least two maytansine moieties linked to each other through a non-degradable bridging group. The payload is attached via at least one linker to at least one functional group capable of reaction with a peptide, protein and/or polymer. For example, the conjugating reagent may comprise a functional group capable of reacting with at least one nucleophile present in a peptide or protein which functional group is attached to the payload via a degradable linker. Advantageously, the bridging group is attached to the linker.

Advantageously, the bridging group bears a pendant connecting group, especially an amine, carboxy, alkyne, azide, hydroxyl or thiol group through which the bridging group is attached, via the linker or linkers to the functional group capable of reaction with a peptide, protein and/or polymer, for example, with at least one nucleophile present in a peptide or protein. The bridging group is as defined above, for example with respect to the maytansine-containing compounds of the first aspect of the invention.

Many conjugating reagents which can be used to conjugate a payload to a protein are known, and the novel conjugating reagents of the second aspect of the invention differ from these known reagents in the nature of the payload they contain. In particular, the conjugating reagents of the invention comprise a novel payload comprising at least two, for example 2, 3 or 4, preferably two, maytansine moieties (D) linked to each other through a bridging group (Bd). The novel payload in the conjugating reagent of the second aspect of the invention is advantageously the maytansine-containing compound of the first aspect of the invention.

The novel conjugating reagents of the second aspect of the present invention may be prepared by methods analogous to known methods. Specific reactions are illustrated in the Examples which follow.

A conjugating reagent according to the second aspect of the invention is capable of reacting with a protein or peptide and/or a polymer and hence becoming chemically bonded thereto to produce a conjugate according to the invention. Any type of known conjugation reaction may be used. For example, the reaction may be carried using the known methods of maleimide bonding, amine conjugation, or click chemistry. For example, the reagent may contain a maleimide group, a click-chemistry group, for example an azide or alkyne group, an amine group or a carboxyl group. Other possible approaches include the use of proteins that have been recombinantly engineered with an amino acid specifically for conjugation such as engineered cysteines or non-natural amino acids, and enzymatic conjugation through a specific enzymatic reaction such as with transglutaminase. The reaction site on the protein may be either nucleophilic or electrophilic in nature. Common protein conjugation sites are at lysine or cysteine amino acid residues or carbohydrate moieties.

One preferred method of conjugation uses maleimide bonding.

A conjugating reagent according to the second aspect of the invention is advantageously capable of reacting with a nucleophile in a protein or peptide and hence becoming chemically bonded to the protein or peptide. As such the conjugating reagent typically includes at least one leaving group which is lost on reaction with a nucleophile. The conjugating reagent may, for example, include two or more leaving groups. Preferably the conjugating reagent according to the second aspect of the invention is capable of reacting with two nucleophiles. Advantageously, the conjugating reagent according to the second aspect of the invention comprises at least two leaving groups. If two or more leaving groups are present, these may be the same or different. Alternatively, a conjugating reagent may contain a single group which is chemically equivalent to two leaving groups and which single group is capable of reacting with two nucleophiles.

One group of reagents is based on the bis-halo-or bis-thio-maleimides and derivatives thereof as described in Smith et at, J. Am. Chem. Sac,. 2010, 132, 1960-1965, and Schumaker et al., Bioconj. Chem., 2011, 22, 132-136. These reagents contain the functional 20 grouping: in which at least one, preferably each, L is a leaving group according to the invention. The nitrogen atom of the maleimide ring may carry a payload, for example a diagnostic, therapeutic or labelling agent, or a binding agent for a diagnostic, therapeutic or labelling agent, for example one of the formula 13213d-Q-mentioned below.

Similarly, maleimides containing a single leaving group L: may be used. Again, the nitrogen atom of the maleimide ring may carry a payload, for example one of the formula D2Bd-O-mentioned below.

Also, maleimides lacking a leaving group: may be used. Again, the nitrogen atom of the maleimide ring may carry a payload, for example one of the formula D2Bd-Q-mentioned below.

In a preferred embodiment of the invention, the conjugating reagent contains the functional grouping:

A-L

B-L (BI) in which W represents an electron-withdrawing group, for example a keto group, an ester group -0-00-, a sulfone group -SO2-, or a cyano group; A represents a C1,5 alkylene or alkenylene chain; B represents a bond or a C14 alkylene or alkenylene chain; and either each L independently represents a leaving group, at least one of which, preferably both of which, must be a novel leaving group according to the invention, or both Is together represent a leaving group according to the invention. Reagents of this type using conventional leaving groups are described in Bioconj. Chem 1990(1), 36-50, Bioconj. Chem 1990(1), 51-59, and J. Am. Chem. Soc. 110, 5211-5212. When reagents containing such groups react with proteins, a first leaving group L is lost to form in situ a conjugating reagent containing a functional grouping of formula: rtfv, W (A-L f-H (BP) in which m is 0 to 4, which reacts with a first nucleophile. The second leaving group L is then lost, and reaction with a second nucleophile occurs. As an alternative to using a reagent containing the functional grouping I as starting material, reagents containing the functional grouping I' may be used as starting material.

Preferably W represents a keto group. Preferably A represents -CH2-and B represents a bond.

Particularly preferred functional groupings of formula BI and BP have the formulae: (BIa) or JUL' W L (BIa') For example, the group may be of the formula:

L

(BIb) or (BIb') Another group of conjugating reagents contains the functional grouping: -W-CR4R41-CR4.LL' (BI1) in which W has the meaning and the preferred meanings given above, and either each R4 represents a hydrogen atom or a Ch.salkyl group, R4' represents a hydrogen atom, and either each L independently represents a leaving group, at least one of which, preferably both of which, must be a novel leaving group according to the invention, or both Ls together represent a leaving group according to the invention; or each R4 represents a hydrogen atom or a Ci_alkyl group, L represents a leaving group according to the present invention, and R4I and Lir together represent a bond.

Mother group of conjugating reagents includes the functional grouping: -W-(CH=CH)p-(CH2)2-L (Bill) or -W-(CH=CH)p-CH=CH2 (Bill) in which W has the meaning and preferred meanings given above and p represents 0 or an integer of from 1 to 4, preferably 0. An especially preferred reagent of this type includes the functional grouping: -NH-CO-Ar-00-(CH2)2-L (Bilk) or -NH-CO-Ar-CO-CH=CH2 (BIIIa) in which Ar represents an optionally substituted aryl, especially phenyl, group.

A leaving group L for example be -SP, -OP, -SO2P, -OSO2P, -N4PR2R3, halogen,-00, in which P represents a hydrogen atom an alkyl (preferably Ch6alkyl), aryl (preferably phenyl),alkyl-aryl (preferably Ci_6alkyl-phenyl) group, or is a group which includes a portion -(CH2CH20)1 in which n is a number of two or more, and each of R2 and R3 independently represents a hydrogen atom, a Ci_alkyl group, or a group P, and 0 represents a substituted aryl, especially phenyl, group, containing at least one electron withdrawing substituent, for example -CN,-NO2, -CO2Ra, -COH, -CH2OH, -CORa, - -000Ra, -00O21r, -SORa, -S021ta, -NHCO Ra, -NRa, CORa, -NHCO2Ra, -NPCO21V, -NO, -NHOH, -NRa OH, -C=N-NHCORa, -C=N-NRa CORa, -N÷Ra 3, -Nlifta 2, -N1-12Ra, halogen, especially chlorine or, especially, fluorine, -CC121, -C=CRa 2 and -C=CHRa, in which each Ra represents a hydrogen atom or an alkyl (preferably Ci_6alkyl), aryl (preferably phenyl), or alkyl-aryl (preferably Ci.6alkyl-phenyl) group.

Conjugating reagents in which P represents a group which includes a portion -(CH2CH20).-30 in which n is a number of two or more are the subject of our copending application ref. 22955. This application discloses the following: "The leaving group may for example include -(CH2CH20)"-R1 where R' is a capping group. A very wide range of capping groups may be used. R' may for example be a hydrogen atom, an alkyl group, especially a Ci_alkyl group, particularly a methyl group, or an optionally substituted aryl group, for example an optionally substituted phenyl group, for example a tolyl group. Alternatively, the capping group may include a functional group such as a carboxyl group or an amine group. Such capping groups may for example have the formula -CH2CH2CO2H or -CH2CH2NH2, and may be prepared by functionalising the terminal unit of a KCH2CH20)"-chain.

Alternatively, rather than being terminated by a capping group, the -(C1-19CH20)"-group may have two points of attachment within the conjugating reagent such that chemically the equivalent of two leaving groups are present,two nucleophiles.

The -(CH2CH20).-portion of the leaving group is based on PEG, polyethylene glycol.

The PEG may be straight-chain or branched, and it may be derivatised or functionalised in any way. n is a number of 2 or more, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10. For example, n may be from 5 to 9. Alternatively, n may be a number of 10 or more. There is no particular upper limit for n. n may for example be 150 or less, for example 120 or less, for example 100 or less. For example n may be from 2 to 150, for example from 7 to 150, for example from 7 to 120. The PEG portion -(CH2CH20)11-of a leaving group may for example have a molecular weight of from 1 to 5 kDa; it may for example be lkDa, 2kDa, 3kDa, 4kDa or 5kDa. A leaving group may if desired contain two or more portions -(CH2CH20)"-separated by one or more spacers.

A leaving group in a according to the invention is suitably of the formula -SP, OP, -SO2P, -0502P, -1\1+PR2R3, in which P is a group which includes a portion -(CH2CH20).-and each of R2 and R3 independently represents a hydrogen atom, a Cj4alkyl group, or a group P. Preferably each of R2 and R3 represents a Ci- 4alkyl group, especially a methyl group, or, especially, a hydrogen atom. Alternatively, the conjugating reagent may include a group of formula -S-P-S-; -S02-P-S02-; -0S02-P-OS02-; and -INT122R3-P-N+R2R3-. Specific groups of this type include -S-(CH2CH20)n-S-, -0-(CH2CH20),,-0-; -S02-(CH2C1120),,-S02- 30; -0S02-(CH2CH20)n-OS02-; or -WR2R1-(CH2CH20),,-N+122R3-. They can also include groups of the type: (C H2CH20)"-R1 (CH2CH20),, R1 (CH2CH20),, -R1 SO2 (CH2CH20)n -R1 ®N R2R3 (C H2C H20)n -R1 ® N R2R3 where the -(CH2CH20),,-group is carried by any suitable linking group, for example an alkyl group. These divalent groups are chemically equivalent to two leaving groupstwo nucleophiles." An especially preferred leaving group L present in a conjugating reagent according to the present invention is -SP or -SO2P, especially -SO2P. Within this group, one preferred embodiment is where P represents a phenyl or, especially, a tosyl group. Another preferred embodiment is where P represents a group which includes a portion -(CH2CH20)", Conjugating reagent according to the second aspect of the invention may contain more than one functional group capable of reaction with a peptide or protein and/or a polymer. For example, the conjugating reagent may comprise a functional group capable of reacting with a polymer. Conjugating reagents according to the invention may, for example, contain more than one functional grouping for reaction with a protein. For example, a reagent may contain a functional grouping of formula I or I' at one end of the molecule, or any other functional grouping containing at least one leaving group, and one or more additional functional groupings, either capable of conjugating with a protein, peptide, polymer or any other molecule, elsewhere in the molecule. Such structures are described in for example Belcheva

O 1 2°

et al, J. Biomater. Sci Polymer Edn. 9(3), 207-226 and are useful in the synthesis of conjugates containing multiple proteins.

Conjugating reagents of the second aspect of the invention containing the unit of formula BI/BP may have the formula (BIc) or (BIc') or, where W represents a cyano group, (BId) or (BId):

A-L

Bd-O-W D A-L Bd-Q-W H (BIc')

D

13-L (BIc) NC.. A-L H D Bd-Q B-L (BM) (BId) in which Q represents a linking group.

Preferred conjugating reagents include the following: DI 0 L (BIe) or D 0

D Q

(Me) or D2Bd-Q-NH-CO-Ar-CO-(CH2)2-L (BIllb) or D2Bd-Q-NH-CO-Ar-CO-C1-1=CH2 (BIM).

The maytansine-containing conjugating reagent of the second aspect of the invention may, for example, be of the general formula: (([132Bd]q-Lki)",-P)p-Lk2-Lk3-P2 (Ha) in which D represents a maytansine moiety; Bd represents a bridging group as defined above; q represents an integer from 1 to 10; Lk' represents a linker; m represents an integer from 1 to 10; P represents a bond or a z-valent group -RI-NH-where z is from 2 to 11 and P1 is a group containing at least one ethylene unit -CH2-CH2-or ethylene glycol unit -0-CH2-CH2-; p represents an integer from 1 to 10; Lk2 represents a bond or a y-valent linker where y is from 2 to 11 and which consists of from 1 to 9 aspartate and/or glutamate residues; Lk3 represents a linker of the general formula: -CO-Ph-X-Y- (MI) in which Ph is an optionally substituted phenyl group; X represents a CO group or a CH.OH group; and Y represents a group of formula: / A- -C H2-C H -C H (AIII) or (Al V) in which each of A and B represents a Ci4alkylene or alkenylene group; and in which L is a leaving group as defined below; the meanings of m, p, q, y and z being chosen such that the conjugating reagent contains from 1 to 9 D2Bd groups.

Any suitable linking group Q or Lk' may be used. In one embodiment, Q or Lk' may for example be a direct bond, an alkylene group (preferably a C1_10 alkylene group), or an optionally-substituted aryl or heteroaryl group, any of which may be terminated or interrupted by one or more oxygen atoms, sulfur atoms, -NR groups (in which R represents a hydrogen atom or an alkyl (preferably Ci.6alkyl), aryl (preferably phenyl), or alkyl-aryl (preferably C1_6alkyl-phenyl) group), keto groups, -0-00-groups, -CO-O-groups, -O-CO-O, -O-CO-NR-, -NR-CO-O-, -CO-NRand/or -NR.CO-groups. Such aryl and heteroaryl groups Q form one preferred embodiment of the invention. Suitable aryl groups include phenyl and naphthyl groups, while suitable heteroaryl groups include pyridine, pyrrole, furan, pyran, imidazole, pyrazole, oxazole, pyridazine, pyrimidine and purine. Especially suitable linking groups Q or Lk' are heteroaryl or, especially, aryl groups, especially phenyl groups. These may have a linking group to the group D2Bd-, for example a group which is, or contains, a -NR.CO-or -CO.NR-group, for example an -NH.CO-or -CO.NH-group.

Substituents which may be present on an optionally substituted aryl, especially phenyl, or heteroaryl group include for example one or more of the same or different substituents selected from alkyl (preferably Chalky', especially methyl, optionally substituted by OH or CO2H), -CN, -NO2, -CO2R, -COH, -CH2OH, -COR, -OR, -OCOR, -OCO2R, -SR, -SOR, -S 02R, -NHCOR, -NRCOR, NHCO2R, -NR.0O2R, -NO, -NHOH, -NR.OH, -C=N-NHCOR, -C=N-NR.COR, -N+HR2, -NEH2R, halogen, for example fluorine or chlorine, -CCR, -C=CR2 and -C=CHR, in which each R independently represents a hydrogen atom or an alkyl (preferably C1.6alkyl), aryl (preferably phenyl), or alkyl-aryl (preferably C1.6alkyl-phenyl) group. The presence of electron withdrawing substituents is especially preferred. Preferred substituents include for example CN, NO2, -OR, -OCOR, SR, -NHCOR, -NR.COR, -NHOH and -NR.COR.

In another embodiment, a linker 0 or Lk' may contain a degradable group, i.e. it may contain a group which breaks under physiological conditions, separating D2Bd from the protein to which, ultimately, it will be bonded. Alternatively, Q or al may be a linker that is not cleavable under physiological conditions.

Suitable degradable linkers are discussed in more detail below. 20 Degradable linkers Conjugation reagents of the second aspect of the invention and conjugates of the third aspect of the invention advantageously comprise a degradable linker. The degradable linker advantageously contains a group which breaks under physiological conditions, separating D2Bd from the protein to which it is, or will ultimately be, bonded. Where a linker breaks under physiological conditions, it is preferably cleavable under intracellular conditions.

Where the target is intracellular, preferably the linker is substantially insensitive to extracellular conditions (i.e. so that delivery to the intracellular target of a sufficient dose of the therapeutic agent is not prohibited). Suitable degradable linkers are discussed in more detail below.

Where Q or Lk' contains a degradable group, this is generally sensitive to hydrolytic conditions, for example it may be a group which degrades at certain pH values (e.g. acidic conditions). Hydrolytic/acidic conditions may for example be found in endosomes or lysosomes. Examples of groups susceptible to hydrolysis under acidic conditions include hydrazones, semicarbazones, thiosemicarbazones, cis-acotinic amides, orthoesters and ketals. Examples of groups susceptible to hydrolytic conditions include: /NON and In a preferred embodiment, Lkd, Q or Lk' is or includes

O 1:1

0 (AV).

For example, Lk", Q or Lki may be:

O

NH

(AVa), in which case it is preferably bonded to D2Bd and P groups as shown: D 0 0 Th(N-\\

O

Lkd, Q or Lk' may also be susceptible to degradation under reducing conditions. For example, Lk1 may contain a disulfide group that is cleavable on exposure to biological reducing agents, such as thiols. Examples of disulfide groups include: S-S and in which R, R', R" and R"' are each independently hydrogen or CiAalkyl. In a preferred embodiment Lkd, 0 or Lki is or includes (AVb) or (AVc).

For example, Lkd, Q or Lk' may be 0 0 (AVd) or (AVe), in which case Lkd, 0 or Lk' is preferably bonded to D2Bd and P groups as shown:

D

I H O 0 D, Bd

Lkd, 0 or Lk' may also contain a group which is susceptible to enzymatic degradation, for example it may be susceptible to cleavage by a protease (e.g. a lysosomal or endosomal protease) or peptidase. For example, Lkd, 0 or Lk' may contain a peptidyl group comprising at least one, for example at least two, or at least three amino acid residues (e.g. Phe-Leu, GlyPhe-Leu-Gly, Val-Ala, Val-Cit, Phe-Lys). For example, Lkd, Q or Lki may be an amino acid chain having from 1 to 5, for example 2 to 4, amino acids. Another example of a group susceptible to enzymatic degradation is:

H O, or

H cO

wherein A represents 1 or two amino acid residues, especially a protease-specific amino acid sequence of two residues, such as Val-Cit.

In a preferred embodiment, Lkd, Q or LIcl is or includes: HNs. (AVf)

For example, Lkd, Q or Lk1 may be

HNC (AVg)

in which case it is preferably bonded to the D2Bd and P groups as shown below H2N","><"0

HNC

D D,Bd

In one embodiment, Lkd, Q or Lk1 carries a single maytansine payload (D2Bd-) (i.e. q=1 in conjugating reagents of the formula (lla)). The specific linkers (AVa), (AVd) and (AVe) shown above are of this type. In another embodiment, Lkd, Q or Lk1 carries multiple maytansine payloads (D2Bd-) (i.e. q>1, for example 2, 3 or 4, in conjugating reagents of the formula (lla)) and the linker Lkd, Q or Lk1 is used as a means of incorporating more than one maytansine payload (D2Bd-) into a conjugate of the invention.

In one embodiment, this may be achieved by the use of a branching linker Lk', which may for example incorporate an aspartate or glutamate or similar residue. This introduces a branching element of formula:

II H

0 (AVI) where b is 1, 2 or 3, b=1 being aspartate and b=2 being glutamate, and b=3 representing one preferred embodiment. Each of the acyl moieties in the formula VI may be coupled to a dimer group D2Bd-via a suitable linker am, where Lk"-is any suitable linker, for example a degradable linker incorporating one of the linkages mentioned above for Lk'. In particular embodiments, Lleld represents the group (AVa), (AVd) or (Aye) shown above. The amino group of the aspartate or glutamate or similar residue may be bonded to P by any suitable means, for example the linkage may be via an amide bond, e.g. the branching group above may be connected to P via a -CO.CH2-group, thus: )1J-Lt) b 0 0 (AVIa) If desired, the aspartate or glutamate or similar residue may be coupled to further aspartate and/or glutamate and/or similar residues, for example: )b )N 0 (AVIIa) or (AVIIb) and so on, up to a maximum of 9 such residues, giving the potential to incorporate up to 10 D groups. As above, each maytansine payload (D2Bd-) may be attached to an aspargate/glutamate or similar residue via any suitable linker Bea.

Polymers The reagents and conjugates of the present invention may contain an oligomer or polymer (jointly referred to herein as "polymer" for convenience), optionally together with a protein or peptide. A polymer is especially a water soluble, synthetic polymer, particularly polyalkylene glycol. A polymer may for example be a polyalkylene glycol, a polyvinylpyrrolidone, a polyacrylate, for example polyacryloyl morpholine, a polymethacrylate, a polyoxazoline, a polyvinylalcohol, a polyacrylamide or polymethacrylamide, for example polycarboxymethacrylamide, or a HPMA copolymer. Additionally, the polymer may be a polymer that is susceptible to enzymatic or hydrolytic degradation. Such polymers, for example, include polyesters, polyacetals, poly(ortho esters), polycarbonates, poly(imino carbonates), and polyamides, such as poly(amino acids). A polymer may be a homopolymer, random copolymer or a structurally defined copolymer such as a block copolymer, for example it may be a block copolymer derived from two or more alkylene oxides, or from poly(alkylene oxide) and either a polyester, polyacetal, poly(ortho ester), or a poly(amino acid). Polyfunctional polymers that may be used include copolymers of divinylether-maleic anhydride and styrene-maleic anhydride.

Naturally occurring polymers may also be used, for example polysaccharides such as chitin, dextran, dextrin, chitosan, starch, cellulose, glycogen, poly(sialylic acid), hyaluronic acid and derivatives thereof. Polymers such as polyglutamic acid may also be used, as may hybrid polymers derived from natural monomers such as saccharides or amino acids and synthetic monomers such as ethylene oxide or methacrylic acid.

If the polymer is a polyalkylene glycol, this is preferably one containing C2 and/or C3 units, and is especially a polyethylene glycol. A polymer, particularly a polyalkylene glycol, may contain a single linear chain, or it may have branched morphology composed of many chains either small or large. The so-called Pluronics are an important class of PEG block copolymers. These are derived from ethylene oxide and propylene oxide blocks. Substituted, or capped, polyalkylene glycols, for example methoxypolyethylene glycol, may be used.

The polymer may, for example, be a comb polymer produced by the method described in WO 2004/113394, the contents of which are incorporated herein by reference. For example, the polymer may be a comb polymer having a general formula: (E)e-(F)f-(G)g where: E, where present, is obtainable by additional polymerisation of one or more olefinically unsaturated monomers which are not as defined in F; F is obtainable by additional polymerisation of a plurality of monomers which are linear, branched, or star-shaped, substituted or non-substituted, and have an olefinically unsaturated moiety; G, where present, is obtainable by additional polymerisation of one or more olefinically-unsaturated monomers which are not as defined in F; e and g are an integer between 0 and 500; f is an integer of 0 to 1000; wherein when A is present, at least one of D, E and F is present.

A polymer may optionally be derivatised or functionalised in any desired way. Reactive groups may be linked at the polymer terminus or end group, or along the polymer chain through pendent linkers; in such case, the polymer is for example a polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate, or a maleic anhydride copolymer. If desired, the polymer may be coupled to a solid support using conventional methods.

The optimum molecular weight of the polymer will of course depend upon the intended application. Long-chain polymers may be used, for example the number average molecular weight may be up to around 75,000, for example up to 50,000, 40,000 or 30,000 g/mole. For example, the number average molecular weight may be in the range of from 500g/mole to around 75,000g/mole. However, very small oligomers, consisting of discrete PEG chains with, for example, as few as 2 repeat units, for example from 2 to 50 repeat units, are useful for some applications, and are present in one preferred embodiment of the invention. For example, the polymer may contain from 2 to 48, for example from 2 to 36, for example from 2 to 24, units may be used. Straight chain or branched PEGs with 12, 20, 24, 36, 40 or 48 repeat units may for example be used. When the protein to be conjugated is intended to leave the circulation and penetrate tissue, for example for use in the treatment of inflammation caused by malignancy, infection or autoimmune disease, or by trauma, it may be advantageous to use a lower molecular weight polymer in the range up to 30,000g/mole. For applications where the polymer-protein conjugate is intended to remain in circulation it may be advantageous to use a higher molecular weight polymer, for example in the range of 20,000 -75,000g/mole.

The polymer to be used should be selected so the conjugate is soluble in the solvent medium for its intended use. For biological applications, particularly for diagnostic applications and therapeutic applications for clinical therapeutic administration to a mammal, the conjugate will be soluble in aqueous media.

Preferably the polymer is a synthetic polymer, and preferably it is a water-soluble polymer. The use of a water-soluble polyethylene glycol is particularly preferred for many applications.

Our copending application reference 23527 relates to the use of PEG-containing linkers of a particular structure, and these may be used in the present invention. That application discloses the following: "The invention provides a conjugate of a protein or peptide with a therapeutic, diagnostic or labelling agent, said conjugate containing a protein or peptide bonding portion and a polyethylene glycol portion; in which said protein or peptide bonding portion has the general formula: V\RW' /A-Nu yr B-Nu (I) in which Pr represents said protein or peptide, each Nu represents a nucleophile present in or attached to the protein or peptide, each of A and B independently represents a Ci.4alkylene or alkenylene chain, and W' represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group; and in which said polyethylene glycol portion is or includes a pendant polyethylene glycol chain which has a terminal end group of formula -CH2CH2OR in which R represents a hydrogen atom, an alkyl group, for example a Ch.:talky! group, especially a methyl group, or an optionally substituted aryl group, especially a phenyl group, especially an unsubstituted phenyl group.

The invention also provides a conjugating reagent capable of reacting with a protein or peptide, and including a therapeutic, diagnostic or labelling agent and a polyethylene glycol portion; said conjugating reagent including a group of the formula: A-L %AAP W VV\ W

B-L

(II) or in which W represents an electron withdrawing group, A and B have the meanings given above, m is 0 to 4, and each L independently represents a leaving group; and in which said polyethylene glycol portion is or includes a pendant polyethylene glycol chain which has a terminal end group of formula -CH2CH2OR in which R represents a hydrogen atom, an alkyl group, for example a Chalky' group, especially a methyl group, or an optionally substituted aryl group, especially a phenyl group, especially an The invention also provides a process for the preparation of a conjugate according to the invention, which comprises reacting a protein or peptide with a conjugating reagent according to the invention.

The conjugate of the invention may be represented schematically by the formula:

PEG (III)

in which D represents the therapeutic, diagnostic or labelling agent, F' represents the group of formula I, and PEG represents the pendant polyethylene glycol chain having a terminal end group of formula -CH2CH2OR.

The reagent of the invention may be represented schematically by the formula:

D

PEG (IV)

in which D represents the therapeutic, diagnostic or labelling agent, F represents the group of formula II or II', and PEG represents the pendant polyethylene glycol chain having a terminal end group of formula -CH2CH2OR. The functional grouping F is capable of reacting with two nucleophiles present in a protein or peptide as explained below.

A polyethylene glycol (PEG) portion of the conjugates and reagents of the invention is or includes a pendant PEG chain which has a terminal end group of formula -CH2CH2OR in which R represents a hydrogen atom, an alkyl group, for example a Ci_diallcyl group, especially a methyl group, or an optionally substituted aryl group, especially a phenyl group, especially an unsubstituted phenyl group.

Preferably R is a methyl group or a hydrogen atom.

The overall size of the PEG portion will of course depend on the intended application. For some applications, high molecular weight PEGs may be used, for example the number average molecular weight may be up to around 75,000, for example up to 50,000, 40,000 or 30,000 g/mole. For example, the number average molecular weight may be in the range of from 500 g/mole to around 75,000. However, smaller PEG portions may be preferred for some applications.

In one preferred embodiment, all of the PEG in the PEG portion is present in the pendant PEG chain. In another embodiment, PEG may also be present in the backbone of the molecule, and this is discussed in more detail below.

As with the PEG portion, the size of the pendant PEG chain will depend on the intended application. For some applications, high molecular weight pendant PEG chains may be used, for example the number average molecular weight may be up to around 75,000, for example up to 50,000, 40,000 or 30,000 g/mole. For example, the number average molecular weight may be in the range of from 500 g/mole to around 75,000. However, for many applications, smaller pendant PEG chains may be used.

For example said PEG chain may have a molecular weight up to 3,000 g/mole. However, very small oligomers, consisting of discrete PEG chains with, for example, as few as 2 repeat units, for example from 2 to 50 repeat units, are useful for some applications, and are present as said PEG chain in one preferred embodiment of the invention. The pendant PEG chain may be straight-chain or branched. PEG chains, for example straight-chain or branched chains with 12, 20, 24, 36, 40 or 48 repeat units may for example be used." Conjugates The conjugate of the third aspect of the invention comprises a protein, peptide and/or polymer attached via a linker to a maytansine-containing that consists of at least two maytansine moieties linked to each other through a non-degradable bridging group. When the conjugate comprises a protein or peptide, the linker attaching the payload to the protein or peptide is advantageously degradable. The protein or peptide (Ab) present in conjugates of the third aspect of the invention may be any desired peptide or protein which contains nucleophilic groups may be conjugated using the process of the present invention. Suitable proteins include for example peptides, polypeptides, antibodies, antibody fragments, enzymes, cytokines, chemokines, receptors, blood factors, peptide hormones, toxin, transcription proteins, or multimeric proteins. Suitable polymers include those mentioned above.

The following gives some specific proteins which may be conjugated using the present invention. Enzymes include carbohydrate-specific enzymes, proteolytic enzymes and the like, for example the oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases disclosed by US 4,179,337. Specific enzymes of interest include asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase, bilirubin oxidase, glucose oxidase, glucuronidase, galactosidase, glucocerbrosidase, glucuronidase, and glutaminase.

Blood proteins include albumin, transferrin, Factor VII, Factor VIII or Factor IX, von Willebrand factor, insulin, ACTH, glucagen, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothalamic releasing factors, antidiuretic hormones, prolactin, interleukins, interferons, for example IFN-a or IFN-i3, colony stimulating factors, hemoglobin, cytokines, antibodies, antibody fragments, chorionicgonadotropin, follicle-stimulating hormone, thyroid stimulating hormone and tissue plasminogen activator.

Other proteins of interest are allergen proteins disclosed by Dreborg et al Crit. Rev. Therap.

Drug Carrier Syst. (1990) 6 315-365 as having reduced allergenicity when conjugated with a polymer such as poly(alkylene oxide) and consequently are suitable for use as tolerance inducers. Among the allergens disclosed are Ragweed antigen E, honeybee venom, mite allergen and the like.

Glycopolypeptides such as immunoglobulins, ovalbumin, lipase, glucocerebrosidase, lectins, tissue plasminogen activator and glycosylated interleukins, interferons and colony stimulating factors are of interest, as are immunoglobulins such as IgG, IgE, IgM, IgA, IgD and fragments thereof.

Of particular interest are receptor and ligand binding proteins and antibodies and antibody fragments which are used in clinical medicine for diagnostic and therapeutic purposes. The binding protein/antibody may be conjugated with a diagnostic, therapeutic or labelling agent, for example a radioisotope or a cytotoxic/antiinfective drug. The use of the invention in the preparation of antibody-drug conjugates where the drug is a cytotoxic drug, for example an auristatin or maytansinoid, is especially preferred.

The protein may be derivatised or functionalised if desired. In particular, prior to conjugation, the protein, for example a native protein, may have been reacted with various blocking groups to protect sensitive groups thereon; or it may have been previously conjugated with one or more polymers or other molecules, either using the process of this invention or using an alternative process. In one embodiment of the invention, it contains a polyhistidine tag, which can be targeted by the conjugating reagent according to the invention.

The maytansine-containing conjugate of the third aspect of the invention may, for example, 10 be of the general formula: (0[D2Bd]q-Lkl)rn-P)p-Lk2-Lk3),,-Ab (IIIa) in which D represents a maytansine moiety; Bd represents a bridging group as defined above; q represents an integer from 1 to 10; Lk' represents a linker; m represents an integer from 1 to 10; P represents a bond or a z-valent group -P1-NH-where z is from 2 to 11 and P1 is a group containing at least one ethylene unit -CH2-CH2-or ethylene glycol unit -0-CH2-CH2-; p represents an integer from 1 to 10; Lk2 represents a bond or a y-valent linker where y is from 2 to 11 and which consists of from 1 to 9 aspartate and/or glutamate residues; Lk3 represents a linker of the general formula -CO-Ph-X-Y- (All) in which Ph is an optionally substituted phenyl group; X represents a CO group or a CH.OH 25 group; and Y represents a group of formula:

A-/

-C H2 -C H (AHD or e (AIV) in which each of A and B represents a Ci4alkylene or alkenylene group; Ab represents a binding protein or peptide capable of binding to a binding partner on a target (such as a protein or peptide described above), said binding protein being bonded to Lk3 via two sulfur atoms derived from a disulfide bond in the binding protein or peptide; and n represents an integer from 1 to s where s is the number of disulfide bonds present in the binding protein or peptide prior to conjugating to L1c3; the meanings of m, n, p, q, y and z being chosen such that the conjugate contains from 1 to 9 D2Bd groups.

Conjugating reagents containing a functional group capable of reacting with a protein, peptide and/or polymer according to the second aspect of the invention may be reacted with a protein, peptide and/or polymer to form a conjugate, and such a reaction forms a further aspect of the invention. In a preferred embodiment of this further aspect of the invention, a conjugating reagent having one of the structures BI, BI', BII or BIII described above (including all of the preferred sub-structures) is reacted with a protein or peptide to form a conjugate. The immediate product of the conjugation process using one of these reagents is a conjugate which contains an electron-withdrawing group W. However, the conjugation process is reversible under suitable conditions. This may be desirable for some applications, for example where rapid release of the protein is required, but for other applications, rapid release of the protein may be undesirable. It may therefore be desirable to stabilise the conjugates by reduction of the electron-withdrawing moiety W to give a moiety which prevents release of the protein. Accordingly, the process described above may comprise an additional optional step of reducing the electron withdrawing group W in the conjugate. The use of a borohydride, for example sodium borohydride, sodium cyanoborohydride, potassium borohydride or sodium triacetoxyborohydride, as reducing agent is particularly preferred. Other reducing agents which may be used include for example tin(II) chloride, alkoxides such as aluminium alkoxide, and lithium aluminium hydride.

Thus, for example, a moiety W containing a keto group may be reduced to a moiety containing a CH(OH) group; an ether group CH.OR may be obtained by the reaction of a hydroxy group with an etherifying agent; an ester group CH.O.C(0)R may be obtained by the reaction of a hydroxy group with an acylating agent; an amine group CH.NH2, CH.NHR or CH.NR2 may be prepared from a ketone by reductive amination; or an amide CH.NHC(0)R or CH.N(C(0)R)2 may be formed by acylation of an amine. A sulfone may be reduced to a sulfoxide, sulfide or thiol ether. A cyano group may be reduced to an amine group.

A key feature of using conjugating reagents of formula BI or BII described above is that an a-methylene leaving group and a double bond are cross-conjugated with an electron withdrawing function that serves as a Michael activating moiety. If the leaving group is prone to elimination in the cross-functional reagent rather than to direct displacement and the electron-withdrawing group is a suitable activating moiety for the Michael reaction then sequential intramolecular bis-alkylation can occur by consecutive Michael and retro Michael reactions. The leaving moiety serves to mask a latent conjugated double bond that is not exposed until after the first alkylation has occurred to give a reagent of formula I' and bisalkylation results from sequential and interactive Michael and retro-Michael reactions. The cross-functional alkylating agents may contain multiple bonds conjugated to the double bond or between the leaving group and the electron withdrawing group.

Where bonding to the protein is via two sulfur atoms derived from a disulfide bond in the protein, the process may be carried out by reducing the disulfide bond in situ following which the reduced product reacts with the reagent having one of the structures I or II. Preferably the disulfide bond is reduced and any excess reducing agent is removed, for example by buffer exchange, before the conjugating reagent is introduced. The disulfide can be reduced, for example, with dithiothreitol, mercaptoethanol, or tris-carboxyethylphosphine using conventional methods.

Conjugation reactions may be carried out under similar conditions to known conjugation processes, including the conditions disclosed in the prior art mentioned above. For example when using conjugating reagents having one of the structures BI, BI' or BII, the conjugation reaction according to the invention may be carried out under reaction conditions similar to those described in WO 2005/007197, WO 2009/047500, WO 2014/064423 and WO 2014/064424._The process may for example be carried out in a solvent or solvent mixture in which all reactants are soluble. For example, the protein may be allowed to react directly with the polymer conjugating reagent in an aqueous reaction medium. This reaction medium may also be buffered, depending on the pH requirements of the nucleophile. The optimum pH for the reaction will generally be at least 4.5, typically between about 5.0 and about 8.5, preferably about 6.0 to 7.5. The optimal reaction conditions will of course depend upon the specific reactants employed.

Reaction temperatures between 3-40 °C are generally suitable when using an aqueous reaction medium. Reactions conducted in organic media (for example THF, ethyl acetate, acetone) are typically conducted at temperatures up to ambient. In one preferred embodiment, the reaction is carried out in aqueous buffer which may contain a proportion of organic solvent, for example up to 20% by volume of organic solvent, typically from 5 to 20 % by volume organic solvent.

The protein can be effectively conjugated using a stoichiometric equivalent or a slight excess of conjugating reagent. However, it is also possible to conduct the conjugation reaction with an excess of conjugating reagent, and this may be desirable for some proteins. The excess reagent can easily be removed by conventional means, for example ion exchange or HPLC chromatography, during subsequent purification of the conjugate.

Of course, it is possible for more than one conjugating reagent to be conjugated to a protein, where the protein contains sufficient suitable attachment points. For example, in a protein which contains two different disulfide bonds, or in a protein which contains one disulfide bond and also carries a polyhistidine tag, it is possible to conjugate two molecules of the reagent per molecule of protein.

The protein or peptide (Ab) present in conjugates of the third aspect of the invention may be any desired peptide or protein which contains nucleophilic groups may be conjugated using the process of the present invention. Suitable proteins include for example peptides, polypeptides, antibodies, antibody fragments, enzymes, cytokines, chemokines, receptors, blood factors, peptide hormones, toxin, transcription proteins, or multimeric proteins.

The following gives some specific proteins which may be conjugated using the present invention. Enzymes include carbohydrate-specific enzymes, proteolytic enzymes and the like, for example the oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases disclosed by US 4,179,337. Specific enzymes of interest include asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, unease, bilirubin oxidase, glucose oxidase, glucuronidase, galactosidase, glucocerbrosidase, glucuronidase, and glutaminase.

Brief description of the drawings

Figures 1 and 2 show the results of Example 7.

The following Examples illustrate the invention.

Example 1: Synthesis of cytotoxic payload 1.

Step 1: Synthesis of compound 2. 2

To a stirred solution of aminohexanoic maytansine (AHX-DM1).TFA salt (29.4 mg) of structure b$ TPA 6 6 in dimethylformamide (DMF) (400 pi) was added a solution of 4-(N-Boc-amino)-1,6-heptanedioic acid bis-pentafluorophenyl ester (10.2 mg) in DMF (200 pi). The solution was cooled to 0 °C before addition of /V,N-diisopropylethYlamine (DIPEA) (13.5 pL). The solution was allowed to warm to room temperature and stirred for 18.5 h. The reaction solution was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give 4-(N-boc-amino)-1,6-heptanediamide bis-AHX-DM1 compound 2 (assumed quantitative yield, 29.7 mg) as a white solid m/z [M+2H-2(H20)-NHCO]2+ 844 (100%), [M+Hr 1767.

Step 2: Synthesis of cytotoxic payload 1.

Compound 2 (assumed quantitative yield, 29.7 mg) was dissolved in formic acid (700 pL) and the solution stirred at room temperature for 1.5 h. Volatiles were removed in vacua and the residue converted to the trifluroacetic acid salt by dissolving in a buffer A:buffer B 50:50 v/v% mixture (1.5 mL, buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid). The solution was stirred at room temperature for 5 min before the solvent was removed by lyophilisation. The process was repeated to give 4-(amino)-1,6-heptanediamide bis-AHX-DM1 cytotoxic payload 1 as an off-white solid (18.0 mg, 60% over 2 steps) m/z [M+2H-2(H20)-NHCO)r 794 (100%), [M+H]+ 1667.

Example 2: Synthesis of a disulfide bridging reagent 3 comprising the cytotoxic payload 1.

Step 1: Synthesis of compound 4.

OH 4

To a stirred solution of 4[2,2-bis[(p-tolylsulfonylkmethyl]acetylThenzoic acid (1.50 g, Nature Protocols, 2006, 1(54), 2241-2252) in DMF (70 mL) was added alpha-methoxyomega-mercapto hepta(ethylene glycol) (3.20 g) and triethylamine (2.50 mL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 19 h, volatiles were removed in vacuo. The resulting residue was dissolved in water (2.4 mL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give 4-[2,2-bis[alpha-methoxy-omega-thio-hepta(ethylene glycop]acetyll-benzoic acid compound 4 as a thick clear colourless oil (1.77 g, 66%) m/z [M+H]' 901.

Step 2: Synthesis of compound 5. 02 7

OH

To a stirred solution of 4 (1.32 g) in methanol:water (18 mL, 9:1 v/v) at room temperature was added Oxone© (2.70 g). After 2.5 h, the volatiles were removed in vacuo and water was azeotropically removed with acetonitrile (2 x 15 mL). The resulting residue was dissolved in dichloromethane (3 x 10 mL), filtered through a column of magnesium sulphate and washed with dichloromethane (2 x 7 mL) The eluent and washings were combined and the volatiles were removed in vacuo to give a thick clear pale yellow oil (1.29 g, 92%). A portion of the residue (700 mg) was dissolved in water:acetonitrile (1.50 mL, 3:1 v/v), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid reagent 5 as a thick clear colourless oil (524 mg, 68%) m/z [M+H]' 965.

Step 3: Synthesis of compound 6. /7sot -(0

Stock solutions of Hbis(dimethylamino)methylene]-111-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (394 mg) in DMF (4 mL) and N-methylmorpholine (NMM) (114 uL) in DMF (4 mL) were prepared. To a DMF (10 mL) solution of compound 5 (250 mg) was added H2N-PEG(24u)-(CO2Bu) (405 mg). The mixture was diluted with DMF (5 mL) and stirred under an inert nitrogen atmosphere at room temperature for 5 min. The mixture was cooled to 0 °C and aliquots of HATU (1 mL) and NMM (1 mL) added every 10 min for a total of 4 additions. After 40 min the reaction mixture was warmed to room temperature. After 2.5 h volatiles were removed in vacuo. The resulting residue was dissolved in water (2 mL), and the product isolated by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v), the organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(24u)2butyl ester compound 6 as a thick clear pale yellow oil (422 mg, 76%) m/z [M-(tBu)+3H]3t 698.46 Da.

Step 4: Synthesis of compound 7.

OH 7

Compound 6 (382 mg) was dissolved in formic acid (5 mL) and the reaction mixture stirred under an inert nitrogen atmosphere at room temperature for 1 h. The formic acid was removed by lyophilisation and the resulting solid dissolved in water (1 mL), prior to being purified directly by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoylbenzamide-PEG(24u) acid compound 7 as a white solid (194 mg, 53%) m/z [M+311]3+ 698.33 Da.

Step 5: Synthesis of compound 8 142N O Compound 8 was synthesised following the procedure described in patent (EP 0 624 377 A2) to give a white solid with spectroscopic data in agreement with that previously reported.

OH

HN

Step 6: Synthesis of compound 9.

Stock solutions of compound 8 (20.0 mg) in DMF (500 pi) and HATU (40.0 mg) in DMF (400 pi) were prepared. To a stirred solution of compound 1 (14.0 mg) in DMF (700 uL) was added aliquots of compound 8 stock solution (126.9 pi) and HATU stock solution (77.8 uL). The reaction solution was cooled to 0 °C before the addition of DIPEA (4.11 uL). The solution was stirred at 0 °C for 50 min before further aliquots of compound 8 stock solution (126.9 uL), HATU stock solution (77.8 uL) and DIPEA (4.11 u1_,) were added. The solution was stirred for 40 min at 0 °C. The reaction solution was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give 4-(Fmoc-val-cit-amido)-1,6-heptanediamide bis-AHX-DM1 compound 9 (assumed quantitative yield, 16.9 mg) as an off-white solid m/z [M+21-1-2(H20)]2* 1055 (100%).

Step 7: Synthesis of compound 10.

To a stirred solution of compound 9 (assumed quantitative yield, 16.9 mg) in DMF (500 FL) was added piperidine (3.04 FL). The reaction solution was stirred at room temperature for 1.5 h before purification by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give 4-(val-citamido)-1,6-heptanediamide bis-AHX-DM1 compound 10 as an off-white solid (8.8 mg, 55% over 2 steps) m/z [M+21-1]2+ 962 (100%).

Step 8: Synthesis of disulfide bridging reagent 3 comprising the cytotoxic payload 1.

Stock solutions of compound 7 (13.5 mg) in DMF (100 FL), HATU (10.0 mg) in DMF (200 FL) and NMM (5.83 FL) in DMF (94.2 FL) were prepared. To a stirred solution of 10 (1.8 mg) in DMF (80 FL) was added aliquots of compound 7 stock solution (16.5 FL) and HATU stock solution (20.2 FL). The reaction solution was cooled to 0 °C before adding an aliquot of NMM stock solution (5 FL). The solution was allowed to stir at 0 °C for 1 h before warming to room temperature. After 3.5 h, the reaction solution was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-val-citamido)-1,6-heptanediamide bis-AHX-DM1 reagent 3 as a solid (1.5 mg, 42%) m/z [M+4H2(H20)]4+ 992 (100%), [M+3H-2(H20)]3+ 1321, [M+2H-2(H20)]2÷ 1982.

Example 3: Synthesis of a disulfide bridging reagent 11 comprising the cytotoxic payload 1.

Step 1: Synthesis of compound 12.

0c k./())--0 NH 12 A solution of Fmoc-L-Glu-(OtBu)-OH (36 mg) in DMF (2 mL) under an argon atmosphere was cooled to 0 °C and (benzotriazol-1-yloxy)tris-(dimethylamino) phosphonium hexafluorophosphate (BOP) (41 mg) was added followed by NH2-PEG(24u)-OMe (100 mg) and DIPEA (19 gL). The solution was allowed to warm to room temperature and after 22 h the volatiles were removed in vacuo. The resulting residue was dissolved in dichloromethane (1 mL) and purified by normal phase column chromatography eluting with dichloromethane.methanol (100:0 v/v to 80:20 v/v). The organic solvent was removed in vacuo to give the Fmoc-Glu-(0tBu)-NH-PEG(24u)-0Me compound 12 as a colourless oil (84 mg, 67%) m/z [M+H]+ (1097, 10%), [M+211]2+ (1035,100%).

Step 2: Synthesis of compound 13 0 0

ON NH2

To a solution of compound 12 (74 mg) in DMF (2 mL) under an argon atmosphere was added piperidine (49 nl.) and the resulting solution was stirred at room temperature. After 22 h, the volatiles were removed in vacuo and the resulting residue was triturated with hexane (3 x 0.7 mL). The organic solvent was decanted each time and resulting residue dried in vacuo to give the Glu-(OtBu)-NH-PEG(24u)-OMe compound 13 as a solid (61 mg, 97%) m/z [M+H]+ (1097, 10%), [M+21-1]2+ (1035,100%).

Step 3: Synthesis of compound 14. 17 0 14

A solution of compound 5 (26.6 mg) in DMF (550 pi) was cooled to 0 °C under an argon atmosphere when HATU (10.5 mg) was added and the solution was stirred for 0.5 h at 0 °C. To this was added a solution of 13 (32 mg) in DMF (550 RC) and the resulting solution was stirred for 5 min at 0 °C before addition of NMM (2.9 nL) and HATU (10.5 mg). The reaction solution was allowed to stir at 0 °C for 2 h before being warmed to room temperature and stirred for a further 3.5 h. After this time the volatiles were removed in vacuo,the resulting residue dissolved in water and acetonitrile (v/v; 1/1, 1.2 mL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-Glu-(0tBu)-NHPEG(24u)-0Me compound 14 as a colourless oil (30.5 mg, 55%) m/z [M+Na]+ (2243, 50%), [M+H]+ (2221, 40%), [M+Na+2H]3+ (747,100%).

Step 4: Synthesis of compound 15. SO2 0

/7 02O 24 0-OH A solution of compound 14 (30 mg) in dichloromethane (2 mL) under an argon atmosphere was cooled to 0 °C after which trifluoroacetic acid (500 up was added and the resulting solution stirred for 1.5 h. The reaction mixture was allowed to warm to room temperature and stirred for a further 1 h, after which time the volatiles were removed in vacuo. The resulting residue was dissolved in water and acetonitrile (v/v; 1/1, 0.6 mL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-Glu-NH-PEG(24u)-OMe compound 15 as a colourless oil (20 mg, 68%) m/z [M+H]+ (2165, 55%), [M+21-1]2+ (1083, 60%), [M+2H+Na]3+ (729, 100%).

Step 5: Synthesis of reagent H. Stock solutions of HATU (10 mg) in DMF (200 uL) and NMM (5.83 aL) in DMF (94.2 u1_,) were prepared. Compound 15 (5.4 mg) was dissolved in a solution of compound 10 (3.6 mg) in DMF (254 ut) with stirring. To the stirred solution was added an aliquot of HATU stock solution (40 aL). The solution was cooled to 0 °C before an aliquot of NMM stock solution (10 uL) was added. After 50 min, further aliquots of HATU stock solution (6.67 uL) and NMM stock solution (1.67 uL) were added. The reaction solution was stirred at 0 °C for a further 20 minutes and purified directly by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give reagent 11 as an off-white solid (3.8 mg, 53%) m/z [M+4H-(H20)-NHC0]4+ 1003 (100%), [M+3H-2(1-120)-NHCO]3+ 1331, [M+2H-2(H20)]2+ 2017.

Example 4: Synthesis of a disulfide bridging reagent 16 comprising the cytotoxic payload 1.

Step 1: Synthesis of compound 17. OMe OMe 17

A stock solution of hydroxybenzotriazole (HOBt, 6.6 mg) in DMF (200 pL) was prepared. To a stirred solution of cytotoxic payload 1 (10 mg) in DMF (500 pL) was added Fmoc-valala-PAB-PNP (3.7 mg) and an aliquot of HOBt stock solution (2 pL). The reaction solution was cooled to 0 °C before DIPEA (2.14 ttL) was added. The reaction solution was then stirred at room temperature for 18 h before purification by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give Fmoc-val-ala-PAB-amido-1,6-heptanediamide bis-AHXDM1 reagent 17.

Step 2: Synthesis of compound 18.

The bis-maytansinoid compound amine-val-ala-PAB-amido-1,6-heptanediamide bis-AHXDM1 compound 18 was synthesised in an analogous way to that described for compound 10 5 in Example 2 (step 7), using compound 17 instead of compound 9.

Step 3: Synthesis of reagent 16.

The bis-maytansinoid reagent bis-mPEG(7u)sulfone-propanoyl-benzamide-G1u1NHPEG(24u)-0Mep [val-ala-PAB-amido-1,6-heptanediamide bis-AHX-DM1] 16 was 10 synthesised in an analogous way to that described for reagent 11 in Example 3, using compound 18 instead of compound 10.

Example 5: Synthesis of a disulfide bridging reagent 19 comprising the cytotoxic payload AHX-DM1 (not according to the invention).

CI 7 _

/24 El 0

N

CI OMe 0 0 I

H2 9--N 5 OMe

HN H2N0

N

H 011E To a stirred solution of val-cit-AHX-DM1 (5.0 mg) in anhydrous DMF (400 ILL) was added reagent 7 (13 mg) and stirred for 5 min at 0 °C. HATU (2.62 mg) and NMM (0.44 mg) were added in succession and the reaction mixture was allowed to stir at 0 °C. After 20 min, an additional amount of HATU (2.62 mg) and NMM (0.44 mg) was added and the reaction mixture was stirred at 0 °C. After 2.5 h, the reaction was cooled to -20 °C for 16 h. The reaction solution was concentrated in vacuo and then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(24u)-val-citAHX-DM1 reagent 19 as thick yellow oil (5.6 mg, 41%) m/z [M-0H+Hj2+ 1571.5.

Example 6: Production of antibody drug conjugate 20 using disulfide bridging reagent 3; antibody drug conjugate 21 using disulfide bridging reagent 11 and antibody drug conjugate 22 using disulfide bridging reagent 16 Antibody drug conjugates were prepared by methods analogous to those described in W02014064423 and W02014064424. Briefly, antibody (trastuzumab) was reduced using tris(2-carboxyethyl)phosphine at 40 °C for 1 h. Conjugation of the antibody with 1.5 molar equivalents of reagent (i.e., 3, 11 or if) per inter-chain disulfide bond was then performed by dissolving reagents to a final concentration of 1.6 mM in either acetonitrile or DMF. The antibody solution was diluted to 4.21 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCI, 20 mM EDTA, pH 7.5. Reagents were added to antibody and the final antibody concentration in the reaction was adjusted to 4 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Each solution was mixed gently and incubated at 22 °C. Antibody drug conjugate product was purified by hydrophobic interaction chromatography for each conjugate to give products with a defined drug to antibody ratio (DAR).

Example 7: In vitro cytotoxicity comparison of antibody drug conjugate 20 comprising the cytotoxic payload 1 with an antibody drug conjugate comprising the cytotoxic payload AHXDM1.

The in vitro efficacy of the antibody drug conjugate 20 (Example 6), comprising the cytotoxic payload 1 was determined by measuring the inhibitory effect on cell growth of HER-2 receptor over-expressing cancer cell lines. A comparator antibody drug conjugate 23 was used in the study comprising the cytotoxic payload AHX-DM1 and prepared from reagent 19 in an analogous way to the preparation of conjugate 20.

Loss of tumour cell viability following treatment with cytotoxic drugs or ADCs in vitro can be measured by growing cell lines in the presence of increasing concentrations of drugs or ADCs and quantifying the loss of proliferation or metabolic activity using CellTiter Glo® Luminescence reagent (Promega Corp. Technical Bulletin TB288; Lewis Phillips G.D, Cancer Res 2008; 68:9280-9290). The protocol describes cell seeding, drug treatment and determination of the cell viability in reference to untreated cells based on ATP synthesis, which is directly related to the number of cells present in the well.

HER2-positive SK-BR-3 cells were trypsinised with 3 mL Trypsin EDTA for -15 min. Trypsinisation was stopped by adding 10 mL complete medium, and cells were transferred to a 50 mL Falcon tube. Cells were counted using a Neubauer haemocytometer and adjusted to a cell density of 5x104/mL. Cells were seeded (100 ttL/well) into poly-D-lysine coated opaque-walled 96-well plates and incubated for 24 h at 37 °C and 5% CO2. Tumour cell lines SKBR-3 (ATCC-HTB-30) were purchased from the American Type Culture Collection (ATCC). SK-BR-3 cells were grown in McCoy's 5A medium (Life Technologies®), 10% fetal bovine serum, 100 ktL/mL Penicillin and 100 µg/mL Streptomycin.

Methods for cell culture were derived from product information sheets for ATCC and references quoted therein, for example, Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney 3rd edition, published by Alan R. Liss, N.Y. 1994, or 5th edition published by Wiley-Liss, N.Y. 2005. Serial dilutions of antibody drug conjugates were made in triplicate by pipetting across a 96 well plate from columns 3-10 with 2-or 3-fold dilutions using the relevant cell culture medium as a diluent. The HER2-positive cell lines of SK-BR-3 were treated with drug concentrations shown in Table 1. Cells were then incubated with the drug at 37 °C and 5% CO2 for a further 72 h. Cell line Drug/drug-conjugate Concentration range SK-BR-3 Antibody drug conjugate 20 1nM -0.457 pM SK-BR-3 Antibody drug conjugate 23 1nM -7.8 pM

Table 1

The cell viability assay was carried out using the Cell-Titer Glo® Luminescence reagent, as described by the manufacturer's instructions, (Promega Corp. Technical Bulletin TB288; Lewis Phillips G D, Cancer Res 2008; 68:9280-9290). Incubation times, e.g. cell lysis and incubation with luminescent reagent, were extended to 3 min and 20 min respectively, for optimal luminescent signal. Luminescence was recorded using a plate reader (e.g. MD Spectramax M3 plate reader), and data subsequently analysed using a four parameter nonlinear regression model.

Figure 1 shows the effect of antibody drug conjugate 20 comprising the cytotoxic payload 1 and the antibody drug conjugate 23 on cell viability of HER2-positive SK-BR-3 cell line.

The results shown in Figure 1 illustrate cell viability responses to treatment with either antibody drug conjugate 20 comprising the cytotoxic payload I or the antibody drug conjugate 23 within SKBR-3 cells. Viability is expressed as % of untreated cells. The % viability (Y-axis) is plotted against the logarithm of drug concentration in nM (x-axis) to determine the ICso values for all conjugates.

Figure 2 is a comparison of ICso values of antibody drug conjugate 20 comprising the cytotoxic payload 1 with the antibody drug conjugate 23 on cell viability of HER2-positive SK-BR-3 cell line. The ICso values for all conjugates are shown in Table 2 and Figure 2.

Sample name IC50 [nM] St. Dev in SK-BR-3 Antibody drug conjugate 20 0.05 0.02 4 Antibody drug conjugate 23 0.23 0.03 3

Table 2

As shown in Figure 2 and Table 2, the antibody drug conjugate 20 comprising the cytotoxic payload 1 is more active in HER2-positive SK-BR-3 cell line than antibody drug conjugate 23.

Claims

Claims 1. A conjugate comprising a protein, peptide and/or polymer attached to a maytansinecontaining payload via a linker, characterised in that the maytansine-containing payload consists of at least two maytansine moieties linked to each other through a non-degradable bridging group, with the proviso that when the conjugate comprises a protein or peptide, the linker attaching the payload to the protein or peptide is degradable.
2. A conjugating reagent which contains a functional group capable of reaction with a peptide or protein and/or a functional group capable of reacting with a polymer, the payload being attached to the functional group(s) via one or more linkers, characterised in that the conjugation reagent comprises a maytansine-containing payload consisting of at least two maytansine moieties linked to each other through a non-degradable bridging group, with the proviso that when the conjugating reagent comprises a functional group capable of reaction with a peptide or protein, the linker attaching the payload to the functional group capable of reaction with at a peptide or protein is degradable.
3. A conjugating reagent of claim 2, which contains a functional group capable of reaction with at least one nucleophile present in a peptide or protein, the functional group including at least one leaving group which is lost on reaction with said nucleophile, wherein the maytansine-containing payload consisting of at least two maytansine moieties linked to each other through a non-degradable bridging group is attached to the functional group capable of reaction with at least one nucleophile present in a peptide or protein via a degradable linker.
4. A conjugate of claim 1 or a conjugating reagent of claim 2 or claim 3, wherein the bridging group has a chain of at least 3 chain carbon atoms; wherein the chain includes at least 2, optionally from 2 to 4, amide linkages; and wherein the chain includes optional poly(ethylene glycol) spacers in addition to the chain carbon atoms.
5. A conjugate of claim 1 or claim 4 or a conjugating reagent of claim 2, claim 3 or claim 4, wherein the bridging group has a chain of at least 3 chain carbon atoms with the proviso that no two heteroatoms are adjacent to one another in the chain and with the proviso that the bridging group does not include the moiety: -C(0)-CH(NR1X)-(CH2)b-C(0)-, in which b is 1, 2 or 3, R1 is selected from hydrogen and C1 to C6 alkyl, and X is any group
6. A maytansine-containing compound consisting of at least two maytansine moieties linked to each other through a bridging group having a chain of at least 3 carbon atoms and optional poly(ethylene glycol) spacers in addition to the chain carbon atoms, with the proviso that no two heteroatoms are adjacent to one another in the chain and with the proviso that the bridging group does not include the moiety: -C(0)-CH(NR1X)-(CH2)b-C(0)-, in which b is 1, 2 or 3, R1 is selected from hydrogen and C1 to C6 alkyl, and X is any group.
7. A maytansine-containing compound of claim 6, wherein the bridging group includes a pendant connecting group selected from amine, carboxy, alkyne, azide, hydroxyl or thiol.
8. A maytansine-containing compound of claim 6 or claim 7, wherein the bridging group includes at least 2, for example from 2 to 4, amide linkages incorporated in the chain.
9. A maytansine-containing compound of any one of claims 6 to 8, wherein the bridging group comprises from 0 to 4 poly(ethylene glycol)-containing units interspersed in the chain each independently including from 2 to 20 ethylene glycol units.
10. A maytansine-containing compound of any one of claims 6 to 9, wherein the bridging group has at least 5 chain carbon atoms substituted with 0 to 8 substituent groups each independently selected from: hydroxyl; halo; * C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl optionally substituted with halo or hydroxyl; C2 to C6 alkyne; * C1 to C6 alkoxy optionally substituted with halo or hydroxyl; NR1R2 in which each of R1 and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl; * azide; * thiol; * carboxylic acid * C1 to C6 alkyl or C1 to C6 haloalkyl ester; and * C3 to C10 aryl or C3 to C8 heteroaryl optionally substituted with 0 to 4 substituent groups each independently selected from hydroxyl; halo; C1 to C6 alkyl optionally substituted with halo or hydroxyl; C2 to C6 alkenyl, C1 to C6 alkoxy optionally substituted with halo or hydroxyl; and -NR'R2 in which each R1 and R2 is independently selected from hydrogen, C1 to C6 alkyl, C1 to C6 haloalkyl and C3 to C10 aryl; and wherein any nitrogen heteroatoms in the chain are unsubstituted or substituted with C1 to C6 alkyl, C2 to C6 alkenyl or C3 to C10 aryl, each optionally substituted with halo or hydroxyl.
11. A maytansine-containing compound of any one of claims 6 to 10, wherein the bridging group has from 7 to 41 chain carbon atoms.
12. A maytansine-containing compound of any one of claims 6 to 11, wherein the bridging group is symmetrical.
13. A maytansine-containing compound of any one of claims 6 to 12, wherein the bridging group incorporates from 0 to 8 carbonyl groups, from 0 to 4 unsaturated carbon-carbon double bonds and from 0 to 4 C3 to C10 aryl or heteroaryl groups, and wherein the at least 3 chain carbon atoms are interspersed with from 0 to 11 chain heteroatoms selected from N, 0 and S
14. A maytansine-containing compound of any one of claims 6 to 12, wherein the bridging group does not include * the moiety: -NH-CH(X)-C(0)-NH-CH(X)-C(0)-in which X is any group, especially a dipeptide portion having 2 or more adjacent amino acid residues, * a group selected from a hydrazone, semicarbazone, thiosemicarboazone, cis-acotinic amide, orthoester or ketal; or S-j( Th< which A is an amino acid residue. in OrHH15. A maytansine-containing compound of any one of claims 6 to 14, wherein the bridging group is non-degradable under physiological conditions.16. A conjugate as claimed in claim 1, wherein the bridging group is as further defined in any one of claims 6 to
15.17. A conjugating reagent as claimed in claim 2 or claim 3, wherein the bridging group is as further defined in any one of claims 6 to 15.18. A conjugating reagent as claimed in any one of claims 1 to 3 and 14, which includes the functional grouping:VANin which L is a leaving group, or which includes the functional grouping: vtArN in which L is a leaving group or which includes the functional grouping: 19. A conjugating reagent as claimed in any one of claims 1 to 3, 14 and 15, which contains the functional grouping BI, Br, BB or BIII:A-L/\./NW (BI) in which W represents an electron-withdrawing group; A represents a C1_5 alkylene or alkenylene chain; B represents a bond or a CiA alkylene or alkenylene chain; and each L is a leaving group;A-LW H m (BI')in which W and A have the meanings given for the general formula BI, m is 0 to 4, and L is a leaving group; -W-CR4R4'-CR4.L.L' (BII) in which W has the meaning given for the general formula BI, and either (i) each R4 represents a hydrogen atom or a Ci_Lialkyl group, R4irepresents a hydrogen atom, and L is a leaving group and L' is a leaving group; or (ii) each R4 represents a hydrogen atom or a C1_ 4alkyl group, L represents a leaving group which includes a portion -(CH2CH20).-in which n is a number of two or more, and R4' and L' together represent a bond; -W-(CH=CH)p-(CH2)2-L (BIII) in which W has the meaning given for the general formula BI, p represents 0 or an integer of from 1 to 4, and L represents a leaving group.20. A conjugating reagent as claimed in claim 19 which has the general formula:D A-L D^D D OfA-LB-L (BIc) or D2Bd-Q-W-CR2R2'-CR2.L.0 (BlIa) D2Bd-Q-(CH=CH)p-(CH2)2-L (BIIIa) or D2Bd-Q-(CH=CH)p-CH=CH2H(BIc') or in which Q represents a linking group and D2Bd represents a maytansine-containing payload consisting of two maytansine moieties linked to each other through a non-degradable bridging group.21. A conjugating reagent as claimed in claim 19, which contains the functional grouping:L(Bib) or (BIb) -NH-CO-Ax-00-(CH2)2-L (BIIIa) or -NH-CO-Ar-CO-CH=CH2 in which Ar represents a phenyl group.22. A conjugating reagent as claimed in claim 21, which has the general formula: D 0 I3dD Q(Bic) or D,Bd Q (Bie) or D2Bd-Q-NH-CO-Ar-00-(CH2)2-L (13111b) or D2Bd-Q-NH-CO-Ar-CO-CH=CH2 (BMW) in which Q represents a linking group and D2Bd represents a maytansine-containing payload consisting of two maytansine moieties linked to each other through a non-degradable bridging group..23. A process for the conjugation of a peptide, protein and/or polymer, which comprises reacting said peptide, protein and/or polymer with a conjugating reagent as claimed in any one of claims 2 to 5 and 16 to 22.24. A process as claimed in claim 23, in which the protein is a receptor or ligand binding protein or an antibody or antibody fragment.25. A protein, peptide and/or polymer conjugate prepared by a process as claimed in either claim 23 or claim 24.